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Sample records for protein phosphatase pp2aa

  1. Overexpression of the protein phosphatase 2A regulatory subunit a gene ZmPP2AA1 improves low phosphate tolerance by remodeling the root system architecture of maize.

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    Jiemin Wang

    Full Text Available Phosphate (Pi limitation is a constraint for plant growth and development in many natural and agricultural ecosystems. In this study, a gene encoding Zea mays L. protein phosphatase 2A regulatory subunit A, designated ZmPP2AA1, was induced in roots by low Pi availability. The function of the ZmPP2AA1 gene in maize was analyzed using overexpression and RNA interference. ZmPP2AA1 modulated root gravitropism, negatively regulated primary root (PR growth, and stimulated the development of lateral roots (LRs. A detailed characterization of the root system architecture (RSA in response to different Pi concentrations with or without indole-3-acetic acid and 1-N-naphthylphthalamic acid revealed that auxin was involved in the RSA response to low Pi availability. Overexpression of ZmPP2AA1 enhanced tolerance to Pi starvation in transgenic maize in hydroponic and soil pot experiments. An increased dry weight (DW, root-to-shoot ratio, and total P content and concentration, along with a delayed and reduced accumulation of anthocyanin in overexpressing transgenic maize plants coincided with their highly branched root system and increased Pi uptake capability under low Pi conditions. Inflorescence development of the ZmPP2AA1 overexpressing line was less affected by low Pi stress, resulting in higher grain yield per plant under Pi deprivation. These data reveal the biological function of ZmPP2AA1, provide insights into a linkage between auxin and low Pi responses, and drive new strategies for the efficient utilization of Pi by maize.

  2. Displacement affinity chromatography of protein phosphatase one (PP1 complexes

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    Gourlay Robert

    2008-11-01

    Full Text Available Abstract Background Protein phosphatase one (PP1 is a ubiquitously expressed, highly conserved protein phosphatase that dephosphorylates target protein serine and threonine residues. PP1 is localized to its site of action by interacting with targeting or regulatory proteins, a majority of which contains a primary docking site referred to as the RVXF/W motif. Results We demonstrate that a peptide based on the RVXF/W motif can effectively displace PP1 bound proteins from PP1 retained on the phosphatase affinity matrix microcystin-Sepharose. Subsequent co-immunoprecipitation experiments confirmed that each identified binding protein was either a direct PP1 interactor or was in a complex that contains PP1. Our results have linked PP1 to numerous new nuclear functions and proteins, including Ki-67, Rif-1, topoisomerase IIα, several nuclear helicases, NUP153 and the TRRAP complex. Conclusion This modification of the microcystin-Sepharose technique offers an effective means of purifying novel PP1 regulatory subunits and associated proteins and provides a simple method to uncover a link between PP1 and additional cellular processes.

  3. Recent insights into Protein Phosphatase 2A structure and regulation: the reasons why PP2A is no longer considered as a lazy passive housekeeping enzyme

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    Martin, M.

    2010-01-01

    Full Text Available Although intracellular signal transduction is often portrayed as a protein kinase "domino effect", the counterbalancing function of phosphatases, and thus the control of phosphatase activity, is equally relevant to proper regulation of cellular function. Protein Phosphatase 2A (PP2A is a widely expressed family of protein phosphatases made of a core dimer, composed of a catalytic (C subunit and a structural (A subunit, in association with a third variable regulatory (B subunit. Although viewed as a constitutive housekeeping enzyme in the past, PP2A is a highly regulated phosphatase and is emerging as an important regulator of multiple cellular processes involving protein phosphorylation. The regulation of PP2A is mainly accomplished by the identity of the regulatory B-type subunit, which determines substrate specificity, subcellular localization and catalytic activity of the PP2A holoenzyme. In agreement with this, recent findings on the structure and post-translational modifications of PP2A emphasize the importance of PP2A holoenzyme composition in its regulation and pleiotropic activities.

  4. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    International Nuclear Information System (INIS)

    Liu, Xin; Zhu, Yanming; Zhai, Hong; Cai, Hua; Ji, Wei; Luo, Xiao; Li, Jing; Bai, Xi

    2012-01-01

    Highlights: ► AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. ► AtPP2CG1 up-regulates the expression of marker genes in different pathways. ► AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2–3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter–GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  5. AtPP2CG1, a protein phosphatase 2C, positively regulates salt tolerance of Arabidopsis in abscisic acid-dependent manner

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Xin, E-mail: fangfei6073@126.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhu, Yanming, E-mail: ymzhu2001@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Zhai, Hong, E-mail: Zhai.h@neigaehrb.ac.cn [Northeast Institute of Geography and Agroecology, Chinese Academy of Sciences, Harbin 150040 (China); Cai, Hua, E-mail: small-big@sohu.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Ji, Wei, E-mail: iwei_j@hotmail.com [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Luo, Xiao, E-mail: luoxiao2010@yahoo.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China); Li, Jing, E-mail: lijing@neau.edu.cn [Plant Secondary Metabolism Laboratory, Northeast Agricultural University, Harbin 150030 (China); Bai, Xi, E-mail: baixi@neau.edu.cn [Plant Bioengineering Laboratory, Northeast Agricultural University, Harbin 150030 (China)

    2012-06-15

    Highlights: Black-Right-Pointing-Pointer AtPP2CG1 positively regulates salt tolerance in ABA-dependent manner. Black-Right-Pointing-Pointer AtPP2CG1 up-regulates the expression of marker genes in different pathways. Black-Right-Pointing-Pointer AtPP2CG1 expresses in the vascular system and trichomes of Arabidopsis. -- Abstract: AtPP2CG1 (Arabidopsis thaliana protein phosphatase 2C G Group 1) was predicted as an abiotic stress candidate gene by bioinformatic analysis in our previous study. The gene encodes a putative protein phosphatase 2C that belongs to Group G of PP2C. There is no report of Group G genes involved in abiotic stress so far. Real-time RT-PCR analysis showed that AtPP2CG1 expression was induced by salt, drought, and abscisic acid (ABA) treatment. The expression levels of AtPP2CG1 in the ABA synthesis-deficient mutant abi2-3 were much lower than that in WT plants under salt stress suggesting that the expression of AtPP2CG1 acts in an ABA-dependent manner. Over-expression of AtPP2CG1 led to enhanced salt tolerance, whereas its loss of function caused decreased salt tolerance. These results indicate that AtPP2CG1 positively regulates salt stress in an ABA-dependent manner. Under salt treatment, AtPP2CG1 up-regulated the expression levels of stress-responsive genes, including RD29A, RD29B, DREB2A and KIN1. GUS activity was detected in roots, leaves, stems, flower, and trichomes of AtPP2CG1 promoter-GUS transgenic plants. AtPP2CG1 protein was localized in nucleus and cytoplasm via AtPP2CG1:eGFP and YFP:AtPP2CG1 fusion approaches.

  6. A novel tetratricopeptide repeat (TPR containing PP5 serine/threonine protein phosphatase in the malaria parasite, Plasmodium falciparum

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    Adams Brian

    2001-11-01

    Full Text Available Abstract Background The malarial parasite, Plasmodium falciparum (Pf, is responsible for nearly 2 million deaths worldwide. However, the mechanisms of cellular signaling in the parasite remain largely unknown. Recent discovery of a few protein kinases and phosphatases point to a thriving reversible phosphorylation system in the parasite, although their function and regulation need to be determined. Results We provide biochemical and sequence evidence for a protein serine/threonine phosphatase type PP5 in Plasmodium falciparum, and named it PfPP5. The 594-amino acid polypeptide was encoded by a 1785 nucleotide long intronless gene in the parasite. The recombinant protein, expressed in bacteria, was indistinguishable from native PfPP5. Sequencing comparison indicated that the extra-long N-terminus of PfPP5 outside the catalytic core contained four tetratricopeptide repeats (TPRs, compared to three such repeats in other PP5 phosphatases. The PfPP5 N-terminus was required for stimulation of the phosphatase activity by polyunsaturated fatty acids. Co-immunoprecipitation demonstrated an interaction between native PfPP5 and Pf heat shock protein 90 (hsp90. PfPP5 was expressed in all the asexual erythrocytic stages of the parasite, and was moderately sensitive to okadaic acid. Conclusions This is the first example of a TPR-domain protein in the Apicomplexa family of parasites. Since TPR domains play important roles in protein-protein interaction, especially relevant to the regulation of PP5 phosphatases, PfPP5 is destined to have a definitive role in parasitic growth and signaling pathways. This is exemplified by the interaction between PfPP5 and the cognate chaperone hsp90.

  7. Protein phosphatase 2A dysfunction in Alzheimer’s disease

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    Jean-Marie eSontag

    2014-03-01

    Full Text Available Protein Phosphatase 2A (PP2A is a large family of enzymes that account for the majority of brain Ser/Thr phosphatase activity. While PP2A enzymes collectively modulate most cellular processes, sophisticated regulatory mechanisms are ultimately responsible for ensuring isoform-specific substrate specificity. Of particular interest to the Alzheimer’s disease (AD field, alterations in PP2A regulators and PP2A catalytic activity, subunit expression, methylation and/or phosphorylation, have been reported in AD-affected brain regions. PP2A dysfunction has been linked to Tau hyperphosphorylation, amyloidogenesis and synaptic deficits that are pathological hallmarks of this neurodegenerative disorder. Deregulation of PP2A enzymes also affects the activity of many Ser/Thr protein kinases implicated in AD. This review will more specifically discuss the role of the PP2A/B holoenzyme and PP2A methylation in AD pathogenesis. The PP2A/B isoform binds to tau and is the primary tau phosphatase. Its deregulation correlates with increased tau phosphorylation in vivo and in AD. Disruption of PP2A/B-Tau protein interactions likely contribute to Tau deregulation in AD. Significantly, alterations in one-carbon metabolism that impair PP2A methylation are associated with increased risk for sporadic AD, and enhanced AD-like pathology in animal models. Experimental studies have linked deregulation of PP2A methylation with down-regulation of PP2A/B, enhanced phosphorylation of Tau and amyloid precursor protein, Tau mislocalization, microtubule destabilization and neuritic defects. While it remains unclear what are the primary events that underlie PP2A dysfunction in AD, deregulation of PP2A enzymes definitely affects key players in the pathogenic process. As such, there is growing interest in developing PP2A-centric therapies for AD, but this may be a daunting task without a better understanding of the regulation and function of specific PP2A enzymes.

  8. Catalytic activity of a novel serine/threonine protein phosphatase PP5 from Leishmania major

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    Norris-Mullins Brianna

    2014-01-01

    Full Text Available Leishmaniasis is a vector-borne disease caused by protozoan parasites of the genus Leishmania. Our knowledge of protein phosphatases (PPs and their implication in signaling events is very limited. Here we report the expression, characterization and mutagenesis analysis of a novel protein phosphatase 5 (PP5 in Leishmania major. Recombinant PP5 is a bona fide phosphatase and is enzymatically active. Site-directed mutagenesis revealed auto-inhibitory roles of the N-terminal region. This is a rational first approach to understand the role of PP5 in the biology of the parasite better as well as its potential future applicability to anti-parasitic intervention.

  9. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    International Nuclear Information System (INIS)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2012-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  10. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

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    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M.H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric (Van Andel); (Scripps); (NWU); (Purdue); (UCR); (Chinese Aca. Sci.); (NU Singapore)

    2014-10-02

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites.

  11. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    Science.gov (United States)

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun; Yong, Eu-Leong; Cutler, Sean; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2013-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, while the conserved ABA-sensing tryptophan of PP2C inserts into the kinase catalytic cleft, thus mimicking receptor-PP2C interactions. These structural results provide a simple mechanism that directly couples ABA binding to SnRK2 kinase activation and highlight a new paradigm of kinase-phosphatase regulation through mutual packing of their catalytic sites. PMID:22116026

  12. Molecular Mimicry Regulates ABA Signaling by SnRK2 Kinases and PP2C Phosphatases

    OpenAIRE

    Soon, Fen-Fen; Ng, Ley-Moy; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Tan, M. H. Eileen; Suino-Powell, Kelly M.; He, Yuanzheng; Xu, Yong; Chalmers, Michael J.; Brunzelle, Joseph S.; Zhang, Huiming; Yang, Huaiyu; Jiang, Hualiang; Li, Jun

    2011-01-01

    Abscisic acid (ABA) is an essential hormone for plants to survive environmental stresses. At the center of the ABA signaling network is a subfamily of type 2C protein phosphatases (PP2Cs), which form exclusive interactions with ABA receptors and subfamily 2 Snfl-related kinase (SnRK2s). Here, we report a SnRK2-PP2C complex structure, which reveals marked similarity in PP2C recognition by SnRK2 and ABA receptors. In the complex, the kinase activation loop docks into the active site of PP2C, wh...

  13. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    Energy Technology Data Exchange (ETDEWEB)

    Boylan, Joan M. [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Salomon, Arthur R. [Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States); Department of Chemistry, Brown University, Providence, RI (United States); Tantravahi, Umadevi [Division of Genetics, Department of Pathology, Brown University and Women and Infants Hospital, Providence, RI (United States); Gruppuso, Philip A., E-mail: philip_gruppuso@brown.edu [Department of Pediatrics, Brown University and Rhode Island Hospital, Providence, RI (United States); Department of Molecular Biology, Cell Biology and Biochemistry, Brown University, Providence, RI (United States)

    2015-07-15

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair.

  14. Adaptation of HepG2 cells to a steady-state reduction in the content of protein phosphatase 6 (PP6) catalytic subunit

    International Nuclear Information System (INIS)

    Boylan, Joan M.; Salomon, Arthur R.; Tantravahi, Umadevi; Gruppuso, Philip A.

    2015-01-01

    Protein phosphatase 6 (PP6) is a ubiquitous Ser/Thr phosphatase involved in an array of cellular processes. To assess the potential of PP6 as a therapeutic target in liver disorders, we attenuated expression of the PP6 catalytic subunit in HepG2 cells using lentiviral-transduced shRNA. Two PP6 knock-down (PP6KD) cell lines (90% reduction of PP6-C protein content) were studied in depth. Both proliferated at a rate similar to control cells. However, flow cytometry indicated G2/M cell cycle arrest that was accounted for by a shift of the cells from a diploid to tetraploid state. PP6KD cells did not show an increase in apoptosis, nor did they exhibit reduced viability in the presence of bleomycin or taxol. Gene expression analysis by microarray showed attenuated anti-inflammatory signaling. Genes associated with DNA replication were downregulated. Mass spectrometry-based phosphoproteomic analysis yielded 80 phosphopeptides representing 56 proteins that were significantly affected by a stable reduction in PP6-C. Proteins involved in DNA replication, DNA damage repair and pre-mRNA splicing were overrepresented among these. PP6KD cells showed intact mTOR signaling. Our studies demonstrated involvement of PP6 in a diverse set of biological pathways and an adaptive response that may limit the effectiveness of targeting PP6 in liver disorders. - Highlights: • Lentiviral-transduced shRNA was used to generate a stable knockdown of PP6 in HepG2 cells. • Cells adapted to reduced PP6; cell proliferation was unaffected, and cell survival was normal. • However, PP6 knockdown was associated with a transition to a tetraploid state. • Genomic profiling showed downregulated anti-inflammatory signaling and DNA replication. • Phosphoproteomic profiling showed changes in proteins associated with DNA replication and repair

  15. Probing protein phosphatase substrate binding

    DEFF Research Database (Denmark)

    Højlys-Larsen, Kim B.; Sørensen, Kasper Kildegaard; Jensen, Knud Jørgen

    2012-01-01

    Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity...... profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides......, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL...

  16. TaPP2C1, a Group F2 Protein Phosphatase 2C Gene, Confers Resistance to Salt Stress in Transgenic Tobacco.

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    Wei Hu

    Full Text Available Group A protein phosphatases 2Cs (PP2Cs are essential components of abscisic acid (ABA signaling in Arabidopsis; however, the function of group F2 subfamily PP2Cs is currently less known. In this study, TaPP2C1 which belongs to group F2 was isolated and characterized from wheat. Expression of the TaPP2C1-GFP fusion protein suggested its ubiquitous localization within a cell. TaPP2C1 expression was downregulated by abscisic acid (ABA and NaCl treatments, but upregulated by H2O2 treatment. Overexpression of TaPP2C1 in tobacco resulted in reduced ABA sensitivity and increased salt resistance of transgenic seedlings. Additionally, physiological analyses showed that improved resistance to salt stress conferred by TaPP2C1 is due to the reduced reactive oxygen species (ROS accumulation, the improved antioxidant system, and the increased transcription of genes in the ABA-independent pathway. Finally, transgenic tobacco showed increased resistance to oxidative stress by maintaining a more effective antioxidant system. Taken together, these results demonstrated that TaPP2C1 negatively regulates ABA signaling, but positively regulates salt resistance. TaPP2C1 confers salt resistance through activating the antioxidant system and ABA-independent gene transcription process.

  17. Quantitative phosphoproteomics reveals new roles for the protein phosphatase PP6 in mitotic cells.

    Science.gov (United States)

    Rusin, Scott F; Schlosser, Kate A; Adamo, Mark E; Kettenbach, Arminja N

    2015-10-13

    Protein phosphorylation is an important regulatory mechanism controlling mitotic progression. Protein phosphatase 6 (PP6) is an essential enzyme with conserved roles in chromosome segregation and spindle assembly from yeast to humans. We applied a baculovirus-mediated gene silencing approach to deplete HeLa cells of the catalytic subunit of PP6 (PP6c) and analyzed changes in the phosphoproteome and proteome in mitotic cells by quantitative mass spectrometry-based proteomics. We identified 408 phosphopeptides on 272 proteins that increased and 298 phosphopeptides on 220 proteins that decreased in phosphorylation upon PP6c depletion in mitotic cells. Motif analysis of the phosphorylated sites combined with bioinformatics pathway analysis revealed previously unknown PP6c-dependent regulatory pathways. Biochemical assays demonstrated that PP6c opposed casein kinase 2-dependent phosphorylation of the condensin I subunit NCAP-G, and cellular analysis showed that depletion of PP6c resulted in defects in chromosome condensation and segregation in anaphase, consistent with dysregulation of condensin I function in the absence of PP6 activity. Copyright © 2015, American Association for the Advancement of Science.

  18. Spatial control of protein phosphatase 2A (de)methylation

    International Nuclear Information System (INIS)

    Longin, Sari; Zwaenepoel, Karen; Martens, Ellen; Louis, Justin V.; Rondelez, Evelien; Goris, Jozef; Janssens, Veerle

    2008-01-01

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2A C ) is an important regulatory mechanism playing a crucial role in the selective recruitment of regulatory B subunits. Here, we investigated the subcellular localization of leucine carboxyl methyltransferase (LCMT1) and protein phosphatase methylesterase (PME-1), the two enzymes catalyzing this process. The results show that PME-1 is predominantly localized in the nucleus and harbors a functional nuclear localization signal, whereas LCMT1 is underrepresented in the nucleus and mainly localizes to the cytoplasm, Golgi region and late endosomes. Indirect immunofluorescence with methylation-sensitive anti-PP2A C antibodies revealed a good correlation with the methylation status of PP2A C , demethylated PP2A C being substantially nuclear. Throughout mitosis, demethylated PP2A C is associated with the mitotic spindle and during cytokinesis with the cleavage furrow. Overexpression of PME-1, but not of an inactive mutant, results in increased demethylation of PP2A C in the nucleus, whereas overexpression of a cytoplasmic PME-1 mutant lacking the NLS results in increased demethylation in the cytoplasm-in all cases, however, without any obvious functional consequences. PME-1 associates with an inactive PP2A population, regardless of its esterase activity or localization. We propose that stabilization of this inactive, nuclear PP2A pool is a major in vivo function of PME-1

  19. Overexpression of PP2A-C5 that encodes the catalytic subunit 5 of protein phosphatase 2A in Arabidopsis confers better root and shoot development under salt conditions

    Science.gov (United States)

    Protein phosphatase 2A (PP2A) is an enzyme consisting of three subunits: a scaffolding A subunit, a regulatory B subunit and a catalytic C subunit. PP2As were shown to play diverse roles in eukaryotes. In this study, the function of the Arabidopsis PP2A-C5 gene that encodes the catalytic subunit 5 o...

  20. Pharmacological Activation of Protein Phosphatase 2 A (PP2A): A Novel Strategy to Fight Against Human Malignancies?

    Science.gov (United States)

    Carratù, Maria Rosaria; Signorile, Anna; De Rasmo, Domenico; Reale, Antonia; Vacca, Angelo

    2016-01-01

    The serine-threonine protein phosphatase 2A (PP2A) regulates multiple cell signaling cascades and its inactivation by viral oncoproteins, mutation of specific structural subunits or upregulation of the cellular endogenous inhibitors may contribute to malignant transformation by regulating specific phosphorylation events. Pharmacological modulation of PP2A activity is becoming an attractive strategy for cancer treatment. Some compounds targeting PP2A are able to induce PP2A reactivation and subsequent cell death in several types of cancer. We undertook a search of bibliographic databases for peer-reviewed articles focusing on the main item of the review. We selected articles published in indexed journals. The quality of retrieved papers was appraised using the standard bibliometric indicators. One hundred and fourteen papers were included in the review. Twenty-seven papers gave an overview of structure and physiological role of PP2A. Twenty-five papers outlined the role of PP2A in tumor suppression. Forty papers analyzed the mechanism involved in PP2A reactivation by synthetic compounds, and twenty-two papers outlined the capability of natural compounds of restoring PP2A activity and how this could be beneficial. Findings analyzed in this review underline the central role of PP2A as a regulator of cell growth and survival, hence its function as tumor suppressor. The discovery that some compounds, either synthetic or natural, are capable of reactivating PP2A opens up new perspectives for future strategies to fully exploit therapeutic potential in human cancer. Thus, this review could also be of particular interest to pharmaceutical or biotechnology companies for drug design and targeted delivery.

  1. ABI1 and PP2CA Phosphatases Are Negative Regulators of Snf1-Related Protein Kinase1 Signaling in Arabidopsis

    OpenAIRE

    Rodrigues, A.; Adamo, M.; Crozet, P.; Margalha, L.; Confraria, A.; Martinho, C.; Elias, A.; Rabissi, A.; Lumbreras, V.; Gonzalez-Guzman, M.; Antoni, R.; Rodriguez, P. L.; Baena-Gonzalez, E.

    2013-01-01

    Plant survival under environmental stress requires the integration of multiple signaling pathways into a coordinated response, but the molecular mechanisms underlying this integration are poorly understood. Stress-derived energy deprivation activates the Snf1-related protein kinases1 (SnRK1s), triggering a vast transcriptional and metabolic reprogramming that restores homeostasis and promotes tolerance to adverse conditions. Here, we show that two clade A type 2C protein phosphatases (PP2Cs),...

  2. Identification of Open Stomata1-Interacting Proteins Reveals Interactions with Sucrose Non-fermenting1-Related Protein Kinases2 and with Type 2A Protein Phosphatases That Function in Abscisic Acid Responses1[OPEN

    Science.gov (United States)

    Waadt, Rainer; Manalansan, Bianca; Rauniyar, Navin; Munemasa, Shintaro; Booker, Matthew A.; Brandt, Benjamin; Waadt, Christian; Nusinow, Dmitri A.; Kay, Steve A.; Kunz, Hans-Henning; Schumacher, Karin; DeLong, Alison; Yates, John R.; Schroeder, Julian I.

    2015-01-01

    The plant hormone abscisic acid (ABA) controls growth and development and regulates plant water status through an established signaling pathway. In the presence of ABA, pyrabactin resistance/regulatory component of ABA receptor proteins inhibit type 2C protein phosphatases (PP2Cs). This, in turn, enables the activation of Sucrose Nonfermenting1-Related Protein Kinases2 (SnRK2). Open Stomata1 (OST1)/SnRK2.6/SRK2E is a major SnRK2-type protein kinase responsible for mediating ABA responses. Arabidopsis (Arabidopsis thaliana) expressing an epitope-tagged OST1 in the recessive ost1-3 mutant background was used for the copurification and identification of OST1-interacting proteins after osmotic stress and ABA treatments. These analyses, which were confirmed using bimolecular fluorescence complementation and coimmunoprecipitation, unexpectedly revealed homo- and heteromerization of OST1 with SnRK2.2, SnRK2.3, OST1, and SnRK2.8. Furthermore, several OST1-complexed proteins were identified as type 2A protein phosphatase (PP2A) subunits and as proteins involved in lipid and galactolipid metabolism. More detailed analyses suggested an interaction network between ABA-activated SnRK2-type protein kinases and several PP2A-type protein phosphatase regulatory subunits. pp2a double mutants exhibited a reduced sensitivity to ABA during seed germination and stomatal closure and an enhanced ABA sensitivity in root growth regulation. These analyses add PP2A-type protein phosphatases as another class of protein phosphatases to the interaction network of SnRK2-type protein kinases. PMID:26175513

  3. Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a.

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    Mariem Bradai

    Full Text Available Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1, which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s were reported to play relevant roles in controlling multiple cellular processes, plant orthologs remain poorly studied. To decipher the role of plant PP1s, we compared PP1 genes from three monocot species, Brachypodium, common wheat and rice at the genomic and transcriptomic levels. To gain more insight into the wheat PP1 proteins, we identified and characterized TdPP1a, the first wheat type one protein phosphatase from a Tunisian durum wheat variety Oum Rabiaa3. TdPP1a is highly conserved in sequence and structure when compared to mammalian, yeast and other plant PP1s. We demonstrate that TdPP1a is an active, metallo-dependent phosphatase in vitro and is able to interact with AtI2, a typical regulator of PP1 functions. Also, TdPP1a is capable to complement the heat stress sensitivity of the yeast mutant indicating that TdPP1a is functional also in vivo. Moreover, transient expression of TdPP1a::GFP in tobacco leaves revealed that it is ubiquitously distributed within the cell, with a strong accumulation in the nucleus. Finally, transcriptional analyses showed similar expression levels in roots and leaves of durum wheat seedlings. Interestingly, the expression in leaves is significantly induced following salinity stress, suggesting a potential role of TdPP1a in wheat salt stress response.

  4. Protein Phosphatase 2A in the Regulation of Wnt Signaling, Stem Cells, and Cancer.

    Science.gov (United States)

    Thompson, Joshua J; Williams, Christopher S

    2018-02-26

    Protein phosphorylation is a ubiquitous cellular process that allows for the nuanced and reversible regulation of protein activity. Protein phosphatase 2A (PP2A) is a heterotrimeric serine-threonine phosphatase-composed of a structural, regulatory, and catalytic subunit-that controls a variety of cellular events via protein dephosphorylation. While much is known about PP2A and its basic biochemistry, the diversity of its components-especially the multitude of regulatory subunits-has impeded the determination of PP2A function. As a consequence of this complexity, PP2A has been shown to both positively and negatively regulate signaling networks such as the Wnt pathway. Wnt signaling modulates major developmental processes, and is a dominant mediator of stem cell self-renewal, cell fate, and cancer stem cells. Because PP2A affects Wnt signaling both positively and negatively and at multiple levels, further understanding of this complex dynamic may ultimately provide insight into stem cell biology and how to better treat cancers that result from alterations in Wnt signaling. This review will summarize literature that implicates PP2A as a tumor suppressor, explore PP2A mutations identified in human malignancy, and focus on PP2A in the regulation of Wnt signaling and stem cells so as to better understand how aberrancy in this pathway can contribute to tumorigenesis.

  5. SAV1 promotes Hippo kinase activation through antagonizing the PP2A phosphatase STRIPAK

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    Bae, Sung Jun [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Ni, Lisheng [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Osinski, Adam [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Tomchick, Diana R. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Brautigam, Chad A. [Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States; Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, United States; Luo, Xuelian [Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas, United States; Department of Biophysics, University of Texas Southwestern Medical Center, Dallas, United States

    2017-10-24

    The Hippo pathway controls tissue growth and homeostasis through a central MST-LATS kinase cascade. The scaffold protein SAV1 promotes the activation of this kinase cascade, but the molecular mechanisms remain unknown. Here, we discover SAV1-mediated inhibition of the PP2A complex STRIPAKSLMAP as a key mechanism of MST1/2 activation. SLMAP binding to autophosphorylated MST2 linker recruits STRIPAK and promotes PP2A-mediated dephosphorylation of MST2 at the activation loop. Our structural and biochemical studies reveal that SAV1 and MST2 heterodimerize through their SARAH domains. Two SAV1–MST2 heterodimers further dimerize through SAV1 WW domains to form a heterotetramer, in which MST2 undergoes trans-autophosphorylation. SAV1 directly binds to STRIPAK and inhibits its phosphatase activity, protecting MST2 activation-loop phosphorylation. Genetic ablation of SLMAP in human cells leads to spontaneous activation of the Hippo pathway and alleviates the need for SAV1 in Hippo signaling. Thus, SAV1 promotes Hippo activation through counteracting the STRIPAKSLMAP PP2A phosphatase complex.

  6. Characterisation and expression of a PP1 serine/threonine protein phosphatase (PfPP1 from the malaria parasite, Plasmodium falciparum: demonstration of its essential role using RNA interference

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    Musiyenko Alla

    2002-04-01

    Full Text Available Abstract Background Reversible protein phosphorylation is relatively unexplored in the intracellular protozoa of the Apicomplexa family that includes the genus Plasmodium, to which belong the causative agents of malaria. Members of the PP1 family represent the most highly conserved protein phosphatase sequences in phylogeny and play essential regulatory roles in various cellular pathways. Previous evidence suggested a PP1-like activity in Plasmodium falciparum, not yet identified at the molecular level. Results We have identified a PP1 catalytic subunit from P. falciparum and named it PfPP1. The predicted primary structure of the 304-amino acid long protein was highly similar to PP1 sequences of other species, and showed conservation of all the signature motifs. The purified recombinant protein exhibited potent phosphatase activity in vitro. Its sensitivity to specific phosphatase inhibitors was characteristic of the PP1 class. The authenticity of the PfPP1 cDNA was further confirmed by mutational analysis of strategic amino acid residues important in catalysis. The protein was expressed in all erythrocytic stages of the parasite. Abrogation of PP1 expression by synthetic short interfering RNA (siRNA led to inhibition of parasite DNA synthesis. Conclusions The high sequence similarity of PfPP1 with other PP1 members suggests conservation of function. Phenotypic gene knockdown studies using siRNA confirmed its essential role in the parasite. Detailed studies of PfPP1 and its regulation may unravel the role of reversible protein phosphorylation in the signalling pathways of the parasite, including glucose metabolism and parasitic cell division. The use of siRNA could be an important tool in the functional analysis of Apicomplexan genes.

  7. Protein phosphatase 2A mediates JS-K-induced apoptosis by affecting Bcl-2 family proteins in human hepatocellular carcinoma HepG2 cells.

    Science.gov (United States)

    Liu, Ling; Huang, Zile; Chen, Jingjing; Wang, Jiangang; Wang, Shuying

    2018-04-25

    Protein phosphatase 2A (PP2A) is an important enzyme within various signal transduction pathways. The present study was investigated PP2A mediates JS-K-induced apoptosis by affecting Bcl-2 family protein. JS-K showed diverse inhibitory effects in five HCC cell lines, especially HepG2 cells. JS-K caused a dose- and time-dependent reduction in cell viability and increased in levels of LDH release. Meanwhile, JS-K- induced apoptosis was characterized by mitochondrial membrane potential reduction, Hoechst 33342 + /PI + dual staining, release of cytochrome c (Cyt c), and activation of cleaved caspase-9/3. Moreover, JS-K-treatment could lead to the activation of protein phosphatase 2A-C (PP2A-C), decrease of anti-apoptotic Bcl-2 family-protein expression including p-Bcl-2 (Ser70), Bcl-2, Bcl-xL, and Mcl-1 as well as the increase of pro-apoptosis Bcl-2 family-protein including Bim, Bad, Bax, and Bak. Furthermore, JS-K caused a marked increase of intracellular NO levels while pre-treatment with Carboxy-PTIO (a NO scavenger) reduced the cytotoxicity effects and the apoptosis rate. Meanwhile, pre-treatment with Carboxy-PTIO attenuated the JS-K-induced up-regulation of PP2A, Cyt c, and cleaved-caspase-9/3 activation. The silencing PP2A-C by siRNA could abolish the activation of PP2A-C, down-regulation of anti-apoptotic Bcl-2 family-protein (p-Bcl-2, Bcl-2, Bcl-xL, and Mcl-1), increase of pro-apoptosis Bcl-2 family-protein (Bim, Bad, Bax, and Bak) and apoptotic-related protein (Cyt c, cleaved caspase-9/3) that were caused by JS-K in HepG2 cells. In addition, pre-treatment with OA (a PP2A inhibitor) also attenuated the above effects induced by JS-K. In summary, NO release from JS-K induces apoptosis through PP2A activation, which contributed to the regulation of Bcl-2 family proteins. © 2018 Wiley Periodicals, Inc.

  8. Phosphatase PP2A and microtubule-mediated pulling forces disassemble centrosomes during mitotic exit

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    Stephen J. Enos

    2018-01-01

    Full Text Available Centrosomes are microtubule-nucleating organelles that facilitate chromosome segregation and cell division in metazoans. Centrosomes comprise centrioles that organize a micron-scale mass of protein called pericentriolar material (PCM from which microtubules nucleate. During each cell cycle, PCM accumulates around centrioles through phosphorylation-mediated assembly of PCM scaffold proteins. During mitotic exit, PCM swiftly disassembles by an unknown mechanism. Here, we used Caenorhabditis elegans embryos to determine the mechanism and importance of PCM disassembly in dividing cells. We found that the phosphatase PP2A and its regulatory subunit SUR-6 (PP2ASUR-6, together with cortically directed microtubule pulling forces, actively disassemble PCM. In embryos depleted of these activities, ∼25% of PCM persisted from one cell cycle into the next. Purified PP2ASUR-6 could dephosphorylate the major PCM scaffold protein SPD-5 in vitro. Our data suggest that PCM disassembly occurs through a combination of dephosphorylation of PCM components and force-driven fragmentation of the PCM scaffold.

  9. Gardenia jasminoides Encodes an Inhibitor-2 Protein for Protein Phosphatase Type 1

    Science.gov (United States)

    Gao, Lan; Li, Hao-Ming

    2017-08-01

    Protein phosphatase-1 (PP1) regulates diverse, essential cellular processes such as cell cycle progression, protein synthesis, muscle contraction, carbohydrate metabolism, transcription and neuronal signaling. Inhibitor-2 (I-2) can inhibit the activity of PP1 and has been found in diverse organisms. In this work, a Gardenia jasminoides fruit cDNA library was constructed, and the GjI-2 cDNA was isolated from the cDNA library by sequencing method. The GjI-2 cDNA contains a predicted 543 bp open reading frame that encodes 180 amino acids. The bioinformatics analysis suggested that the GjI-2 has conserved PP1c binding motif, and contains a conserved phosphorylation site, which is important in regulation of its activity. The three-dimensional model structure of GjI-2 was buite, its similar with the structure of I-2 from mouse. The results suggest that GjI-2 has relatively conserved RVxF, FxxR/KxR/K and HYNE motif, and these motifs are involved in interaction with PP1.

  10. Arctigenin inhibits triple-negative breast cancers by targeting CIP2A to reactivate protein phosphatase 2A.

    Science.gov (United States)

    Huang, Qiuyue; Qin, Shanshan; Yuan, Xiaoning; Zhang, Liang; Ji, Juanli; Liu, Xuewen; Ma, Wenjing; Zhang, Yunfei; Liu, Pengfei; Sun, Zhiting; Zhang, Jingxuan; Liu, Ying

    2017-07-01

    We have shown that a novel STAT3 inhibitor arctigenin (Atn) induces significant cytotoxicity in triple-negative breast cancer (TNBC) cells. This study further delineated molecular mechanisms where by Atn triggered cytotoxicity in TNBC cells. We found Atn can also inhibit metastasis in TNBC cells through cancerous inhibitor of protein phosphatase 2A (CIP2A) pathway. CIP2A is an endogenous inhibitor of protein phosphatase 2A (PP2A), which can increase the migration and invasion of various cancer cells. PP2A is a tumor suppressor, which is functionally defective in various cancers. Atn-induced metastasis inhibition was associated with reactivation of PP2A, downregulation of CIP2A and Akt phosphorylation. Silencing CIP2A enhanced Atn-induced metastasis inhibition and apoptosis in TNBCs. Furthermore, ectopic expression of CIP2A or inhibition of PP2A in TNBC cells abolished the effects of Atn. In conclusion, we found that enhancement of PP2A activity by inhibition of CIP2A, at least in part, promotes the anti-metastasis effect induced by Atn. Our findings disclose the novel therapeutic mechanism of this targeted agent, and suggest the therapeutic potential and feasibility of developing PP2A enhancers as a novel anticancer strategy.

  11. An extract of Urtica dioica L. mitigates obesity induced insulin resistance in mice skeletal muscle via protein phosphatase 2A (PP2A).

    Science.gov (United States)

    Obanda, Diana N; Ribnicky, David; Yu, Yongmei; Stephens, Jacqueline; Cefalu, William T

    2016-02-26

    The leaf extract of Urtica dioica L. (UT) has been reported to improve glucose homeostasis in vivo, but definitive studies on efficacy and mechanism of action are lacking. We investigated the effects of UT on obesity- induced insulin resistance in skeletal muscle. Male C57BL/6J mice were divided into three groups: low-fat diet (LFD), high-fat diet (HFD) and HFD supplemented with UT. Body weight, body composition, plasma glucose and plasma insulin were monitored. Skeletal muscle (gastrocnemius) was analyzed for insulin sensitivity, ceramide accumulation and the post translational modification and activity of protein phosphatase 2A (PP2A). PP2A is activated by ceramides and dephosphorylates Akt. C2C12 myotubes exposed to excess free fatty acids with or without UT were also evaluated for insulin signaling and modulation of PP2A. The HFD induced insulin resistance, increased fasting plasma glucose, enhanced ceramide accumulation and PP2A activity in skeletal muscle. Supplementation with UT improved plasma glucose homeostasis and enhanced skeletal muscle insulin sensitivity without affecting body weight and body composition. In myotubes, UT attenuated the ability of FFAs to induce insulin resistance and PP2A hyperactivity without affecting ceramide accumulation and PP2A expression. UT decreased PP2A activity through posttranslational modification that was accompanied by a reduction in Akt dephosphorylation.

  12. Small G proteins Rac1 and Ras regulate serine/threonine protein phosphatase 5 (PP5)·extracellular signal-regulated kinase (ERK) complexes involved in the feedback regulation of Raf1.

    Science.gov (United States)

    Mazalouskas, Matthew D; Godoy-Ruiz, Raquel; Weber, David J; Zimmer, Danna B; Honkanen, Richard E; Wadzinski, Brian E

    2014-02-14

    Serine/threonine protein phosphatase 5 (PP5, PPP5C) is known to interact with the chaperonin heat shock protein 90 (HSP90) and is involved in the regulation of multiple cellular signaling cascades that control diverse cellular processes, such as cell growth, differentiation, proliferation, motility, and apoptosis. Here, we identify PP5 in stable complexes with extracellular signal-regulated kinases (ERKs). Studies using mutant proteins reveal that the formation of PP5·ERK1 and PP5·ERK2 complexes partially depends on HSP90 binding to PP5 but does not require PP5 or ERK1/2 activity. However, PP5 and ERK activity regulates the phosphorylation state of Raf1 kinase, an upstream activator of ERK signaling. Whereas expression of constitutively active Rac1 promotes the assembly of PP5·ERK1/2 complexes, acute activation of ERK1/2 fails to influence the phosphatase-kinase interaction. Introduction of oncogenic HRas (HRas(V12)) has no effect on PP5-ERK1 binding but selectively decreases the interaction of PP5 with ERK2, in a manner that is independent of PP5 and MAPK/ERK kinase (MEK) activity, yet paradoxically requires ERK2 activity. Additional studies conducted with oncogenic variants of KRas4B reveal that KRas(L61), but not KRas(V12), also decreases the PP5-ERK2 interaction. The expression of wild type HRas or KRas proteins fails to reduce PP5-ERK2 binding, indicating that the effect is specific to HRas(V12) and KRas(L61) gain-of-function mutations. These findings reveal a novel, differential responsiveness of PP5-ERK1 and PP5-ERK2 interactions to select oncogenic Ras variants and also support a role for PP5·ERK complexes in regulating the feedback phosphorylation of PP5-associated Raf1.

  13. Protein phosphatase 5 promotes hepatocarcinogenesis through interaction with AMP-activated protein kinase.

    Science.gov (United States)

    Chen, Yao-Li; Hung, Man-Hsin; Chu, Pei-Yi; Chao, Tzu-I; Tsai, Ming-Hsien; Chen, Li-Ju; Hsiao, Yung-Jen; Shih, Chih-Ting; Hsieh, Feng-Shu; Chen, Kuen-Feng

    2017-08-15

    The serine-threonine protein phosphatase family members are known as critical regulators of various cellular functions, such as survival and transformation. Growing evidence suggests that pharmacological manipulation of phosphatase activity exhibits therapeutic benefits. Ser/Thr protein phosphatase 5 (PP5) is known to participate in glucocorticoid receptor (GR) and stress-induced signaling cascades that regulate cell growth and apoptosis, and has been shown to be overexpressed in various human malignant diseases. However, the role of PP5 in hepatocellular carcinoma (HCC) and whether PP5 may be a viable therapeutic target for HCC treatment are unknown. Here, by analyzing HCC clinical samples obtained from 215 patients, we found that overexpression of PP5 is tumor specific and associated with worse clinical outcomes. We further characterized the oncogenic properties of PP5 in HCC cells. Importantly, both silencing of PP5 with lentiviral-mediated short hairpin RNA (shRNA) and chemical inhibition of PP5 phosphatase activity using the natural compound cantharidin/norcantharidin markedly suppressed the growth of HCC cells and tumors in vitro and in vivo. Moreover, we identified AMP-activated protein kinase (AMPK) as a novel downstream target of oncogenic PP5 and demonstrated that the antitumor mechanisms underlying PP5 inhibition involve activation of AMPK signaling. Overall, our results establish a pathological function of PP5 in hepatocarcinogenesis via affecting AMPK signaling and suggest that PP5 inhibition is an attractive therapeutic approach for HCC. Copyright © 2017 Elsevier Inc. All rights reserved.

  14. Protein phosphatase 2A regulates central sensitization in the spinal cord of rats following intradermal injection of capsaicin

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    Fang Li

    2006-03-01

    Full Text Available Abstract Background Intradermal injection of capsaicin into the hind paw of rats induces spinal cord central sensititzation, a process in which the responsiveness of central nociceptive neurons is amplified. In central sensitization, many signal transduction pathways composed of several cascades of intracellular enzymes are involved. As the phosphorylation state of neuronal proteins is strictly controlled and balanced by the opposing activities of protein kinases and phosphatases, the involvement of phosphatases in these events needs to be investigated. This study is designed to determine the influence of serine/threonine protein phosphatase type 2A (PP2A on the central nociceptive amplification process, which is induced by intradermal injection of capsaicin in rats. Results In experiment 1, the expression of PP2A protein in rat spinal cord at different time points following capsaicin or vehicle injection was examined using the Western blot method. In experiment 2, an inhibitor of PP2A (okadaic acid, 20 nM or fostriecin, 30 nM was injected into the subarachnoid space of the spinal cord, and the spontaneous exploratory activity of the rats before and after capsaicin injection was recorded with an automated photobeam activity system. The results showed that PP2A protein expression in the spinal cord was significantly upregulated following intradermal injection of capsaicin in rats. Capsaicin injection caused a significant decrease in exploratory activity of the rats. Thirty minutes after the injection, this decrease in activity had partly recovered. Infusion of a phosphatase inhibitor into the spinal cord intrathecal space enhanced the central sensitization induced by capsaicin by making the decrease in movement last longer. Conclusion These findings indicate that PP2A plays an important role in the cellular mechanisms of spinal cord central sensitization induced by intradermal injection of capsaicin in rats, which may have implications in

  15. The Ebola Virus Nucleoprotein Recruits the Host PP2A-B56 Phosphatase to Activate Transcriptional Support Activity of VP30

    DEFF Research Database (Denmark)

    Kruse, Thomas; Biedenkopf, Nadine; Hertz, Emil Peter Thrane

    2018-01-01

    Transcription of the Ebola virus genome depends on the viral transcription factor VP30 in its unphosphorylated form, but the underlying molecular mechanism of VP30 dephosphorylation is unknown. Here we show that the Ebola virus nucleoprotein (NP) recruits the host PP2A-B56 protein phosphatase......A-B56 and show that it suppresses Ebola virus transcription and infection. This work dissects the molecular mechanism of VP30 dephosphorylation by PP2A-B56, and it pinpoints this phosphatase as a potential target for therapeutic intervention....

  16. Characterization of protein phosphatase 5 from three lepidopteran insects: Helicoverpa armigera, Mythimna separata and Plutella xylostella.

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    Xi'en Chen

    Full Text Available Protein phosphatase 5 (PP5, a unique member of serine/threonine phosphatases, regulates a variety of biological processes. We obtained full-length PP5 cDNAs from three lepidopteran insects, Helicoverpa armigera, Mythimna separata and Plutella xylostella, encoding predicted proteins of 490 (55.98 kDa, 490 (55.82 kDa and 491 (56.07 kDa amino acids, respectively. These sequences shared a high identity with other insect PP5s and contained the TPR (tetratricopeptide repeat domains at N-terminal regions and highly conserved C-terminal catalytic domains. Tissue- and stage-specific expression pattern analyses revealed these three PP5 genes were constitutively expressed in all stages and in tested tissues with predominant transcription occurring at the egg and adult stages. Activities of Escherichia coli-produced recombinant PP5 proteins could be enhanced by almost 2-fold by a known PP5 activator: arachidonic acid. Kinetic parameters of three recombinant proteins against substrate pNPP were similar both in the absence or presence of arachidonic acid. Protein phosphatases inhibitors, okadaic acid, cantharidin, and endothall strongly impeded the activities of the three recombinant PP5 proteins, as well as exerted an inhibitory effect on crude protein phosphatases extractions from these three insects. In summary, lepidopteran PP5s share similar characteristics and are all sensitive to the protein phosphatases inhibitors. Our results also imply protein phosphatase inhibitors might be used in the management of lepidopteran pests.

  17. Characterization of protein phosphatase 2A acting on phosphorylated plasma membrane aquaporin of tulip petals.

    Science.gov (United States)

    Azad, Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2004-05-01

    A protein phosphatase holo-type enzyme (38, 65, and 75 kDa) preparation and a free catalytic subunit (38 kDa) purified from tulip petals were characterized as protein phosphatase 2A (PP2A) by immunological and biochemical approaches. The plasma membrane containing the putative plasma membrane aquaporin (PM-AQP) was prepared from tulip petals, phosphorylated in vitro, and used as the substrate for both of the purified PP2A preparations. Although both preparations dephosphorylated the phosphorylated PM-AQP at 20 degrees C, only the holo-type enzyme preparation acted at 5 degrees C on the phosphorylated PM-AQP with higher substrate specificity, suggesting that regulatory subunits are required for low temperature-dependent dephosphorylation of PM-AQP in tulip petals.

  18. Differential regulation of protein phosphatase 1 (PP1) isoforms in human heart failure and atrial fibrillation.

    Science.gov (United States)

    Meyer-Roxlau, Stefanie; Lämmle, Simon; Opitz, Annett; Künzel, Stephan; Joos, Julius P; Neef, Stefan; Sekeres, Karolina; Sossalla, Samuel; Schöndube, Friedrich; Alexiou, Konstantin; Maier, Lars S; Dobrev, Dobromir; Guan, Kaomei; Weber, Silvio; El-Armouche, Ali

    2017-07-01

    Protein phosphatase 1 (PP1) is a key regulator of important cardiac signaling pathways. Dysregulation of PP1 has been heavily implicated in cardiac dysfunctions. Accordingly, pharmacological targeting of PP1 activity is considered for therapeutic intervention in human cardiomyopathies. Recent evidence from animal models implicated previously unrecognized, isoform-specific activities of PP1 in the healthy and diseased heart. Therefore, this study examined the expression of the distinct PP1 isoforms PP1α, β, and γ in human heart failure (HF) and atrial fibrillation (AF) and addressed the consequences of β-adrenoceptor blocker (beta-blocker) therapy for HF patients with reduced ejection fraction on PP1 isoform expression. Using western blot analysis, we found greater abundance of PP1 isoforms α and γ but unaltered PP1β levels in left ventricular myocardial tissues from HF patients as compared to non-failing controls. However, expression of all three PP1 isoforms was higher in atrial appendages from patients with AF compared to patients with sinus rhythm. Moreover, we found that in human failing ventricles, beta-blocker therapy was associated with lower PP1α abundance and activity, as indicated by higher phosphorylation of the PP1α-specific substrate eIF2α. Greater eIF2α phosphorylation is a known repressor of protein translation, and accordingly, we found lower levels of the endoplasmic reticulum (ER) stress marker Grp78 in the very same samples. We propose that isoform-specific targeting of PP1α activity may be a novel and innovative therapeutic strategy for the treatment of human cardiac diseases by reducing ER stress conditions.

  19. Marine Longilenes, Oxasqualenoids with Ser-Thr Protein Phosphatase 2A Inhibition Activity

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    Francisco Cen-Pacheco

    2018-04-01

    Full Text Available The red seaweed Laurencia viridis is a rich source of oxygenated secondary metabolites that were derived from squalene. We report here the structures of three novel compounds, (+-longilene peroxide (1, longilene (2, and (+-prelongilene (3 that were isolated from this alga, in addition to other substances, 4 and 5, resulting from their acid-mediated degradation. The effect of compounds 1 and 3 against Ser-Thr protein phosphatase type 2A (PP2A was evaluated, showing that (+-longilene peroxide (1 inhibited PP2A (IC50 11.3 μM. In order to explain the interaction between PP2A and compounds 1 and 3, molecular docking simulations onto the PP2A enzyme-binding region were used.

  20. Genome wide identification of wheat and Brachypodium type one protein phosphatases and functional characterization of durum wheat TdPP1a

    OpenAIRE

    Bradai, Mariem; Mahjoubi, Habib; Chini, Andrea; Chabouté, Marie-Edith; Hanin, Moez; Ebel, Chantal

    2018-01-01

    Reversible phosphorylation is an essential mechanism regulating signal transduction during development and environmental stress responses. An important number of dephosphorylation events in the cell are catalyzed by type one protein phosphatases (PP1), which catalytic activity is driven by the binding of regulatory proteins that control their substrate specificity or subcellular localization. Plants harbor several PP1 isoforms accounting for large functional redundancies. While animal PP1s we...

  1. Regulation of protein phosphatase 2A during embryonic diapause process in the silkworm, Bombyx mori.

    Science.gov (United States)

    Gu, Shi-Hong; Hsieh, Hsiao-Yen; Lin, Pei-Ling

    2017-11-01

    Regulation of protein phosphorylation requires coordinated interactions between protein kinases and protein phosphatases. In the present study, we investigated regulation of protein phosphatase 2A (PP2A) during the embryonic diapause process of B. mori. An immunoblotting analysis showed that Bombyx eggs contained a catalytic C subunit, a major regulatory B subunit (B55/PR55 subunit), and a structural A subunit, with the A and B subunits undergoing differential changes between diapause and non-diapause eggs during embryonic process. In non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling of diapausing eggs at 5°C for 70days and then were transferred to 25°C, protein levels of the A and B subunits of PP2A gradually increased toward embryonic development. However, protein levels of the A and B subunits in diapause eggs remained at low levels during the first 8days after oviposition. The direct determination of PP2A enzymatic activity showed that the activity remained at low levels in diapause eggs during the first 8days after oviposition. However, in non-diapause eggs, eggs whose diapause initiation was prevented by HCl, and eggs in which diapause had been terminated by chilling, PP2A enzymatic activity sharply increased during the first several days, reached a peak during the middle embryonic development, and then greatly decreased 3 or 4days before hatching. Examination of temporal changes in mRNA expression levels of the catalytic β subunit and regulatory subunit of PP2A showed high levels in eggs whose diapause initiation was prevented by HCl compared to those in diapause eggs. These results demonstrate that the higher PP2A gene expression and PP2A A and B subunit protein levels and increased enzymatic activity are related to embryonic development of B. mori. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Protein phosphatases decrease their activity during capacitation: a new requirement for this event.

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    Janetti R Signorelli

    Full Text Available There are few reports on the role of protein phosphatases during capacitation. Here, we report on the role of PP2B, PP1, and PP2A during human sperm capacitation. Motile sperm were resuspended in non-capacitating medium (NCM, Tyrode's medium, albumin- and bicarbonate-free or in reconstituted medium (RCM, NCM plus 2.6% albumin/25 mM bicarbonate. The presence of the phosphatases was evaluated by western blotting and the subcellular localization by indirect immunofluorescence. The function of these phosphatases was analyzed by incubating the sperm with specific inhibitors: okadaic acid, I2, endothall, and deltamethrin. Different aliquots were incubated in the following media: 1 NCM; 2 NCM plus inhibitors; 3 RCM; and 4 RCM plus inhibitors. The percent capacitated sperm and phosphatase activities were evaluated using the chlortetracycline assay and a phosphatase assay kit, respectively. The results confirm the presence of PP2B and PP1 in human sperm. We also report the presence of PP2A, specifically, the catalytic subunit and the regulatory subunits PR65 and B. PP2B and PP2A were present in the tail, neck, and postacrosomal region, and PP1 was present in the postacrosomal region, neck, middle, and principal piece of human sperm. Treatment with phosphatase inhibitors rapidly (≤1 min increased the percent of sperm depicting the pattern B, reaching a maximum of ∼40% that was maintained throughout incubation; after 3 h, the percent of capacitated sperm was similar to that of the control. The enzymatic activity of the phosphatases decreased during capacitation without changes in their expression. The pattern of phosphorylation on threonine residues showed a sharp increase upon treatment with the inhibitors. In conclusion, human sperm express PP1, PP2B, and PP2A, and the activity of these phosphatases decreases during capacitation. This decline in phosphatase activities and the subsequent increase in threonine phosphorylation may be an important

  3. A Role for Protein Phosphatase 2A in Regulating p38 Mitogen Activated Protein Kinase Activation and Tumor Necrosis Factor-Alpha Expression during Influenza Virus Infection

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    Anna H. Y. Law

    2013-04-01

    Full Text Available Influenza viruses of avian origin continue to pose pandemic threats to human health. Some of the H5N1 and H9N2 virus subtypes induce markedly elevated cytokine levels when compared with the seasonal H1N1 virus. We previously showed that H5N1/97 hyperinduces tumor necrosis factor (TNF-alpha through p38 mitogen activated protein kinase (MAPK. However, the detailed mechanisms of p38MAPK activation and TNF-alpha hyperinduction following influenza virus infections are not known. Negative feedback regulations of cytokine expression play important roles in avoiding overwhelming production of proinflammatory cytokines. Here we hypothesize that protein phosphatases are involved in the regulation of cytokine expressions during influenza virus infection. We investigated the roles of protein phosphatases including MAPK phosphatase-1 (MKP-1 and protein phosphatase type 2A (PP2A in modulating p38MAPK activation and downstream TNF-alpha expressions in primary human monocyte-derived macrophages (PBMac infected with H9N2/G1 or H1N1 influenza virus. We demonstrate that H9N2/G1 virus activated p38MAPK and hyperinduced TNF-alpha production in PBMac when compared with H1N1 virus. H9N2/G1 induced PP2A activity in PBMac and, with the treatment of a PP2A inhibitor, p38MAPK phosphorylation and TNF-alpha production were further increased in the virus-infected macrophages. However, H9N2/G1 did not induce the expression of PP2A indicating that the activation of PP2A is not mediated by p38MAPK in virus-infected PBMac. On the other hand, PP2A may not be the targets of H9N2/G1 in the upstream of p38MAPK signaling pathways since H1N1 also induced PP2A activation in primary macrophages. Our results may provide new insights into the control of cytokine dysregulation.

  4. Purification and characterization of protein phosphatase 2A from petals of the tulip Tulipa gesnerina.

    Science.gov (United States)

    Azad, Md Abul Kalam; Sawa, Yoshihiro; Ishikawa, Takahiro; Shibata, Hitoshi

    2006-11-30

    The holoenzyme of protein phosphatase (PP) from tulip petals was purified by using hydrophobic interaction, anion exchange and microcystin affinity chromatography to analyze activity towards p-nitrophenyl phosphate (p-NPP). The catalytic subunit of PP was released from its endogenous regulatory subunits by ethanol precipitation and further purified. Both preparations were characterized by immunological and biochemical approaches to be PP2A. On SDS-PAGE, the final purified holoenzyme preparation showed three protein bands estimated at 38, 65, and 75 kDa while the free catalytic subunit preparation showed only the 38 kDa protein. In both preparations, the 38 kDa protein was identified immunologically as the catalytic subunit of PP2A by using a monoclonal antibody against the PP2A catalytic subunit. The final 623- and 748- fold purified holoenzyme and the free catalytic preparations, respectively, exhibited high sensitivity to inhibition by 1 nM okadaic acid when activity was measured with p-NPP. The holoenzyme displayed higher stimulation in the presence of ammonium sulfate than the free catalytic subunit did by protamine, thereby suggesting different enzymatic behaviors.

  5. Antihelminthic drug niclosamide inhibits CIP2A and reactivates tumor suppressor protein phosphatase 2A in non-small cell lung cancer cells.

    Science.gov (United States)

    Kim, Myeong-Ok; Choe, Min Ho; Yoon, Yi Na; Ahn, Jiyeon; Yoo, Minjin; Jung, Kwan-Young; An, Sungkwan; Hwang, Sang-Gu; Oh, Jeong Su; Kim, Jae-Sung

    2017-11-15

    Protein phosphatase 2A (PP2A) is a critical tumor suppressor complex responsible for the inactivation of various oncogenes. Recently, PP2A reactivation has emerged asan anticancer strategy. Cancerous inhibitor of protein phosphatase 2A (CIP2A), an endogenous inhibitor of PP2A, is upregulated in many cancer cells, including non-small cell lung cancer (NSCLC) cells. We demonstrated that the antihelminthic drug niclosamide inhibited the expression of CIP2A and reactivated the tumor suppressor PP2A in NSCLC cells. We performed a drug-repurposing screen and identified niclosamide asa CIP2A suppressor in NSCLC cells. Niclosamide inhibited cell proliferation, colony formation, and tumor sphere formation, and induced mitochondrial dysfunction through increased mitochondrial ROS production in NSCLC cells; however, these effects were rescued by CIP2A overexpression, which indicated that the antitumor activity of niclosamide was dependent on CIP2A. We found that niclosamide increased PP2A activity through CIP2A inhibition, which reduced the phosphorylation of several oncogenic proteins. Moreover, we found that a niclosamide analog inhibited CIP2A expression and increased PP2A activity in several types of NSCLC cells. Finally, we showed that other well-known PP2A activators, including forskolin and FTY720, did not inhibit CIP2A and that their activities were not dependent on CIP2A. Collectively, our data suggested that niclosamide effectively suppressed CIP2A expression and subsequently activated PP2A in NSCLC cells. This provided strong evidence for the potential use of niclosamide asa PP2A-activating drug in the clinical treatment of NSCLC. Copyright © 2017 Elsevier Inc. All rights reserved.

  6. Protein phosphatase 2ACα gene knock-out results in cortical atrophy through activating hippo cascade in neuronal progenitor cells.

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    Liu, Bo; Sun, Li-Hua; Huang, Yan-Fei; Guo, Li-Jun; Luo, Li-Shu

    2018-02-01

    Protein phosphatase 2ACα (PP2ACα), a vital member of the protein phosphatase family, has been studied primarily as a regulator for the development, growth and protein synthesis of a lot of cell types. Dysfunction of PP2ACα protein results in neurodegenerative disease; however, this finding has not been directly confirmed in the mouse model with PP2ACα gene knock-out. Therefore, in this study presented here, we generated the PP2ACα gene knock-out mouse model by the Cre-loxP targeting gene system, with the purpose to directly observe the regulatory role of PP2ACα gene in the development of mouse's cerebral cortex. We observe that knocking-out PP2ACα gene in the central nervous system (CNS) results in cortical neuronal shrinkage, synaptic plasticity impairments, and learning/memory deficits. Further study reveals that PP2ACα gene knock-out initiates Hippo cascade in cortical neuroprogenitor cells (NPCs), which blocks YAP translocation into the nuclei of NPCs. Notably, p73, directly targeted by Hippo cascade, can bind to the promoter of glutaminase2 (GLS2) that plays a dominant role in the enzymatic regulation of glutamate/glutamine cycle. Finally, we find that PP2ACα gene knock-out inhibits the glutamine synthesis through up-regulating the activity of phosphorylated-p73 in cortical NPCs. Taken together, it concludes that PP2ACα critically supports cortical neuronal growth and cognitive function via regulating the signaling transduction of Hippo-p73 cascade. And PP2ACα indirectly modulates the glutamine synthesis of cortical NPCs through targeting p73 that plays a direct transcriptional regulatory role in the gene expression of GLS2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  7. Proteomic analysis of human norepinephrine transporter complexes reveals associations with protein phosphatase 2A anchoring subunit and 14-3-3 proteins

    International Nuclear Information System (INIS)

    Sung, Uhna; Jennings, Jennifer L.; Link, Andrew J.; Blakely, Randy D.

    2005-01-01

    The norepinephrine transporter (NET) terminates noradrenergic signals by clearing released NE at synapses. NET regulation by receptors and intracellular signaling pathways is supported by a growing list of associated proteins including syntaxin1A, protein phosphatase 2A (PP2A) catalytic subunit (PP2A-C), PICK1, and Hic-5. In the present study, we sought evidence for additional partnerships by mass spectrometry-based analysis of proteins co-immunoprecipitated with human NET (hNET) stably expressed in a mouse noradrenergic neuroblastoma cell line. Our initial proteomic analyses reveal multiple peptides derived from hNET, peptides arising from the mouse PP2A anchoring subunit (PP2A-Ar) and peptides derived from 14-3-3 proteins. We verified physical association of NET with PP2A-Ar via co-immunoprecipitation studies using mouse vas deferens extracts and with 14-3-3 via a fusion pull-down approach, implicating specifically the hNET NH 2 -terminus for interactions. The transporter complexes described likely support mechanisms regulating transporter activity, localization, and trafficking

  8. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development.

    Science.gov (United States)

    Beier, Anna; Teichert, Ines; Krisp, Christoph; Wolters, Dirk A; Kück, Ulrich

    2016-06-21

    The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK) complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general. The striatin-interacting phosphatase and kinase (STRIPAK) complex is highly conserved from yeasts to humans and is an important regulator of numerous eukaryotic developmental processes, such as cellular signaling and cell development. Although functional insights into the STRIPAK complex are accumulating, the detailed molecular mechanisms of single subunits are only partially understood

  9. Protein phosphatase 5 is necessary for ATR-mediated DNA repair

    International Nuclear Information System (INIS)

    Kang, Yoonsung; Cheong, Hyang-Min; Lee, Jung-Hee; Song, Peter I.; Lee, Kwang-Ho; Kim, Sang-Yong; Jun, Jae Yeoul; You, Ho Jin

    2011-01-01

    Research highlights: → Serine/threonine protein phosphatase 5 (PP5) has been shown to participate in ataxia telangiectasia-mutated (ATM)- and ATR (ATM- and Rad3-related)-mediated checkpoint pathways, which plays an important role in the DNA damage response and maintenance of genomic stability. → However, it is not clear exactly how PP5 participates in this process. → Our results indicate that PP5 is more closely related with ATR-mediated pathway than ATM-mediated pathway in DNA damage repair. -- Abstract: Several recent studies have shown that protein phosphatase 5 (PP5) participates in cell cycle arrest after DNA damage, but its roles in DNA repair have not yet been fully characterized. We investigated the roles of PP5 in the repair of ultraviolet (UV)- and neocarzinostatin (NCS)-induced DNA damage. The results of comet assays revealed different repair patterns in UV- and NCS-exposed U2OS-PS cells. PP5 is only essential for Rad3-related (ATR)-mediated DNA repair. Furthermore, the phosphorylation of 53BP1 and BRCA1, important mediators of DNA damage repair, and substrates of ATR and ATM decreased in U2OS-PS cells exposed to UV radiation. In contrast, the cell cycle arrest proteins p53, CHK1, and CHK2 were normally phosphorylated in U2OS and U2OS-PS cells exposed to UV radiation or treated with NCS. In view of these results, we suggest that PP5 plays a crucial role in ATR-mediated repair of UV-induced DNA damage.

  10. Protein phosphatase 2a (PP2A binds within the oligomerization domain of striatin and regulates the phosphorylation and activation of the mammalian Ste20-Like kinase Mst3

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    Jones Candace A

    2011-10-01

    Full Text Available Abstract Background Striatin, a putative protein phosphatase 2A (PP2A B-type regulatory subunit, is a multi-domain scaffolding protein that has recently been linked to several diseases including cerebral cavernous malformation (CCM, which causes symptoms ranging from headaches to stroke. Striatin association with the PP2A A/C (structural subunit/catalytic subunit heterodimer alters PP2A substrate specificity, but targets and roles of striatin-associated PP2A are not known. In addition to binding the PP2A A/C heterodimer to form a PP2A holoenzyme, striatin associates with cerebral cavernous malformation 3 (CCM3 protein, the mammalian Mps one binder (MOB homolog, Mob3/phocein, the mammalian sterile 20-like (Mst kinases, Mst3, Mst4 and STK25, and several other proteins to form a large signaling complex. Little is known about the molecular architecture of the striatin complex and the regulation of these sterile 20-like kinases. Results To help define the molecular organization of striatin complexes and to determine whether Mst3 might be negatively regulated by striatin-associated PP2A, a structure-function analysis of striatin was performed. Two distinct regions of striatin are capable of stably binding directly or indirectly to Mob3--one N-terminal, including the coiled-coil domain, and another more C-terminal, including the WD-repeat domain. In addition, striatin residues 191-344 contain determinants necessary for efficient association of Mst3, Mst4, and CCM3. PP2A associates with the coiled-coil domain of striatin, but unlike Mob3 and Mst3, its binding appears to require striatin oligomerization. Deletion of the caveolin-binding domain on striatin abolishes striatin family oligomerization and PP2A binding. Point mutations in striatin that disrupt PP2A association cause hyperphosphorylation and activation of striatin-associated Mst3. Conclusions Striatin orchestrates the regulation of Mst3 by PP2A. It binds Mst3 likely as a dimer with CCM3 via

  11. Molecular mechanism of serine/threonine protein phosphatase 1 (PP1cα-PP1r7) in spermatogenesis of Toxocara canis.

    Science.gov (United States)

    Ma, Guang Xu; Zhou, Rong Qiong; Song, Zhen Hui; Zhu, Hong Hong; Zhou, Zuo Yong; Zeng, Yuan Qin

    2015-09-01

    Toxocariasis is one of the most important, but neglected, zoonoses, which is mainly caused by Toxocara canis. To better understand the role of serine/threonine protein phosphatase 1 (PP1) in reproductive processes of male adult T. canis, differential expression analysis was used to reveal the profiles of PP1 catalytic subunit α (PP1cα) gene Tc-stp-1 and PP1 regulatory subunit 7 (PP1r7) gene TcM-1309. Indirect fluorescence immunocytochemistry was carried out to determine the subcellular distribution of PP1cα. Double-stranded RNA interference (RNAi) assays were employed to illustrate the function and mechanism of PP1cα in male adult reproduction. Real-time quantitative PCR (qPCR) showed transcriptional consistency of Tc-stp-1 and TcM-1309 in sperm-producing germline tissues and localization research showed cytoplasmic distribution of PP1cα in sf9 cells, which indicated relevant involvements of PP1cα and PP1r7 in spermatogenesis. Moreover, spatiotemporal transcriptional differences of Tc-stp-1 were determined by gene knockdown analysis, which revealed abnormal morphologies and blocked meiotic divisions of spermatocytes by phenotypic aberration scanning, thereby highlighting the crucial involvement of PP1cα in spermatogenesis. These results revealed a PP1cα-PP1r7 mechanism by which PP1 regulates kinetochore-microtubule interactions in spermatogenesis and provided important clues to identify novel drug or vaccine targets for toxocariasis control. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Dephosphorylation of chicken cardiac myofibril C-protein by protein phosphatases 1 and 2A

    International Nuclear Information System (INIS)

    Thysseril, T.J.; Hegazy, M.G.; Schlender, K.K.

    1987-01-01

    C-Protein, which is a regulatory component of cardiac muscle myofibrils, is phosphorylated in response to β-adrenergic agonists by a cAMP-dependent mechanism and dephosphorylated in response to cholinergic agonists. It is believed that the cAMP-dependent phosphorylation is due to cAMP-dependent protein kinase. The protein phosphatase(s) involved in the dephosphorylation of C-protein has not been determined. In this study, chicken cardiac C-protein was phosphorylated with the cAMP-dependent protein kinase to about 3 mol phosphate/mol C-protein. Incubation of [ 32 P]C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of 32 [P]. Phosphopeptide maps and phosphoamino acid analysis revealed that the major site(s) dephosphorylated by either phosphatase was a phosphothreonine residue(s) located on the same tryptic peptide and on the same CNBr fragment. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A completely dephosphorylated C-protein. Preliminary studies showed that the major protein phosphatase associated with the myofibril was phosphatase 2A. These results indicate the phosphatase 2A may be important in the regulation of the phosphorylation state of C-protein

  13. Protein phosphatase 2Cm is a critical regulator of branched-chain amino acid catabolism in mice and cultured cells.

    Science.gov (United States)

    Lu, Gang; Sun, Haipeng; She, Pengxiang; Youn, Ji-Youn; Warburton, Sarah; Ping, Peipei; Vondriska, Thomas M; Cai, Hua; Lynch, Christopher J; Wang, Yibin

    2009-06-01

    The branched-chain amino acids (BCAA) are essential amino acids required for protein homeostasis, energy balance, and nutrient signaling. In individuals with deficiencies in BCAA, these amino acids can be preserved through inhibition of the branched-chain-alpha-ketoacid dehydrogenase (BCKD) complex, the rate-limiting step in their metabolism. BCKD is inhibited by phosphorylation of its E1alpha subunit at Ser293, which is catalyzed by BCKD kinase. During BCAA excess, phosphorylated Ser293 (pSer293) becomes dephosphorylated through the concerted inhibition of BCKD kinase and the activity of an unknown intramitochondrial phosphatase. Using unbiased, proteomic approaches, we have found that a mitochondrial-targeted phosphatase, PP2Cm, specifically binds the BCKD complex and induces dephosphorylation of Ser293 in the presence of BCKD substrates. Loss of PP2Cm completely abolished substrate-induced E1alpha dephosphorylation both in vitro and in vivo. PP2Cm-deficient mice exhibited BCAA catabolic defects and a metabolic phenotype similar to the intermittent or intermediate types of human maple syrup urine disease (MSUD), a hereditary disorder caused by defects in BCKD activity. These results indicate that PP2Cm is the endogenous BCKD phosphatase required for nutrient-mediated regulation of BCKD activity and suggest that defects in PP2Cm may be responsible for a subset of human MSUD.

  14. Polo-like kinase 1 (PLK1) and protein phosphatase 6 (PP6) regulate DNA-dependent protein kinase catalytic subunit (DNA-PKcs) phosphorylation in mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Trinkle-Mulcahy, Laura; Neal, Jessica A; De Wever, Veerle; Morrice, Nick A; Meek, Katheryn; Lees-Miller, Susan P

    2014-06-25

    The protein kinase activity of the DNA-PKcs (DNA-dependent protein kinase catalytic subunit) and its autophosphorylation are critical for DBS (DNA double-strand break) repair via NHEJ (non-homologous end-joining). Recent studies have shown that depletion or inactivation of DNA-PKcs kinase activity also results in mitotic defects. DNA-PKcs is autophosphorylated on Ser2056, Thr2647 and Thr2609 in mitosis and phosphorylated DNA-PKcs localize to centrosomes, mitotic spindles and the midbody. DNA-PKcs also interacts with PP6 (protein phosphatase 6), and PP6 has been shown to dephosphorylate Aurora A kinase in mitosis. Here we report that DNA-PKcs is phosphorylated on Ser3205 and Thr3950 in mitosis. Phosphorylation of Thr3950 is DNA-PK-dependent, whereas phosphorylation of Ser3205 requires PLK1 (polo-like kinase 1). Moreover, PLK1 phosphorylates DNA-PKcs on Ser3205 in vitro and interacts with DNA-PKcs in mitosis. In addition, PP6 dephosphorylates DNA-PKcs at Ser3205 in mitosis and after IR (ionizing radiation). DNA-PKcs also phosphorylates Chk2 on Thr68 in mitosis and both phosphorylation of Chk2 and autophosphorylation of DNA-PKcs in mitosis occur in the apparent absence of Ku and DNA damage. Our findings provide mechanistic insight into the roles of DNA-PKcs and PP6 in mitosis and suggest that DNA-PKcs' role in mitosis may be mechanistically distinct from its well-established role in NHEJ.

  15. Protein phosphatase 2A interacts with the Na,K-ATPase and modulates its trafficking by inhibition of its association with arrestin.

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    Toru Kimura

    Full Text Available The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na⁺,K⁺-ATPase. The catalytic subunit of the Na⁺,K⁺-ATPase includes several functional domains that determine its enzymatic and trafficking properties.Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A catalytic C-subunit is a specific Na⁺,K⁺-ATPase interacting protein. PP-2A C-subunit interacted with the Na⁺,K⁺-ATPase, but not with the homologous sequences of the H⁺,K⁺-ATPase. We confirmed that the Na⁺,K⁺-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs are important regulators of G-protein coupled receptor (GPCR signaling, and they also regulate Na⁺,K⁺-ATPase trafficking through direct association. PP2A inhibits association between the Na⁺,K⁺-ATPase and arrestin, and diminishes the effect of arrestin on Na⁺,K⁺-ATPase trafficking. GRK phosphorylates the Na⁺,K⁺-ATPase and PP2A can at least partially reverse this phosphorylation.Taken together, these data demonstrate that the sodium pump belongs to a growing list of ion transport proteins that are regulated through direct interactions with the catalytic subunit of a protein phosphatase.

  16. PP2A-Mediated Anticancer Therapy

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    Weibo Chen

    2013-01-01

    Full Text Available PP2A is a family of mammalian serine/threonine phosphatases that is involved in the control of many cellular functions including protein synthesis, cellular signaling, cell cycle determination, apoptosis, metabolism, and stress responses through the negative regulation of signaling pathways initiated by protein kinases. Rapid progress is being made in the understanding of PP2A complex and its functions. Emerging studies have correlated changes in PP2A with human diseases, especially cancer. PP2A is comprised of 3 subunits: a catalytic subunit, a scaffolding subunit, and a regulatory subunit. The alternations of the subunits have been shown to be in association with many human malignancies. Therapeutic agents targeting PP2A inhibitors or activating PP2A directly have shed light on the therapy of cancers. This review focuses on PP2A structure, cancer-associated mutations, and the targeting of PP2A-related molecules to restore or reactivate PP2A in anticancer therapy, especially in digestive system cancer therapy.

  17. Interaction of Protein Phosphatase 1δ with Nucleophosmin in Human Osteoblastic Cells

    International Nuclear Information System (INIS)

    Haneji, Tatsuji; Teramachi, Jumpei; Hirashima, Kanji; Kimura, Koji; Morimoto, Hiroyuki

    2012-01-01

    Protein phosphorylation and dephosphorylation has been recognized as an essential mechanism in the regulation of cellular metabolism and function in various tissues. Serine and threonine protein phosphatases (PP) are divided into four categories: PP1, PP2A, PP2B, and PP2C. At least four isoforms of PP1 catalytic subunit in rat, PP1α, PP1γ1, PP2, and PP1δ, were isolated. In the present study, we examined the localization and expression of PP1δ in human osteoblastic Saos-2 cells. Anti-PP1δ antibody recognized a protein present in the nucleolar regions in Saos-2 cells. Cellular fractionation revealed that PP1δ is a 37 kDa protein localized in the nucleolus. Nucleophosmin is a nucleolar phosphoprotein and located mainly in the nucleolus. Staining pattern of nucleophosmin in Saos-2 cells was similar to that of PP1δ. PP1δ and nucleophosmin were specifically stained as dots in the nucleus. Dual fluorescence images revealed that PP1δ and nucleophosmin were localized in the same regions in the nucleolus. Similar distribution patterns of PP1δ and nucleophosmin were observed in osteoblastic MG63 cells. The interaction of PP1δ and nucleophosmin was also shown by immunoprecipitation and Western analysis. These results indicated that PP1δ associate with nucleophosmin directly in the nucleolus and suggested that nucleophosmin is one of the candidate substrate for PP

  18. The PP2AB56 phosphatase promotes the association of Cdc20 with APC/C in mitosis.

    Science.gov (United States)

    Lee, Sun Joo; Rodriguez-Bravo, Veronica; Kim, Hyunjung; Datta, Sutirtha; Foley, Emily A

    2017-05-15

    PP2A comprising B56 regulatory subunit isoforms (PP2A B56 ) is a serine/threonine phosphatase essential for mitosis. At the kinetochore, PP2A B56 both stabilizes microtubule binding and promotes silencing of the spindle assembly checkpoint (SAC) through its association with the SAC protein BubR1. Cells depleted of the B56 regulatory subunits of PP2A are delayed in activation of Cdc20-containing APC/C (APC/C Cdc20 ), which is an essential step for mitotic exit. It has been hypothesized that this delay arises from increased production of the mitotic checkpoint complex (MCC), an APC/C Cdc20 inhibitor formed at unattached kinetochores through SAC signaling. In contrast to this prediction, we show that depletion of B56 subunits does not increase the amount or stability of the MCC. Rather, delays in APC/C Cdc20 activation in B56-depleted cells correlate with impaired Cdc20 binding to APC/C. Stimulation of APC/C Cdc20 assembly does not require binding between PP2A B56 and BubR1, and thus this contribution of PP2A B56 towards mitotic exit is distinct from its functions at kinetochores. PP2A B56 associates with APC/C constitutively in a BubR1-independent manner. A mitotic phosphorylation site on Cdc20, known to be a substrate of PP2A B56 , modulates APC/C Cdc20 assembly. These results elucidate the contributions of PP2A B56 towards completion of mitosis. © 2017. Published by The Company of Biologists Ltd.

  19. Type 2C Phosphatase 1 of Artemisia annua L. Is a Negative Regulator of ABA Signaling

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    Fangyuan Zhang

    2014-01-01

    Full Text Available The phytohormone abscisic acid (ABA plays an important role in plant development and environmental stress response. Additionally, ABA also regulates secondary metabolism such as artemisinin in the medicinal plant Artemisia annua L. Although an earlier study showed that ABA receptor, AaPYL9, plays a positive role in ABA-induced artemisinin content improvement, many components in the ABA signaling pathway remain to be elucidated in Artemisia annua L. To get insight of the function of AaPYL9, we isolated and characterized an AaPYL9-interacting partner, AaPP2C1. The coding sequence of AaPP2C1 encodes a deduced protein of 464 amino acids, with all the features of plant type clade A PP2C. Transcriptional analysis showed that the expression level of AaPP2C1 is increased after ABA, salt, and drought treatments. Yeast two-hybrid and bimolecular fluorescence complementation assays (BiFC showed that AaPYL9 interacted with AaPP2C1. The P89S, H116A substitution in AaPYL9 as well as G199D substitution or deletion of the third phosphorylation site-like motif in AaPP2C1 abolished this interaction. Furthermore, constitutive expression of AaPP2C1 conferred ABA insensitivity compared with the wild type. In summary, our data reveals that AaPP2C1 is an AaPYL9-interacting partner and involved in the negative modulation of the ABA signaling pathway in A. annua L.

  20. Identification of Plasmodium falciparum translation initiation eIF2β subunit: direct interaction with Protein Phosphatase type 1

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    Géraldine eTellier

    2016-05-01

    Full Text Available Protein phosphatase 1 (PP1c is one of the main phosphatases whose function is shaped by many regulators to confer a specific location and a selective function for this enzyme. Here, we report that eukaryotic initiation factor 2 of P. falciparum (PfeIF2β is an interactor of PfPP1c. Sequence analysis of PfeIF2 revealed a deletion of 111 amino acids when compared to its human counterpart and the presence of two potential binding motifs to PfPP1 (29FGEKKK34, 103KVAW106. As expected, we showed that PfeIF2 binds PfeIF2 and PfeIF5, confirming its canonical interaction with partners of the translation complex. Studies of the PfeIF2-PfPP1 interaction using wild-type, single and double mutated versions of PfeIF2β revealed that both binding motifs are critical. We next showed that PfeIF2 is able to induce Germinal Vesicle BreakDown (GVBD when expressed in Xenopus oocytes, an indicator of its capacity to regulate PP1. Only combined mutations of both binding motifs abolished the interaction with PP1 and the induction of GVBD. In P. falciparum, although the locus is accessible for genetic manipulation, PfeIF2 seems to play an essential role in intraerythrocytic cycle as no viable knockout parasites were detectable. Interestingly, as for PfPP1, the subcellular fractionation of P. falciparum localized PfeIF2β in cytoplasm and nuclear extracts, suggesting a potential effect on PfPP1 in both compartments and raising the question of a non-canonical function of PfeIf2 in the nucleus. Hence, the role played by PfeIF2 in blood stage parasites could occur at multiple levels involving the binding to proteins of the translational complex and to PfPP1.

  1. AR-v7 protein expression is regulated by protein kinase and phosphatase

    Science.gov (United States)

    Li, Yinan; Xie, Ning; Gleave, Martin E.; Rennie, Paul S.; Dong, Xuesen

    2015-01-01

    Failure of androgen-targeted therapy and progression of castration-resistant prostate cancer (CRPC) are often attributed to sustained expression of the androgen receptor (AR) and its major splice variant, AR-v7. Although the new generation of anti-androgens such as enzalutamide effectively inhibits AR activity, accumulating pre-clinical and clinical evidence indicates that AR-v7 remains constitutively active in driving CRPC progression. However, molecular mechanisms which control AR-v7 protein expression remain unclear. We apply multiple prostate cancer cell models to demonstrate that enzalutamide induces differential activation of protein phosphatase-1 (PP-1) and Akt kinase depending on the gene context of cancer cells. The balance between PP-1 and Akt activation governs AR phosphorylation status and activation of the Mdm2 ubiquitin ligase. Mdm2 recognizes phosphorylated serine 213 of AR-v7, and induces AR-v7 ubiquitination and protein degradation. These findings highlight the decisive roles of PP-1 and Akt for AR-v7 protein expression and activities when AR is functionally blocked. PMID:26378044

  2. Disruption of a Guard Cell–Expressed Protein Phosphatase 2A Regulatory Subunit, RCN1, Confers Abscisic Acid Insensitivity in Arabidopsis

    Science.gov (United States)

    Kwak, June M.; Moon, Ji-Hye; Murata, Yoshiyuki; Kuchitsu, Kazuyuki; Leonhardt, Nathalie; DeLong, Alison; Schroeder, Julian I.

    2002-01-01

    Pharmacological studies have led to a model in which the phytohormone abscisic acid (ABA) may be positively transduced via protein phosphatases of the type 1 (PP1) or type 2A (PP2A) families. However, pharmacological evidence also exists that PP1s or PP2As may function as negative regulators of ABA signaling. Furthermore, recessive disruption mutants in protein phosphatases that function in ABA signal transduction have not yet been identified. A guard cell–expressed PP2A gene, RCN1, which had been characterized previously as a molecular component affecting auxin transport and gravity response, was isolated. A T-DNA disruption mutation in RCN1 confers recessive ABA insensitivity to Arabidopsis. The rcn1 mutation impairs ABA-induced stomatal closing and ABA activation of slow anion channels. Calcium imaging analyses show a reduced sensitivity of ABA-induced cytosolic calcium increases in rcn1, whereas mechanisms downstream of cytosolic calcium increases show wild-type responses, suggesting that RCN1 functions in ABA signal transduction upstream of cytosolic Ca2+ increases. Furthermore, rcn1 shows ABA insensitivity in ABA inhibition of seed germination and ABA-induced gene expression. The PP1 and PP2A inhibitor okadaic acid phenocopies the rcn1 phenotype in wild-type plants both in ABA-induced cytosolic calcium increases and in seed germination, and the wild-type RCN1 genomic DNA complements rcn1 phenotypes. These data show that RCN1 functions as a general positive transducer of early ABA signaling. PMID:12417706

  3. PP2A regulates kinetochore-microtubule attachment during meiosis I in oocyte.

    Science.gov (United States)

    Tang, An; Shi, Peiliang; Song, Anying; Zou, Dayuan; Zhou, Yue; Gu, Pengyu; Huang, Zan; Wang, Qinghua; Lin, Zhaoyu; Gao, Xiang

    2016-06-02

    Studies using in vitro cultured oocytes have indicated that the protein phosphatase 2A (PP2A), a major serine/threonine protein phosphatase, participates in multiple steps of meiosis. Details of oocyte maturation regulation by PP2A remain unclear and an in vivo model can provide more convincing information. Here, we inactivated PP2A by mutating genes encoding for its catalytic subunits (PP2Acs) in mouse oocytes. We found that eliminating both PP2Acs caused female infertility. Oocytes lacking PP2Acs failed to complete 1(st) meiotic division due to chromosome misalignment and abnormal spindle assembly. In mitosis, PP2A counteracts Aurora kinase B/C (AurkB/C) to facilitate correct kinetochore-microtubule (KT-MT) attachment. In meiosis I in oocyte, we found that PP2Ac deficiency destabilized KT-MT attachments. Chemical inhibition of AurkB/C in PP2Ac-null oocytes partly restored the formation of lateral/merotelic KT-MT attachments but not correct KT-MT attachments. Taken together, our findings demonstrate that PP2Acs are essential for chromosome alignments and regulate the formation of correct KT-MT attachments in meiosis I in oocytes.

  4. The heterotrimeric G protein Gβ1 interacts with the catalytic subunit of protein phosphatase 1 and modulates G protein-coupled receptor signaling in platelets.

    Science.gov (United States)

    Pradhan, Subhashree; Khatlani, Tanvir; Nairn, Angus C; Vijayan, K Vinod

    2017-08-11

    Thrombosis is caused by the activation of platelets at the site of ruptured atherosclerotic plaques. This activation involves engagement of G protein-coupled receptors (GPCR) on platelets that promote their aggregation. Although it is known that protein kinases and phosphatases modulate GPCR signaling, how serine/threonine phosphatases integrate with G protein signaling pathways is less understood. Because the subcellular localization and substrate specificity of the catalytic subunit of protein phosphatase 1 (PP1c) is dictated by PP1c-interacting proteins, here we sought to identify new PP1c interactors. GPCRs signal via the canonical heterotrimeric Gα and Gβγ subunits. Using a yeast two-hybrid screen, we discovered an interaction between PP1cα and the heterotrimeric G protein Gβ 1 subunit. Co-immunoprecipitation studies with epitope-tagged PP1c and Gβ 1 revealed that Gβ 1 interacts with the PP1c α, β, and γ1 isoforms. Purified PP1c bound to recombinant Gβ 1 -GST protein, and PP1c co-immunoprecipitated with Gβ 1 in unstimulated platelets. Thrombin stimulation of platelets induced the dissociation of the PP1c-Gβ 1 complex, which correlated with an association of PP1c with phospholipase C β3 (PLCβ3), along with a concomitant dephosphorylation of the inhibitory Ser 1105 residue in PLCβ3. siRNA-mediated depletion of GNB1 (encoding Gβ 1 ) in murine megakaryocytes reduced protease-activated receptor 4, activating peptide-induced soluble fibrinogen binding. Thrombin-induced aggregation was decreased in PP1cα -/- murine platelets and in human platelets treated with a small-molecule inhibitor of Gβγ. Finally, disruption of PP1c-Gβ 1 complexes with myristoylated Gβ 1 peptides containing the PP1c binding site moderately decreased thrombin-induced human platelet aggregation. These findings suggest that Gβ 1 protein enlists PP1c to modulate GPCR signaling in platelets.

  5. Suppressing Type 2C Protein Phosphatases Alters Fruit Ripening and the Stress Response in Tomato.

    Science.gov (United States)

    Zhang, Yushu; Li, Qian; Jiang, Li; Kai, Wenbin; Liang, Bin; Wang, Juan; Du, Yangwei; Zhai, Xiawan; Wang, Jieling; Zhang, Yingqi; Sun, Yufei; Zhang, Lusheng; Leng, Ping

    2018-01-01

    Although ABA signaling has been widely studied in Arabidopsis, the roles of core ABA signaling components in fruit remain poorly understood. Herein, we characterize SlPP2C1, a group A type 2C protein phosphatase that negatively regulates ABA signaling and fruit ripening in tomato. The SlPP2C1 protein was localized in the cytoplasm close to AtAHG3/AtPP2CA. The SlPP2C1 gene was expressed in all tomato tissues throughout development, particularly in flowers and fruits, and it was up-regulated by dehydration and ABA treatment. SlPP2C1 expression in fruits was increased at 30 d after full bloom and peaked at the B + 1 stage. Suppression of SlPP2C1 expression significantly accelerated fruit ripening which was associated with higher levels of ABA signaling genes that are reported to alter the expression of fruit ripening genes involved in ethylene release and cell wall catabolism. SlPP2C1-RNAi (RNA interference) led to increased endogenous ABA accumulation and advanced release of ethylene in transgenic fruits compared with wild-type (WT) fruits. SlPP2C1-RNAi also resulted in abnormal flowers and obstructed the normal abscission of pedicels. SlPP2C1-RNAi plants were hypersensitized to ABA, and displayed delayed seed germination and primary root growth, and increased resistance to drought stress compared with WT plants. These results demonstrated that SlPP2C1 is a functional component in the ABA signaling pathway which participates in fruit ripening, ABA responses and drought tolerance. © The Author 2017. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.

  6. Methylation-regulated decommissioning of multimeric PP2A complexes

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    Wu, Cheng-Guo; Zheng, Aiping; Jiang, Li; Rowse, Michael; Stanevich, Vitali; Chen, Hui; Li, Yitong; Satyshur, Kenneth A.; Johnson, Benjamin; Gu, Ting-Jia; Liu, Zuojia; Xing, Yongna

    2017-12-01

    Dynamic assembly/disassembly of signaling complexes are crucial for cellular functions. Specialized latency and activation chaperones control the biogenesis of protein phosphatase 2A (PP2A) holoenzymes that contain a common scaffold and catalytic subunits and a variable regulatory subunit. Here we show that the butterfly-shaped TIPRL (TOR signaling pathway regulator) makes highly integrative multibranching contacts with the PP2A catalytic subunit, selective for the unmethylated tail and perturbing/inactivating the phosphatase active site. TIPRL also makes unusual wobble contacts with the scaffold subunit, allowing TIPRL, but not the overlapping regulatory subunits, to tolerate disease-associated PP2A mutations, resulting in reduced holoenzyme assembly and enhanced inactivation of mutant PP2A. Strikingly, TIPRL and the latency chaperone, α4, coordinate to disassemble active holoenzymes into latent PP2A, strictly controlled by methylation. Our study reveals a mechanism for methylation-responsive inactivation and holoenzyme disassembly, illustrating the complexity of regulation/signaling, dynamic complex disassembly, and disease mutations in cancer and intellectual disability.

  7. Antitumor effects of metformin via indirect inhibition of protein phosphatase 2A in patients with endometrial cancer.

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    Shinsuke Hanawa

    Full Text Available Metformin, an antidiabetic drug, inhibits the endometrial cancer cell growth in vivo by improving the insulin resistance; however, its mechanism of action is not completely understood. Protein phosphatase 2A (PP2A is a serine/threonine phosphatase associated with insulin resistance and type 2 diabetes, and its inhibition restores the insulin resistance. This study investigated the antitumor effect of metformin on endometrial cancer with a focus on PP2A.Metformin (1,500-2,250 mg/day was preoperatively administered to patients with endometrial cancer for 4 to 6 weeks. Expression of the PP2A regulatory subunits, 4 (PPP2R4 and B (PP2A-B, was evaluated using real-time polymerase chain reaction (RT-PCR and immunohistochemistry (IHC using paired specimens obtained before and after metformin treatment. The effect of PPP2R4 inhibition with small interfering RNA was evaluated in the endometrial cancer cell lines HEC265 and HEC1B. P values of < .05 were considered statistically significant.Preoperative metformin treatment significantly reduced the expression of PP2A-B, as determined using IHC, and the mRNA expression of PPP2R4, as determined using RT-PCR, in the patients with endometrial cancer. However, metformin could not directly alter the PPP2R4 mRNA levels in the endometrial cancer cell lines in vitro. PPP2R4 knockdown reduced the proliferation and induced the apoptosis by activating caspases 3/7 in HEC265 and HEC1B cells.Downregulation of the PP2A-B subunit, including PPP2R4, is an important indirect target of metformin. Inhibition of PP2A may be an option for the treatment of endometrial cancer patients with insulin resistance.This trial is registered with UMIN-CTR (number UMIN000004852.

  8. The PP4R1 sub-unit of protein phosphatase PP4 is essential for inhibition of NF-κB by merkel polyomavirus small tumour antigen.

    Science.gov (United States)

    Abdul-Sada, Hussein; Müller, Marietta; Mehta, Rajni; Toth, Rachel; Arthur, J Simon C; Whitehouse, Adrian; Macdonald, Andrew

    2017-04-11

    Merkel cell carcinoma (MCC) is a highly aggressive skin cancer with a high metastatic potential. The majority of MCC cases are caused by the Merkel cell polyomavirus (MCPyV), through expression of the virus-encoded tumour antigens. Whilst mechanisms attributing tumour antigen expression to transformation are being uncovered, little is known of the mechanisms by which MCPyV persists in the host. We previously identified the MCPyV small T antigen (tAg) as a novel inhibitor of nuclear factor kappa B (NF-kB) signalling and a modulator of the host anti-viral response. Here we demonstrate that regulation of NF-kB activation involves a previously undocumented interaction between tAg and regulatory sub-unit 1 of protein phosphatase 4 (PP4R1). Formation of a complex with PP4R1 and PP4c is required to bridge MCPyV tAg to the NEMO adaptor protein, allowing deactivation of the NF-kB pathway. Mutations in MCPyV tAg that fail to interact with components of this complex, or siRNA depletion of PP4R1, prevents tAg-mediated inhibition of NF-kB and pro-inflammatory cytokine production. Comparison of tAg binding partners from other human polyomavirus demonstrates that interactions with NEMO and PP4R1 are unique to MCPyV. Collectively, these data identify PP4R1 as a novel target for virus subversion of the host anti-viral response.

  9. Catalytic Subunit 1 of Protein Phosphatase 2A Is a Subunit of the STRIPAK Complex and Governs Fungal Sexual Development

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    Anna Beier

    2016-06-01

    Full Text Available The generation of complex three-dimensional structures is a key developmental step for most eukaryotic organisms. The details of the molecular machinery controlling this step remain to be determined. An excellent model system to study this general process is the generation of three-dimensional fruiting bodies in filamentous fungi like Sordaria macrospora. Fruiting body development is controlled by subunits of the highly conserved striatin-interacting phosphatase and kinase (STRIPAK complex, which has been described in organisms ranging from yeasts to humans. The highly conserved heterotrimeric protein phosphatase PP2A is a subunit of STRIPAK. Here, catalytic subunit 1 of PP2A was functionally characterized. The Δpp2Ac1 strain is sterile, unable to undergo hyphal fusion, and devoid of ascogonial septation. Further, PP2Ac1, together with STRIPAK subunit PRO22, governs vegetative and stress-related growth. We revealed in vitro catalytic activity of wild-type PP2Ac1, and our in vivo analysis showed that inactive PP2Ac1 blocks the complementation of the sterile deletion strain. Tandem affinity purification, followed by mass spectrometry and yeast two-hybrid analysis, verified that PP2Ac1 is a subunit of STRIPAK. Further, these data indicate links between the STRIPAK complex and other developmental signaling pathways, implying the presence of a large interconnected signaling network that controls eukaryotic developmental processes. The insights gained in our study can be transferred to higher eukaryotes and will be important for understanding eukaryotic cellular development in general.

  10. Analysis of nucleo-cytoplasmic shuttling of the proto-oncogene SET/I2PP2A

    NARCIS (Netherlands)

    Lam, B. Daniel; Anthony, Eloise C.; Hordijk, Peter L.

    2012-01-01

    SET/I2PP2A is a nuclear protein that was initially identified as an oncogene in human undifferentiated acute myeloid leukemia, fused to the nuclear porin Nup-214. In addition, SET is a potent inhibitior of the phosphatase PP2A. Previously, we proposed a model in which the small GTPase Rac1 recruits

  11. Protein phosphatase 2A inhibition and subsequent cytoskeleton reorganization contributes to cell migration caused by microcystin-LR in human laryngeal epithelial cells (Hep-2).

    Science.gov (United States)

    Wang, Beilei; Liu, Jinghui; Huang, Pu; Xu, Kailun; Wang, Hanying; Wang, Xiaofeng; Guo, Zonglou; Xu, Lihong

    2017-03-01

    The major toxic mechanism of Microcystin-LR is inhibition of the activity of protein phosphatase 2A (PP2A), resulting in a series of cytotoxic effects. Our previous studies have demonstrated that microcystin-LR (MCLR) induced very different molecular effects in normal cells and the tumor cell line SMMC7721. To further explore the MCLR toxicity mechanism in tumor cells, human laryngeal epithelial cells (Hep-2) was examined in this study. Western blot, immunofluorescence, immunoprecipitation, and transwell migration assay were used to detect the effects of MCLR on PP2A activity, PP2A substrates, cytoskeleton, and cell migration. The results showed that the protein level of PP2A subunits and the posttranslational modification of the catalytic subunit were altered and that the binding of the AC core enzyme as well as the binding of PP2A/C and α4, was also affected. As PP2A substrates, the phosphorylation of MAPK pathway members, p38, ERK1/2, and the cytoskeleton-associated proteins, Hsp27, VASP, Tau, and Ezrin were increased. Furthermore, MCLR induced reorganization of the cytoskeleton and promoted cell migration. Taken together, direct covalent binding to PP2A/C, alteration of the protein levels and posttranslational modification, as well as the binding of subunits, are the main pattern for the effects of MCLR on PP2A in Hep-2. A dose-dependent change in p-Tau and p-Ezrin due to PP2A inhibition may contribute to the changes in the cytoskeleton and be related to the cell migration in Hep-2. Our data provide a comprehensive exposition of the MCLR mechanism on tumor cells. © 2016 Wiley Periodicals, Inc. Environ Toxicol 32: 890-903, 2017. © 2016 Wiley Periodicals, Inc.

  12. A role for protein phosphatase-2A in p38 mitogen-activated protein kinase-mediated regulation of the c-Jun NH(2)-terminal kinase pathway in human neutrophils.

    Science.gov (United States)

    Avdi, Natalie J; Malcolm, Kenneth C; Nick, Jerry A; Worthen, G Scott

    2002-10-25

    Human neutrophil accumulation in inflammatory foci is essential for the effective control of microbial infections. Although exposure of neutrophils to cytokines such as tumor necrosis factor-alpha (TNFalpha), generated at sites of inflammation, leads to activation of MAPK pathways, mechanisms responsible for the fine regulation of specific MAPK modules remain unknown. We have previously demonstrated activation of a TNFalpha-mediated JNK pathway module, leading to apoptosis in adherent human neutrophils (Avdi, N. J., Nick, J. A., Whitlock, B. B., Billstrom, M. A., Henson, P. M., Johnson, G. L., and Worthen, G. S. (2001) J. Biol. Chem. 276, 2189-2199). Herein, evidence is presented linking regulation of the JNK pathway to p38 MAPK and the Ser/Thr protein phosphatase-2A (PP2A). Inhibition of p38 MAPK by SB 203580 and M 39 resulted in significant augmentation of TNFalpha-induced JNK and MKK4 (but not MKK7 or MEKK1) activation, whereas prior exposure to a p38-activating agent (platelet-activating factor) diminished the TNFalpha-induced JNK response. TNFalpha-induced apoptosis was also greatly enhanced upon p38 inhibition. Studies with a reconstituted cell-free system indicated the absence of a direct inhibitory effect of p38 MAPK on the JNK module. Neutrophil exposure to the Ser/Thr phosphatase inhibitors okadaic acid and calyculin A induced JNK activation. Increased phosphatase activity following TNFalpha stimulation was shown to be PP2A-associated and p38-dependent. Furthermore, PP2A-induced dephosphorylation of MKK4 resulted in its inactivation. Thus, in neutrophils, p38 MAPK, through a PP2A-mediated mechanism, regulates the JNK pathway, thus determining the extent and nature of subsequent responses such as apoptosis.

  13. Effects of light and the regulatory B-subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae) infestation.

    Science.gov (United States)

    Rasool, Brwa; Karpinska, Barbara; Konert, Grzegorz; Durian, Guido; Denessiouk, Konstantin; Kangasjärvi, Saijaliisa; Foyer, Christine H

    2014-01-01

    The interactions between biotic and abiotic stress signaling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP)2A regulatory subunit B'γ (gamma; pp2a-b'γ) or B'ζ (zeta; pp2a-b'ζ1-1 and pp2a-b'ζ 1-2) and in gamma zeta double mutants (pp2a-b'γζ) lacking both subunits. All the mutants except for pp2a-b'ζ 1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b'γ mutant relative to the wild type but not in the pp2a-b'γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b'γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of B-subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonization, depending on the prevailing abiotic stress environment.

  14. Effects of light and the regulatory Beta subunit composition of protein phosphatase 2A on the susceptibility of Arabidopsis thaliana to aphid (Myzus persicae infestation

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    Brwa eRasool

    2014-08-01

    Full Text Available The interactions between biotic and abiotic stress signalling pathways are complex and poorly understood but protein kinase/phosphatase cascades are potentially important components. Aphid fecundity and susceptibility to Pseudomonas syringae infection were determined in the low light-grown Arabidopsis thaliana wild type and in mutant lines defective in either the protein phosphatase (PP2A regulatory subunit B’γ (gamma; pp2a-b’γ or B’ζ (zeta; pp2a-b’ζ1-1 and pp2a-b’ζ1-2 and in gamma zeta double mutants (pp2a-b’γζ lacking both subunits. All the mutants except for pp2a-b’ζ1-1 had significantly lower leaf areas than the wild type. Susceptibility to P. syringae was similar in all genotypes. In contrast, aphid fecundity was significantly decreased in the pp2a-b’γ mutant relative to the wild type but not in the pp2a-b’γζ double mutant. A high light pre-treatment, which led to a significant increase in rosette growth in all mutant lines but not in the wild type, led to a significant decrease in aphid fecundity in all genotypes. The high light pre-treatment abolished the differences in aphid resistance observed in the pp2a-b’γ mutant relative to the wild type. The light and CO2 response curves for photosynthesis were changed in response to the high light pre-treatment, but the high light effects were similar in all genotypes. These data demonstrate that a pre-exposure to high light and the composition of subunits on the trimeric PP2A holoenzymes are important in regulating plant resistance to aphids. The functional specificity for the individual regulatory B-subunits may therefore limit aphid colonisation, depending on the prevailing abiotic stress environment.

  15. PP2A contributes to endothelial death in high glucose: inhibition by benfotiamine.

    Science.gov (United States)

    Du, Y; Kowluru, A; Kern, T S

    2010-12-01

    Endothelial death is critical in diabetic vascular diseases, but regulating factors have been only partially elucidated. Phosphatases play important regulatory roles in cell metabolism, but have not previously been implicated in hyperglycemia-induced cell death. We investigated the role of the phosphatase, type 2A protein phosphatase (PP2A), in hyperglycemia-induced changes in signaling and death in bovine aortic endothelial cells (BAEC). We explored also the influence of benfotiamine on this phosphatase. Activation of PP2A was assessed in BAEC by the extent of methylation and measurement of activity, and the enzyme was inhibited using selective pharmacological (okadaic acid, sodium fostriecin) and molecular (small interfering RNA) approaches. BAECs cultured in 30 mM glucose significantly increased PP2A methylation and activity, and PP2A inhibitors blocked these abnormalities. PP2A activity was increased also in aorta and retina from diabetic rats. NF-κB activity and cell death in BAEC were significantly increased in 30 mM glucose and inhibited by PP2A inhibition. NF-κB played a role in the hyperglycemia-induced death of BAEC, since blocking its translocation with SN50 also inhibited cell death. Inhibition of PP2A blocked the hyperglycemia-induced dephosphorylation of NF-κB and Bad, thus favoring cell survival. Incubation of benfotiamine with BAEC inhibited the high glucose-induced activation of PP2A and NF-κB and cell death, as well as several other metabolic defects, which likewise were inhibited by inhibitors of PP2A. Activation of PP2A contributes to endothelial cell death in high glucose, and beneficial actions of benfotiamine are due, at least in part, to inhibition of PP2A activation.

  16. NMDA-induced potentiation of mGluR5 is mediated by activation of protein phosphatase 2B/calcineurin

    Science.gov (United States)

    Alagarsamy, Sudar; Saugstad, Julie; Warren, Lee; Mansuy, Isabelle M.; Gereau, Robert W.; Conn, P. Jeffrey

    2010-01-01

    Previous reports have shown that activation of N-methyl-D-aspartate (NMDA) receptors potentiates responses to activation of the group I metabotropic glutamate receptor mGluR5 by reversing PKC-mediated desensitization of this receptor. NMDA-induced reversal of mGluR5 desensitization is dependent on activation of protein phosphatases. However, the specific protein phosphatase involved and the precise mechanism by which NMDA receptor activation reduces mGluR desensitization are not known. We have performed a series of molecular, biochemical, and genetic studies to show that NMDA-induced regulation of mGluR5 is dependent on activation of calcium-dependent protein phosphatase 2B/calcineurin (PP2B/CaN). Furthermore, we report that purified calcineurin directly dephosphorylates the C-terminal tail of mGluR5 at sites that are phosphorylated by PKC. Finally, immunoprecipitation and GST fusion protein pull-down experiments reveal that calcineurin interacts with mGluR5, suggesting that these proteins could be colocalized in a signaling complex. Taken together with previous studies, these data suggest that activation of NMDA receptors leads to activation of calcineurin and that calcineurin modulates mGluR5 function by directly dephosphorylating mGluR5 at PKC sites that are involved in desensitization of this receptor. 2005 Elsevier Ltd. All rights reserved. PMID:16005030

  17. Protein phosphatase 2A inhibition and circumvention of cisplatin cross-resistance by novel TCM-platinum anticancer agents containing demethylcantharidin.

    Science.gov (United States)

    To, Kenneth K W; Wang, Xinning; Yu, Chun Wing; Ho, Yee-Ping; Au-Yeung, Steve C F

    2004-09-01

    Novel TCM-platinum compounds [Pt(C(8)H(8)O(5))(NH(2)R)(2)] 1-5, derived from integrating demethylcantharidin, a modified component from a traditional Chinese medicine (TCM) with a platinum moiety, possess anticancer and protein phosphatase 2A inhibition properties. The compounds are able to circumvent cisplatin resistance by apparently targeting the DNA repair mechanism. Novel isosteric analogues [Pt(C(9)H(10)O(4))(NH(2)R)(2)] A and B, devoid of PP2A-inhibitory activity, were found to suffer from an enhanced DNA repair and were cross-resistant to cisplatin. The results advocate a well-defined structure-activity requirement associating the PP2A-inhibiting demethylcantharidin with the circumvention of cisplatin cross-resistance demonstrated by TCM-Pt compounds 1-5.

  18. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition.

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-06-14

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition.

  19. Repression of class I transcription by cadmium is mediated by the protein phosphatase 2A

    Science.gov (United States)

    Zhou, Lei; Le Roux, Gwenaëlle; Ducrot, Cécile; Chédin, Stéphane; Labarre, Jean; Riva, Michel; Carles, Christophe

    2013-01-01

    Toxic metals are part of our environment, and undue exposure to them leads to a variety of pathologies. In response, most organisms adapt their metabolism and have evolved systems to limit this toxicity and to acquire tolerance. Ribosome biosynthesis being central for protein synthesis, we analyzed in yeast the effects of a moderate concentration of cadmium (Cd2+) on Pol I transcription that represents >60% of the transcriptional activity of the cells. We show that Cd2+ rapidly and drastically shuts down the expression of the 35S rRNA. Repression does not result from a poisoning of any of the components of the class I transcriptional machinery by Cd2+, but rather involves a protein phosphatase 2A (PP2A)-dependent cellular signaling pathway that targets the formation/dissociation of the Pol I–Rrn3 complex. We also show that Pol I transcription is repressed by other toxic metals, such as Ag+ and Hg2+, which likewise perturb the Pol I–Rrn3 complex, but through PP2A-independent mechanisms. Taken together, our results point to a central role for the Pol I–Rrn3 complex as molecular switch for regulating Pol I transcription in response to toxic metals. PMID:23640330

  20. Phosphatase-regulated recruitment of the spindle- and kinetochore-associated (Ska complex to kinetochores

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    Sushama Sivakumar

    2017-11-01

    Full Text Available Kinetochores move chromosomes on dynamic spindle microtubules and regulate signaling of the spindle checkpoint. The spindle- and kinetochore-associated (Ska complex, a hexamer composed of two copies of Ska1, Ska2 and Ska3, has been implicated in both roles. Phosphorylation of kinetochore components by the well-studied mitotic kinases Cdk1, Aurora B, Plk1, Mps1, and Bub1 regulate chromosome movement and checkpoint signaling. Roles for the opposing phosphatases are more poorly defined. Recently, we showed that the C terminus of Ska1 recruits protein phosphatase 1 (PP1 to kinetochores. Here we show that PP1 and protein phosphatase 2A (PP2A both promote accumulation of Ska at kinetochores. Depletion of PP1 or PP2A by siRNA reduces Ska binding at kinetochores, impairs alignment of chromosomes to the spindle midplane, and causes metaphase delay or arrest, phenotypes that are also seen after depletion of Ska. Artificial tethering of PP1 to the outer kinetochore protein Nuf2 promotes Ska recruitment to kinetochores, and it reduces but does not fully rescue chromosome alignment and metaphase arrest defects seen after Ska depletion. We propose that Ska has multiple functions in promoting mitotic progression and that kinetochore-associated phosphatases function in a positive feedback cycle to reinforce Ska complex accumulation at kinetochores.

  1. ARPP-16 Is a Striatal-Enriched Inhibitor of Protein Phosphatase 2A Regulated by Microtubule-Associated Serine/Threonine Kinase 3 (Mast 3 Kinase).

    Science.gov (United States)

    Andrade, Erika C; Musante, Veronica; Horiuchi, Atsuko; Matsuzaki, Hideo; Brody, A Harrison; Wu, Terence; Greengard, Paul; Taylor, Jane R; Nairn, Angus C

    2017-03-08

    ARPP-16 (cAMP-regulated phospho-protein of molecular weight 16 kDa) is one of several small acid-soluble proteins highly expressed in medium spiny neurons of striatum that are phosphorylated in response to dopamine acting via D1 receptor/protein kinase A (PKA) signaling. We show here that ARPP-16 is also phosphorylated in vitro and in vivo by microtubule-associated serine/threonine kinase 3 (MAST3 kinase), an enzyme of previously unknown function that is enriched in striatum. We find that ARPP-16 interacts directly with the scaffolding A subunit of the serine/threonine protein phosphatase, PP2A, and that phosphorylation of ARPP-16 at Ser46 by MAST3 kinase converts the protein into a selective inhibitor of B55α- and B56δ-containing heterotrimeric forms of PP2A. Ser46 of ARPP-16 is phosphorylated to a high basal stoichiometry in striatum, suggestive of basal inhibition of PP2A in striatal neurons. In support of this hypothesis, conditional knock-out of ARPP-16 in CaMKIIα::cre/floxed ARPP-16/19 mice results in dephosphorylation of a subset of PP2A substrates including phospho-Thr75-DARPP-32, phospho-T308-Akt, and phospho-T202/Y204-ERK. Conditional knock-out of ARPP-16/19 is associated with increased motivation measured on a progressive ratio schedule of food reinforcement, yet an attenuated locomotor response to acute cocaine. Our previous studies have shown that ARPP-16 is phosphorylated at Ser88 by PKA. Activation of PKA in striatal slices leads to phosphorylation of Ser88, and this is accompanied by marked dephosphorylation of Ser46. Together, these studies suggest that phospho-Ser46-ARPP-16 acts to basally control PP2A in striatal medium spiny neurons but that dopamine acting via PKA inactivates ARPP-16 leading to selective potentiation of PP2A signaling. SIGNIFICANCE STATEMENT We describe a novel mechanism of signal transduction enriched in medium spiny neurons of striatum that likely mediates effects of the neurotransmitter dopamine acting on these cells. We

  2. Nuclear protein phosphatase-1: an epigenetic regulator of fear memory and amygdala long-term potentiation.

    Science.gov (United States)

    Koshibu, K; Gräff, J; Mansuy, I M

    2011-01-26

    Complex brain diseases and neurological disorders in human generally result from the disturbance of multiple genes and signaling pathways. These disturbances may derive from mutations, deletions, translocations or rearrangements of specific gene(s). However, over the past years, it has become clear that such disturbances may also derive from alterations in the epigenome affecting several genes simultaneously. Our work recently demonstrated that epigenetic mechanisms in the adult brain are in part regulated by protein phosphatase 1 (PP1), a protein Ser/Thr phosphatase that negatively regulates hippocampus-dependent long-term memory (LTM) and synaptic plasticity. PP1 is abundant in brain structures involved in emotional processing like the amygdala, it may therefore be involved in the regulation of fear memory, a form of memory related to post-traumatic stress disorder (PTSD) in human. Here, we demonstrate that PP1 is a molecular suppressor of fear memory and synaptic plasticity in the amygdala that can control chromatin remodeling in neurons. We show that the selective inhibition of the nuclear pool of PP1 in amygdala neurons significantly alters posttranslational modifications (PTMs) of histones and the expression of several memory-associated genes. These alterations correlate with enhanced fear memory, and with an increase in long-term potentiation (LTP) that is transcription-dependent. Our results underscore the importance of nuclear PP1 in the amygdala as an epigenetic regulator of emotional memory, and the relevance of protein phosphatases as potential targets for therapeutic treatment of brain disorders like PTSD. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved.

  3. Reciprocal regulation of ARPP-16 by PKA and MAST3 kinases provides a cAMP-regulated switch in protein phosphatase 2A inhibition

    Science.gov (United States)

    Musante, Veronica; Li, Lu; Kanyo, Jean; Lam, Tukiet T; Colangelo, Christopher M; Cheng, Shuk Kei; Brody, A Harrison; Greengard, Paul; Le Novère, Nicolas; Nairn, Angus C

    2017-01-01

    ARPP-16, ARPP-19, and ENSA are inhibitors of protein phosphatase PP2A. ARPP-19 and ENSA phosphorylated by Greatwall kinase inhibit PP2A during mitosis. ARPP-16 is expressed in striatal neurons where basal phosphorylation by MAST3 kinase inhibits PP2A and regulates key components of striatal signaling. The ARPP-16/19 proteins were discovered as substrates for PKA, but the function of PKA phosphorylation is unknown. We find that phosphorylation by PKA or MAST3 mutually suppresses the ability of the other kinase to act on ARPP-16. Phosphorylation by PKA also acts to prevent inhibition of PP2A by ARPP-16 phosphorylated by MAST3. Moreover, PKA phosphorylates MAST3 at multiple sites resulting in its inhibition. Mathematical modeling highlights the role of these three regulatory interactions to create a switch-like response to cAMP. Together, the results suggest a complex antagonistic interplay between the control of ARPP-16 by MAST3 and PKA that creates a mechanism whereby cAMP mediates PP2A disinhibition. DOI: http://dx.doi.org/10.7554/eLife.24998.001 PMID:28613156

  4. Protein phosphatases 2A as well as reactive oxygen species involved in tributyltin-induced apoptosis in mouse livers.

    Science.gov (United States)

    Zhang, Yali; Chen, Yonggang; Sun, Lijun; Liang, Jing; Guo, Zonglou; Xu, Lihong

    2014-02-01

    Tributyltin (TBT), a highly toxic environmental contaminant, has been shown to induce caspase-3-dependent apoptosis in human amniotic cells through protein phosphatase 2A (PP2A) inhibition and consequent JNK activation. This in vivo study was undertaken to further verify the results derived from our previous in vitro study. Mice were orally dosed with 0, 10, 20, and 60 mg/kg of body weight TBT, and levels of PP2A, reactive oxygen species (ROS), mitogen-activated protein kinase (MAPK), Bax/Bcl-2, and caspase-3 were detected in the mouse livers. Apoptosis was also evaluated using the TUNEL assay. The results showed that PP2A activity was inhibited, ROS levels were elevated, and MAPKs including ERK, JNK, and p38 were activated in mouse livers treated with the highest dose of TBT. Additionally, the ratio of Bax/Bcl-2 was increased, caspase-3 was activated, and apoptosis in mouse livers could be detected in the highest dose group. Therefore, a possible signaling pathway in TBT-induced apoptosis in mouse livers involves PP2A inhibition and ROS elevation serving a pivotal function as upstream activators of MAPKs; activation of MAPKs in turn leads to an increase in the Bax/Bcl-2 ratio, ultimately leading to the activation of caspase-3. The results give a comprehensive and novel description of the mechanism of TBT-induced toxicity. Copyright © 2011 Wiley Periodicals, Inc., A Wiley Company.

  5. The PP2A Regulatory Subunit Tap46, a Component of the TOR Signaling Pathway, Modulates Growth and Metabolism in Plants[W

    Science.gov (United States)

    Ahn, Chang Sook; Han, Jeong-A; Lee, Ho-Seok; Lee, Semi; Pai, Hyun-Sook

    2011-01-01

    Tap42/α4, a regulatory subunit of protein phosphatase 2A, is a downstream effector of the target of rapamycin (TOR) protein kinase, which regulates cell growth in coordination with nutrient and environmental conditions in yeast and mammals. In this study, we characterized the functions and phosphatase regulation of plant Tap46. Depletion of Tap46 resulted in growth arrest and acute plant death with morphological markers of programmed cell death. Tap46 interacted with PP2A and PP2A-like phosphatases PP4 and PP6. Tap46 silencing modulated cellular PP2A activities in a time-dependent fashion similar to TOR silencing. Immunoprecipitated full-length and deletion forms of Arabidopsis thaliana TOR phosphorylated recombinant Tap46 protein in vitro, supporting a functional link between Tap46 and TOR. Tap46 depletion reproduced the signature phenotypes of TOR inactivation, such as dramatic repression of global translation and activation of autophagy and nitrogen mobilization, indicating that Tap46 may act as a positive effector of TOR signaling in controlling those processes. Additionally, Tap46 silencing in tobacco (Nicotiana tabacum) BY-2 cells caused chromatin bridge formation at anaphase, indicating its role in sister chromatid segregation. These findings suggest that Tap46, in conjunction with associated phosphatases, plays an essential role in plant growth and development as a component of the TOR signaling pathway. PMID:21216945

  6. Beyond the Dopamine Receptor: Regulation and Roles of Serine/Threonine Protein Phosphatases

    Directory of Open Access Journals (Sweden)

    Sven I Walaas

    2011-08-01

    Full Text Available Dopamine plays an important modulatory role in the central nervous system, helping to control critical aspects of motor function and reward learning. Alteration in normal dopaminergic neurotransmission underlies multiple neurological diseases including schizophrenia, Huntington's disease and Parkinson's disease. Modulation of dopamine-regulated signaling pathways is also important in the addictive actions of most drugs of abuse. Our studies over the last 30 years have focused on the molecular actions of dopamine acting on medium spiny neurons, the predominant neurons of the neostriatum. Striatum-enriched phosphoproteins, particularly DARPP-32, RCS (Regulator of Calmodulin Signaling and ARPP-16, mediate pleiotropic actions of dopamine. Notably, each of these proteins, either directly or indirectly, regulates the activity of one of the three major subclasses of serine/threonine protein phosphatases, PP1, PP2B and PP2A, respectively. For example, phosphorylation of DARPP-32 at Thr34 by protein kinase A results in potent inhibition of PP1, leading to potentiation of dopaminergic signaling at multiple steps from the dopamine receptor to the nucleus. The discovery of DARPP-32 and its emergence as a critical molecular integrator of striatal signaling will be discussed, as will more recent studies that highlight novel roles for RCS and ARPP-16 in dopamine-regulated striatal signaling pathways.

  7. Phosphatase control of 4E-BP1 phosphorylation state is central for glycolytic regulation of retinal protein synthesis.

    Science.gov (United States)

    Gardner, Thomas W; Abcouwer, Steven F; Losiewicz, Mandy K; Fort, Patrice E

    2015-09-15

    Control of protein synthesis in insulin-responsive tissues has been well characterized, but relatively little is known about how this process is regulated in nervous tissues. The retina exhibits a relatively high protein synthesis rate, coinciding with high basal Akt and metabolic activities, with the majority of retinal ATP being derived from aerobic glycolysis. We examined the dependency of retinal protein synthesis on the Akt-mTOR signaling and glycolysis using ex vivo rat retinas. Akt inhibitors significantly reduced retinal protein synthesis but did not affect glycolytic lactate production. Surprisingly, the glycolytic inhibitor 2-deoxyglucose (2-DG) markedly inhibited Akt1 and Akt3 activities, as well as protein synthesis. The effects of 2-DG, and 2-fluorodeoxyglucose (2-FDG) on retinal protein synthesis correlated with inhibition of lactate production and diminished ATP content, with all these effects reversed by provision of d-mannose. 2-DG treatment was not associated with increased AMPK, eEF2, or eIF2α phosphorylation; instead, it caused rapid dephosphorylation of 4E-BP1. 2-DG reduced total mTOR activity by 25%, but surprisingly, it did not reduce mTORC1 activity, as indicated by unaltered raptor-associated mTOR autophosphorylation and ribosomal protein S6 phosphorylation. Dephosphorylation of 4E-BP1 was largely prevented by inhibition of PP1/PP2A phosphatases with okadaic acid and calyculin A, and inhibition of PPM1 phosphatases with cadmium. Thus, inhibition of retinal glycolysis diminished Akt and protein synthesis coinciding with accelerated dephosphorylation of 4E-BP1 independently of mTORC1. These results demonstrate a novel mechanism regulating protein synthesis in the retina involving an mTORC1-independent and phosphatase-dependent regulation of 4E-BP1. Copyright © 2015 the American Physiological Society.

  8. Overexpression of a Protein Phosphatase 2C from Beech Seeds in Arabidopsis Shows Phenotypes Related to Abscisic Acid Responses and Gibberellin Biosynthesis1

    Science.gov (United States)

    Reyes, David; Rodríguez, Dolores; González-García, Mary Paz; Lorenzo, Oscar; Nicolás, Gregorio; García-Martínez, José Luis; Nicolás, Carlos

    2006-01-01

    A functional abscisic acid (ABA)-induced protein phosphatase type 2C (PP2C) was previously isolated from beech (Fagus sylvatica) seeds (FsPP2C2). Because transgenic work is not possible in beech, in this study we overexpressed this gene in Arabidopsis (Arabidopsis thaliana) to provide genetic evidence on FsPP2C2 function in seed dormancy and other plant responses. In contrast with other PP2Cs described so far, constitutive expression of FsPP2C2 in Arabidopsis, under the cauliflower mosaic virus 35S promoter, produced enhanced sensitivity to ABA and abiotic stress in seeds and vegetative tissues, dwarf phenotype, and delayed flowering, and all these effects were reversed by gibberellic acid application. The levels of active gibberellins (GAs) were reduced in 35S:FsPP2C2 plants, although transcript levels of AtGA20ox1 and AtGA3ox1 increased, probably as a result of negative feedback regulation, whereas the expression of GASA1 was induced by GAs. Additionally, FsPP2C2-overexpressing plants showed a strong induction of the Responsive to ABA 18 (RAB18) gene. Interestingly, FsPP2C2 contains two nuclear targeting sequences, and transient expression assays revealed that ABA directed this protein to the nucleus. Whereas other plant PP2Cs have been shown to act as negative regulators, our results support the hypothesis that FsPP2C2 is a positive regulator of ABA. Moreover, our results indicate the existence of potential cross-talk between ABA signaling and GA biosynthesis. PMID:16815952

  9. Storage Stability Improvement of Copolymer Grafted Polypropylene-AcrylicAcid (PP-AA), by means of Various After Treatment Processes

    International Nuclear Information System (INIS)

    Gitopadmojo, Isminingsih

    2000-01-01

    Polypropylene yams that have been subjected to irradiation induced graftco-polymerization with acrylic acid, have gained its moisture regain and dyeability, that fulfilled the requirement as textile material for garment.However, the copolymer grafted PP-AA has suffered from degradation in thestorage, which was indicated in the previous study that the strengthretention has dropped tremendously by photo-oxidation or photo-degradation.After treatments of PP-AA yams with chemical compound that was able toprevent further photo-oxidation, will be expected to improve the stability ofPP-AA in storage. In this research activity, the polypropylene (PP) yams weresubjected to irradiation induced graft co-polymerization by means ofγ-Ray Co-60 as irradiation source with acrylic acid (AA) as monomer.Various after treatments were subjected to the grafted PP-AA yams such asalkalisation process; dyeing (anionic dyes, cationic dyes and nonionic dyes);as well as processing with optical brightening agent and UV stabilizer,separately. The PP-AA yams (before and after treatment) were subjected tostorage from 1 month up to 42 months, and then being tested for theirmoisture regain, strength retention and elongation at breaks. The samplesbeing stored for 12 months were subjected to radical analysis. It isconcluded from the experiment that after treatment of grafted PP-AA by meansof those various processes were able to improve the stability of copolymergrafted PP-AA in storage. The presence of peroxide radical in the ESR(electron spin resonance) spectrum on PP-AA yams before treatment and theones after treated with alkaline and being stored for 12 months haveindicated the presence of photo oxidation or photo degradation, while thepresence of poly enyl radical in the ESR spectrum of after treated PP-AA withdyes having azo and azine compound as chromophore, as well as with UVstabilizer with carbonyl as chromophore and being stored for 12 months haveproved that its presence have protected such

  10. The composition and function of the striatin-interacting phosphatases and kinases (STRIPAK) complex in fungi.

    Science.gov (United States)

    Kück, Ulrich; Beier, Anna M; Teichert, Ines

    2016-05-01

    The striatin-interacting phosphatases and kinases (STRIPAK) complex is a highly conserved eukaryotic protein complex that was recently described for diverse animal and fungal species. Here, we summarize our current knowledge about the composition and function of the STRIPAK complex from the ascomycete Sordaria macrospora, which we discovered by investigating sexually sterile mutants (pro), having a defect in fruiting body development. Mass spectrometry and yeast two-hybrid analysis defined core subunits of the STRIPAK complex, which have structural homologs in animal and other fungal organisms. These subunits (and their mammalian homologs) are PRO11 (striatin), PRO22 (STRIP1/2), SmMOB3 (Mob3), PRO45 (SLMAP), and PP2AA, the structural, and PP2Ac, the catalytic subunits of protein phosphatase 2A (PP2A). Beside fruiting body formation, the STRIPAK complex controls vegetative growth and hyphal fusion in S. macrospora. Although the contribution of single subunits to diverse cellular and developmental processes is not yet fully understood, functional analysis has already shown that mammalian homologs are able to substitute the function of distinct fungal STRIPAK subunits. This underscores the view that fungal model organisms serve as useful tools to get a molecular insight into cellular and developmental processes of eukaryotes in general. Future work will unravel the precise localization of single subunits within the cell and decipher their STRIPAK-related and STRIPAK-independent functions. Finally, evidence is accumulating that there is a crosstalk between STRIPAK and various signaling pathways, suggesting that eukaryotic development is dependent on STRIPAK signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. ROTUNDA3 function in plant development by phosphatase 2A-mediated regulation of auxin transporter recycling.

    Science.gov (United States)

    Karampelias, Michael; Neyt, Pia; De Groeve, Steven; Aesaert, Stijn; Coussens, Griet; Rolčík, Jakub; Bruno, Leonardo; De Winne, Nancy; Van Minnebruggen, Annemie; Van Montagu, Marc; Ponce, María Rosa; Micol, José Luis; Friml, Jiří; De Jaeger, Geert; Van Lijsebettens, Mieke

    2016-03-08

    The shaping of organs in plants depends on the intercellular flow of the phytohormone auxin, of which the directional signaling is determined by the polar subcellular localization of PIN-FORMED (PIN) auxin transport proteins. Phosphorylation dynamics of PIN proteins are affected by the protein phosphatase 2A (PP2A) and the PINOID kinase, which act antagonistically to mediate their apical-basal polar delivery. Here, we identified the ROTUNDA3 (RON3) protein as a regulator of the PP2A phosphatase activity in Arabidopsis thaliana. The RON3 gene was map-based cloned starting from the ron3-1 leaf mutant and found to be a unique, plant-specific gene coding for a protein with high and dispersed proline content. The ron3-1 and ron3-2 mutant phenotypes [i.e., reduced apical dominance, primary root length, lateral root emergence, and growth; increased ectopic stages II, IV, and V lateral root primordia; decreased auxin maxima in indole-3-acetic acid (IAA)-treated root apical meristems; hypergravitropic root growth and response; increased IAA levels in shoot apices; and reduced auxin accumulation in root meristems] support a role for RON3 in auxin biology. The affinity-purified PP2A complex with RON3 as bait suggested that RON3 might act in PIN transporter trafficking. Indeed, pharmacological interference with vesicle trafficking processes revealed that single ron3-2 and double ron3-2 rcn1 mutants have altered PIN polarity and endocytosis in specific cells. Our data indicate that RON3 contributes to auxin-mediated development by playing a role in PIN recycling and polarity establishment through regulation of the PP2A complex activity.

  12. [Effect of inhibitors serine/threonine protein kinases and protein phosphatases on mitosis progression of synchronized tobacco by-2 cells].

    Science.gov (United States)

    Sheremet, Ia A; Emets, A I; Azmi, A; Vissenberg, K; Verbelen, J-P; Blium, Ia B

    2012-01-01

    In order to investigate the role of various serine/ threonine protein kinases and protein phosphatases in the regulation of mitosis progression in plant cells the influence of cyclin-dependent (olomoucine) and Ca2+ -calmodulin-dependent (W7) protein kinases inhibitors, as well as protein kinase C inhibitors (H7 and staurosporine) and protein phosphatases inhibitor (okadaic acid) on mitosis progression in synchronized tobacco BY-2 cells has been studied. It was found that BY-2 culture treatment with inhibitors of cyclin dependent protein kinases and protein kinase C causes prophase delay, reduces the mitotic index and displaces of mitotic peak as compare with control cells. Inhibition of Ca2+ -calmodulin dependent protein kinases enhances the cell entry into prophase and delays their exit from mitosis. Meanwhile inhibition of serine/threonine protein phosphatases insignificantly enhances of synchronized BY-2 cells entering into all phases of mitosis.

  13. Calyculins and Related Marine Natural Products as Serine- Threonine Protein Phosphatase PP1 and PP2A Inhibitors and Total Syntheses of Calyculin A, B, and C

    Directory of Open Access Journals (Sweden)

    Ari M. P. Koskinen

    2010-01-01

    Full Text Available Calyculins, highly cytotoxic polyketides, originally isolated from the marine sponge Discodermia calyx by Fusetani and co-workers, belong to the lithistid sponges group. These molecules have become interesting targets for cell biologists and synthetic organic chemists. The serine/threonine protein phosphatases play an essential role in the cellular signalling, metabolism, and cell cycle control. Calyculins express potent protein phosphatase 1 and 2A inhibitory activity, and have therefore become valuable tools for cellular biologists studying intracellular processes and their control by reversible phosphorylation. Calyculins might also play an important role in the development of several diseases such as cancer, neurodegenerative diseases, and type 2-diabetes mellitus. The fascinating structures of calyculins have inspired various groups of synthetic organic chemists to develop total syntheses of the most abundant calyculins A and C. However, with fifteen chiral centres, a cyano-capped tetraene unit, a phosphate-bearing spiroketal, an anti, anti, anti dipropionate segment, an α-chiral oxazole, and a trihydroxylated γ-amino acid, calyculins reach versatility that only few natural products can surpass, and truly challenge modern chemists’ asymmetric synthesis skills.

  14. Distinct functional domains within the acidic cluster of tegument protein pp28 required for trafficking and cytoplasmic envelopment of human cytomegalovirus.

    Science.gov (United States)

    Seo, Jun-Young; Jeon, Hyejin; Hong, Sookyung; Britt, William J

    2016-10-01

    Human cytomegalovirus UL99-encoded tegument protein pp28 contains a 16 aa acidic cluster that is required for pp28 trafficking to the assembly compartment (AC) and the virus assembly. However, functional signals within the acidic cluster of pp28 remain undefined. Here, we demonstrated that an acidic cluster rather than specific sorting signals was required for trafficking to the AC. Recombinant viruses with chimeric pp28 proteins expressing non-native acidic clusters exhibited delayed viral growth kinetics and decreased production of infectious virus, indicating that the native acidic cluster of pp28 was essential for wild-type virus assembly. These results suggested that the acidic cluster of pp28 has distinct functional domains required for trafficking and for efficient virus assembly. The first half (aa 44-50) of the acidic cluster was sufficient for pp28 trafficking, whereas the native acidic cluster consisting of aa 51-59 was required for the assembly of wild-type levels of infectious virus.

  15. Andrographolide induces vascular smooth muscle cell apoptosis through a SHP-1-PP2A-p38MAPK-p53 cascade.

    Science.gov (United States)

    Chen, Yu-Ying; Hsieh, Cheng-Ying; Jayakumar, Thanasekaran; Lin, Kuan-Hung; Chou, Duen-Suey; Lu, Wan-Jung; Hsu, Ming-Jen; Sheu, Joen-Rong

    2014-07-10

    The abnormal growth of vascular smooth muscle cells (VSMCs) is considered a critical pathogenic process in inflammatory vascular diseases. We have previously demonstrated that protein phosphatase 2 A (PP2A)-mediated NF-κB dephosphorylation contributes to the anti-inflammatory properties of andrographolide, a novel NF-κB inhibitor. In this study, we investigated whether andrographolide causes apoptosis, and characterized its apoptotic mechanisms in rat VSMCs. Andrographolide activated the p38 mitogen-activated protein kinase (p38MAPK), leading to p53 phosphorylation. Phosphorylated p53 subsequently transactivated the expression of Bax, a pro-apoptotic protein. Transfection with pp2a small interfering RNA (siRNA) suppressed andrographolide-induced p38MAPK activation, p53 phosphorylation, and caspase 3 activation. Andrographolide also activated the Src homology 1 domain-containing protein tyrosine phosphatase (SHP-1), and induced PP2A dephosphorylation, both of which were inhibited by the SHP-1 inhibitor sodium stibogluconate (SSG) or shp-1 siRNA. SSG or shp-1 siRNA prevented andrographolide-induced apoptosis. These results suggest that andrographolide activates the PP2A-p38MAPK-p53-Bax cascade, causing mitochondrial dysfunction and VSMC death through an SHP-1-dependent mechanism.

  16. Lipid rafts regulate PCB153-induced disruption of occludin and brain endothelial barrier function through protein phosphatase 2A and matrix metalloproteinase-2

    Energy Technology Data Exchange (ETDEWEB)

    Eum, Sung Yong, E-mail: seum@miami.edu; Jaraki, Dima; András, Ibolya E.; Toborek, Michal

    2015-09-15

    Occludin is an essential integral transmembrane protein regulating tight junction (TJ) integrity in brain endothelial cells. Phosphorylation of occludin is associated with its localization to TJ sites and incorporation into intact TJ assembly. The present study is focused on the role of lipid rafts in polychlorinated biphenyl (PCB)-induced disruption of occludin and endothelial barrier function. Exposure of human brain endothelial cells to 2,2′,4,4′,5,5′-hexachlorobiphenyl (PCB153) induced dephosphorylation of threonine residues of occludin and displacement of occludin from detergent-resistant membrane (DRM)/lipid raft fractions within 1 h. Moreover, lipid rafts modulated the reduction of occludin level through activation of matrix metalloproteinase 2 (MMP-2) after 24 h PCB153 treatment. Inhibition of protein phosphatase 2A (PP2A) activity by okadaic acid or fostriecin markedly protected against PCB153-induced displacement of occludin and increased permeability of endothelial cells. The implication of lipid rafts and PP2A signaling in these processes was further defined by co-immunoprecipitation of occludin with PP2A and caveolin-1, a marker protein of lipid rafts. Indeed, a significant MMP-2 activity was observed in lipid rafts and was increased by exposure to PCB153. The pretreatment of MMP-2 inhibitors protected against PCB153-induced loss of occludin and disruption of lipid raft structure prevented the increase of endothelial permeability. Overall, these results indicate that lipid raft-associated processes, such as PP2A and MMP-2 activation, participate in PCB153-induced disruption of occludin function in brain endothelial barrier. This study contributes to a better understanding of the mechanisms leading to brain endothelial barrier dysfunction in response to exposure to environmental pollutants, such as ortho-substituted PCBs. - Highlights: • PCB153 disturbed human brain endothelial barrier through disruption of occludin. • Lipid raft-associated PP

  17. Liver carbohydrates metabolism: A new islet-neogenesis associated protein peptide (INGAP-PP) target.

    Science.gov (United States)

    Villagarcía, Hernán Gonzalo; Román, Carolina Lisi; Castro, María Cecilia; González, Luisa Arbeláez; Ronco, María Teresa; Francés, Daniel Eleazar; Massa, María Laura; Maiztegui, Bárbara; Flores, Luis Emilio; Gagliardino, Juan José; Francini, Flavio

    2018-03-01

    Islet-Neogenesis Associated Protein-Pentadecapeptide (INGAP-PP) increases β-cell mass and enhances glucose and amino acids-induced insulin secretion. Our aim was to demonstrate its effect on liver metabolism. For that purpose, adult male Wistar rats were injected twice-daily (10 days) with saline solution or INGAP-PP (250 μg). Thereafter, serum glucose, triglyceride and insulin levels were measured and homeostasis model assessment (HOMA-IR) and hepatic insulin sensitivity (HIS) were determined. Liver glucokinase and glucose-6-phosphatase (G-6-Pase) expression and activity, phosphoenolpyruvate carboxykinase (PEPCK) expression, phosphofructokinase-2 (PFK-2) protein content, P-Akt/Akt and glycogen synthase kinase-3β (P-GSK3/GSK3) protein ratios and glycogen deposit were also determined. Additionally, glucokinase activity and G-6-Pase and PEPCK gene expression were also determined in isolated hepatocytes from normal rats incubated with INGAP-PP (5 μg/ml). INGAP-PP administration did not modify any of the serum parameters tested but significantly increased activity of liver glucokinase and the protein level of its cytosolic activator, PFK-2. Conversely, INGAP-PP treated rats decreased gene expression and enzyme activity of gluconeogenic enzymes, G-6-Pase and PEPCK. They also showed a higher glycogen deposit and P-GSK3/GSK3 and P-Akt/Akt ratio. In isolated hepatocytes, INGAP-PP increased GK activity and decreased G-6-Pase and PEPCK expression. These results demonstrate a direct effect of INGAP-PP on the liver acting through P-Akt signaling pathway. INGAP-PP enhances liver glucose metabolism and deposit and reduces its production/output, thereby contributing to maintain normal glucose homeostasis. These results reinforce the concept that INGAP-PP might become a useful tool to treat people with impaired islet/liver glucose metabolism as it occurs in T2D. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. A Tenebrio molitor GPI-anchored alkaline phosphatase is involved in binding of Bacillus thuringiensis Cry3Aa to brush border membrane vesicles.

    Science.gov (United States)

    Zúñiga-Navarrete, Fernando; Gómez, Isabel; Peña, Guadalupe; Bravo, Alejandra; Soberón, Mario

    2013-03-01

    Bacillus thuringiensis Cry toxins recognizes their target cells in part by the binding to glycosyl-phosphatidyl-inositol (GPI) anchored proteins such as aminopeptidase-N (APN) or alkaline phosphatases (ALP). Treatment of Tenebrio molitor brush border membrane vesicles (BBMV) with phospholipase C that cleaves out GPI-anchored proteins from the membranes, showed that GPI-anchored proteins are involved in binding of Cry3Aa toxin to BBMV. A 68 kDa GPI-anchored ALP was shown to bind Cry3Aa by toxin overlay assays. The 68 kDa GPI-anchored ALP was preferentially expressed in early instar larvae in comparison to late instar larvae. Our work shows for the first time that GPI-anchored ALP is important for Cry3Aa binding to T. molitor BBMV suggesting that the mode of action of Cry toxins is conserved in different insect orders. Copyright © 2012 Elsevier Inc. All rights reserved.

  19. PP2A: The Achilles Heal in MDS with 5q Deletion

    Directory of Open Access Journals (Sweden)

    David eSallman

    2014-09-01

    Full Text Available Myelodysplastic syndromes (MDS represent a hematologically diverse group of myeloid neoplasms, however, one subtype characterized by an isolated deletion of chromosome 5q (del(5q is pathologically and clinically distinct. Patients with del(5q MDS share biological features that account for the profound hypoplastic anemia and unique sensitivity to treatment with lenalidomide. Ineffective erythropoiesis in del(5q MDS arises from allelic deletion of the ribosomal processing S-14 (RPS14 gene, which leads to MDM2 sequestration with consequent p53 activation and erythroid cell death. Since its approval in 2005, lenalidomide has changed the natural course of the disease. Patients who achieve transfusion independence and/or a cytogenetic response with lenalidomide have a decreased risk of progression to AML and an improved overall survival compared to non-responders. Elucidation of the mechanisms of action of lenalidomide in del(5q MDS has advanced therapeutic strategies for this disease. The selective cytotoxicity of lenalidomide in del(5q clones derives from inhibition of a haplodeficient phosphatase whose catalytic domain is encoded within the common deleted region on chromosome 5q, i.e., protein phosphatase 2A (PP2Acα. PP2A is a highly conserved, dual specificity phosphatase that plays an essential role in regulation of the G2/M checkpoint. Inhibition of PP2Acα results in cell cycle arrest and apoptosis in del(5q cells. Targeted knockdown of PP2Acα using siRNA is sufficient to sensitize non-del(5q clones to lenalidomide. Through its inhibitory effect on PP2A, lenalidomide stabilizes MDM2 to restore p53 degradation in erythroid precursors, with subsequent arrest in G2/M. Unfortunately, the majority of patients with del(5q MDS develop resistance to lenalidomide over time associated with PP2Acα overexpression. Targeted inhibition of PP2A with a more potent inhibitor has emerged as an attractive therapeutic approach for patients with del(5q MDS.

  20. Membrane Topology and Biochemical Characterization of the Escherichia coli BacA Undecaprenyl-Pyrophosphate Phosphatase.

    Directory of Open Access Journals (Sweden)

    Guillaume Manat

    Full Text Available Several integral membrane proteins exhibiting undecaprenyl-pyrophosphate (C55-PP phosphatase activity were previously identified in Escherichia coli that belonged to two distinct protein families: the BacA protein, which accounts for 75% of the C55-PP phosphatase activity detected in E. coli cell membranes, and three members of the PAP2 phosphatidic acid phosphatase family, namely PgpB, YbjG and LpxT. This dephosphorylation step is required to provide the C55-P carrier lipid which plays a central role in the biosynthesis of various cell wall polymers. We here report detailed investigations of the biochemical properties and membrane topology of the BacA protein. Optimal activity conditions were determined and a narrow-range substrate specificity with a clear preference for C55-PP was observed for this enzyme. Alignments of BacA protein sequences revealed two particularly well-conserved regions and several invariant residues whose role in enzyme activity was questioned by using a site-directed mutagenesis approach and complementary in vitro and in vivo activity assays. Three essential residues Glu21, Ser27, and Arg174 were identified, allowing us to propose a catalytic mechanism for this enzyme. The membrane topology of the BacA protein determined here experimentally did not validate previous program-based predicted models. It comprises seven transmembrane segments and contains in particular two large periplasmic loops carrying the highly-conserved active site residues. Our data thus provide evidence that all the different E. coli C55-PP phosphatases identified to date (BacA and PAP2 catalyze the dephosphorylation of C55-PP molecules on the same (outer side of the plasma membrane.

  1. Synemin promotes AKT-dependent glioblastoma cell proliferation by antagonizing PP2A.

    Science.gov (United States)

    Pitre, Aaron; Davis, Nathan; Paul, Madhumita; Orr, A Wayne; Skalli, Omar

    2012-04-01

    The intermediate filament protein synemin is present in astrocyte progenitors and glioblastoma cells but not in mature astrocytes. Here we demonstrate a role for synemin in enhancing glioblastoma cell proliferation and clonogenic survival, as synemin RNA interference decreased both behaviors by inducing G1 arrest along with Rb hypophosphorylation and increased protein levels of the G1/S inhibitors p21(Cip1) and p27(Kip1). Akt involvement was demonstrated by decreased phosphorylation of its substrate, p21(Cip1), and reduced Akt catalytic activity and phosphorylation at essential activation sites. Synemin silencing, however, did not affect the activities of PDPK1 and mTOR complex 2, which directly phosphorylate Akt activation sites, but instead enhanced the activity of the major regulator of Akt dephosphorylation, protein phosphatase type 2A (PP2A). This was accompanied by changes in PP2A subcellular distribution resulting in increased physical interactions between PP2A and Akt, as shown by proximity ligation assays (PLAs). PLAs and immunoprecipitation experiments further revealed that synemin and PP2A form a protein complex. In addition, treatment of synemin-silenced cells with the PP2A inhibitor cantharidic acid resulted in proliferation and pAkt and pRb levels similar to those of controls. Collectively these results indicate that synemin positively regulates glioblastoma cell proliferation by helping sequester PP2A away from Akt, thereby favoring Akt activation.

  2. Protein Phosphatase 1 Recruitment by Rif1 Regulates DNA Replication Origin Firing by Counteracting DDK Activity

    Directory of Open Access Journals (Sweden)

    Anoushka Davé

    2014-04-01

    Full Text Available The firing of eukaryotic origins of DNA replication requires CDK and DDK kinase activities. DDK, in particular, is involved in setting the temporal program of origin activation, a conserved feature of eukaryotes. Rif1, originally identified as a telomeric protein, was recently implicated in specifying replication timing in yeast and mammals. We show that this function of Rif1 depends on its interaction with PP1 phosphatases. Mutations of two PP1 docking motifs in Rif1 lead to early replication of telomeres in budding yeast and misregulation of origin firing in fission yeast. Several lines of evidence indicate that Rif1/PP1 counteract DDK activity on the replicative MCM helicase. Our data suggest that the PP1/Rif1 interaction is downregulated by the phosphorylation of Rif1, most likely by CDK/DDK. These findings elucidate the mechanism of action of Rif1 in the control of DNA replication and demonstrate a role of PP1 phosphatases in the regulation of origin firing.

  3. EKPD: a hierarchical database of eukaryotic protein kinases and protein phosphatases.

    Science.gov (United States)

    Wang, Yongbo; Liu, Zexian; Cheng, Han; Gao, Tianshun; Pan, Zhicheng; Yang, Qing; Guo, Anyuan; Xue, Yu

    2014-01-01

    We present here EKPD (http://ekpd.biocuckoo.org), a hierarchical database of eukaryotic protein kinases (PKs) and protein phosphatases (PPs), the key molecules responsible for the reversible phosphorylation of proteins that are involved in almost all aspects of biological processes. As extensive experimental and computational efforts have been carried out to identify PKs and PPs, an integrative resource with detailed classification and annotation information would be of great value for both experimentalists and computational biologists. In this work, we first collected 1855 PKs and 347 PPs from the scientific literature and various public databases. Based on previously established rationales, we classified all of the known PKs and PPs into a hierarchical structure with three levels, i.e. group, family and individual PK/PP. There are 10 groups with 149 families for the PKs and 10 groups with 33 families for the PPs. We constructed 139 and 27 Hidden Markov Model profiles for PK and PP families, respectively. Then we systematically characterized ∼50,000 PKs and >10,000 PPs in eukaryotes. In addition, >500 PKs and >400 PPs were computationally identified by ortholog search. Finally, the online service of the EKPD database was implemented in PHP + MySQL + JavaScript.

  4. Progesterone-associated proteins PP12 and PP14 in the human endometrium.

    Science.gov (United States)

    Rutanen, E M; Koistinen, R; Seppälä, M; Julkunen, M; Suikkari, A M; Huhtala, M L

    1987-01-01

    Two proteins, designated as PP12 and PP14 were originally isolated from soluble extracts of the human placenta and its adjacent membranes. We have shown that they are synthesized by decidualized/secretory endometrium and not by placenta. Both proteins occur at high concentrations in human amniotic fluid, which is therefore an excellent source for purification. PP12 is a 34-kDa glycoprotein, which has an N-terminal amino acid sequence of Ala-Pro-Trp-Gln-Cys-Ala-Pro-Cys-Ser-Ala. This is identical with that of somatomedin-binding protein purified from the amniotic fluid. PP12 too binds somatomedin-C, or IGF-I (insulin-like growth factor-I). Human secretory endometrium synthesizes and secretes PP12, and progesterone stimulates its secretion. PP14 is a 28-kDa glycoprotein. Its N-terminal sequence shows homology to that of beta-lactoglobulins from various species. We have found PP14 in the human endometrium, serum and milk. Immunologically, PP14 is related to progestagen-associated endometrial protein (PEP), alpha-2 pregnancy-associated endometrial protein (alpha-2, PEG), endometrial protein 15 (EP15), alpha-uterine protein (AUP) and chorionic alpha-2 microglobulin (CAG-2). In ovulatory menstrual cycles, the concentration of PP14 increases in endometrial tissue as the secretory changes advance. In serum, the PP14 concentration begins to rise later than the progesterone levels, and high serum PP14 levels are maintained for the first days of the next cycle. By contrast, no elevation of serum PP14 level is seen in anovulatory cycles. Our results show that progesterone-associated proteins are synthesized by the human endometrium and appear in the peripheral circulation, where they can be quantitatively measured using immunochemical techniques.

  5. Statin-activated nuclear receptor PXR promotes SGK2 dephosphorylation by scaffolding PP2C to induce hepatic gluconeogenesis.

    Science.gov (United States)

    Gotoh, Saki; Negishi, Masahiko

    2015-09-22

    Statin therapy is known to increase blood glucose levels in humans. Statins utilize pregnane X receptor (PXR) and serum/glucocorticoid regulated kinase 2 (SGK2) to activate phosphoenolpyruvate carboxykinase 1 (PEPCK1) and glucose-6-phosphatase (G6Pase) genes, thereby increasing glucose production in human liver cells. Here, the novel statin/PXR/SGK2-mediated signaling pathway has now been characterized for hepatic gluconeogenesis. Statin-activated PXR scaffolds the protein phosphatase 2C (PP2C) and SGK2 to stimulate PP2C to dephosphorylate SGK2 at threonine 193. Non-phosphorylated SGK2 co-activates PXR-mediated trans-activation of promoters of gluconeogenic genes in human liver cells, thereby enhancing gluconeogenesis. This gluconeogenic statin-PXR-SGK2 signal is not present in mice, in which statin treatment suppresses hepatic gluconeogenesis. These findings provide the basis for statin-associated side effects such as an increased risk for Type 2 diabetes.

  6. beta2-adaptin is constitutively de-phosphorylated by serine/threonine protein phosphatase PP2A and phosphorylated by a staurosporine-sensitive kinase

    DEFF Research Database (Denmark)

    Lauritsen, Jens Peter Holst; Menné, C; Kastrup, J

    2000-01-01

    Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2-ada...... the hypothesis that phosphorylation/de-phosphorylation of coat proteins plays a regulatory role in the assembly/disassembly cycle of clathrin-coated vesicles.......Clathrin-mediated endocytosis includes cycles of assembly and disassembly of the clathrin-coated vesicle constituents. How these cycles are regulated is still not fully known but previous studies have indicated that phosphorylation of coat subunits may play a role. Here we describe that beta2......-adaptin undergoes cycles of phosphorylation/de-phosphorylation in intact cells. Thus, beta2-adaptin was constitutively de-phosphorylated by serine/threonine protein phosphatase 2A and phosphorylated by a staurosporine-sensitive kinase in vivo. Confocal laser scanning microscopy demonstrated...

  7. Glucohexaose-induced protein phosphatase 2C regulates cell redox ...

    Indian Academy of Sciences (India)

    Q M Chen

    2018-02-13

    Feb 13, 2018 ... glucohexaose, CsPP2C80s play a positive regulatory role in process of ABA combined with ABA receptors ..... protein kinases (SnRKs) involve in the stress responses .... In this work, the endogenous ABA content increased.

  8. Synergistic apoptosis induction in leukemic cells by the phosphatase inhibitor salubrinal and proteasome inhibitors.

    Directory of Open Access Journals (Sweden)

    Hannes C A Drexler

    Full Text Available Cells adapt to endoplasmic reticulum (ER-stress by arresting global protein synthesis while simultaneously activating specific transcription factors and their downstream targets. These processes are mediated in part by the phosphorylation-dependent inactivation of the translation initiation factor eIF2alpha. Following restoration of homeostasis protein synthesis is resumed when the serine/threonine-protein phosphatase PP1 dephosphorylates and reactivates eIF2alpha. Proteasome inhibitors, used to treat multiple myeloma patients evoke ER-stress and apoptosis by blocking the ER-associated degradation of misfolded proteins (ERAD, however, the role of eIF2alpha phosphorylation in leukemic cells under conditions of proteasome inhibitor-mediated ER stress is currently unclear.Bcr-Abl-positive and negative leukemic cell lines were used to investigate the functional implications of PP1-related phosphatase activities on eIF2alpha phosphorylation in proteasome inhibitor-mediated ER stress and apoptosis. Rather unexpectedly, salubrinal, a recently identified PP1 inhibitor capable to protect against ER stress in various model systems, strongly synergized with proteasome inhibitors to augment apoptotic death of different leukemic cell lines. Salubrinal treatment did not affect the phosphorlyation status of eIF2alpha. Furthermore, the proapoptotic effect of salubrinal occurred independently from the chemical nature of the proteasome inhibitor, was recapitulated by a second unrelated phosphatase inhibitor and was unaffected by overexpression of a dominant negative eIF2alpha S51A variant that can not be phosphorylated. Salubrinal further aggravated ER-stress and proteotoxicity inflicted by the proteasome inhibitors on the leukemic cells since characteristic ER stress responses, such as ATF4 and CHOP synthesis, XBP1 splicing, activation of MAP kinases and eventually apoptosis were efficiently abrogated by the translational inhibitor cycloheximide.Although PP1

  9. A protein-tyrosine phosphatase with sequence similarity to the SH2 domain of the protein-tyrosine kinases.

    Science.gov (United States)

    Shen, S H; Bastien, L; Posner, B I; Chrétien, P

    1991-08-22

    The phosphorylation of proteins at tyrosine residues is critical in cellular signal transduction, neoplastic transformation and control of the mitotic cycle. These mechanisms are regulated by the activities of both protein-tyrosine kinases (PTKs) and protein-tyrosine phosphatases (PTPases). As in the PTKs, there are two classes of PTPases: membrane associated, receptor-like enzymes and soluble proteins. Here we report the isolation of a complementary DNA clone encoding a new form of soluble PTPase, PTP1C. The enzyme possesses a large noncatalytic region at the N terminus which unexpectedly contains two adjacent copies of the Src homology region 2 (the SH2 domain) found in various nonreceptor PTKs and other cytoplasmic signalling proteins. As with other SH2 sequences, the SH2 domains of PTP1C formed high-affinity complexes with the activated epidermal growth factor receptor and other phosphotyrosine-containing proteins. These results suggest that the SH2 regions in PTP1C may interact with other cellular components to modulate its own phosphatase activity against interacting substrates. PTPase activity may thus directly link growth factor receptors and other signalling proteins through protein-tyrosine phosphorylation.

  10. Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

    International Nuclear Information System (INIS)

    Chuang, Jian-Ying; Hung, Jan-Jong

    2011-01-01

    Highlights: → Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. → Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. → Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

  11. Overexpression of HDAC1 induces cellular senescence by Sp1/PP2A/pRb pathway

    Energy Technology Data Exchange (ETDEWEB)

    Chuang, Jian-Ying [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Hung, Jan-Jong, E-mail: petehung@mail.ncku.edu.tw [Department of Pharmacology, National Cheng-Kung University, Tainan 701, Taiwan (China); Institute of Bioinformatics and Biosignal Transduction, National Cheng-Kung University, Tainan 701, Taiwan (China)

    2011-04-15

    Highlights: {yields} Overexpression of HDAC1 induces Sp1 deacetylation and raises Sp1/p300 complex formation to bind to PP2Ac promoter. {yields} Overexpression of HDAC1 strongly inhibits the phosphorylation of pRb through up-regulation of PP2A. {yields} Overexpressed HDAC1 restrains cell proliferaction and induces cell senescence though a novel Sp1/PP2A/pRb pathway. -- Abstract: Senescence is associated with decreased activities of DNA replication, protein synthesis, and cellular division, which can result in deterioration of cellular functions. Herein, we report that the growth and division of tumor cells were significantly repressed by overexpression of histone deacetylase (HDAC) 1 with the Tet-off induced system or transient transfection. In addition, HDAC1 overexpression led to senescence through both an accumulation of hypophosphorylated active retinoblastoma protein (pRb) and an increase in the protein level of protein phosphatase 2A catalytic subunit (PP2Ac). HDAC1 overexpression also increased the level of Sp1 deacetylation and elevated the interaction between Sp1 and p300, and subsequently that Sp1/p300 complex bound to the promoter of PP2Ac, thus leading to induction of PP2Ac expression. Similar results were obtained in the HDAC1-Tet-off stable clone. Taken together, these results indicate that HDAC1 overexpression restrained cell proliferation and induced premature senescence in cervical cancer cells through a novel Sp1/PP2A/pRb pathway.

  12. Purification and characterization of a phosphotyrosyl-protein phosphatase from wheat seedlings.

    Science.gov (United States)

    Cheng, H F; Tao, M

    1989-10-19

    A neutral phosphatase which catalyzes the hydrolysis of p-nitrophenylphosphate has been purified to homogeneity from wheat seedlings. The enzyme is a monomeric glycoprotein exhibiting a molecular weight of 35,000, frictional ratio of 1.22, Stokes' radius of 260 nm, and sedimentation coefficient of 3.2 S. That the enzyme is a glycoprotein is surmised from its chromatographic property on Concanavalin A-Sepharose column. An examination of the substrate specificity indicates that the enzyme exhibits a preference for phosphotyrosine over a number of phosphocompounds, including p-nitrophenylphosphate and several glycolytic intermediates. Both phosphoserine and phosphothreonine are not hydrolyzed by the enzyme. The phosphatase activity is not affected by high concentrations of chelating agents and does not require metal ions. Molybdate, orthovanadate, Zn2+, and Hg2+ are all potent inhibitors of the phosphatase activity. The ability of the phosphatase to dephosphorylate protein phosphotyrosine has been investigated. [32P-Tyr]poly(Glu,Tyr)n, [32P-Tyr]alkylated bovine serum albumin, [32P-Tyr]angiotensin-I, and [32P-Tyr]band 3 (from human erythrocyte) are all substrates of the phosphatase. On the other hand, the enzyme has no activity toward protein phosphoserine and phosphothreonine. Our result further indicates that the neutral phosphatase is distinct from the wheat germ acid phosphatase. The latter enzyme is found to dephosphorylate phosphotyrosyl as well as phosphoseryl and phosphothreonyl groups in proteins. In light of the many similarities in properties to phosphotyrosyl protein phosphatases isolated from several sources, it is suggested that the wheat seedling phosphatase may participate in cellular regulation involving protein tyrosine phosphorylation.

  13. Dipalmitoleoylphosphoethanolamine as a PP2A enhancer obstructs insulin signaling by promoting Ser/Thr dephosphorylation of Akt.

    Science.gov (United States)

    Tsuchiya, Ayako; Kanno, Takeshi; Nishizaki, Tomoyuki

    2014-01-01

    The phospholipid phosphatidylethanolamine is implicated in the regulation of a variety of cellular processes. The present study investigated the effect of phosphatidylethanolamines such as 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE), 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE), and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE) on protein phosphatases, Akt1/2 activity, GLUT4 mobilizations, and glucose uptake into cells. Activity of protein phosphatase 2A (PP2A) was assayed under the cell-free conditions, and Western blotting, intracellular GLUT4 trafficking, and glucose uptake into cells were monitored using differentiated 3T3-L1-GLUT4myc adipocytes. Of the investigated phosphatidylethanolamines, DLPE and DPPE significantly enhanced PP2A activity. DPPE inhibited insulin-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in differentiated 3T3-L1-GLUT4myc adipocytes. DPPE also inhibited insulin-stimulated GLUT4 translocation to the cell surface and reduced insulin-stimulated glucose uptake into adipocytes. The results of the present study indicate that the PP2A enhancer DPPE obstructs insulin signaling by promoting serine/threonine dephosphorylation of Akt1/2, resulting in the suppression of GLUT4 translocation to the cell surface and glucose uptake into adipocytes. © 2014 S. Karger AG, Basel.

  14. Dipalmitoleoylphosphoethanolamine as a PP2A Enhancer Obstructs Insulin Signaling by Promoting Ser/Thr Dephosphorylation of Akt

    Directory of Open Access Journals (Sweden)

    Ayako Tsuchiya

    2014-08-01

    Full Text Available Background/Aims: The phospholipid phosphatidylethanolamine is implicated in the regulation of a variety of cellular processes. The present study investigated the effect of phosphatidylethanolamines such as 1,2-diarachidonoyl-sn-glycero-3-phosphoethanolamine (DAPE, 1,2-dilinoleoyl-sn-glycero-3-phosphoethanolamine (DLPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE, and 1,2-dipalmitoleoyl-sn-glycero-3-phosphoethanolamine (DPPE on protein phosphatases, Akt1/2 activity, GLUT4 mobilizations, and glucose uptake into cells. Methods: Activity of protein phosphatase 2A (PP2A was assayed under the cell-free conditions, and Western blotting, intracellular GLUT4 trafficking, and glucose uptake into cells were monitored using differentiated 3T3-L1-GLUT4myc adipocytes. Results: Of the investigated phosphatidylethanolamines, DLPE and DPPE significantly enhanced PP2A activity. DPPE inhibited insulin-induced phosphorylation of Akt1/2 at Thr308/309 and Ser473/474 in differentiated 3T3-L1-GLUT4myc adipocytes. DPPE also inhibited insulin-stimulated GLUT4 translocation to the cell surface and reduced insulin-stimulated glucose uptake into adipocytes. Conclusion: The results of the present study indicate that the PP2A enhancer DPPE obstructs insulin signaling by promoting serine/threonine dephosphorylation of Akt1/2, resulting in the suppression of GLUT4 translocation to the cell surface and glucose uptake into adipocytes.

  15. and alanine (EC. 2.6.1.2) transaminases, and alkaline phosphatase

    African Journals Online (AJOL)

    The activities of aspartate (E.C. 2.6.1.1) and alanine (E.C. 2.6.1.2) transaminases (AST and ALT, respectively), and alkaline phosphatase (ALP) (E.C. 3.1.3.1) were determined in erythrocytes obtained from 20 HbAA, 15 HbAS and 12 HbSS human subjects. The results showed that the three enzymes had different levels of ...

  16. HONSU, a protein phosphatase 2C, regulates seed dormancy by inhibiting ABA signaling in Arabidopsis.

    Science.gov (United States)

    Kim, Woohyun; Lee, Yeon; Park, Jeongmoo; Lee, Nayoung; Choi, Giltsu

    2013-04-01

    Seed dormancy, a seed status that prohibits germination even in the presence of inductive germination signals, is a poorly understood process. To identify molecular components that regulate seed dormancy, we screened T-DNA insertion lines and identified a mutant designated honsu (hon). HON loss-of-function mutants display deep seed dormancy, whereas HON-overexpressing lines display shallow seed dormancy. HON encodes a seed-specific group A phosphatase 2C (PP2C) and is one of the major negative regulators of seed dormancy among group A PP2Cs. Like other PP2C family members, HON interacts with PYR1/RCAR11 in the presence of ABA. Our analysis indicates that HON inhibits ABA signaling and activates gibberellic acid signaling, and both of these conditions must be satisfied to promote the release of seed dormancy. However, HON mRNA levels are increased in mutants displaying deep seed dormancy or under conditions that deepen seed dormancy, and decreased in mutants displaying shallow seed dormancy or under conditions that promote the release of seed dormancy. Taken together, our results indicate that the expression of HON mRNA is homeostatically regulated by seed dormancy.

  17. Protein Phosphatase 2A Signaling in Human Prostate Cancer

    Science.gov (United States)

    2014-08-01

    phosphatidylinositol 3’-kinase and Akt/protein kinase B. Cancer Res 1999;59:1449-53. (14) Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad...growth and sig- nalling. Biochem J 2001;353:417–39. 15. Grethe S, Porn -Ares MI. p38 MAPK regulates phosphorylation of Bad via PP2A-dependent suppression of

  18. Tissue- and environmental response-specific expression of 10 PP2C transcripts in Mesembryanthemum crystallinum.

    Science.gov (United States)

    Miyazaki, S; Koga, R; Bohnert, H J; Fukuhara, T

    1999-03-01

    Ten transcripts (Mpc1-10) homologous to protein phosphatases of the 2C family have been isolated from the halophyte Mesembryanthemum crystallinum (common ice plant). Transcripts range in size from 1.6 to 2.6 kb, and encode proteins whose catalytic domains are between 24% and 62% identical to that of the Arabidopsis PP2C, ABI1. Transcript expression is tissue specific. Two isoforms are present only in roots (Mpc1 and Mpc5), three in young leaves (Mpc6, 8 and 9), two in old leaves (Mpc6 and Mpc8), and two in post-flowering leaves (Mpc8 and Mpc9). Mpc2 is strongly expressed in roots and also in seeds, meristematic tissues and mature flowers. Mpc3 is specific for leaf meristems, and Mpc4 is found in root and leaf meristems. Mpc7 is restricted to meristematic tissues. Mpc10 is only present in mature flowers. Mpc2 (in roots and leaves), Mpc5 (in roots) and Mpc8 (weakly in leaves) are induced by salinity stress and drought conditions with different kinetics in different tissues, but other Mpcs are downregulated by stress. Cold stress (4 degrees C) leads to a decline in Mpc5 and Mp6, but low temperature provoked a long-term (days) increase in Mpc2 levels in leaves and a transient increase (less than 24 h) in roots. Four full-length transcripts have been obtained. In each case, after over-expression in E. coli, the isolated proteins exhibited (Mg2+-dependent, okadeic acid-insensitive) protein phosphatase activity, although activity against 32P-phosphocasein varied among different PP2Cs. Determination of tissue developmental and stress response specificity of PP2C will facilitate functional studies of signal-transducing enzymes in this halophytic organism.

  19. Abscisic acid affects transcription of chloroplast genes via protein phosphatase 2C-dependent activation of nuclear genes: repression by guanosine-3'-5'-bisdiphosphate and activation by sigma factor 5.

    Science.gov (United States)

    Yamburenko, Maria V; Zubo, Yan O; Börner, Thomas

    2015-06-01

    Abscisic acid (ABA) represses the transcriptional activity of chloroplast genes (determined by run-on assays), with the exception of psbD and a few other genes in wild-type Arabidopsis seedlings and mature rosette leaves. Abscisic acid does not influence chloroplast transcription in the mutant lines abi1-1 and abi2-1 with constitutive protein phosphatase 2C (PP2C) activity, suggesting that ABA affects chloroplast gene activity by binding to the pyrabactin resistance (PYR)/PYR1-like or regulatory component of ABA receptor protein family (PYR/PYL/RCAR) and signaling via PP2Cs and sucrose non-fermenting protein-related kinases 2 (SnRK2s). Further we show by quantitative PCR that ABA enhances the transcript levels of RSH2, RSH3, PTF1 and SIG5. RelA/SpoT homolog 2 (RSH2) and RSH3 are known to synthesize guanosine-3'-5'-bisdiphosphate (ppGpp), an inhibitor of the plastid-gene-encoded chloroplast RNA polymerase. We propose, therefore, that ABA leads to an inhibition of chloroplast gene expression via stimulation of ppGpp synthesis. On the other hand, sigma factor 5 (SIG5) and plastid transcription factor 1 (PTF1) are known to be necessary for the transcription of psbD from a specific light- and stress-induced promoter (the blue light responsive promoter, BLRP). We demonstrate that ABA activates the psbD gene by stimulation of transcription initiation at BLRP. Taken together, our data suggest that ABA affects the transcription of chloroplast genes by a PP2C-dependent activation of nuclear genes encoding proteins involved in chloroplast transcription. © 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.

  20. Shugoshin-1 balances Aurora B kinase activity via PP2A to promote chromosome bi-orientation

    NARCIS (Netherlands)

    Meppelink, Amanda; Kabeche, Lilian; Vromans, Martijn J M; Compton, Duane A; Lens, Susanne M A

    2015-01-01

    Correction of faulty kinetochore-microtubule attachments is essential for faithful chromosome segregation and dictated by the opposing activities of Aurora B kinase and PP1 and PP2A phosphatases. How kinase and phosphatase activities are appropriately balanced is less clear. Here, we show that a

  1. E2/ER β Enhances Calcineurin Protein Degradation and PI3K/Akt/MDM2 Signal Transduction to Inhibit ISO-Induced Myocardial Cell Apoptosis

    Directory of Open Access Journals (Sweden)

    Kuan-Ho Lin

    2017-04-01

    Full Text Available Secretion of multifunctional estrogen and its receptor has been widely considered as the reason for markedly higher frequency of heart disease in men than in women. 17β-Estradiol (E2, for instance, has been reported to prevent development of cardiac apoptosis via activation of estrogen receptors (ERs. In addition, protein phosphatase such as protein phosphatase 1 (PP1 and calcineurin (PP2B are also involved in cardiac hypertrophy and cell apoptosis signaling. However, the mechanism by which E2/ERβ suppresses apoptosis is not fully understood, and the role of protein phosphatase in E2/ERβ action also needs further investigation. In this study, we observed that E2/ERβ inhibited isoproterenol (ISO-induced myocardial cell apoptosis, cytochrome c release and downstream apoptotic markers. Moreover, we found that E2/ERβ blocks ISO-induced apoptosis in H9c2 cells through the enhancement of calcineurin protein degradation through PI3K/Akt/MDM2 signaling pathway. Our results suggest that supplementation with estrogen and/or overexpression of estrogen receptor β gene may prove to be effective means to treat stress-induced myocardial damage.

  2. Physiological and Pathological Roles of CaMKII-PP1 Signaling in the Brain

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-12-01

    Full Text Available Ca2+/calmodulin (CaM-dependent protein kinase II (CaMKII, a multifunctional serine (Ser/threonine (Thr protein kinase, regulates diverse activities related to Ca2+-mediated neuronal plasticity in the brain, including synaptic activity and gene expression. Among its regulators, protein phosphatase-1 (PP1, a Ser/Thr phosphatase, appears to be critical in controlling CaMKII-dependent neuronal signaling. In postsynaptic densities (PSDs, CaMKII is required for hippocampal long-term potentiation (LTP, a cellular process correlated with learning and memory. In response to Ca2+ elevation during hippocampal LTP induction, CaMKIIα, an isoform that translocates from the cytosol to PSDs, is activated through autophosphorylation at Thr286, generating autonomous kinase activity and a prolonged Ca2+/CaM-bound state. Moreover, PP1 inhibition enhances Thr286 autophosphorylation of CaMKIIα during LTP induction. By contrast, CaMKII nuclear import is regulated by Ser332 phosphorylation state. CaMKIIδ3, a nuclear isoform, is dephosphorylated at Ser332 by PP1, promoting its nuclear translocation, where it regulates transcription. In this review, we summarize physio-pathological roles of CaMKII/PP1 signaling in neurons. CaMKII and PP1 crosstalk and regulation of gene expression is important for neuronal plasticity as well as survival and/or differentiation.

  3. Serine/threonine-protein phosphatase 1 α levels are paralleling olfactory memory formation in the CD1 mouse.

    Science.gov (United States)

    Winding, Christiana; Sun, Yanwei; Höger, Harald; Bubna-Littitz, Hermann; Pollak, Arnold; Schmidt, Peter; Lubec, Gert

    2011-06-01

    Although olfactory discrimination has already been studied in several mouse strains, data on protein levels linked to olfactory memory are limited. Wild mouse strains Mus musculus musculus, Mus musculus domesticus and CD1 laboratory outbred mice were tested in a conditioned odor preference task and trained to discriminate between two odors, Rose and Lemon, by pairing one odor with a sugar reward. Six hours following the final test, mice were sacrificed and olfactory bulbs (OB) were taken for gel-based proteomics analyses and immunoblotting. OB proteins were extracted, separated by 2-DE and quantified using specific software (Proteomweaver). Odor-trained mice showed a preference for the previously rewarded odor suggesting that conditioned odor preference occurred. In CD1 mice levels, one out of 482 protein spots was significantly increased in odor-trained mice as compared with the control group; it was in-gel digested by trypsin and chymotrypsin and analyzed by tandem mass spectrometry (nano-ESI-LC-MS/MS). The spot was unambiguously identified as serine/threonine-protein phosphatase PP1-α catalytic subunit (PP-1A) and differential levels observed in gel-based proteomic studies were verified by immunoblotting. PP-1A is a key signalling element in synaptic plasticity and memory processes and is herein shown to be paralleling olfactory discrimination representing olfactory memory. Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  4. Tributyltin induces a G2/M cell cycle arrest in human amniotic cells via PP2A inhibition-mediated inactivation of the ERK1/2 cascades.

    Science.gov (United States)

    Zhang, Yali; Guo, Zonglou; Xu, Lihong

    2014-03-01

    The molecular mechanisms underlying the cell cycle alterations induced by tributyltin (TBT), a highly toxic environmental contaminant, remain elusive. In this study, cell cycle progression and some key regulators in G2/M phase were investigated in human amniotic cells treated with TBT. Furthermore, protein phosphatase (PP) 2A and the ERK cascades were examined. The results showed that TBT caused a G2/M cell cycle arrest that was accompanied by a decrease in the total cdc25C protein level and an increase in the p-cdc2 level in the nucleus. TBT caused a decrease in PP2A activity and inhibited the ERK cascade by inactivating Raf-1, resulting in the dephosphorylation of MEK1/2, ERK1/2, and c-Myc. Taken together, TBT leads to a G2/M cell cycle arrest in FL cells, an increase in p-cdc2 and a decrease in the levels of total cdc25C protein, which may be caused by the PP2A inhibition-mediated inactivation of the ERK1/2 cascades. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Receptor tyrosine phosphatase R-PTP-alpha is tyrosine-phosphorylated and associated with the adaptor protein Grb2

    DEFF Research Database (Denmark)

    Su, J; Batzer, A; Sap, J

    1994-01-01

    Receptor tyrosine phosphatases (R-PTPases) have generated interest because of their suspected involvement in cellular signal transduction. The adaptor protein Grb2 has been implicated in coupling receptor tyrosine kinases to Ras. We report that a ubiquitous R-PTPase, R-PTP-alpha, is tyrosine......-phosphorylated and associated in vivo with the Grb2 protein. This association can be reproduced in stably and transiently transfected cells, as well as in vitro using recombinant Grb2 protein. Association requires the presence of an intact SH2 domain in Grb2, as well as tyrosine phosphorylation of R-PTP-alpha. This observation...... links a receptor tyrosine phosphatase with a key component of a central cellular signalling pathway and provides a basis for addressing R-PTP-alpha function....

  6. The homeodomain-leucine zipper (HD-Zip) class I transcription factors ATHB7 and ATHB12 modulate abscisic acid signalling by regulating protein phosphatase 2C and abscisic acid receptor gene activities.

    Science.gov (United States)

    Valdés, Ana Elisa; Overnäs, Elin; Johansson, Henrik; Rada-Iglesias, Alvaro; Engström, Peter

    2012-11-01

    Plants perceiving drought activate multiple responses to improve survival, including large-scale alterations in gene expression. This article reports on the roles in the drought response of two Arabidopsis thaliana homeodomain-leucine zipper class I genes; ATHB7 and ATHB12, both strongly induced by water-deficit and abscisic acid (ABA). ABA-mediated transcriptional regulation of both genes is shown to depend on the activity of protein phosphatases type 2C (PP2C). ATHB7 and ATHB12 are, thus, targets of the ABA signalling mechanism defined by the PP2Cs and the PYR/PYL family of ABA receptors, with which the PP2C proteins interact. Our results from chromatin immunoprecipitation and gene expression analyses demonstrate that ATHB7 and ATHB12 act as positive transcriptional regulators of PP2C genes, and thereby as negative regulators of abscisic acid signalling. In support of this notion, our results also show that ATHB7 and ATHB12 act to repress the transcription of genes encoding the ABA receptors PYL5 and PYL8 in response to an ABA stimulus. In summary, we demonstrate that ATHB7 and ATHB12 have essential functions in the primary response to drought, as mediators of a negative feedback effect on ABA signalling in the plant response to water deficit.

  7. Rapamycin causes activation of protein phosphatase-2A1 and nuclear translocation of PCNA in CD4+ T cells

    International Nuclear Information System (INIS)

    Morrow, Peter W.; Tung, H.Y. Lim; Hemmings, Hugh C.

    2004-01-01

    Rapamycin is a powerful immunosuppressant that causes cell cycle arrest in T cells and several other cell types. Despite its important clinical role, the mechanism of action of rapamycin is not fully understood. Here, we show that rapamycin causes the activation of protein phosphatase-2A 1 which forms a complex with proliferation cell nuclear antigen (PCNA) in a CD 4+ T cell line. Rapamycin also induces PCNA translocation from the cytoplasm to the nucleus, an effect which is antagonized by okadaic acid, an inhibitor of type 2A protein phosphatases. These findings provide evidence for the existence of a signal transduction pathway that links a rapamycin-activated type 2A protein phosphatase to the control of DNA synthesis, DNA repair, cell cycle, and cell death via PCNA

  8. Functional analysis of the glycogen binding subunit CG9238/Gbs-70E of protein phosphatase 1 in Drosophila melanogaster.

    Science.gov (United States)

    Kerekes, Éva; Kókai, Endre; Páldy, Ferenc Sándor; Dombrádi, Viktor

    2014-06-01

    The product of the CG9238 gene that we termed glycogen binding subunit 70E (Gbs-70E) was characterized by biochemical and molecular genetics methods. The interaction between Gbs-70E and all catalytic subunits of protein phosphatase 1 (Pp1-87B, Pp1-9C, Pp1-96A and Pp1-13C) of Drosophila melanogaster was confirmed by pairwise yeast two-hybrid tests, co-immunoprecipitation and pull down experiments. The binding of Gbs-70E to glycogen was demonstrated by sedimentation analysis. With RT-PCR we found that the mRNAs coding for the longer Gbs-70E PB/PC protein were expressed in all developmental stages of the fruit flies while the mRNA for the shorter Gbs-70E PA was restricted to the eggs and the ovaries of the adult females. The development specific expression of the shorter splice variant was not conserved in different Drosophila species. The expression level of the gene was manipulated by P-element insertions and gene deletion to analyze the functions of the gene product. A small or moderate reduction in the gene expression resulted in no significant changes, however, a deletion mutant expressing very low level of the transcript lived shorter and exhibited reduced glycogen content in the imagos. In addition, the gene deletion decreased the fertility of the fruit flies. Our results prove that Gbs-70E functions as the glycogen binding subunit of protein phosphatase 1 that regulates glycogen content and plays a role in the development of eggs in D. melanogaster. Copyright © 2014 Elsevier Ltd. All rights reserved.

  9. Membrane-bound 2,3-diphosphoglycerate phosphatase of human erythrocytes.

    Science.gov (United States)

    Schröter, W; Neuvians, M

    1970-12-01

    Gradual osmotic hemolysis of human erythrocytes reduces the cell content of whole protein, hemoglobin, 2,3-diphosphoglycerate and triosephosphate isomerase extensively, but not that of membrane protein and 2,3-diphosphoglycerate phosphatase. After the refilling of the ghosts with 2,3-diphosphoglycerate and reconstitution of the membrane, the 2,3-diphosphoglycerate phosphatase activity equals that of intact red cells. The membrane-bound 2,3-diphosphoglycerate phosphatase can be activated by sodium hyposulfite. The enzyme system of ghosts seems to differ from that of intact red cells with regard to the optima of pH and temperature. It remains to be elucidated if the membrane binding of the 2,3-diphosphoglycerate phosphatase is related to the transfer of inorganic phosphate across the red cell membrane.

  10. Modulation of catalytic activity in multi-domain protein tyrosine phosphatases.

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    Lalima L Madan

    Full Text Available Signaling mechanisms involving protein tyrosine phosphatases govern several cellular and developmental processes. These enzymes are regulated by several mechanisms which include variation in the catalytic turnover rate based on redox stimuli, subcellular localization or protein-protein interactions. In the case of Receptor Protein Tyrosine Phosphatases (RPTPs containing two PTP domains, phosphatase activity is localized in their membrane-proximal (D1 domains, while the membrane-distal (D2 domain is believed to play a modulatory role. Here we report our analysis of the influence of the D2 domain on the catalytic activity and substrate specificity of the D1 domain using two Drosophila melanogaster RPTPs as a model system. Biochemical studies reveal contrasting roles for the D2 domain of Drosophila Leukocyte antigen Related (DLAR and Protein Tyrosine Phosphatase on Drosophila chromosome band 99A (PTP99A. While D2 lowers the catalytic activity of the D1 domain in DLAR, the D2 domain of PTP99A leads to an increase in the catalytic activity of its D1 domain. Substrate specificity, on the other hand, is cumulative, whereby the individual specificities of the D1 and D2 domains contribute to the substrate specificity of these two-domain enzymes. Molecular dynamics simulations on structural models of DLAR and PTP99A reveal a conformational rationale for the experimental observations. These studies reveal that concerted structural changes mediate inter-domain communication resulting in either inhibitory or activating effects of the membrane distal PTP domain on the catalytic activity of the membrane proximal PTP domain.

  11. Purification and molecular cloning of SH2- and SH3-containing inositol polyphosphate-5-phosphatase, which is involved in the signaling pathway of granulocyte-macrophage colony-stimulating factor, erythropoietin, and Bcr-Abl.

    Science.gov (United States)

    Odai, H; Sasaki, K; Iwamatsu, A; Nakamoto, T; Ueno, H; Yamagata, T; Mitani, K; Yazaki, Y; Hirai, H

    1997-04-15

    Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine

  12. Proteomic analysis of protein phosphatase Z1 from Candida albicans.

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    Bernadett Márkus

    Full Text Available Protein phosphatase Z is a "novel type" fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0 that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly

  13. Proteomic analysis of protein phosphatase Z1 from Candida albicans

    Science.gov (United States)

    Pfliegler, Walter P.; Petrényi, Katalin; Boros, Enikő; Pócsi, István; Tőzsér, József; Dombrádi, Viktor

    2017-01-01

    Protein phosphatase Z is a “novel type” fungus specific serine/threonine protein phosphatase. Previously our research group identified the CaPPZ1 gene in the opportunistic pathogen Candida albicans and reported that the gene deletion had several important physiological consequences. In order to reveal the protein targets and the associated mechanisms behind the functions of the phosphatase a proteomic method was adopted for the comparison of the cappz1 deletion mutant and the genetically matching QMY23 control strain. Proteins extracted from the control and deletion mutant strains were separated by two-dimensional gel electrophoresis and the protein spots were stained with RuBPS and Pro-Q Diamond in order to visualize the total proteome and the phosphoproteome, respectively. The alterations in spot intensities were determined by densitometry and were analysed with the Delta2D (Decodon) software. Spots showing significantly different intensities between the mutant and control strains were excised from the gels and were digested with trypsin. The resulting peptides were identified by LC-MS/MS mass spectrometry. As many as 15 protein spots were found that exhibited significant changes in their intensity upon the deletion of the phosphatase and 20 phosphoproteins were identified in which the level of phosphorylation was modified significantly in the mutant. In agreement with previous findings we found that the affected proteins function in protein synthesis, oxidative stress response, regulation of morphology and metabolism. Among these proteins we identified two potential CaPpz1 substrates (Eft2 and Rpp0) that may regulate the elongation step of translation. RT-qPCR experiments revealed that the expression of the genes coding for the affected proteins was not altered significantly. Thus, the absence of CaPpz1 exerted its effects via protein synthesis/degradation and phosphorylation/dephosphorylation. In addition, our proteomics data strongly suggested a role for

  14. The protein histidine phosphatase LHPP is a tumour suppressor.

    Science.gov (United States)

    Hindupur, Sravanth K; Colombi, Marco; Fuhs, Stephen R; Matter, Matthias S; Guri, Yakir; Adam, Kevin; Cornu, Marion; Piscuoglio, Salvatore; Ng, Charlotte K Y; Betz, Charles; Liko, Dritan; Quagliata, Luca; Moes, Suzette; Jenoe, Paul; Terracciano, Luigi M; Heim, Markus H; Hunter, Tony; Hall, Michael N

    2018-03-29

    Histidine phosphorylation, the so-called hidden phosphoproteome, is a poorly characterized post-translational modification of proteins. Here we describe a role of histidine phosphorylation in tumorigenesis. Proteomic analysis of 12 tumours from an mTOR-driven hepatocellular carcinoma mouse model revealed that NME1 and NME2, the only known mammalian histidine kinases, were upregulated. Conversely, expression of the putative histidine phosphatase LHPP was downregulated specifically in the tumours. We demonstrate that LHPP is indeed a protein histidine phosphatase. Consistent with these observations, global histidine phosphorylation was significantly upregulated in the liver tumours. Sustained, hepatic expression of LHPP in the hepatocellular carcinoma mouse model reduced tumour burden and prevented the loss of liver function. Finally, in patients with hepatocellular carcinoma, low expression of LHPP correlated with increased tumour severity and reduced overall survival. Thus, LHPP is a protein histidine phosphatase and tumour suppressor, suggesting that deregulated histidine phosphorylation is oncogenic.

  15. Drosophila Syd-1, liprin-α, and protein phosphatase 2A B' subunit Wrd function in a linear pathway to prevent ectopic accumulation of synaptic materials in distal axons.

    Science.gov (United States)

    Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J; Levitan, Edwin S; Wu, Chunlai

    2014-06-18

    During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B' [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). Copyright © 2014 the authors 0270-6474/14/348474-14$15.00/0.

  16. Drosophila Syd-1, Liprin-α, and Protein Phosphatase 2A B′ Subunit Wrd Function in a Linear Pathway to Prevent Ectopic Accumulation of Synaptic Materials in Distal Axons

    Science.gov (United States)

    Li, Long; Tian, Xiaolin; Zhu, Mingwei; Bulgari, Dinara; Böhme, Mathias A.; Goettfert, Fabian; Wichmann, Carolin; Sigrist, Stephan J.; Levitan, Edwin S.

    2014-01-01

    During synaptic development, presynaptic differentiation occurs as an intrinsic property of axons to form specialized areas of plasma membrane [active zones (AZs)] that regulate exocytosis and endocytosis of synaptic vesicles. Genetic and biochemical studies in vertebrate and invertebrate model systems have identified a number of proteins involved in AZ assembly. However, elucidating the molecular events of AZ assembly in a spatiotemporal manner remains a challenge. Syd-1 (synapse defective-1) and Liprin-α have been identified as two master organizers of AZ assembly. Genetic and imaging analyses in invertebrates show that Syd-1 works upstream of Liprin-α in synaptic assembly through undefined mechanisms. To understand molecular pathways downstream of Liprin-α, we performed a proteomic screen of Liprin-α-interacting proteins in Drosophila brains. We identify Drosophila protein phosphatase 2A (PP2A) regulatory subunit B′ [Wrd (Well Rounded)] as a Liprin-α-interacting protein, and we demonstrate that it mediates the interaction of Liprin-α with PP2A holoenzyme and the Liprin-α-dependent synaptic localization of PP2A. Interestingly, loss of function in syd-1, liprin-α, or wrd shares a common defect in which a portion of synaptic vesicles, dense-core vesicles, and presynaptic cytomatrix proteins ectopically accumulate at the distal, but not proximal, region of motoneuron axons. Strong genetic data show that a linear syd-1/liprin-α/wrd pathway in the motoneuron antagonizes glycogen synthase kinase-3β kinase activity to prevent the ectopic accumulation of synaptic materials. Furthermore, we provide data suggesting that the syd-1/liprin-α/wrd pathway stabilizes AZ specification at the nerve terminal and that such a novel function is independent of the roles of syd-1/liprin-α in regulating the morphology of the T-bar structural protein BRP (Bruchpilot). PMID:24948803

  17. Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes

    Science.gov (United States)

    Haj Slimane, Zeineb; Bedioune, Ibrahim; Lechêne, Patrick; Varin, Audrey; Lefebvre, Florence; Mateo, Philippe; Domergue-Dupont, Valérie; Dewenter, Matthias; Richter, Wito; Conti, Marco; El-Armouche, Ali; Zhang, Jin; Fischmeister, Rodolphe; Vandecasteele, Grégoire

    2014-01-01

    Aims The cAMP-dependent protein kinase (PKA) mediates β-adrenoceptor (β-AR) regulation of cardiac contraction and gene expression. Whereas PKA activity is well characterized in various subcellular compartments of adult cardiomyocytes, its regulation in the nucleus remains largely unknown. The aim of the present study was to compare the modalities of PKA regulation in the cytoplasm and nucleus of cardiomyocytes. Methods and results Cytoplasmic and nuclear cAMP and PKA activity were measured with targeted fluorescence resonance energy transfer probes in adult rat ventricular myocytes. β-AR stimulation with isoprenaline (Iso) led to fast cAMP elevation in both compartments, whereas PKA activity was fast in the cytoplasm but markedly slower in the nucleus. Iso was also more potent and efficient in activating cytoplasmic than nuclear PKA. Similar slow kinetics of nuclear PKA activation was observed upon adenylyl cyclase activation with L-858051 or phosphodiesterase (PDE) inhibition with 3-isobutyl-1-methylxantine. Consistently, pulse stimulation with Iso (15 s) maximally induced PKA and myosin-binding protein C phosphorylation in the cytoplasm, but marginally activated PKA and cAMP response element-binding protein phosphorylation in the nucleus. Inhibition of PDE4 or ablation of the Pde4d gene in mice prolonged cytoplasmic PKA activation and enhanced nuclear PKA responses. In the cytoplasm, phosphatase 1 (PP1) and 2A (PP2A) contributed to the termination of PKA responses, whereas only PP1 played a role in the nucleus. Conclusion Our study reveals a differential integration of cytoplasmic and nuclear PKA responses to β-AR stimulation in cardiac myocytes. This may have important implications in the physiological and pathological hypertrophic response to β-AR stimulation. PMID:24550350

  18. Protein phosphatases active on acetyl-CoA carboxylase phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase

    International Nuclear Information System (INIS)

    Witters, L.A.; Bacon, G.W.

    1985-01-01

    The protein phosphatases in rat liver cytosol, active on rat liver acetyl-CoA carboxylase (ACC) phosphorylated by casein kinase I, casein kinase II and the cAMP-dependent protein kinase, have been partially purified by anion-exchange and gel filtration chromatography. The major phosphatase activities against all three substrates copurify through fractionation and appear to be identical to protein phosphatases 2A1 and 2A2. No unique protein phosphatase active on 32 P-ACC phosphorylated by the casein kinases was identified

  19. Protein kinase and phosphatase activities of thylakoid membranes

    International Nuclear Information System (INIS)

    Michel, H.; Shaw, E.K.; Bennett, J.

    1987-01-01

    Dephosphorylation of the 25 and 27 kDa light-harvesting Chl a/b proteins (LHCII) of the thylakoid membranes is catalyzed by a phosphatase which differs from previously reported thylakoid-bound phosphatases in having an alkaline pH optimum (9.0) and a requirement for Mg 2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg 2+ ion concentration more than 200 fold higher than that for dephosphorylation of LHC II. The 8.3 kDa and 27 kDa proteins appear to be phosphorylated by two distinct kinases, which differ in substrate specificity and sensitivity to inhibitors. The plastoquinone antagonist 2,5-dibromo-3-methyl-6-isopropyl-benzoquinone (DBMIB) inhibits phosphorylation of the 27 kDa LHC II much more readily than phosphorylation of the 8.3 kDa protein. A similar pattern of inhibition is seen for two synthetic oligopeptides (MRKSATTKKAVC and ATQTLESSSRC) which are analogs of the phosphorylation sites of the two proteins. Possible modes of action of DBMIB are discussed. 45 refs., 7 figs., 3 tabs

  20. PDGF activates K-Cl cotransport through phosphoinositide 3-kinase and protein phosphatase-1 in primary cultures of vascular smooth muscle cells.

    Science.gov (United States)

    Zhang, Jing; Lauf, Peter K; Adragna, Norma C

    2005-07-15

    K-Cl cotransport (K-Cl COT, KCC) is an electroneutrally coupled movement of K and Cl present in most cells. In this work, we studied the pathways of regulation of K-Cl COT by platelet-derived growth factor (PDGF) in primary cultures of vascular smooth muscle cells (VSMCs). Wortmannin and LY 294002 blocked the PDGF-induced K-Cl COT activation, indicating that the phosphoinositide 3-kinase (PI 3-K) pathway is involved. However, PD 98059 had no effect on K-Cl COT activation by PDGF, suggesting that the mitogen-activated protein kinase pathway is not involved under the experimental conditions tested. Involvement of phosphatases was also examined. Sodium orthovanadate, cyclosporin A and okadaic acid had no effect on PDGF-stimulated K-Cl COT. Calyculin A blocked the PDGF-stimulated K-Cl COT by 60%, suggesting that protein phosphatase-1 (PP-1) is a mediator in the PDGF signaling pathway/s. In conclusion, our results indicate that the PDGF-mediated pathways of K-Cl COT regulation involve the signaling molecules PI 3-K and PP-1.

  1. Regulation of cortical contractility and spindle positioning by the protein phosphatase 6 PPH-6 in one-cell stage C. elegans embryos

    Science.gov (United States)

    Afshar, Katayoun; Werner, Michael E.; Tse, Yu Chung; Glotzer, Michael; Gönczy, Pierre

    2010-01-01

    Modulation of the microtubule and the actin cytoskeleton is crucial for proper cell division. Protein phosphorylation is known to be an important regulatory mechanism modulating these cytoskeletal networks. By contrast, there is a relative paucity of information regarding how protein phosphatases contribute to such modulation. Here, we characterize the requirements for protein phosphatase PPH-6 and its associated subunit SAPS-1 in one-cell stage C. elegans embryos. We establish that the complex of PPH-6 and SAPS-1 (PPH-6/SAPS-1) is required for contractility of the actomyosin network and proper spindle positioning. Our analysis demonstrates that PPH-6/SAPS-1 regulates the organization of cortical non-muscle myosin II (NMY-2). Accordingly, we uncover that PPH-6/SAPS-1 contributes to cytokinesis by stimulating actomyosin contractility. Furthermore, we demonstrate that PPH-6/SAPS-1 is required for the proper generation of pulling forces on spindle poles during anaphase. Our results indicate that this requirement is distinct from the role in organizing the cortical actomyosin network. Instead, we uncover that PPH-6/SAPS-1 contributes to the cortical localization of two positive regulators of pulling forces, GPR-1/2 and LIN-5. Our findings provide the first insights into the role of a member of the PP6 family of phosphatases in metazoan development. PMID:20040490

  2. A new fluorescence-based method identifies protein phosphatases regulating lipid droplet metabolism.

    Directory of Open Access Journals (Sweden)

    Bruno L Bozaquel-Morais

    Full Text Available In virtually every cell, neutral lipids are stored in cytoplasmic structures called lipid droplets (LDs and also referred to as lipid bodies or lipid particles. We developed a rapid high-throughput assay based on the recovery of quenched BODIPY-fluorescence that allows to quantify lipid droplets. The method was validated by monitoring lipid droplet turnover during growth of a yeast culture and by screening a group of strains deleted in genes known to be involved in lipid metabolism. In both tests, the fluorimetric assay showed high sensitivity and good agreement with previously reported data using microscopy. We used this method for high-throughput identification of protein phosphatases involved in lipid droplet metabolism. From 65 yeast knockout strains encoding protein phosphatases and its regulatory subunits, 13 strains revealed to have abnormal levels of lipid droplets, 10 of them having high lipid droplet content. Strains deleted for type I protein phosphatases and related regulators (ppz2, gac1, bni4, type 2A phosphatase and its related regulator (pph21 and sap185, type 2C protein phosphatases (ptc1, ptc4, ptc7 and dual phosphatases (pps1, msg5 were catalogued as high-lipid droplet content strains. Only reg1, a targeting subunit of the type 1 phosphatase Glc7p, and members of the nutrient-sensitive TOR pathway (sit4 and the regulatory subunit sap190 were catalogued as low-lipid droplet content strains, which were studied further. We show that Snf1, the homologue of the mammalian AMP-activated kinase, is constitutively phosphorylated (hyperactive in sit4 and sap190 strains leading to a reduction of acetyl-CoA carboxylase activity. In conclusion, our fast and highly sensitive method permitted us to catalogue protein phosphatases involved in the regulation of LD metabolism and present evidence indicating that the TOR pathway and the SNF1/AMPK pathway are connected through the Sit4p-Sap190p pair in the control of lipid droplet biogenesis.

  3. Rif1 acts through Protein Phosphatase 1 but independent of replication timing to suppress telomere extension in budding yeast.

    Science.gov (United States)

    Kedziora, Sylwia; Gali, Vamsi K; Wilson, Rosemary H C; Clark, Kate R M; Nieduszynski, Conrad A; Hiraga, Shin-Ichiro; Donaldson, Anne D

    2018-05-04

    The Rif1 protein negatively regulates telomeric TG repeat length in the budding yeast Saccharomyces cerevisiae, but how it prevents telomere over-extension is unknown. Rif1 was recently shown to control DNA replication by acting as a Protein Phosphatase 1 (PP1)-targeting subunit. Therefore, we investigated whether Rif1 controls telomere length by targeting PP1 activity. We find that a Rif1 mutant defective for PP1 interaction causes a long-telomere phenotype, similar to that of rif1Δ cells. Tethering PP1 at a specific telomere partially substitutes for Rif1 in limiting TG repeat length, confirming the importance of PP1 in telomere length control. Ablating Rif1-PP1 interaction is known to cause precocious activation of telomere-proximal replication origins and aberrantly early telomere replication. However, we find that Rif1 still limits telomere length even if late replication is forced through deletion of nearby replication origins, indicating that Rif1 can control telomere length independent of replication timing. Moreover we find that, even at a de novo telomere created after DNA synthesis during a mitotic block, Rif1-PP1 interaction is required to suppress telomere lengthening and prevent inappropriate recruitment of Tel1 kinase. Overall, our results show that Rif1 controls telomere length by recruiting PP1 to directly suppress telomerase-mediated TG repeat lengthening.

  4. Nuclear localization of CPI-17, a protein phosphatase-1 inhibitor protein, affects histone H3 phosphorylation and corresponds to proliferation of cancer and smooth muscle cells

    Energy Technology Data Exchange (ETDEWEB)

    Eto, Masumi, E-mail: masumi.eto@jefferson.edu [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kirkbride, Jason A.; Chugh, Rishika; Karikari, Nana Kofi [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Kim, Jee In [Department of Molecular Physiology and Biophysics, and Kimmel Cancer Center, Thomas Jefferson University, 1020 Locust Street, PA 19107 (United States); Cardiovascular Research Institute, Kyungpook National University School of Medicine, Daegu 700-422 (Korea, Republic of)

    2013-04-26

    Highlights: •Non-canonical roles of the myosin phosphatase inhibitor (CPI-17) were studied. •CPI-17 is localized in the nucleus of hyperplastic cancer and smooth muscle cells. •CPI-17 Ser12 phosphorylation may regulate the nuclear import. •CPI-17 regulates histone H3 phosphorylation and cell proliferation. •The nuclear CPI-17-PP1 axis plays a proliferative role in cells. -- Abstract: CPI-17 (C-kinase-activated protein phosphatase-1 (PP1) inhibitor, 17 kDa) is a cytoplasmic protein predominantly expressed in mature smooth muscle (SM) that regulates the myosin-associated PP1 holoenzyme (MLCP). Here, we show CPI-17 expression in proliferating cells, such as pancreatic cancer and hyperplastic SM cells. Immunofluorescence showed that CPI-17 was concentrated in nuclei of human pancreatic cancer (Panc1) cells. Nuclear accumulation of CPI-17 was also detected in the proliferating vascular SM cell culture and cells at neointima of rat vascular injury model. The N-terminal 21-residue tail domain of CPI-17 was necessary for the nuclear localization. Phospho-mimetic Asp-substitution of CPI-17 at Ser12 attenuated the nuclear import. CPI-17 phosphorylated at Ser12 was not localized at nuclei, suggesting a suppressive role of Ser12 phosphorylation in the nuclear import. Activated CPI-17 bound to all three isoforms of PP1 catalytic subunit in Panc1 nuclear extracts. CPI-17 knockdown in Panc1 resulted in dephosphorylation of histone H3 at Thr3, Ser10 and Thr11, whereas it had no effects on the phosphorylation of myosin light chain and merlin, the known targets of MLCP. In parallel, CPI-17 knockdown suppressed Panc1 proliferation. We propose that CPI-17 accumulated in the nucleus through the N-terminal tail targets multiple PP1 signaling pathways regulating cell proliferation.

  5. Response to DNA damage: why do we need to focus on protein phosphatases?

    Directory of Open Access Journals (Sweden)

    Midori eShimada

    2013-01-01

    Full Text Available Eukaryotic cells are continuously threatened by unavoidable errors during normal DNA replication or various sources of genotoxic stresses that cause DNA damage or stalled replication. To maintain genomic integrity, cells have developed a coordinated signaling network, known as the DNA damage response (DDR. Following DNA damage, sensor molecules detect the presence of DNA damage and transmit signals to downstream transducer molecules. This in turn conveys the signals to numerous effectors, which initiate a large number of specific biological responses, including transient cell cycle arrest mediated by checkpoints, DNA repair, and apoptosis. It is recently becoming clear that dephosphorylation events are involved in keeping DDR factors inactive during normal cell growth. Moreover, dephosphorylation is required to shut off checkpoint arrest following DNA damage and has been implicated in the activation of the DDR. Spatial and temporal regulation of phosphorylation events is essential for the DDR, and fine-tuning of phosphorylation is partly mediated by protein phosphatases. While the role of kinases in the DDR has been well documented, the complex roles of protein dephosphorylation have only recently begun to be investigated. Therefore, it is important to focus on the role of phosphatases and to determine how their activity is regulated upon DNA damage. In this work, we summarize current knowledge on the involvement of serine/threonine phosphatases, especially the protein phosphatase 1, protein phosphatase 2A, and protein phosphatase Mg2+/Mn2+-dependent families, in the DDR.

  6. A type 2C protein phosphatase FgPtc3 is involved in cell wall integrity, lipid metabolism, and virulence in Fusarium graminearum.

    Directory of Open Access Journals (Sweden)

    Jinhua Jiang

    Full Text Available Type 2C protein phosphatases (PP2Cs play important roles in regulating many biological processes in eukaryotes. Currently, little is known about functions of PP2Cs in filamentous fungi. The causal agent of wheat head blight, Fusarium graminearum, contains seven putative PP2C genes, FgPTC1, -3, -5, -5R, -6, -7 and -7R. In order to investigate roles of these PP2Cs, we constructed deletion mutants for all seven PP2C genes in this study. The FgPTC3 deletion mutant (ΔFgPtc3-8 exhibited reduced aerial hyphae formation and deoxynivalenol (DON production, but increased production of conidia. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents on potato dextrose agar plates. Pathogencity assays showed that ΔFgPtc3-8 is unable to infect flowering wheat head. All of the defects were restored when ΔFgPtc3-8 was complemented with the wild-type FgPTC3 gene. Additionally, the FgPTC3 partially rescued growth defect of a yeast PTC1 deletion mutant under various stress conditions. Ultrastructural and histochemical analyses showed that conidia of ΔFgPtc3-8 contained an unusually high number of large lipid droplets. Furthermore, the mutant accumulated a higher basal level of glycerol than the wild-type progenitor. Quantitative real-time PCR assays showed that basal expression of FgOS2, FgSLT2 and FgMKK1 in the mutant was significantly higher than that in the wild-type strain. Serial analysis of gene expression in ΔFgPtc3-8 revealed that FgPTC3 is associated with various metabolic pathways. In contrast to the FgPTC3 mutant, the deletion mutants of FgPTC1, FgPTC5, FgPTC5R, FgPTC6, FgPTC7 or FgPTC7R did not show aberrant phenotypic features when grown on PDA medium or inoculated on wheat head. These results indicate FgPtc3 is the key PP2C that plays a critical role in a variety of cellular and biological functions, including cell wall integrity, lipid and secondary metabolisms, and virulence in F. graminearum.

  7. The muscle-specific protein phosphatase PP1G/R(GL)(G(M))is essential for activation of glycogen synthase by exercise

    DEFF Research Database (Denmark)

    Aschenbach, W G; Suzuki, Y; Breeden, K

    2001-01-01

    In skeletal muscle both insulin and contractile activity are physiological stimuli for glycogen synthesis, which is thought to result in part from the dephosphorylation and activation of glycogen synthase (GS). PP1G/R(GL)(G(M)) is a glycogen/sarcoplasmic reticulum-associated type 1 phosphatase...... that was originally postulated to mediate insulin control of glycogen metabolism. However, we recently showed (Suzuki, Y., Lanner, C., Kim, J.-H., Vilardo, P. G., Zhang, H., Jie Yang, J., Cooper, L. D., Steele, M., Kennedy, A., Bock, C., Scrimgeour, A., Lawrence, J. C. Jr., L., and DePaoli-Roach, A. A. (2001) Mol....... Cell. Biol. 21, 2683-2694) that insulin activates GS in muscle of R(GL)(G(M)) knockout (KO) mice similarly to the wild type (WT). To determine whether PP1G is involved in glycogen metabolism during muscle contractions, R(GL) KO and overexpressors (OE) were subjected to two models of contraction...

  8. Protein tyrosine phosphatases: regulatory mechanisms.

    NARCIS (Netherlands)

    den Hertog, J.; Ostman, A.; Bohmer, F.D.

    2008-01-01

    Protein-tyrosine phosphatases are tightly controlled by various mechanisms, ranging from differential expression in specific cell types to restricted subcellular localization, limited proteolysis, post-translational modifications affecting intrinsic catalytic activity, ligand binding and

  9. The PP2C Alphabet is a negative regulator of stress-activated protein kinase signaling in Drosophila.

    Science.gov (United States)

    Baril, Caroline; Sahmi, Malha; Ashton-Beaucage, Dariel; Stronach, Beth; Therrien, Marc

    2009-02-01

    The Jun N-terminal kinase and p38 pathways, also known as stress-activated protein kinase (SAPK) pathways, are signaling conduits reiteratively used throughout the development and adult life of metazoans where they play central roles in the control of apoptosis, immune function, and environmental stress responses. We recently identified a Drosophila Ser/Thr phosphatase of the PP2C family, named Alphabet (Alph), which acts as a negative regulator of the Ras/ERK pathway. Here we show that Alph also plays an inhibitory role with respect to Drosophila SAPK signaling during development as well as under stress conditions such as oxidative or genotoxic stresses. Epistasis experiments suggest that Alph acts at a step upstream of the MAPKKs Hep and Lic. Consistent with this interpretation, biochemical experiments identify the upstream MAPKKKs Slpr, Tak1, and Wnd as putative substrates. Together with previous findings, this work identifies Alph as a general attenuator of MAPK signaling in Drosophila.

  10. Cloning and expression profiling of the PacSnRK2 and PacPP2C gene families during fruit development, ABA treatment, and dehydration stress in sweet cherry.

    Science.gov (United States)

    Shen, Xinjie; Guo, Xiao; Zhao, Di; Zhang, Qiang; Jiang, Yuzhuang; Wang, Yantao; Peng, Xiang; Wei, Yan; Zhai, Zefeng; Zhao, Wei; Li, Tianhong

    2017-10-01

    Plant SNF1-related protein kinase 2 (SnRK2) and protein phosphatase 2C (PP2C) family members are core components of the ABA signal transduction pathway. SnRK2 and PP2C proteins have been suggested to play crucial roles in fruit ripening and improving plant tolerance to drought stress, but supporting genetic information has been lacking in sweet cherry (Prunus avium L.). Here, we cloned six full-length SnRK2 genes and three full-length PP2C genes from sweet cherry cv. Hong Deng. Quantitative PCR analysis revealed that PacSnRK2.2, PacSnRK2.3, PacSnRK2.6, and PacPP2C1-3 were negatively regulated in fruits in response to exogenous ABA treatment, PacSnRK2.4 and PacSnRK2.5 were upregulated, and PacSnRK2.1 expression was not affected. The ABA treatment also significantly promoted the accumulation of anthocyanins in sweet cherry fruit. The expression of all PacSnRK2 and PacPP2C genes was induced by dehydration stress, which also promoted the accumulation of drought stress signaling molecules in the sweet cherry fruits, including ABA, soluble sugars, and anthocyanin. Furthermore, a yeast two-hybrid analysis demonstrated that PacPP2C1 interacts with all six PacSnRK2s, while PacPP2C3 does not interact with PacSnRK2.5. PacPP2C2 does not interact with PacSnRK2.1 or PacSnRK2.4. These results indicate that PacSnRK2s and PacPP2Cs may play a variety of roles in the sweet cherry ABA signaling pathway and the fruit response to drought stress. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  11. Transcription and translation of phloem protein (PP2) during phloem differentiation in Cucurbita maxima.

    Science.gov (United States)

    Sham, M H; Northcote, D H

    1987-03-01

    The synthesis of a major phloem protein, PP2, was investigated by measurement of the mRNA at various stages of phloem development in Cucurbita. Quantitative assays with immuno-electrophoresis showed that the amounts of PP2 in hypocotyls of Cucurbita seedlings increased with the age of seedlings. An increase in mRNA for PP2 during the early stages of seedling growth was also observed by immunoprecipitation of the invitro translation products of hypocotyl polyadenylated RNA. There was close timing in the variations of PP2 synthesised in vivo and in the changes in amounts of translatable PP2-mRNA during the course of seedling growth. A complementary-DNA (cDNA) library to polyadenylated RNA from hypocotyls of 3-d-old Cucurbita seedlings has been constructed. Two cDNA clones, A and B, have been identified by hybrid-release translation to be complementary to the mRNA coding for PP2. The levels of total mRNA for PP2 measured with clone A were found to increase in the first 4 d of seedling growth but decreased to lower levels in older seedlings. Regulatory controls on both transcription and modification of transcripts appeared to occur during the synthesis of PP2.

  12. Rif1 controls DNA replication by directing Protein Phosphatase 1 to reverse Cdc7-mediated phosphorylation of the MCM complex.

    Science.gov (United States)

    Hiraga, Shin-Ichiro; Alvino, Gina M; Chang, Fujung; Lian, Hui-Yong; Sridhar, Akila; Kubota, Takashi; Brewer, Bonita J; Weinreich, Michael; Raghuraman, M K; Donaldson, Anne D

    2014-02-15

    Initiation of eukaryotic DNA replication requires phosphorylation of the MCM complex by Dbf4-dependent kinase (DDK), composed of Cdc7 kinase and its activator, Dbf4. We report here that budding yeast Rif1 (Rap1-interacting factor 1) controls DNA replication genome-wide and describe how Rif1 opposes DDK function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. Deleting RIF1 partially compensates for the limited DDK activity in a cdc7-1 mutant strain by allowing increased, premature phosphorylation of Mcm4. PP1 interaction motifs within the Rif1 N-terminal domain are critical for its repressive effect on replication. We confirm that Rif1 interacts with PP1 and that PP1 prevents premature Mcm4 phosphorylation. Remarkably, our results suggest that replication repression by Rif1 is itself also DDK-regulated through phosphorylation near the PP1-interacting motifs. Based on our findings, we propose that Rif1 is a novel PP1 substrate targeting subunit that counteracts DDK-mediated phosphorylation during replication. Fission yeast and mammalian Rif1 proteins have also been implicated in regulating DNA replication. Since PP1 interaction sites are evolutionarily conserved within the Rif1 sequence, it is likely that replication control by Rif1 through PP1 is a conserved mechanism.

  13. Identification of Plasmodium falciparum Translation Initiation eIF2β Subunit: Direct Interaction with Protein Phosphatase Type 1

    Czech Academy of Sciences Publication Activity Database

    Tellier, G.; Lenne, A.; Cailliau-Maggio, K.; Cabezas-Cruz, A.; Valdés, James J.; Martoriati, A.; Aliouat, El M.; Gosset, P.; Delaire, B.; Fréville, A.; Pierrot, C.; Khalife, J.

    2016-01-01

    Roč. 7, MAY 26 (2016), č. článku 777. ISSN 1664-302X Institutional support: RVO:60077344 Keywords : Plasmodium falciparum * Protein Phosphatase type1 * eIF2b * protein-protein interaction * translation complex Subject RIV: EE - Microbiology, Virology Impact factor: 4.076, year: 2016

  14. MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis.

    Directory of Open Access Journals (Sweden)

    Julija Umbrasaite

    2010-12-01

    Full Text Available In plant post-embryonic epidermis mitogen-activated protein kinase (MAPK signaling promotes differentiation of pavement cells and inhibits initiation of stomata. Stomata are cells specialized to modulate gas exchange and water loss. Arabidopsis MAPKs MPK3 and MPK6 are at the core of the signaling cascade; however, it is not well understood how the activity of these pleiotropic MAPKs is constrained spatially so that pavement cell differentiation is promoted only outside the stomata lineage. Here we identified a PP2C-type phosphatase termed AP2C3 (Arabidopsis protein phosphatase 2C that is expressed distinctively during stomata development as well as interacts and inactivates MPK3, MPK4 and MPK6. AP2C3 co-localizes with MAPKs within the nucleus and this localization depends on its N-terminal extension. We show that other closely related phosphatases AP2C2 and AP2C4 are also MAPK phosphatases acting on MPK6, but have a distinct expression pattern from AP2C3. In accordance with this, only AP2C3 ectopic expression is able to stimulate cell proliferation leading to excess stomata development. This function of AP2C3 relies on the domains required for MAPK docking and intracellular localization. Concomitantly, the constitutive and inducible AP2C3 expression deregulates E2F-RB pathway, promotes the abundance and activity of CDKA, as well as changes of CDKB1;1 forms. We suggest that AP2C3 downregulates the MAPK signaling activity to help maintain the balance between differentiation of stomata and pavement cells.

  15. The phosphorylated form of FTY720 activates PP2A, represses inflammation and is devoid of S1P agonism in A549 lung epithelial cells.

    Science.gov (United States)

    Rahman, Md Mostafizur; Prünte, Laura; Lebender, Leonard F; Patel, Brijeshkumar S; Gelissen, Ingrid; Hansbro, Philip M; Morris, Jonathan C; Clark, Andrew R; Verrills, Nicole M; Ammit, Alaina J

    2016-11-16

    Protein phosphatase 2A (PP2A) activity can be enhanced pharmacologically by PP2A-activating drugs (PADs). The sphingosine analog FTY720 is the best known PAD and we have shown that FTY720 represses production of pro-inflammatory cytokines responsible for respiratory disease pathogenesis. Whether its phosphorylated form, FTY720-P, also enhances PP2A activity independently of the sphingosine 1-phosphate (S1P) pathway was unknown. Herein, we show that FTY720-P enhances TNF-induced PP2A phosphatase activity and significantly represses TNF-induced interleukin 6 (IL-6) and IL-8 mRNA expression and protein secretion from A549 lung epithelial cells. Comparing FTY720 and FTY720-P with S1P, we show that unlike S1P, the sphingosine analogs do not induce cytokine production on their own. In fact, FTY720 and FTY720-P significantly repress S1P-induced IL-6 and IL-8 production. We then examined their impact on expression of cyclooxygenase 2 (COX-2) and resultant prostaglandin E 2 (PGE 2) production. S1P did not increase production of this pro-inflammatory enzyme because COX-2 mRNA gene expression is NF-κB-dependent, and unlike TNF, S1P did not activate NF-κB. However, TNF-induced COX-2 mRNA expression and PGE 2 secretion is repressed by FTY720 and FTY720-P. Hence, FTY720-P enhances PP2A activity and that PADs can repress production of pro-inflammatory cytokines and enzymes in A549 lung epithelial cells in a manner devoid of S1P agonism.

  16. THE UNCOVERING OF A NOVEL REGULATORY MECHANISM FOR PLD2: FORMATION OF A TERNARY COMPLEX WITH PROTEIN TYROSINE PHOSPHATASE PTP1B AND GROWTH FACTOR RECEPTOR-BOUND PROTEIN GRB2

    Science.gov (United States)

    Horn, Jeff; Lopez, Isabel; Miller, Mill; Gomez-Cambronero, Julian

    2011-01-01

    The regulation of PLD2 activation is poorly understood at present. Transient transfection of COS-7 with a mycPLD2 construct results in elevated levels of PLD2 enzymatic activity and tyrosyl phosphorylation. To investigate whether this phosphorylation affects PLD2 enzymatic activity, anti-myc immunoprecipitates were treated with recombinant protein tyrosine phosphatase PTP1B. Surprisingly, lipase activity and PY levels both increased over a range of PTP1B concentrations. These increases occurred in parallel to a measurable PTP1B-associated phosphatase activity. Inhibitor studies demonstrated that an EGF-receptor type kinase is involved in phosphorylation. In a COS-7 cell line created in the laboratory that stably expressed myc-PLD2, PTP1B induced a robust (>6-fold) augmentation of myc-PLD2 phosphotyrosine content. The addition of growth factor receptor-bound protein 2 (Grb2) to cell extracts also elevated PY levels of myc-PLD (>10-fold). Systematic co-immunoprecipitation-immunoblotting experiments pointed at a physical association between PLD2, Grb2 and PTP1B in both physiological conditions and in overexpressed cells. This is the first report of a demonstration of the mammalian isoform PLD2 existing in a ternary complex with a protein tyrosine phosphatase, PTP1b, and the docking protein Grb2 which greatly enhances tyrosyl phosphorylation of the lipase. PMID:15896299

  17. A widespread amino acid polymorphism at codon 905 of the glycogen-associated regulatory subunit of protein phosphatase-1 is associated with insulin resistance and hypersecretion of insulin

    DEFF Research Database (Denmark)

    Hansen, L; Hansen, T; Vestergaard, H

    1995-01-01

    The regulatory G-subunit of the glycogen-associated form of protein phosphatase 1 (PP1) plays a crucial part in muscle tissue glycogen synthesis and breakdown. As impaired insulin stimulated glycogen synthesis in peripheral tissues is considered to be a pathogenic factor in subsets of non-insulin...

  18. MECHANISM OF PROTEIN TYROSINE PHOSPHATASE INHIBITION IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZN2+

    Science.gov (United States)

    A number of studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to Zn2+ inhibits protein tyrosine phosphatase (PTP) activity and leads to activation of epidermal growth factor receptor (EGFR) signaling in ...

  19. A novel strategy for the development of selective active-site inhibitors of the protein tyrosine phosphatase-like proteins islet-cell antigen 512 (IA-2) and phogrin (IA-2beta).

    NARCIS (Netherlands)

    Drake, P.G.; Peters, G.H.; Andersen, H.S.; Hendriks, W.J.A.J.; Moller, N.P.

    2003-01-01

    Islet-cell antigen 512 (IA-2) and phogrin (IA-2beta) are atypical members of the receptor protein tyrosine phosphatase (PTP) family that are characterized by a lack of activity against conventional PTP substrates. The physiological role(s) of these proteins remain poorly defined, although recent

  20. Structure determination of T-cell protein-tyrosine phosphatase

    DEFF Research Database (Denmark)

    Iversen, L.F.; Møller, K. B.; Pedersen, A.K.

    2002-01-01

    Protein-tyrosine phosphatase 1B (PTP1B) has recently received much attention as a potential drug target in type 2 diabetes. This has in particular been spurred by the finding that PTP1B knockout mice show increased insulin sensitivity and resistance to diet-induced obesity. Surprisingly, the highly...... homologous T cell protein-tyrosine phosphatase (TC-PTP) has received much less attention, and no x-ray structure has been provided. We have previously co-crystallized PTP1B with a number of low molecular weight inhibitors that inhibit TC-PTP with similar efficiency. Unexpectedly, we were not able to co...... the high degree of functional and structural similarity between TC-PTP and PTP1B, we have been able to identify areas close to the active site that might be addressed to develop selective inhibitors of each enzyme....

  1. Identification of residues within the African swine fever virus DP71L protein required for dephosphorylation of translation initiation factor eIF2α and inhibiting activation of pro-apoptotic CHOP

    Energy Technology Data Exchange (ETDEWEB)

    Barber, Claire; Netherton, Chris; Goatley, Lynnette [The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF (United Kingdom); Moon, Alice; Goodbourn, Steve [Institute for Infection and Immunity, St. George' s, University of London, London SW17 0RE (United Kingdom); Dixon, Linda, E-mail: linda.dixon@pirbright.ac.uk [The Pirbright Institute, Ash Road, Pirbright, Woking, Surrey GU24 0NF (United Kingdom)

    2017-04-15

    The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate the translation initiation factor 2α (eIF2α) and avoid shut-off of global protein synthesis and downstream activation of the pro-apoptotic factor CHOP. Residues V16 and F18A were critical for binding of DP71L to PP1. Mutation of this PP1 binding motif or deletion of residues between 52 and 66 reduced the ability of DP71L to cause dephosphorylation of eIF2α and inhibit CHOP induction. The residues LSAVL, between 57 and 61, were also required. PP1 was co-precipitated with wild type DP71L and the mutant lacking residues 52- 66 or the LSAVL motif, but not with the PP1 binding motif mutant. The residues in the LSAVL motif play a critical role in DP71L function but do not interfere with binding to PP1. Instead we propose these residues are important for DP71L binding to eIF2α. - Highlights: •The African swine fever virus DP71L protein recruits protein phosphatase 1 (PP1) to dephosphorylate translation initiation factor eIF2α (eIF2α). •The residues V{sup 16}, F{sup 18} of DP71L are required for binding to the α, β and γ isoforms of PP1 and for DP71L function. •The sequence LSAVL downstream from the PP1 binding site (residues 57–61) are also important for DP71L function. •DP71L mutants of the LSAVL sequence retain ability to co-precipitate with PP1 showing these sequences have a different role to PP1 binding.

  2. Cyclin G2 is a centrosome-associated nucleocytoplasmic shuttling protein that influences microtubule stability and induces a p53-dependent cell cycle arrest

    International Nuclear Information System (INIS)

    Arachchige Don, Aruni S.; Dallapiazza, Robert F.; Bennin, David A.; Brake, Tiffany; Cowan, Colleen E.; Horne, Mary C.

    2006-01-01

    Cyclin G2 is an atypical cyclin that associates with active protein phosphatase 2A. Cyclin G2 gene expression correlates with cell cycle inhibition; it is significantly upregulated in response to DNA damage and diverse growth inhibitory stimuli, but repressed by mitogenic signals. Ectopic expression of cyclin G2 promotes cell cycle arrest, cyclin dependent kinase 2 inhibition and the formation of aberrant nuclei [Bennin, D. A., Don, A. S., Brake, T., McKenzie, J. L., Rosenbaum, H., Ortiz, L., DePaoli-Roach, A. A., and Horne, M. C. (2002). Cyclin G2 associates with protein phosphatase 2A catalytic and regulatory B' subunits in active complexes and induces nuclear aberrations and a G 1 /S-phase cell cycle arrest. J Biol Chem 277, 27449-67]. Here we report that endogenous cyclin G2 copurifies with centrosomes and microtubules (MT) and that ectopic G2 expression alters microtubule stability. We find exogenous and endogenous cyclin G2 present at microtubule organizing centers (MTOCs) where it colocalizes with centrosomal markers in a variety of cell lines. We previously reported that cyclin G2 forms complexes with active protein phosphatase 2A (PP2A) and colocalizes with PP2A in a detergent-resistant compartment. We now show that cyclin G2 and PP2A colocalize at MTOCs in transfected cells and that the endogenous proteins copurify with isolated centrosomes. Displacement of the endogenous centrosomal scaffolding protein AKAP450 that anchors PP2A at the centrosome resulted in the depletion of centrosomal cyclin G2. We find that ectopic expression of cyclin G2 induces microtubule bundling and resistance to depolymerization, inhibition of polymer regrowth from MTOCs and a p53-dependent cell cycle arrest. Furthermore, we determined that a 100 amino acid carboxy-terminal region of cyclin G2 is sufficient to both direct GFP localization to centrosomes and induce cell cycle inhibition. Colocalization of endogenous cyclin G2 with only one of two GFP-centrin-tagged centrioles, the

  3. Identification and characterization of an ATP.Mg-dependent protein phosphatase from pig brain

    International Nuclear Information System (INIS)

    Yang, S.D.; Fong, Y.L.

    1985-01-01

    Substantial amounts of ATP.Mg-dependent phosphorylase phosphatase (Fc. M) and its activator (kinase FA) were identified and extensively purified from pig brain, in spite of the fact that glycogen metabolism in the brain is of little importance. The brain Fc.M was completely inactive and could only be activated by ATP.Mg and FA, isolated either from rabbit muscle or pig brain. Kinetical analysis of the dephosphorylation of endogenous brain protein indicates that Fc.M could dephosphorylate 32 P-labeled myelin basic protein (MBP) and [ 32 P]phosphorylase alpha at a comparable rate and moreover, this associated MBP phosphatase activity was also strictly kinase FA/ATP.Mg-dependent, demonstrating that MBP is a potential substrate for Fc.M in the brain. By manipulating MBP and inhibitor-2 as specific potent phosphorylase phosphatase inhibitors, we further demonstrate that 1) Fc.M contains two distinct catalytic sites to dephosphorylate different substrates, and 2) brain MBP may be a physiological trigger involved in the regulation of protein phosphatase substrate specificity in mammalian nervous tissues

  4. Development of a gene therapy strategy to target hepatocellular carcinoma based inhibition of protein phosphatase 2A using the α-fetoprotein promoter enhancer and pgk promoter: an in vitro and in vivo study

    International Nuclear Information System (INIS)

    Li, Wei; Tao, Min; Li, Dao-Ming; Chen, Kai; Chen, Zheng; Zong, Yang; Yin, Hong; Xu, Ze-Kuan; Zhu, Yi; Gong, Fei-Ran

    2012-01-01

    Hepatocellular carcinoma (HCC) is one of the leading causes of cancer-related deaths worldwide. Current therapies are insufficient, making HCC an intractable disease. Our previous studies confirmed that inhibition of protein phosphatase 2A (PP2A) may provide a promising therapeutic strategy for cancer. Unfortunately, constitutive expression of PP2A in normal tissues limits the application of PP2A inhibition. Thus, a HCC-specific gene delivery system should be developed. The α-fetoprotein (AFP) promoter is commonly used in HCC-specific gene therapy strategies; however, the utility of this approach is limited due to the weak activity of the AFP promoter. It has been shown that linking the AFP enhancer with the promoter of the non-tissue-specific, human housekeeping phosphoglycerate kinase (pgk) gene can generate a strong and HCC-selective promoter. We constructed a HCC-specific gene therapy system to target PP2A using the AFP enhancer/pgk promoter, and evaluated the efficiency and specificity of this system both in vitro and in vivo. AFP enhancer/pgk promoter-driven expression of the dominant negative form of the PP2A catalytic subunit α (DN-PP2Acα) exerted cytotoxic effects against an AFP-positive human hepatoma cell lines (HepG2 and Hep3B), but did not affect AFP-negative human hepatoma cells (SK-HEP-1) or normal human liver cells (L-02). Moreover, AFP enhancer/pgk promoter driven expression of DN-PP2Acα inhibited the growth of AFP-positive HepG2 tumors in nude mice bearing solid tumor xenografts, but did not affect AFP-negative SK-HEP-1 tumors. The novel approach of AFP enhancer/pgk promoter-driven expression of DN-PP2Acα may provide a useful cancer gene therapy strategy to selectively target HCC

  5. Sp1 transcriptional activity is up-regulated by phosphatase 2A in dividing T lymphocytes.

    Science.gov (United States)

    Lacroix, Isabelle; Lipcey, Carol; Imbert, Jean; Kahn-Perlès, Brigitte

    2002-03-15

    We have followed Sp1 expression in primary human T lymphocytes induced, via CD2 plus CD28 costimulation, to sustained proliferation and subsequent return to quiescence. Binding of Sp1 to wheat germ agglutinin lectin was not modified following activation, indicating that the overall glycosylation of the protein was unchanged. Sp1 underwent, instead, a major dephosphorylation that correlated with cyclin A expression and, thus, with cell cycle progression. A similar change was observed in T cells that re-entered cell cycle following secondary interleukin-2 stimulation, as well as in serum-induced proliferating NIH/3T3 fibroblasts. Phosphatase 2A (PP2A) appears involved because 1) treatment of dividing cells with okadaic acid or cantharidin inhibited Sp1 dephosphorylation and 2) PP2A dephosphorylated Sp1 in vitro and strongly interacted with Sp1 in vivo. Sp1 dephosphorylation is likely to increase its transcriptional activity because PP2A overexpression potentiated Sp1 site-driven chloramphenicol acetyltransferase expression in dividing Kit225 T cells and okadaic acid reversed this effect. This increase might be mediated by a stronger affinity of dephosphorylated Sp1 for DNA, as illustrated by the reduced DNA occupancy by hyperphosphorylated Sp factors from cantharidin- or nocodazole-treated cells. Finally, Sp1 dephosphorylation appears to occur throughout cell cycle except for mitosis, a likely common feature to all cycling cells.

  6. Molecular cloning and chromosome mapping of the human gene encoding protein phosphotyrosyl phosphatase 1B

    International Nuclear Information System (INIS)

    Brown-Shimer, S.; Johnson, K.A.; Bruskin, A.; Green, N.R.; Hill, D.E.; Lawrence, J.B.; Johnson, C.

    1990-01-01

    The inactivation of growth suppressor genes appears to play a major role in the malignant process. To assess whether protein phosphotyrosyl phosphatases function as growth suppressors, the authors have isolated a cDNA clone encoding human protein phosphotyrosyl phosphatase 1B for structural and functional characterization. The translation product deduced from the 1,305-nucleotide open reading frame predicts a protein containing 435 amino acids and having a molecular mass of 49,966 Da. The amino-terminal 321 amino acids deduced from the cDNA sequence are identical to the empirically determined sequence of protein phosphotyrosyl phosphatase 1B. A genomic clone has been isolated and used in an in situ hybridization to banded metaphase chromosomes to determine that the gene encoding protein phosphotyrosyl phosphatase 1B maps as a single-copy gene to the long arm of chromosome 20 in the region q13.1-q13.2

  7. Adsorption of Insecticidal Crystal Protein Cry11Aa onto Nano-Mg(OH)2: Effects on Bioactivity and Anti-Ultraviolet Ability.

    Science.gov (United States)

    Pan, Xiaohong; Xu, Zhangyan; Li, Lan; Shao, Enshi; Chen, Saili; Huang, Tengzhou; Chen, Zhi; Rao, Wenhua; Huang, Tianpei; Zhang, Lingling; Wu, Songqing; Guan, Xiong

    2017-11-01

    The traditional Bacillus thuringiensis (Bt) formulations for field applications are not resistant to harsh environmental conditions. Hence, the active ingredients of the Bt bioinsecticides could degrade quickly and has low anti-ultraviolet ability in the field, which significantly limits its practical application. In the present study, we developed an efficient and stable delivery system for Bt Cry11Aa toxins. We coated Cry11Aa proteins with Mg(OH) 2 nanoparticles (MHNPs), and then assessed the effects of MHNPs on bioactivity and anti-ultraviolet ability of the Cry11Aa proteins. Our results indicated that MHNPs, like "coating clothes", could effectively protect the Cry protein and enhance the insecticidal bioactivity after UV radiation (the degradation rate was decreased from 64.29% to 16.67%). In addtion, MHNPs could improve the proteolysis of Cry11Aa in the midgut and aggravate the damage of the Cry protein to the gut epithelial cells, leading to increased insecticidal activity against Culex quinquefasciatus. Our results revealed that MHNPs, as an excellent nanocarrier, could substantially improve the insecticidal bioactivity and anti-ultraviolet ability of Cry11Aa.

  8. Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans.

    Science.gov (United States)

    Lange, Karen I; Heinrichs, Jeffrey; Cheung, Karen; Srayko, Martin

    2013-01-15

    Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A) enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B', B″) but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly) is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts), and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

  9. Suppressor mutations identify amino acids in PAA-1/PR65 that facilitate regulatory RSA-1/B″ subunit targeting of PP2A to centrosomes in C. elegans

    Directory of Open Access Journals (Sweden)

    Karen I. Lange

    2012-11-01

    Protein phosphorylation and dephosphorylation is a key mechanism for the spatial and temporal regulation of many essential developmental processes and is especially prominent during mitosis. The multi-subunit protein phosphatase 2A (PP2A enzyme plays an important, yet poorly characterized role in dephosphorylating proteins during mitosis. PP2As are heterotrimeric complexes comprising a catalytic, structural, and regulatory subunit. Regulatory subunits are mutually exclusive and determine subcellular localization and substrate specificity of PP2A. At least 3 different classes of regulatory subunits exist (termed B, B′, B″ but there is no obvious similarity in primary sequence between these classes. Therefore, it is not known how these diverse regulatory subunits interact with the same holoenzyme to facilitate specific PP2A functions in vivo. The B″ family of regulatory subunits is the least understood because these proteins lack conserved structural domains. RSA-1 (regulator of spindle assembly is a regulatory B″ subunit required for mitotic spindle assembly in Caenorhabditis elegans. In order to address how B″ subunits interact with the PP2A core enzyme, we focused on a conditional allele, rsa-1(or598ts, and determined that this mutation specifically disrupts the protein interaction between RSA-1 and the PP2A structural subunit, PAA-1. Through genetic screening, we identified a putative interface on the PAA-1 structural subunit that interacts with a defined region of RSA-1/B″. In the context of previously published results, these data propose a mechanism of how different PP2A B-regulatory subunit families can bind the same holoenzyme in a mutually exclusive manner, to perform specific tasks in vivo.

  10. Coupling TOR to the Cell Cycle by the Greatwall–Endosulfine–PP2A-B55 Pathway

    Directory of Open Access Journals (Sweden)

    Livia Pérez-Hidalgo

    2017-08-01

    Full Text Available Cell growth and division are two processes tightly coupled in proliferating cells. While Target of Rapamycin (TOR is the master regulator of growth, the cell cycle is dictated by the activity of the cyclin-dependent kinases (CDKs. A long-standing question in cell biology is how these processes may be connected. Recent work has highlighted that regulating the phosphatases that revert CDK phosphorylations is as important as regulating the CDKs for cell cycle progression. At mitosis, maintaining a low level of protein phosphatase 2A (PP2A-B55 activity is essential for CDK substrates to achieve the correct level of phosphorylation. The conserved Greatwall–Endosulfine pathway has been shown to be required for PP2A-B55 inhibition at mitosis in yeasts and multicellular organisms. Interestingly, in yeasts, the Greatwall–Endosulfine pathway is negatively regulated by TOR Complex 1 (TORC1. Moreover, Greatwall–Endosulfine activation upon TORC1 inhibition has been shown to regulate the progression of the cell cycle at different points: the G1 phase in budding yeast, the G2/M transition and the differentiation response in fission yeast, and the entry into quiescence in both budding and fission yeasts. In this review, we discuss the recent findings on how the Greatwall–Endosulfine pathway may provide a connection between cell growth and the cell cycle machinery.

  11. Dephosphorylation of microtubule-binding sites at the neurofilament-H tail domain by alkaline, acid, and protein phosphatases.

    Science.gov (United States)

    Hisanaga, S; Yasugawa, S; Yamakawa, T; Miyamoto, E; Ikebe, M; Uchiyama, M; Kishimoto, T

    1993-06-01

    The dephosphorylation-induced interaction of neurofilaments (NFs) with microtubules (MTs) was investigated by using several phosphatases. Escherichia coli alkaline and wheat germ acid phosphatases increased the electrophoretic mobility of NF-H and NF-M by dephosphorylation, and induced the binding of NF-H to MTs. The binding of NFs to MTs was observed only after the electrophoretic mobility of NF-H approached the exhaustively dephosphorylated level when alkaline phosphatase was used. The number of phosphate remaining when NF-H began to bind to MTs was estimated by measuring phosphate bound to NF-H. NF-H did not bind to MTs even when about 40 phosphates from the total of 51 had been removed by alkaline phosphatase. The removal of 6 further phosphates finally resulted in the association of NF-H with MTs. A similar finding, that the restricted phosphorylation sites in the NF-H tail domain, but not the total amount of phosphates, were important for binding to MTs, was also obtained with acid phosphatases. In contrast to alkaline and acid phosphatases, four classes of protein phosphatases (protein phosphatases 1, 2A, 2B, and 2C) were ineffective for shifting the electrophoretic mobility of NF proteins and for inducing the association of NFs to MTs.

  12. Resonance production in p+p, p+A and A+A collisions measured with HADES

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    Reshetin A.

    2012-11-01

    Full Text Available The knowledge of baryonic resonance properties and production cross sections plays an important role for the extraction and understanding of medium modifications of mesons in hot and/or dense nuclear matter. We present and discuss systematics on dielectron and strangeness production obtained with HADES on p+p, p+A and A+A collisions in the few GeV energy regime with respect to these resonances.

  13. SH2 domain-containing protein tyrosine phosphatase 2 and focal adhesion kinase protein interactions regulate pulmonary endothelium barrier function.

    Science.gov (United States)

    Chichger, Havovi; Braza, Julie; Duong, Huetran; Harrington, Elizabeth O

    2015-06-01

    Enhanced protein tyrosine phosphorylation is associated with changes in vascular permeability through formation and dissolution of adherens junctions and regulation of stress fiber formation. Inhibition of the protein tyrosine phosphorylase SH2 domain-containing protein tyrosine phosphatase 2 (SHP2) increases tyrosine phosphorylation of vascular endothelial cadherin and β-catenin, resulting in disruption of the endothelial monolayer and edema formation in the pulmonary endothelium. Vascular permeability is a hallmark of acute lung injury (ALI); thus, enhanced SHP2 activity offers potential therapeutic value for the pulmonary vasculature in diseases such as ALI, but this has not been characterized. To assess whether SHP2 activity mediates protection against edema in the endothelium, we assessed the effect of molecular activation of SHP2 on lung endothelial barrier function in response to the edemagenic agents LPS and thrombin. Both LPS and thrombin reduced SHP2 activity, correlated with decreased focal adhesion kinase (FAK) phosphorylation (Y(397) and Y(925)) and diminished SHP2 protein-protein associations with FAK. Overexpression of constitutively active SHP2 (SHP2(D61A)) enhanced baseline endothelial monolayer resistance and completely blocked LPS- and thrombin-induced permeability in vitro and significantly blunted pulmonary edema formation induced by either endotoxin (LPS) or Pseudomonas aeruginosa exposure in vivo. Chemical inhibition of FAK decreased SHP2 protein-protein interactions with FAK concomitant with increased permeability; however, overexpression of SHP2(D61A) rescued the endothelium and maintained FAK activity and FAK-SHP2 protein interactions. Our data suggest that SHP2 activation offers the pulmonary endothelium protection against barrier permeability mediators downstream of the FAK signaling pathway. We postulate that further studies into the promotion of SHP2 activation in the pulmonary endothelium may offer a therapeutic approach for patients

  14. A Global Protein Kinase and Phosphatase Interaction Network in Yeast

    Science.gov (United States)

    Breitkreutz, Ashton; Choi, Hyungwon; Sharom, Jeffrey R.; Boucher, Lorrie; Neduva, Victor; Larsen, Brett; Lin, Zhen-Yuan; Breitkreutz, Bobby-Joe; Stark, Chris; Liu, Guomin; Ahn, Jessica; Dewar-Darch, Danielle; Reguly, Teresa; Tang, Xiaojing; Almeida, Ricardo; Qin, Zhaohui Steve; Pawson, Tony; Gingras, Anne-Claude; Nesvizhskii, Alexey I.; Tyers, Mike

    2011-01-01

    The interactions of protein kinases and phosphatases with their regulatory subunits and substrates underpin cellular regulation. We identified a kinase and phosphatase interaction (KPI) network of 1844 interactions in budding yeast by mass spectrometric analysis of protein complexes. The KPI network contained many dense local regions of interactions that suggested new functions. Notably, the cell cycle phosphatase Cdc14 associated with multiple kinases that revealed roles for Cdc14 in mitogen-activated protein kinase signaling, the DNA damage response, and metabolism, whereas interactions of the target of rapamycin complex 1 (TORC1) uncovered new effector kinases in nitrogen and carbon metabolism. An extensive backbone of kinase-kinase interactions cross-connects the proteome and may serve to coordinate diverse cellular responses. PMID:20489023

  15. The TriTryp Phosphatome: analysis of the protein phosphatase catalytic domains

    Directory of Open Access Journals (Sweden)

    Huxley-Jones Julie

    2007-11-01

    Full Text Available Abstract Background The genomes of the three parasitic protozoa Trypanosoma cruzi, Trypanosoma brucei and Leishmania major are the main subject of this study. These parasites are responsible for devastating human diseases known as Chagas disease, African sleeping sickness and cutaneous Leishmaniasis, respectively, that affect millions of people in the developing world. The prevalence of these neglected diseases results from a combination of poverty, inadequate prevention and difficult treatment. Protein phosphorylation is an important mechanism of controlling the development of these kinetoplastids. With the aim to further our knowledge of the biology of these organisms we present a characterisation of the phosphatase complement (phosphatome of the three parasites. Results An ontology-based scan of the three genomes was used to identify 86 phosphatase catalytic domains in T. cruzi, 78 in T. brucei, and 88 in L. major. We found interesting differences with other eukaryotic genomes, such as the low proportion of tyrosine phosphatases and the expansion of the serine/threonine phosphatase family. Additionally, a large number of atypical protein phosphatases were identified in these species, representing more than one third of the total phosphatase complement. Most of the atypical phosphatases belong to the dual-specificity phosphatase (DSP family and show considerable divergence from classic DSPs in both the domain organisation and sequence features. Conclusion The analysis of the phosphatome of the three kinetoplastids indicates that they possess orthologues to many of the phosphatases reported in other eukaryotes, including humans. However, novel domain architectures and unusual combinations of accessory domains, suggest distinct functional roles for several of the kinetoplastid phosphatases, which await further experimental exploration. These distinct traits may be exploited in the selection of suitable new targets for drug development to prevent

  16. The protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation and increases sarcoplasmic/endoplasmic reticulum calcium ATPase 2 levels

    International Nuclear Information System (INIS)

    King, Taj D.; Gandy, Johanna C.; Bijur, Gautam N.

    2006-01-01

    The ubiquitously expressed protein glycogen synthase kinase-3 (GSK3) is constitutively active, however its activity is markedly diminished following phosphorylation of Ser21 of GSK3α and Ser9 of GSK3β. Although several kinases are known to phosphorylate Ser21/9 of GSK3, for example Akt, relatively much less is known about the mechanisms that cause the dephosphorylation of GSK3 at Ser21/9. In the present study KCl-induced plasma membrane depolarization of SH-SY5Y cells, which increases intracellular calcium concentrations caused a transient decrease in the phosphorylation of Akt at Thr308 and Ser473, and GSK3 at Ser21/9. Overexpression of the selective protein phosphatase-1 inhibitor protein, inhibitor-2, increased basal GSK3 phosphorylation at Ser21/9 and significantly blocked the KCl-induced dephosphorylation of GSK3β, but not GSK3α. The phosphorylation of Akt was not affected by the overexpression of inhibitor-2. GSK3 activity is known to affect sarcoplasmic/endoplasmic reticulum calcium ATPase 2 (SERCA2) levels. Overexpression of inhibitor-2 or treatment of cells with the GSK3 inhibitors lithium and SB216763 increased the levels of SERCA2. These results indicate that the protein phosphatase-1/inhibitor-2 complex differentially regulates GSK3 dephosphorylation induced by KCl and that GSK3 activity regulates SERCA2 levels

  17. The PP1 binding code: a molecular-lego strategy that governs specificity.

    Science.gov (United States)

    Heroes, Ewald; Lesage, Bart; Görnemann, Janina; Beullens, Monique; Van Meervelt, Luc; Bollen, Mathieu

    2013-01-01

    Ser/Thr protein phosphatase 1 (PP1) is a single-domain hub protein with nearly 200 validated interactors in vertebrates. PP1-interacting proteins (PIPs) are ubiquitously expressed but show an exceptional diversity in brain, testis and white blood cells. The binding of PIPs is mainly mediated by short motifs that dock to surface grooves of PP1. Although PIPs often contain variants of the same PP1 binding motifs, they differ in the number and combination of docking sites. This molecular-lego strategy for binding to PP1 creates holoenzymes with unique properties. The PP1 binding code can be described as specific, universal, degenerate, nonexclusive and dynamic. PIPs control associated PP1 by interference with substrate recruitment or access to the active site. In addition, some PIPs have a subcellular targeting domain that promotes dephosphorylation by increasing the local concentration of PP1. The diversity of the PP1 interactome and the properties of the PP1 binding code account for the exquisite specificity of PP1 in vivo. © 2012 The Authors Journal compilation © 2012 FEBS.

  18. Cloning and characterization of rat density-enhanced phosphatase-1, a protein tyrosine phosphatase expressed by vascular cells.

    Science.gov (United States)

    Borges, L G; Seifert, R A; Grant, F J; Hart, C E; Disteche, C M; Edelhoff, S; Solca, F F; Lieberman, M A; Lindner, V; Fischer, E H; Lok, S; Bowen-Pope, D F

    1996-09-01

    We have cloned from cultured vascular smooth muscle cells a protein tyrosine phosphatase, rat density-enhanced phosphatase-1 (rDEP-1), which is a probable rat homologue of DEP-1/HPTP eta. rDEP-1 is encoded by an 8.7-kb transcript and is expressed as a 180- to 220-kD protein. The rDEP-1 gene is located on human chromosome 11 (region p11.2) and on mouse chromosome 2 (region 2E). The cDNA sequence predicts a transmembrane protein consisting of a single phosphatase catalytic domain in the intracellular region, a single transmembrane domain, and eight fibronectin type III repeats in the extracellular region (GenBank accession number U40790). In situ hybridization analysis demonstrates that rDEP-1 is widely expressed in vivo but that expression is highest in cells that form epithelioid monolayers. In cultured cells with epitheliod morphology, including endothelial cells and newborn smooth muscle cells, but not in fibroblast-like cells, rDEP-1 transcript levels are dramatically upregulated as population density increases. In vivo, quiescent endothelial cells in normal arteries express relatively high levels of rDEP-1. During repair of vascular injury, expression of rDEP-1 is downregulated in migrating and proliferating endothelial cells. In vivo, rDEP-1 transcript levels are present in very high levels in megakaryocytes, and circulating plates have high levels of the rDEP-1 protein. In vitro, initiation of differentiation of the human megakaryoblastic cell line CHRF-288-11 with phorbol 12-myristate 13-acetate leads to a very strong upregulation of rDEP-1 transcripts. The deduced structure and the regulation of expression of rDEP-1 suggest that it may play a role in adhesion and/or signaling events involving cell-cell and cell-matrix contact.

  19. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    Energy Technology Data Exchange (ETDEWEB)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M. [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Iuliano, Rodolfo [Dipartimento di Medicina Sperimentale e Clinica, Facoltà di Medicina e Chirurgia, Università di Catanzaro, 88100 Catanzaro (Italy); Fusco, Alfredo [Dipartimento di Biologia e Patologia Cellulare e Molecolare, c/o Instituto di Endocrinologia ed Oncologia Sperimentale del CNR, Facolta di Medicina e Chirurgia, Università degli Studi di Napoli ‘Federico II’, Via Pansini 5, 80131 Naples (Italy); NOGEC (Naples Oncogenomocs Center)-CEINGE, Biotecnologie Avanzate, Via Comunale Margherita 482, 80145 Naples (Italy); Polikarpov, Igor, E-mail: ipolikarpov@if.sc.usp.br [Instituto de Física de São Carlos, Departamento de Física e Informática, Universidade de São Paulo, Avenida Trabalhador São Carlense 400, CEP 13566-590 São Carlos, SP (Brazil); Laboratório Nacional de Luz Síncrotron, Campinas, SP (Brazil)

    2006-09-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2{sub 1}2{sub 1}2{sub 1}, with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit.

  20. Crystallization and preliminary X-ray diffraction analysis of rat protein tyrosine phosphatase η

    International Nuclear Information System (INIS)

    Matozo, Huita C.; Nascimento, Alessandro S.; Santos, Maria A. M.; Iuliano, Rodolfo; Fusco, Alfredo; Polikarpov, Igor

    2006-01-01

    In this study, the catalytic domain of rat protein tyrosine phosphatase η was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. The rat protein tyrosine phosphatase η (rPTPη) is a cysteine-dependent phosphatase which hydrolyzes phosphoester bonds in proteins and other molecules. rPTPη and its human homologue DEP-1 are involved in neoplastic transformations. Thus, expression of the protein is reduced in all oncogene-transformed thyroid cell lines and is absent in highly malignant thyroid cells. Moreover, consistent with the suggested tumour suppression role of PTPη, inhibition of the tumorigenic process occurs after its exogenous reconstitution, suggesting that PTPη might be important for gene therapy of cancers. In this study, the catalytic domain of rPTPη was produced in Escherichia coli in soluble form and purified to homogeneity. Crystals were obtained by the hanging-drop vapour-diffusion method. Diffraction data were collected to 1.87 Å resolution. The crystal belongs to space group P2 1 2 1 2 1 , with unit-cell parameters a = 46.46, b = 63.07, c = 111.64 Å, and contains one molecule per asymmetric unit

  1. Identification of a novel Leucine-rich repeat protein and candidate PP1 regulatory subunit expressed in developing spermatids

    Directory of Open Access Journals (Sweden)

    Sperry Ann O

    2008-01-01

    Full Text Available Abstract Background Spermatogenesis is comprised of a series of highly regulated developmental changes that transform the precursor germ cell into a highly specialized spermatozoon. The last phase of spermatogenesis, termed spermiogenesis, involves dramatic morphological change including formation of the acrosome, elongation and condensation of the nucleus, formation of the flagella, and disposal of unnecessary cytoplasm. A prominent cytoskeletal component of the developing spermatid is the manchette, a unique microtubular structure that surrounds the nucleus of the developing spermatid and is thought to assist in both the reshaping of the nucleus and redistribution of spermatid cytoplasm. Although the molecular motor KIFC1 has been shown to associate with the manchette, its precise role in function of the manchette and the identity of its testis specific protein partners are unknown. The purpose of this study was to identify proteins in the testis that interact with KIFC1 using a yeast 2 hybrid screen of a testis cDNA library. Results Thirty percent of the interacting clones identified in our screen contain an identical cDNA encoding a 40 kD protein. This interacting protein has 4 leucine-rich repeats in its amino terminal half and is expressed primarily in the testis; therefore we have named this protein testis leucine-rich repeat protein or TLRR. TLRR was also found to associate tightly with the KIFC1 targeting domain using affinity chromatography. In addition to the leucine-rich repeats, TLRR contains a consensus-binding site for protein phosphatase-1 (PP1. Immunocytochemistry using a TLRR specific antibody demonstrates that this protein is found near the manchette of developing spermatids. Conclusion We have identified a previously uncharacterized leucine-rich repeat protein that is expressed abundantly in the testis and associates with the manchette of developing spermatids, possibly through its interaction with the KIFC1 molecular motor

  2. Comparison of Protein Phosphatase Inhibition Assay with LC-MS/MS for Diagnosis of Microcystin Toxicosis in Veterinary Cases

    Directory of Open Access Journals (Sweden)

    Caroline E. Moore

    2016-03-01

    Full Text Available Microcystins are acute hepatotoxins of increasing global concern in drinking and recreational waters and are a major health risk to humans and animals. Produced by cyanobacteria, microcystins inhibit serine/threonine protein phosphatase 1 (PP1. A cost-effective PP1 assay using p-nitrophenyl phosphate was developed to quickly assess water and rumen content samples. Significant inhibition was determined via a linear model, which compared increasing volumes of sample to the log-transformed ratio of the exposed rate over the control rate of PP1 activity. To test the usefulness of this model in diagnostic case investigations, samples from two veterinary cases were tested. In August 2013 fifteen cattle died around two ponds in Kentucky. While one pond and three tested rumen contents had significant PP1 inhibition and detectable levels of microcystin-LR, the other pond did not. In August 2013, a dog became fatally ill after swimming in Clear Lake, California. Lake water samples collected one and four weeks after the dog presented with clinical signs inhibited PP1 activity. Subsequent analysis using liquid chromatography-mass spectrometry (LC-MS/MS detected microcystin congeners -LR, -LA, -RR and -LF but not -YR. These diagnostic investigations illustrate the advantages of using functional assays in combination with LC-MS/MS.

  3. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    Energy Technology Data Exchange (ETDEWEB)

    Manet, Oliva-Trastoy; Berthonaud, V.; Chevalier, A.; Ducrot, C.; Marsolier-Kergoat, M.C.; Mann, C.; Leteurtre, F. [CEA Saclay, DSV, DBJC, SBGM, Lab. du Controle du Cycle Cellulaire, 91 - Gif-sur-Yvette (France)

    2006-07-01

    adaptation). Our group previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in S. cerevisiae. Here we show the conservation of this pathway in mammalian cells. In response to DNA damage, ATM (ataxia telangiectasia mutated) phosphorylates the Chk2 tumor suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by auto-phosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and de-phosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. The recombinant Chk2 protein is fully phosphorylated and activated, due to the high protein concentrations obtained during production. In vitro, Wip 1 de-phosphorylates the phospho-T68 of Chk2, but does not reduce Chk2 kinase activity on its usual GST-CDC25C substrate. These observations suggest that Wip1 phosphatase controls Chk2 activation rather than its enzymatic activity that relies on phosphorylations in the T-loop. The physiological consequences of Wip1 overexpression were tested in human adenocarcinoma cells: the HCT15 cell line. The specificities of this cell line are (i ) the absence of functional p53 proteins, leading to a G2 delay in response to a genotoxic stress, and (ii) the absence of functional Chk2 proteins, because of one CHK2 allele being unexpressed and because the second allele codes for a mutated protein that is unstable and inactive. The HCT15 cell line was complemented by a functional form of HA-Chk2 and the selected clone expresses the protein to a level similar to that observed in other cell lines. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip 1 modest overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to

  4. The wip1 phosphatase (PPM1D) antagonizes activation of the CHK2 tumor suppressor kinase

    International Nuclear Information System (INIS)

    Manet, Oliva-Trastoy; Berthonaud, V.; Chevalier, A.; Ducrot, C.; Marsolier-Kergoat, M.C.; Mann, C.; Leteurtre, F.

    2006-01-01

    ). Our group previously demonstrated that type 2C protein phosphatases (PP2C) Ptc2 and Ptc3 are required for DNA checkpoint inactivation after DNA double-strand break repair or adaptation in S. cerevisiae. Here we show the conservation of this pathway in mammalian cells. In response to DNA damage, ATM (ataxia telangiectasia mutated) phosphorylates the Chk2 tumor suppressor kinase at threonine 68 (Thr68), allowing Chk2 kinase dimerization and activation by auto-phosphorylations in the T-loop. The oncogenic protein Wip1, a PP2C phosphatase, binds Chk2 and de-phosphorylates phospho-Thr68. Consequently, Wip1 opposes Chk2 activation by ATM after ionizing irradiation of cells. The recombinant Chk2 protein is fully phosphorylated and activated, due to the high protein concentrations obtained during production. In vitro, Wip 1 de-phosphorylates the phospho-T68 of Chk2, but does not reduce Chk2 kinase activity on its usual GST-CDC25C substrate. These observations suggest that Wip1 phosphatase controls Chk2 activation rather than its enzymatic activity that relies on phosphorylations in the T-loop. The physiological consequences of Wip1 overexpression were tested in human adenocarcinoma cells: the HCT15 cell line. The specificities of this cell line are (i ) the absence of functional p53 proteins, leading to a G2 delay in response to a genotoxic stress, and (ii) the absence of functional Chk2 proteins, because of one CHK2 allele being unexpressed and because the second allele codes for a mutated protein that is unstable and inactive. The HCT15 cell line was complemented by a functional form of HA-Chk2 and the selected clone expresses the protein to a level similar to that observed in other cell lines. In HCT15 colorectal cancer cells corrected for functional Chk2 activity, Wip 1 modest overexpression suppressed the contribution of Chk2 to the G2/M DNA damage checkpoint. These results indicate that Wip1 is one of the phosphatases regulating the activity of Chk2 in response to DNA

  5. Activity of vegetative insecticidal proteins Vip3Aa58 and Vip3Aa59 of Bacillus thuringiensis against lepidopteran pests.

    Science.gov (United States)

    Baranek, Jakub; Kaznowski, Adam; Konecka, Edyta; Naimov, Samir

    2015-09-01

    Vegetative insecticidal proteins (Vips) secreted by some isolates of Bacillus thuringiensis show activity against insects and are regarded as insecticides against pests. A number of B. thuringiensis strains harbouring vip3A genes were isolated from different sources and identified by using a PCR based approach. The isolates with the highest insecticidal activity were indicated in screening tests, and their vip genes were cloned and sequenced. The analysis revealed two polymorphic Vip protein forms, which were classified as Vip3Aa58 and Vip3Aa59. After expression of the vip genes, the proteins were isolated and characterized. The activity of both toxins was estimated against economically important lepidopteran pests of woodlands (Dendrolimus pini), orchards (Cydia pomonella) and field crops (Spodoptera exigua). Vip3Aa58 and Vip3Aa59 were highly toxic and their potency surpassed those of many Cry proteins used in commercial bioinsecticides. Vip3Aa59 revealed similar larvicidal activity as Vip3Aa58 against S. exigua and C. pomonella. Despite 98% similarity of amino acid sequences of both proteins, Vip3Aa59 was significantly more active against D. pini. Additionally the effect of proteolytic activation of Vip58Aa and Vip3Aa59 on toxicity of D. pini and S. exigua was studied. Both Vip3Aa proteins did not show any activity against Tenebrio molitor (Coleoptera) larvae. The results suggest that the Vip3Aa58 and Vip3Aa59 toxins might be useful for controlling populations of insect pests of crops and forests. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Nuclear trafficking of the human cytomegalovirus pp71 (ppUL82) tegument protein

    International Nuclear Information System (INIS)

    Shen Weiping; Westgard, Elizabeth; Huang Liqun; Ward, Michael D.; Osborn, Jodi L.; Chau, Nha H.; Collins, Lindsay; Marcum, Benjamin; Koach, Margaret A.; Bibbs, Jennifer; Semmes, O. John; Kerry, Julie A.

    2008-01-01

    The human cytomegalovirus tegument protein pp71 localizes to the nucleus immediately upon infection, and functions to initiate viral gene expression. Analysis of a series of random insertion mutations revealed that sequences within the mid region (MR) of pp71 are important for localization to the nucleus. Fusion of MR sequences with eGFP revealed that amino acids 94 to 300 were sufficient to target proteins to the nucleus. Random substitution mutagenesis within this domain resulted in two double substitution mutants, pp71P203T/T223M and pp71T228M/L275Q, with a predominantly cytoplasmic localization. Disruption of nuclear targeting resulted in relocalization of the fusion proteins to a distinct perinuclear region. Using tandem mass spectrometry, we determined that threonine 223 can be phosphorylated. Mutation of this residue to a phosphomimetic amino acid resulted in abrogation of nuclear targeting. These results strongly suggest that the intracellular trafficking of pp71 is regulated by phosphorylation

  7. ABA signaling in guard cells entails a dynamic protein-protein interaction relay from the PYL-RCAR family receptors to ion channels.

    Science.gov (United States)

    Lee, Sung Chul; Lim, Chae Woo; Lan, Wenzhi; He, Kai; Luan, Sheng

    2013-03-01

    Plant hormone abscisic acid (ABA) serves as an integrator of environmental stresses such as drought to trigger stomatal closure by regulating specific ion channels in guard cells. We previously reported that SLAC1, an outward anion channel required for stomatal closure, was regulated via reversible protein phosphorylation events involving ABA signaling components, including protein phosphatase 2C members and a SnRK2-type kinase (OST1). In this study, we reconstituted the ABA signaling pathway as a protein-protein interaction relay from the PYL/RCAR-type receptors, to the PP2C-SnRK2 phosphatase-kinase pairs, to the ion channel SLAC1. The ABA receptors interacted with and inhibited PP2C phosphatase activity against the SnRK2-type kinase, releasing active SnRK2 kinase to phosphorylate, and activate the SLAC1 channel, leading to reduced guard cell turgor and stomatal closure. Both yeast two-hybrid and bimolecular fluorescence complementation assays were used to verify the interactions among the components in the pathway. These biochemical assays demonstrated activity modifications of phosphatases and kinases by their interaction partners. The SLAC1 channel activity was used as an endpoint readout for the strength of the signaling pathway, depending on the presence of different combinations of signaling components. Further study using transgenic plants overexpressing one of the ABA receptors demonstrated that changing the relative level of interacting partners would change ABA sensitivity.

  8. Central regulation of metabolism by protein tyrosine phosphatases

    Directory of Open Access Journals (Sweden)

    Ryan eTsou

    2013-01-01

    Full Text Available Protein tyrosine phosphatases (PTPs are important regulators of intracellular signaling pathways via the dephosphorylation of phosphotyrosyl residues on various receptor and non-receptor substrates. The phosphorylation state of central nervous system (CNS signaling components underlies the molecular mechanisms of a variety of physiological functions including the control of energy balance and glucose homeostasis. In this review, we summarize the current evidence implicating PTPs as central regulators of metabolism, specifically highlighting their interactions with the neuronal leptin and insulin signaling pathways. We discuss the role of a number of PTPs (PTP1B, SHP2, TCPTP, RPTPe, and PTEN, reviewing the findings from genetic mouse models and in vitro studies which highlight these phosphatases as key central regulators of energy homeostasis.

  9. Photon and pomeron-induced production of dijets in pp, pA and AA collisions

    Energy Technology Data Exchange (ETDEWEB)

    Basso, E. [Universidade Federal da Grande Dourados (UFGD), Faculdade de Ciencias Exatas e Tecnologias, Dourados, MS (Brazil); Goncalves, V.P. [Universidade Federal de Pelotas (UFPel), Instituto de Fisica e Matematica, Pelotas, RS (Brazil); Kohara, A.K. [Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Fisica, Rio de Janeiro, RJ (Brazil); Centro Brasileiro de Pesquisas Fisicas (CBPF), Rio de Janeiro, RJ (Brazil); Rangel, M.S. [Universidade Federal do Rio de Janeiro (UFRJ), Instituto de Fisica, Rio de Janeiro, RJ (Brazil)

    2017-09-15

    In this paper we present a detailed comparison of the dijet production by photon-photon, photon-pomeron and pomeron-pomeron interactions in pp, pA and AA collisions at the LHC energy. The transverse momentum, pseudo-rapidity and angular dependencies of the cross sections are calculated at LHC energy using the Forward Physics Monte Carlo (FPMC), which allows one to obtain realistic predictions for the dijet production with two leading intact hadrons. We see that the γIP channel is dominant at forward rapidities in pp collisions and in the full kinematical range in the nuclear collisions of heavy nuclei. Our results indicate that the analysis of dijet production at the LHC can be useful to test the resolved pomeron model as well as to constrain the magnitude of the absorption effects. (orig.)

  10. TORC1 regulates Pah1 phosphatidate phosphatase activity via the Nem1/Spo7 protein phosphatase complex.

    Directory of Open Access Journals (Sweden)

    Emmanuelle Dubots

    Full Text Available The evolutionarily conserved target of rapamycin complex 1 (TORC1 controls growth-related processes such as protein, nucleotide, and lipid metabolism in response to growth hormones, energy/ATP levels, and amino acids. Its deregulation is associated with cancer, type 2 diabetes, and obesity. Among other substrates, mammalian TORC1 directly phosphorylates and inhibits the phosphatidate phosphatase lipin-1, a central enzyme in lipid metabolism that provides diacylglycerol for the synthesis of membrane phospholipids and/or triacylglycerol as neutral lipid reserve. Here, we show that yeast TORC1 inhibits the function of the respective lipin, Pah1, to prevent the accumulation of triacylglycerol. Surprisingly, TORC1 regulates Pah1 in part indirectly by controlling the phosphorylation status of Nem1 within the Pah1-activating, heterodimeric Nem1-Spo7 protein phosphatase module. Our results delineate a hitherto unknown TORC1 effector branch that controls lipin function in yeast, which, given the recent discovery of Nem1-Spo7 orthologous proteins in humans, may be conserved.

  11. Molecular characterization of a phloem-specific gene encoding the filament protein, phloem protein 1 (PP1), from Cucurbita maxima.

    Science.gov (United States)

    Clark, A M; Jacobsen, K R; Bostwick, D E; Dannenhoffer, J M; Skaggs, M I; Thompson, G A

    1997-07-01

    Sieve elements in the phloem of most angiosperms contain proteinaceous filaments and aggregates called P-protein. In the genus Cucurbita, these filaments are composed of two major proteins: PP1, the phloem filament protein, and PP2, the phloem lactin. The gene encoding the phloem filament protein in pumpkin (Cucurbita maxima Duch.) has been isolated and characterized. Nucleotide sequence analysis of the reconstructed gene gPP1 revealed a continuous 2430 bp protein coding sequence, with no introns, encoding an 809 amino acid polypeptide. The deduced polypeptide had characteristics of PP1 and contained a 15 amino acid sequence determined by N-terminal peptide sequence analysis of PP1. The sequence of PP1 was highly repetitive with four 200 amino acid sequence domains containing structural motifs in common with cysteine proteinase inhibitors. Expression of the PP1 gene was detected in roots, hypocotyls, cotyledons, stems, and leaves of pumpkin plants. PP1 and its mRNA accumulated in pumpkin hypocotyls during the period of rapid hypocotyl elongation after which mRNA levels declined, while protein levels remained elevated. PP1 was immunolocalized in slime plugs and P-protein bodies in sieve elements of the phloem. Occasionally, PP1 was detected in companion cells. PP1 mRNA was localized by in situ hybridization in companion cells at early stages of vascular differentiation. The developmental accumulation and localization of PP1 and its mRNA paralleled the phloem lactin, further suggesting an interaction between these phloem-specific proteins.

  12. Enzyme kinetic characterization of protein tyrosine phosphatases

    DEFF Research Database (Denmark)

    Peters, Günther H.J.; Branner, S.; Møller, K. B.

    2003-01-01

    Protein tyrosine phosphatases (PTPs) play a central role in cellular signaling processes, resulting in an increased interest in modulating the activities of PTPs. We therefore decided to undertake a detailed enzyme kinetic evaluation of various transmembrane and cytosolic PTPs (PTPalpha, PTPbeta...

  13. Voltage sensitive phosphatases: emerging kinship to protein tyrosine phosphatases from structure-function research

    Directory of Open Access Journals (Sweden)

    Kirstin eHobiger

    2015-02-01

    Full Text Available The transmembrane protein Ci-VSP from the ascidian Ciona intestinalis was described as first member of a fascinating family of enzymes, the voltage sensitive phosphatases (VSPs. Ci-VSP and its voltage-activated homologs from other species are stimulated by positive membrane potentials and dephosphorylate the head groups of negatively charged phosphoinositide phosphates (PIPs. In doing so, VSPs act as control centers at the cytosolic membrane surface, because they intervene in signaling cascades that are mediated by PIP lipids. The characteristic motif CX5RT/S in the active site classifies VSPs as members of the huge family of cysteine-based protein tyrosine phosphatases (PTPs. Although PTPs have already been well characterized regarding both, structure and function, their relationship to VSPs has drawn only limited attention so far. Therefore, the intention of this review is to give a short overview about the extensive knowledge about PTPs in relation to the facts known about VSPs. Here, we concentrate on the structural features of the catalytic domain which are similar between both classes of phosphatases and their consequences for the enzymatic function. By discussing results obtained from crystal structures, molecular dynamics simulations, and mutagenesis studies, a possible mechanism for the catalytic cycle of VSPs is presented based on that one proposed for PTPs. In this way, we want to link the knowledge about the catalytic activity of VSPs and PTPs.

  14. Role of protein phosphatase 1 in dephosphorylation of Ebola virus VP30 protein and its targeting for the inhibition of viral transcription.

    Science.gov (United States)

    Ilinykh, Philipp A; Tigabu, Bersabeh; Ivanov, Andrey; Ammosova, Tatiana; Obukhov, Yuri; Garron, Tania; Kumari, Namita; Kovalskyy, Dmytro; Platonov, Maxim O; Naumchik, Vasiliy S; Freiberg, Alexander N; Nekhai, Sergei; Bukreyev, Alexander

    2014-08-15

    The filovirus Ebola (EBOV) causes the most severe hemorrhagic fever known. The EBOV RNA-dependent polymerase complex includes a filovirus-specific VP30, which is critical for the transcriptional but not replication activity of EBOV polymerase; to support transcription, VP30 must be in a dephosphorylated form. Here we show that EBOV VP30 is phosphorylated not only at the N-terminal serine clusters identified previously but also at the threonine residues at positions 143 and 146. We also show that host cell protein phosphatase 1 (PP1) controls VP30 dephosphorylation because expression of a PP1-binding peptide cdNIPP1 increased VP30 phosphorylation. Moreover, targeting PP1 mRNA by shRNA resulted in the overexpression of SIPP1, a cytoplasm-shuttling regulatory subunit of PP1, and increased EBOV transcription, suggesting that cytoplasmic accumulation of PP1 induces EBOV transcription. Furthermore, we developed a small molecule compound, 1E7-03, that targeted a non-catalytic site of PP1 and increased VP30 dephosphorylation. The compound inhibited the transcription but increased replication of the viral genome and completely suppressed replication of EBOV in cultured cells. Finally, mutations of Thr(143) and Thr(146) of VP30 significantly inhibited EBOV transcription and strongly induced VP30 phosphorylation in the N-terminal Ser residues 29-46, suggesting a novel mechanism of regulation of VP30 phosphorylation. Our findings suggest that targeting PP1 with small molecules is a feasible approach to achieve dysregulation of the EBOV polymerase activity. This novel approach may be used for the development of antivirals against EBOV and other filovirus species. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  15. [Exposure degree of important non-target arthropods to Cry2Aa in Bt rice fields].

    Science.gov (United States)

    Zhang, Qing-Ling; Li, Yun-He; Hua, Hong-Xia; Yang, Chang-Ju; Wu, Hong-Jin; Peng, Yu-Fa

    2013-06-01

    Based on the principle of "risk = hazard x exposure", the selected representative nontarget organisms in the assessment of the potential effects of insect-resistant genetically modified (GM) crops on non-target arthropods in laboratory are generally the arthropod species highly exposed to the insecticidal proteins expressed by the GM crops in farmland ecosystem. In order to understand the exposure degree of the important arthropod species to Cry proteins in Bt rice fields, and to select the appropriate non-target arthropods in the risk assessment of insect-resistant GM crops, the enzyme-linked immunosorbent assay (ELISA) was conducted to measure the Cry2Aa protein concentration in the arthropods collected from the cry2Aa rice fields at different rice growth stages. The results showed that there was a significant difference in the Cry2Aa content protein concentration in different arthropod species. Some species did not contain Cry2Aa protein, while some species contained larger amounts of Cry2Aa protein. Relative to the arthropods colleted after rice anthesis, the arthropods colleted in rice anthesis contained relative higher concentrations of Cry2Aa protein, especially for the predacious arthropods. No Cry proteins were detected in parasitic arthropods. This study provided references for the laboratory assessment of the effects of GM rice on nontarget arthropods.

  16. 3' Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP).

    Science.gov (United States)

    Kurokawa, Tatsuki; Takasuga, Shunsuke; Sakata, Souhei; Yamaguchi, Shinji; Horie, Shigeo; Homma, Koichi J; Sasaki, Takehiko; Okamura, Yasushi

    2012-06-19

    Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5' position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P(3)] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P(2)] upon voltage depolarization. However, it is unclear whether VSPs also have 3' phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P(3). TLC assay showed that the 3' phosphate of PI(3,4,5)P(3) was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P(2)] was removed by VSPs. Monitoring of PI(3,4)P(2) levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PH(TAPP1)-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5' phosphatase activity of VSP toward PI(3,4,5)P(3). However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P(3) is dephosphorylated at the 5' position, PI(3,4)P(2) is then dephosphorylated at the 3' position. These results suggest that substrate specificity of the VSP changes with membrane potential.

  17. The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

    Energy Technology Data Exchange (ETDEWEB)

    Kubota, Akira [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Bainy, Afonso C.D. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Departamento de Bioquímica, CCB, Universidade Federal de Santa Catarina, Florianopolis, SC 88040-900 (Brazil); Woodin, Bruce R.; Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543 (United States)

    2013-10-01

    The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

  18. The cytochrome P450 2AA gene cluster in zebrafish (Danio rerio): Expression of CYP2AA1 and CYP2AA2 and response to phenobarbital-type inducers

    International Nuclear Information System (INIS)

    Kubota, Akira; Bainy, Afonso C.D.; Woodin, Bruce R.; Goldstone, Jared V.; Stegeman, John J.

    2013-01-01

    The cytochrome P450 (CYP) 2 gene family is the largest and most diverse CYP gene family in vertebrates. In zebrafish, we have identified 10 genes in a new subfamily, CYP2AA, which does not show orthology to any human or other mammalian CYP genes. Here we report evolutionary and structural relationships of the 10 CYP2AA genes and expression of the first two genes, CYP2AA1 and CYP2AA2. Parsimony reconstruction of the tandem duplication pattern for the CYP2AA cluster suggests that CYP2AA1, CYP2AA2 and CYP2AA3 likely arose in the earlier duplication events and thus are most diverged in function from the other CYP2AAs. On the other hand, CYP2AA8 and CYP2AA9 are genes that arose in the latest duplication event, implying functional similarity between these two CYPs. A molecular model of CYP2AA1 showing the sequence conservation across the CYP2AA cluster reveals that the regions with the highest variability within the cluster map onto CYP2AA1 near the substrate access channels, suggesting differing substrate specificities. Zebrafish CYP2AA1 transcript was expressed predominantly in the intestine, while CYP2AA2 was most highly expressed in the kidney, suggesting differing roles in physiology. In the liver CYP2AA2 expression but not that of CYP2AA1, was increased by 1,4-bis [2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and, to a lesser extent, by phenobarbital (PB). In contrast, pregnenolone 16α-carbonitrile (PCN) increased CYP2AA1 expression, but not CYP2AA2 in the liver. The results identify a CYP2 subfamily in zebrafish that includes genes apparently induced by PB-type chemicals and PXR agonists, the first concrete in vivo evidence for a PB-type response in fish. - Highlights: • A tandemly duplicated cluster of ten CYP2AA genes was described in zebrafish. • Parsimony and duplication analyses suggest pathways to CYP2AA diversity. • Homology models reveal amino acid positions possibly related to functional diversity. • The CYP2AA locus does not share synteny with

  19. Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

    International Nuclear Information System (INIS)

    Rantanen, Mika K.; Lehtiö, Lari; Rajagopal, Lakshmi; Rubens, Craig E.; Goldman, Adrian

    2006-01-01

    Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail

  20. Crystallization and preliminary crystallographic analysis of two Streptococcus agalactiae proteins: the family II inorganic pyrophosphatase and the serine/threonine phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Rantanen, Mika K.; Lehtiö, Lari [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland); Rajagopal, Lakshmi; Rubens, Craig E. [Division of Infectious Disease, Children’s Hospital and Regional Medical Center, Seattle, Washington 98105 (United States); Goldman, Adrian, E-mail: adrian.goldman@helsinki.fi [Institute of Biotechnology, University of Helsinki, PO Box 65, FIN-00014, Helsinki (Finland)

    2006-09-01

    Two S. agalactiae proteins, the inorganic pyrophosphatase and the serine/threonine phosphatase, were crystallized and diffraction data were collected and processed from these crystals. The data from the two protein crystals extended to 2.80 and 2.65 Å, respectively. Streptococcus agalactiae, which infects human neonates and causes sepsis and meningitis, has recently been shown to possess a eukaryotic-like serine/threonine protein phosphorylation signalling cascade. Through their target proteins, the S. agalactiae Ser/Thr kinase and Ser/Thr phosphatase together control the growth as well as the morphology and virulence of this organism. One of the targets is the S. agalactiae family II inorganic pyrophosphatase. The inorganic pyrophosphatase and the serine/threonine phosphatase have therefore been purified and crystallized and diffraction data have been collected from their crystals. The data were processed using XDS. The inorganic pyrosphosphatase crystals diffracted to 2.80 Å and the Ser/Thr phosphatase crystals to 2.65 Å. Initial structure-solution experiments indicate that structure solution will be successful in both cases. Solving the structure of the proteins involved in this cascade is the first step towards understanding this phenomenon in atomic detail.

  1. Mechanism of protein tyrosine phosphatase 1B-mediated inhibition of leptin signalling

    DEFF Research Database (Denmark)

    Lund, I K; Hansen, J A; Andersen, H S

    2005-01-01

    Upon leptin binding, the leptin receptor is activated, leading to stimulation of the JAK/STAT signal transduction cascade. The transient character of the tyrosine phosphorylation of JAK2 and STAT3 suggests the involvement of protein tyrosine phosphatases (PTPs) as negative regulators...

  2. Serine/threonine phosphatase tapp2cs might be served as an early signal molecule for water stress in wheat

    International Nuclear Information System (INIS)

    Song, K. H.; Tian, W. L.; Hou, B. Z.; Guo, J. X.; Mei, X. R.; Li, Y. Z.

    2015-01-01

    Much progress has been made towards understanding the role of serine/threonine phosphatases type 2C (PP2Cs) in abscisic acid (ABA) signaling transduction. However, how the negative regulator, PP2Cs, responds to plant water loss remains unclear. Here, we used a series of relative soil moisture (RSM: 85 percentage (well watered), 65 percentage (moderate stress), 45 percentage (severe stress) potted winter wheat (Triticum aestivum L.) and the detached leaves to detect ABA levels and transcripts of PP2Cs, including PP2C40, PP2C45, PP2C59 and PP2C6 as well as the core downstream signals of ABA, including ABF, SnRK2.4 and SnRK2.5. The results showed that the continual loss of water led to a consistent increase in ABA levels, and that the mRNA expression levels of PP2Cs were dependent on plant water condition. PP2Cs expression could be induced by a slight loss of water, and inhibited under severe loss of water. These results were further confirmed by the transcripts of ABF, SnRK2.4 and SnRK2.5. Furthermore, in slight loss of water, 100 μM exogenous ABA could promote PP2Cs expression; in severe loss of water, it inhibited PP2Cs expression. In conclusion, ABA accumulation is controlled by water condition and the PP2C expression is dependent on plant water condition, suggesting that PP2Cs might be served as an early signal molecule for water stress in wheat. (author)

  3. Human liver phosphatase 2A: cDNA and amino acid sequence of two catalytic subunit isotypes

    International Nuclear Information System (INIS)

    Arino, J.; Woon, Chee Wai; Brautigan, D.L.; Miller, T.B. Jr.; Johnson, G.L.

    1988-01-01

    Two cDNA clones were isolated from a human liver library that encode two phosphatase 2A catalytic subunits. The two cDNAs differed in eight amino acids (97% identity) with three nonconservative substitutions. All of the amino acid substitutions were clustered in the amino-terminal domain of the protein. Amino acid sequence of one human liver clone (HL-14) was identical to the rabbit skeletal muscle phosphatase 2A cDNA (with 97% nucleotide identity). The second human liver clone (HL-1) is encoded by a separate gene, and RNA gel blot analysis indicates that both mRNAs are expressed similarly in several human clonal cell lines. Sequence comparison with phosphatase 1 and 2A indicates highly divergent amino acid sequences at the amino and carboxyl termini of the proteins and identifies six highly conserved regions between the two proteins that are predicted to be important for phosphatase enzymatic activity

  4. Detection of AA76, a Common Form of Amyloid A Protein, as a Way of Diagnosing AA Amyloidosis.

    Science.gov (United States)

    Sato, Junji; Okuda, Yasuaki; Kuroda, Takeshi; Yamada, Toshiyuki

    2016-01-01

    Reactive amyloid deposits consist of amyloid A (AA) proteins, the degradation products of serum amyloid A (SAA). Since the most common species of AA is the amino terminal portion produced by cleavage between residues 76 and 77 of SAA (AA76), the presence of AA76 in tissues could be a consequence of AA amyloid deposition. This study assessed the diagnostic significance of the detection of AA76 for AA amyloidosis using two different approaches. Biopsy specimens (n=130 from 54 subjects) from gastroduodenal mucosa or abdominal fat (n=9 from 9 subjects) of patients who had already been diagnosed with or were suspected of having AA amyloidosis were used. Fixed mucosal sections were subjected to immunohistochemistry using a newly developed antibody recognizing the carboxyl terminal end of AA76 (anti-AA76). The non-fixed materials from gastroduodenal mucosa or abdominal fat were subjected to immunoblotting for detection of the size of AA76. Among the gastroduodenal specimens (n=115) from already diagnosed patients, the positive rates of Congo red staining, immunohistochemistry using anti-AA76, and immunoblotting were 68.4%, 73.0%, and 92.2%, respectively. The anti-AA76 did not stain the supposed SAA in the blood or leakage, which was stained by anti-SAA antibody. AA76 was not detected either by immunohistochemistry or by immunoblot in the materials from patients in whom AA amyloidosis had been ruled out. In the abdominal fat, the immunoblot detected AA76 in 8 materials from 8 already diagnosed patients and did not in 1 patient whose gastroduodenal mucosa was negative. In conclusion, the detection of AA76 may alter the ability to diagnose AA amyloidosis. In immunohistochemistry for fixed specimens, the new anti-AA76 antibody can improve the specificity. Immunoblot for non-fixed materials, which can considerably improve the sensitivity, should be beneficial for small materials like abdominal fat. © 2016 by the Association of Clinical Scientists, Inc.

  5. Mitogen activated protein kinase phosphatase-1 prevents the development of tactile sensitivity in a rodent model of neuropathic pain

    Directory of Open Access Journals (Sweden)

    Ndong Christian

    2012-04-01

    Full Text Available Abstract Background Neuropathic pain due to nerve injury is one of the most difficult types of pain to treat. Following peripheral nerve injury, neuronal and glial plastic changes contribute to central sensitization and perpetuation of mechanical hypersensitivity in rodents. The mitogen activated protein kinase (MAPK family is pivotal in this spinal cord plasticity. MAPK phosphatases (MKPs limit inflammatory processes by dephosphorylating MAPKs. For example, MKP-1 preferentially dephosphorylates p-p38. Since spinal p-p38 is pivotal for the development of chronic hypersensitivity in rodent models of pain, and p-p38 inhibitors have shown clinical potential in acute and chronic pain patients, we hypothesize that induction of spinal MKP-1 will prevent the development of peripheral nerve-injury-induced hypersensitivity and p-p38 overexpression. Results We cloned rat spinal cord MKP-1 and optimize MKP-1 cDNA in vitro using transfections to BV-2 cells. We observed that in vitro overexpression of MKP-1 blocked lipopolysaccharide-induced phosphorylation of p38 (and other MAPKs as well as release of pro-algesic effectors (i.e., cytokines, chemokines, nitric oxide. Using this cDNA MKP-1 and a non-viral, in vivo nanoparticle transfection approach, we found that spinal cord overexpression of MKP-1 prevented development of peripheral nerve-injury-induced tactile hypersensitivity and reduced pro-inflammatory cytokines and chemokines and the phosphorylated form of p38. Conclusions Our results indicate that MKP-1, the natural regulator of p-p38, mediates resolution of the spinal cord pro-inflammatory milieu induced by peripheral nerve injury, resulting in prevention of chronic mechanical hypersensitivity. We propose that MKP-1 is a potential therapeutic target for pain treatment or prevention.

  6. Transcriptional regulation of SlPYL, SlPP2C, and SlSnRK2 gene families encoding ABA signal core components during tomato fruit development and drought stress.

    Science.gov (United States)

    Sun, Liang; Wang, Yan-Ping; Chen, Pei; Ren, Jie; Ji, Kai; Li, Qian; Li, Ping; Dai, Sheng-Jie; Leng, Ping

    2011-11-01

    In order to characterize the potential transcriptional regulation of core components of abscisic acid (ABA) signal transduction in tomato fruit development and drought stress, eight SlPYL (ABA receptor), seven SlPP2C (type 2C protein phosphatase), and eight SlSnRK2 (subfamily 2 of SNF1-related kinases) full-length cDNA sequences were isolated from the tomato nucleotide database of NCBI GenBank. All SlPYL, SlPP2C, and SlSnRK2 genes obtained are homologous to Arabidopsis AtPYL, AtPP2C, and AtSnRK2 genes, respectively. Based on phylogenetic analysis, SlPYLs and SlSnRK2s were clustered into three subfamilies/subclasses, and all SlPP2Cs belonged to PP2C group A. Within the SlPYL gene family, SlPYL1, SlPYL2, SlPYL3, and SlPYL6 were the major genes involved in the regulation of fruit development. Among them, SlPYL1 and SlPYL2 were expressed at high levels throughout the process of fruit development and ripening; SlPYL3 was strongly expressed at the immature green (IM) and mature green (MG) stages, while SlPYL6 was expressed strongly at the IM and red ripe (RR) stages. Within the SlPP2C gene family, the expression of SlPP2C, SlPP2C3, and SlPP2C4 increased after the MG stage; SlPP2C1 and SlPP2C5 peaked at the B3 stage, while SlPP2C2 and SlPP2C6 changed little during fruit development. Within the SlSnRK2 gene family, the expression of SlSnRK2.2, SlSnRK2.3, SlSnRK2.4, and SlSnRK2C was higher than that of other members during fruit development. Additionally, most SlPYL genes were down-regulated, while most SlPP2C and SlSnRK2 genes were up-regulated by dehydration in tomato leaf.

  7. Effects of Src Kinase Inhibition on Expression of Protein Tyrosine Phosphatase 1B after Brain Hypoxia in a Piglet Animal Model

    Directory of Open Access Journals (Sweden)

    Dimitrios Angelis

    2017-01-01

    Full Text Available Background. Protein tyrosine phosphatases (PTPs in conjunction with protein tyrosine kinases (PTKs regulate cellular processes by posttranslational modifications of signal transduction proteins. PTP nonreceptor type 1B (PTP-1B is an enzyme of the PTP family. We have previously shown that hypoxia induces an increase in activation of a class of nonreceptor PTK, the Src kinases. In the present study, we investigated the changes that occur in the expression of PTP-1B in the cytosolic component of the brain of newborn piglets acutely after hypoxia as well as long term for up to 2 weeks. Methods. Newborn piglets were divided into groups: normoxia, hypoxia, hypoxia followed by 1 day and 15 days in FiO2 0.21, and hypoxia pretreated with Src kinase inhibitor PP2, prior to hypoxia followed by 1 day and 15 days. Hypoxia was achieved by providing 7% FiO2 for 1 hour and PTP-1B expression was measured via immunoblotting. Results. PTP-1B increased posthypoxia by about 30% and persisted for 2 weeks while Src kinase inhibition attenuated the expected PTP-1B-increased expression. Conclusions. Our study suggests that Src kinase mediates a hypoxia-induced increased PTP-1B expression.

  8. Receptor protein tyrosine phosphatase alpha is essential for hippocampal neuronal migration and long-term potentiation

    DEFF Research Database (Denmark)

    Petrone, Angiola; Battaglia, Fortunato; Wang, Cheng

    2003-01-01

    Despite clear indications of their importance in lower organisms, the contributions of protein tyrosine phosphatases (PTPs) to development or function of the mammalian nervous system have been poorly explored. In vitro studies have indicated that receptor protein tyrosine phosphatase alpha...

  9. Structural basis for inhibition of the protein tyrosine phosphatase 1B by phosphotyrosine peptide mimetics

    NARCIS (Netherlands)

    Groves, M R; Yao, Z J; Roller, P P; Burke, T R; Barford, D

    1998-01-01

    Protein tyrosine phosphatases regulate diverse cellular processes and represent important targets for therapeutic intervention in a number of diseases. The crystal structures of protein tyrosine phosphatase 1B (PTP1B) in complex with small molecule inhibitors based upon two classes of

  10. Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo.

    Science.gov (United States)

    Jing, Yue; Wang, Gang; Xiao, Qi; Zhou, Yachun; Wei, Yingjie; Gong, Zhunan

    2018-01-01

    Angiogenesis plays a vital role in many physiological and pathological processes and several diseases are connected with its dysregulation. Asiatic acid (AA) has demonstrated anticancer properties and we suspect this might be attributable to an effect on angio-genesis. A modified derivative of AA, N-(2α,3β,23-acetoxyurs-12-en-28-oyl)-L-proline methyl ester (AA-PMe), has improved efficacy over its parent compound, but its effect on blood vessel development remains unclear. In this study, we investigated the antiangiogenic activity of AA and AA-PMe in zebrafish embryos and human umbilical vein endothelial cells (HUVECs). First of all, we treated HUVECs with increasing concentrations of AA-PMe or AA, with or without vascular endothelial growth factor (VEGF) present, and assessed cell viability, tube formation, and cell migration and invasion. Quantitative real-time polymerase chain reaction and Western blot analysis were later used to determine the role of vascular endothelial growth factor receptor 2 (VEGFR2)-mediated signaling in AA-PMe inhibition of angiogenesis. We extended these studies to follow angiogenesis using Tg(fli:EGFP) transgenic zebrafish embryos. For these experiments, embryos were treated with varying concentrations of AA-PMe or AA from 24 to 72 hours postfertilization prior to morphological observation, angiogenesis assessment, and endogenous alkaline phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined. We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling. For the first time

  11. cAMP response element binding protein (CREB activates transcription via two distinct genetic elements of the human glucose-6-phosphatase gene

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    Stefano Luisa

    2005-01-01

    Full Text Available Abstract Background The enzyme glucose-6-phosphatase catalyzes the dephosphorylation of glucose-6-phosphatase to glucose, the final step in the gluconeogenic and glycogenolytic pathways. Expression of the glucose-6-phosphatase gene is induced by glucocorticoids and elevated levels of intracellular cAMP. The effect of cAMP in regulating glucose-6-phosphatase gene transcription was corroborated by the identification of two genetic motifs CRE1 and CRE2 in the human and murine glucose-6-phosphatase gene promoter that resemble cAMP response elements (CRE. Results The cAMP response element is a point of convergence for many extracellular and intracellular signals, including cAMP, calcium, and neurotrophins. The major CRE binding protein CREB, a member of the basic region leucine zipper (bZIP family of transcription factors, requires phosphorylation to become a biologically active transcriptional activator. Since unphosphorylated CREB is transcriptionally silent simple overexpression studies cannot be performed to test the biological role of CRE-like sequences of the glucose-6-phosphatase gene. The use of a constitutively active CREB2/CREB fusion protein allowed us to uncouple the investigation of target genes of CREB from the variety of signaling pathways that lead to an activation of CREB. Here, we show that this constitutively active CREB2/CREB fusion protein strikingly enhanced reporter gene transcription mediated by either CRE1 or CRE2 derived from the glucose-6-phosphatase gene. Likewise, reporter gene transcription was enhanced following expression of the catalytic subunit of cAMP-dependent protein kinase (PKA in the nucleus of transfected cells. In contrast, activating transcription factor 2 (ATF2, known to compete with CREB for binding to the canonical CRE sequence 5'-TGACGTCA-3', did not transactivate reporter genes containing CRE1, CRE2, or both CREs derived from the glucose-6-phosphatase gene. Conclusions Using a constitutively active CREB2

  12. Emerging issues in receptor protein tyrosine phosphatase function: lifting fog or simply shifting?

    DEFF Research Database (Denmark)

    Petrone, A; Sap, J

    2000-01-01

    Transmembrane (receptor) tyrosine phosphatases are intimately involved in responses to cell-cell and cell-matrix contact. Several important issues regarding the targets and regulation of this protein family are now emerging. For example, these phosphatases exhibit complex interactions with signal...

  13. Study on expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice

    International Nuclear Information System (INIS)

    Huang Dingde; Chen Qi; Han Ling; Cai Jianming; Li Bailong; Huang Yuecheng; Gao Jianguo; Sun Suping

    2003-01-01

    Objective: To investigate the expression of SH2 domain containing-protein tyrosine phosphatase SHP-1 and SHP-2 in γ-ray irradiation-induced thymus lymphoma in mice. Methods: Altogether 338 BALB/c mice were randomly divided into irradiation groups and controls. Irradiation groups which were irradiated with γ-rays included canceration groups confirmed with histology and uncanceration groups. The controls were fed synchronistically with irradiation groups. The expression of SHP-1 and SHP-2 was detected with Western blot in thymus cells. Results: The expression of SHP-1 in canceration groups was much higher than that in uncanceration groups and controls significantly, while the expression of SHP-2 in canceration groups was higher than that in uncanceration groups and controls. When authors detected the expression of SHP-2 with Western blot, the authors found another protein with a molecular weight of 55x10 3 , which expression in canceration groups was higher than that in uncanceration groups and controls. Conclusion: The expression of SH2 domain-containing protein tyrosine phosphatase SHP-1 and SHP-2 is significantly increased in canceration groups, suggesting that SHP-1 and SHP-2 may be related with γ-ray induced thymus lymphoma in mice. Further research is expected on the relationship between development of cancer and SHP-1 and SHP-2

  14. Protein Phosphatase 1 Down Regulates ZYG-1 Levels to Limit Centriole Duplication.

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    Nina Peel

    2017-01-01

    Full Text Available In humans perturbations of centriole number are associated with tumorigenesis and microcephaly, therefore appropriate regulation of centriole duplication is critical. The C. elegans homolog of Plk4, ZYG-1, is required for centriole duplication, but our understanding of how ZYG-1 levels are regulated remains incomplete. We have identified the two PP1 orthologs, GSP-1 and GSP-2, and their regulators I-2SZY-2 and SDS-22 as key regulators of ZYG-1 protein levels. We find that down-regulation of PP1 activity either directly, or by mutation of szy-2 or sds-22 can rescue the loss of centriole duplication associated with a zyg-1 hypomorphic allele. Suppression is achieved through an increase in ZYG-1 levels, and our data indicate that PP1 normally regulates ZYG-1 through a post-translational mechanism. While moderate inhibition of PP1 activity can restore centriole duplication to a zyg-1 mutant, strong inhibition of PP1 in a wild-type background leads to centriole amplification via the production of more than one daughter centriole. Our results thus define a new pathway that limits the number of daughter centrioles produced each cycle.

  15. Memantine Attenuates Alzheimer's Disease-Like Pathology and Cognitive Impairment.

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    Xiaochuan Wang

    Full Text Available Deficiency of protein phosphatase-2A is a key event in Alzheimer's disease. An endogenous inhibitor of protein phosphatase-2A, inhibitor-1, I1PP2A, which inhibits the phosphatase activity by interacting with its catalytic subunit protein phosphatase-2Ac, is known to be upregulated in Alzheimer's disease brain. In the present study, we overexpressed I1PP2A by intracerebroventricular injection with adeno-associated virus vector-1-I1PP2A in Wistar rats. The I1PP2A rats showed a decrease in brain protein phosphatase-2A activity, abnormal hyperphosphorylation of tau, neurodegeneration, an increase in the level of activated glycogen synthase kinase-3beta, enhanced expression of intraneuronal amyloid-beta and spatial reference memory deficit; littermates treated identically but with vector only, i.e., adeno-associated virus vector-1-enhanced GFP, served as a control. Treatment with memantine, a noncompetitive NMDA receptor antagonist which is an approved drug for treatment of Alzheimer's disease, rescued protein phosphatase-2A activity by decreasing its demethylation at Leu309 selectively and attenuated Alzheimer's disease-like pathology and cognitive impairment in adeno-associated virus vector-1-I1PP2A rats. These findings provide new clues into the possible mechanism of the beneficial therapeutic effect of memantine in Alzheimer's disease patients.

  16. Interaction of Myosin Phosphatase Target Subunit (MYPT1) with Myosin Phosphatase-RhoA Interacting Protein (MRIP): A Role of Glutamic Acids in the Interaction.

    Science.gov (United States)

    Lee, Eunhee; Stafford, Walter F

    2015-01-01

    Scaffold proteins bind to and functionally link protein members of signaling pathways. Interaction of the scaffold proteins, myosin phosphatase target subunit (MYPT1) and myosin phosphatase-RhoA interacting protein (MRIP), causes co-localization of myosin phosphatase and RhoA to actomyosin. To examine biophysical properties of interaction of MYPT1 with MRIP, we employed analytical ultracentrifugation and surface plasmon resonance. In regard to MRIP, its residues 724-837 are sufficient for the MYPT1/MRIP interaction. Moreover, MRIP binds to MYPT1 as either a monomer or a dimer. With respect to MYPT1, its leucine repeat region, LR (residues 991-1030) is sufficient to account for the MYPT1/MRIP interaction. Furthermore, point mutations that replace glutamic acids 998-1000 within LR reduced the binding affinity toward MRIP. This suggests that the glutamic acids of MYPT1 play an important role in the interaction.

  17. Comparative studies on the carbohydrate, protein and acid phosphatase contents in seeds of some rye (Secale cereale varieties

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    B. Morawiecka

    2015-01-01

    Full Text Available The contents of sugars. proteins and acid phosphatase extracted with 0.1 M acetate buffer, pH 5.1, from some rye varieties were determined. The total sugar level amounted to 3.25-9.70 g per 100 g of seeds; the estimates for pentoses were 1.7-2.9 g and those for proteins 0.91-1.60 g per 100 g of seeds. Acid phosphatase showed and activity level between 0.18 and 1.26 units/mg protein. After disc electrophoresis proteins were separated into 10 to 11 bands: at pH 9.4 or into 4-7 bands at pH 3.8. Essential variety differences were expressed in protein patterns after electrophoresis at pH 3.8. Acid phosphatase was separated into 5 and 4 activity bands at pH 9.4 and 3.3,. respectively. No variations in zymogram patterns were observed in respect to variety differences or cultivation in various climate and soil conditions.

  18. ETS1 mediates MEK1/2-dependent overexpression of cancerous inhibitor of protein phosphatase 2A (CIP2A in human cancer cells.

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    Anchit Khanna

    2011-03-01

    Full Text Available EGFR-MEK-ERK signaling pathway has an established role in promoting malignant growth and disease progression in human cancers. Therefore identification of transcriptional targets mediating the oncogenic effects of the EGFR-MEK-ERK pathway would be highly relevant. Cancerous inhibitor of protein phosphatase 2A (CIP2A is a recently characterized human oncoprotein. CIP2A promotes malignant cell growth and is over expressed at high frequency (40-80% in most of the human cancer types. However, the mechanisms inducing its expression in cancer still remain largely unexplored. Here we present systematic analysis of contribution of potential gene regulatory mechanisms for high CIP2A expression in cancer. Our data shows that evolutionary conserved CpG islands at the proximal CIP2A promoter are not methylated both in normal and cancer cells. Furthermore, sequencing of the active CIP2A promoter region from altogether seven normal and malignant cell types did not reveal any sequence alterations that would increase CIP2A expression specifically in cancer cells. However, treatment of cancer cells with various signaling pathway inhibitors revealed that CIP2A mRNA expression was sensitive to inhibition of EGFR activity as well as inhibition or activation of MEK-ERK pathway. Moreover, MEK1/2-specific siRNAs decreased CIP2A protein expression. Series of CIP2A promoter-luciferase constructs were created to identify proximal -27 to -107 promoter region responsible for MEK-dependent stimulation of CIP2A expression. Additional mutagenesis and chromatin immunoprecipitation experiments revealed ETS1 as the transcription factor mediating stimulation of CIP2A expression through EGFR-MEK pathway. Thus, ETS1 is probably mediating high CIP2A expression in human cancers with increased EGFR-MEK1/2-ERK pathway activity. These results also suggest that in addition to its established role in invasion and angiogenesis, ETS1 may support malignant cellular growth via regulation of

  19. TGF-β1/Smad3 Pathway Targets PP2A-AMPK-FoxO1 Signaling to Regulate Hepatic Gluconeogenesis*

    Science.gov (United States)

    Yadav, Hariom; Devalaraja, Samir; Chung, Stephanie T.; Rane, Sushil G.

    2017-01-01

    Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. PMID:28069811

  20. Analysis of the humoral immune response to Chlamydia outer membrane protein 2

    DEFF Research Database (Denmark)

    Mygind, P; Christiansen, Gunna; Persson, K

    1998-01-01

    The humoral immune response to Chlamydia outer membrane protein 2 (Omp2) was studied. Omp2 is a highly genus-conserved structural protein of all Chlamydia species, containing a variable N-terminal fragment. To analyze where the immunogenic parts were localized, seven highly purified truncated...... fusion proteins constituting different regions of the protein were produced (Chlamydia pneumoniae-Omp2aa23-aa93, Chlamydia psittaci-Omp2aa23-aa94, and Chlamydia trachomatis-Omp2aa23-aa84, aa87-aa547, aa23-aa182, aa167-aa434, aa420-aa547). By an enzyme-linked immunosorbent assay with serologically defined...... patient sera, Omp2 was found to be a major immunogen of both C. pneumoniae and C. trachomatis infections (P species-specific anti-Omp2 immunoglobulins were detected....

  1. Low molecular weight protein tyrosine phosphatases control antibiotic production in Streptomyces coelicolor A3(2)

    DEFF Research Database (Denmark)

    Sohoni, Sujata Vijay; Lieder, Sarah; Bapat, Prashant Madhusudhan

    2014-01-01

    3700 was established usingpara-nitrophenyl phosphate and the tyrosine-phosphorylated protein PtkA from Bacillus subtilis as substrates. Theoptimum pH for the Sco3700 phosphatase activity was 6.8, and KM for pNPP was 14.3 mM compared to pH 6.0and KM0.75 mM for PtpA. The potential of Sco3700...... of ACT in the ptpA over expression strain. Furthermore, a significantly earlier onset of ACT productionwas observed when ptpA was over expressed. Sco3700 overexpression had a pleiotropic effect on the cell, and thestrain exhibited lower productivities and final concentrations of antibiotics. We conclude...... that Sco3700 is indeed atyrosine phosphatase, and it contributes to regulation of antibiotic production in S. coelicolor affecting the timing ofonset of the antibiotic production...

  2. The immunoglobulin-like domains 1 and 2 of the protein tyrosine phosphatase LAR adopt an unusual horseshoe-like conformation

    Science.gov (United States)

    Biersmith, Bridget H.; Hammel, Michal; Geisbrecht, Erika R.; Bouyain, Samuel

    2011-01-01

    Neurogenesis depends on exquisitely regulated interactions between macromolecules on the cell surface and in the extracellular matrix. In particular, interactions between proteoglycans and members of the type IIa subgroup of receptor protein tyrosine phosphatases underlie critical developmental processes such as the formation of synapses at the neuromuscular junction and the migration of axons to their appropriate targets. We report here the crystal structures of the first and second immunoglobulin-like domains of the Drosophila type IIa receptor Dlar and its mouse homologue LAR. These two domains adopt an unusual antiparallel arrangement that has not been previously observed in tandem repeats of immunoglobulin-like domains and that is presumably conserved in all type IIa receptor protein tyrosine phosphatases. PMID:21402080

  3. Tight regulation between cell survival and programmed cell death in GBM stem-like cells by EGFR/GSK3b/PP2A signaling.

    Science.gov (United States)

    Gürsel, Demirkan B; Banu, Matei A; Berry, Nicholas; Marongiu, Roberta; Burkhardt, Jan-Karl; Kobylarz, Keith; Kaplitt, Michael G; Rafii, Shahin; Boockvar, John A

    2015-01-01

    Malignant gliomas represent one of the most aggressive forms of cancer, displaying high mortality rates and limited treatment options. Specific subpopulations of cells residing in the tumor niche with stem-like characteristics have been postulated to initiate and maintain neoplasticity while resisting conventional therapies. The study presented here aims to define the role of glycogen synthase kinase 3 beta (GSK3b) in patient-derived glioblastoma (GBM) stem-like cell (GSC) proliferation, apoptosis and invasion. To evaluate the potential role of GSK3b in GBM, protein profiles from 68 GBM patients and 20 normal brain samples were analyzed for EGFR-mediated PI3kinase/Akt and GSK3b signaling molecules including protein phosphatase 2A (PP2A). To better understand the function of GSK3b in GBM, GSCs were isolated from GBM patient samples. Blocking GSK3b phosphorylation at Serine 9 attenuated cell proliferation while concomitantly stimulating apoptosis through activation of Caspase-3 in patient-derived GSCs. Increasing GSK3b protein content resulted in the inhibition of cell proliferation, colony formation and stimulated programmed cell death. Depleting GSK3b in GSCs down regulated PP2A. Furthermore, knocking down PP2A or blocking its activity by okadaic acid inactivated GSK3b by increasing GSK3b phosphorylation at Serine 9. Our data suggests that GSK3b may function as a regulator of apoptosis and tumorigenesis in GSCs. Therapeutic approaches targeting GSK3b in glioblastoma stem-like cells may be a useful addition to our current therapeutic armamentarium.

  4. A peptide export-import control circuit modulating bacterial development regulates protein phosphatases of the phosphorelay.

    Science.gov (United States)

    Perego, M

    1997-08-05

    The phosphorelay signal transduction system activates developmental transcription in sporulation of Bacillus subtilis by phosphorylation of aspartyl residues of the Spo0F and Spo0A response regulators. The phosphorylation level of these response regulators is determined by the opposing activities of protein kinases and protein aspartate phosphatases that interpret positive and negative signals for development in a signal integration circuit. The RapA protein aspartate phosphatase of the phosphorelay is regulated by a peptide that directly inhibits its activity. This peptide is proteolytically processed from an inactive pre-inhibitor protein encoded in the phrA gene. The pre-inhibitor is cleaved by the protein export apparatus to a putative pro-inhibitor that is further processed to the active inhibitor peptide and internalized by the oligopeptide permease. This export-import circuit is postulated to be a mechanism for timing phosphatase activity where the processing enzymes regulate the rate of formation of the active inhibitor. The processing events may, in turn, be controlled by a regulatory hierarchy. Chromosome sequencing has revealed several other phosphatase-prepeptide gene pairs in B. subtilis, suggesting that the use of this mechanism may be widespread in signal transduction.

  5. Functional Crosstalk between the PP2A and SUMO Pathways Revealed by Analysis of STUbL Suppressor, razor 1-1.

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    Minghua Nie

    2016-07-01

    Full Text Available Posttranslational modifications (PTMs provide dynamic regulation of the cellular proteome, which is critical for both normal cell growth and for orchestrating rapid responses to environmental stresses, e.g. genotoxins. Key PTMs include ubiquitin, the Small Ubiquitin-like MOdifier SUMO, and phosphorylation. Recently, SUMO-targeted ubiquitin ligases (STUbLs were found to integrate signaling through the SUMO and ubiquitin pathways. In general, STUbLs are recruited to target proteins decorated with poly-SUMO chains to ubiquitinate them and drive either their extraction from protein complexes, and/or their degradation at the proteasome. In fission yeast, reducing or preventing the formation of SUMO chains can circumvent the essential and DNA damage response functions of STUbL. This result indicates that whilst some STUbL "targets" have been identified, the crucial function of STUbL is to antagonize SUMO chain formation. Herein, by screening for additional STUbL suppressors, we reveal crosstalk between the serine/threonine phosphatase PP2A-Pab1B55 and the SUMO pathway. A hypomorphic Pab1B55 mutant not only suppresses STUbL dysfunction, but also mitigates the phenotypes associated with deletion of the SUMO protease Ulp2, or mutation of the STUbL cofactor Rad60. Together, our results reveal a novel role for PP2A-Pab1B55 in modulating SUMO pathway output, acting in parallel to known critical regulators of SUMOylation homeostasis. Given the broad evolutionary functional conservation of the PP2A and SUMO pathways, our results could be relevant to the ongoing attempts to therapeutically target these factors.

  6. Role of tyrosine phosphatase inhibitors in cancer treatment with emphasis on SH2 domain-containing tyrosine phosphatases (SHPs)

    NARCIS (Netherlands)

    Irandoust, Mahban; van den Berg, Timo K.; Kaspers, Gertjan J. L.; Cloos, Jacqueline

    2009-01-01

    Protein tyrosine phosphorylation is one of the key mechanisms involved in signal transduction pathways. This modification is regulated by concerted action of protein tyrosine phosphatases and protein tyrosine kinases. Deregulation of either of these key regulators lead to abnormal cellular

  7. Therapeutic relevance of the PP2A-B55 inhibitory kinase MASTL/Greatwall in breast cancer.

    Science.gov (United States)

    Álvarez-Fernández, Mónica; Sanz-Flores, María; Sanz-Castillo, Belén; Salazar-Roa, María; Partida, David; Zapatero-Solana, Elisabet; Ali, H Raza; Manchado, Eusebio; Lowe, Scott; VanArsdale, Todd; Shields, David; Caldas, Carlos; Quintela-Fandino, Miguel; Malumbres, Marcos

    2017-12-11

    PP2A is a major tumor suppressor whose inactivation is frequently found in a wide spectrum of human tumors. In particular, deletion or epigenetic silencing of genes encoding the B55 family of PP2A regulatory subunits is a common feature of breast cancer cells. A key player in the regulation of PP2A/B55 phosphatase complexes is the cell cycle kinase MASTL (also known as Greatwall). During cell division, inhibition of PP2A-B55 by MASTL is required to maintain the mitotic state, whereas inactivation of MASTL and PP2A reactivation is required for mitotic exit. Despite its critical role in cell cycle progression in multiple organisms, its relevance as a therapeutic target in human cancer and its dependence of PP2A activity is mostly unknown. Here we show that MASTL overexpression predicts poor survival and shows prognostic value in breast cancer patients. MASTL knockdown or knockout using RNA interference or CRISPR/Cas9 systems impairs proliferation of a subset of breast cancer cells. The proliferative function of MASTL in these tumor cells requires its kinase activity and the presence of PP2A-B55 complexes. By using a new inducible CRISPR/Cas9 system in breast cancer cells, we show that genetic ablation of MASTL displays a significant therapeutic effect in vivo. All together, these data suggest that the PP2A inhibitory kinase MASTL may have both prognostic and therapeutic value in human breast cancer.

  8. Requirement for tyrosine phosphatase during serotonergic neuromodulation by protein kinase C.

    Science.gov (United States)

    Catarsi, S; Drapeau, P

    1997-08-01

    Tyrosine kinases and phosphatases are abundant in the nervous system, where they signal cellular differentiation, mediate the responses to growth factors, and direct neurite outgrowth during development. Tyrosine phosphorylation can also alter ion channel activity, but its physiological significance remains unclear. In an identified leech mechanosensory neuron, the ubiquitous neuromodulator serotonin increases the activity of a cation channel by activating protein kinase C (PKC), resulting in membrane depolarization and modulation of the receptive field properties. We observed that the effects on isolated neurons and channels were blocked by inhibiting tyrosine phosphatases. Serotonergic stimulation of PKC thus activates a tyrosine phosphatase activity associated with the channels, which reverses their constitutive inhibition by tyrosine phosphorylation, representing a novel form of neuromodulation.

  9. Ca2+-regulatory proteins in cardiomyocytes from the right ventricle in children with congenital heart disease

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    Wu Yihe

    2012-04-01

    Full Text Available Abstract Background Hypoxia and hypertrophy are the most frequent pathophysiological consequence of congenital heart disease (CHD which can induce the alteration of Ca2+-regulatory proteins and inhibit cardiac contractility. Few studies have been performed to examine Ca2+-regulatory proteins in human cardiomyocytes from the hypertrophic right ventricle with or without hypoxia. Methods Right ventricle tissues were collected from children with tetralogy of Fallot [n = 25, hypoxia and hypertrophy group (HH group], pulmonary stenosis [n = 25, hypertrophy group (H group], or small isolated ventricular septal defect [n = 25, control group (C group] during open-heart surgery. Paraffin sections of tissues were stained with 3,3′-dioctadecyloxacarbocyanine perchlorate to measure cardiomyocyte size. Expression levels of Ca2+-regulatory proteins [sarcoplasmic reticulum Ca2+-ATPase (SERCA2a, ryanodine receptor (RyR2, sodiumcalcium exchanger (NCX, sarcolipin (SLN and phospholamban (PLN] were analysed by means of real-time PCR, western blot, or immunofluorescence. Additionally, phosphorylation level of RyR and PLN and activity of protein phosphatase (PP1 were evaluated using western blot. Results Mild cardiomyocyte hypertrophy of the right ventricle in H and HH groups was confirmed by comparing cardiomyocyte size. A significant reduction of SERCA2a in mRNA (P16-phosphorylated PLN was down-regulated (PP Conclusions The decreased SERCA2a mRNA may be a biomarker of the pathological process in the early stage of cyanotic CHD with the hypertrophic right ventricle. A combination of hypoxia and hypertrophy can induce the adverse effect of PLN-Ser16 dephosphorylation. Increased PP1 could result in the decreased PLN-Ser16 and inhibition of PP1 is a potential therapeutic target for heart dysfunction in pediatrics.

  10. The structure of the protein phosphatase 2A PR65/A subunit reveals the conformation of its 15 tandemly repeated HEAT motifs

    NARCIS (Netherlands)

    Groves, M R; Hanlon, N; Turowski, P; Hemmings, B A; Barford, D

    1999-01-01

    The PR65/A subunit of protein phosphatase 2A serves as a scaffolding molecule to coordinate the assembly of the catalytic subunit and a variable regulatory B subunit, generating functionally diverse heterotrimers. Mutations of the beta isoform of PR65 are associated with lung and colon tumors. The

  11. Effects of SOV-induced phosphatase inhibition and expression of protein tyrosine phosphatases in rat corneal endothelial cells.

    Science.gov (United States)

    Chen, Wei-Li; Harris, Deshea L; Joyce, Nancy C

    2005-11-01

    Contact inhibition is an important mechanism for maintaining corneal endothelium in a non-replicative state. Protein tyrosine phosphatases (PTPs) play a role in regulating the integrity of cell-cell contacts, differentiation, and growth. In this study, we aimed to evaluate whether phosphatases are involved in the maintenance of contact-dependent inhibition of proliferation in corneal endothelial cells and to identify candidate PTPs that are expressed in these cells and might be involved in regulation of contact inhibition. Confluent cultures of rat corneal endothelial cells or endothelium in ex vivo corneas were treated with the general phosphatase inhibitor, sodium orthovanadate (SOV). Immunocytochemistry (ICC) evaluated the effect of SOV on cell-cell contacts by staining for ZO-1, and on cell cycle progression by staining for Ki67. Transverse sections of rat cornea and cultured rat corneal endothelial cells were used to test for expression of the candidate PTPs: PTP-mu, PTP-LAR, PTP1B, SHP-1, SHP-2, and PTEN using ICC and either Western blots or RT-PCR. ZO-1 staining demonstrated that SOV induced a time-dependent release of cell-cell contacts in confluent cultures of corneal endothelial cells and in the endothelium of ex vivo corneas. Staining for Ki67 indicated that SOV promoted limited cell cycle progression in the absence of serum. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN, but not PTP-LAR, were expressed in rat corneal endothelial cells in situ and in culture. The subcellular location of PTP-mu and PTP1B differed in subconfluent and confluent cells, while that of SHP-1, SHP-2, and PTEN was similar, regardless of confluent status. Western blots confirmed the expression of PTP1B, SHP-1, SHP-2, and PTEN. RT-PCR confirmed expression of PTP-mu mRNA. Phosphatases are involved in regulation of junctional integrity and of cell proliferation in corneal endothelial cells. PTP-mu, PTP1B, SHP-1, SHP-2, and PTEN are expressed in rat corneal endothelium and may be involved in

  12. Adaptor protein GRB2 promotes Src tyrosine kinase activation and podosomal organization by protein-tyrosine phosphatase ϵ in osteoclasts.

    Science.gov (United States)

    Levy-Apter, Einat; Finkelshtein, Eynat; Vemulapalli, Vidyasiri; Li, Shawn S-C; Bedford, Mark T; Elson, Ari

    2014-12-26

    The non-receptor isoform of protein-tyrosine phosphatase ϵ (cyt-PTPe) supports adhesion of bone-resorbing osteoclasts by activating Src downstream of integrins. Loss of cyt-PTPe reduces Src activity in osteoclasts, reduces resorption of mineralized matrix both in vivo and in cell culture, and induces mild osteopetrosis in young female PTPe KO mice. Activation of Src by cyt-PTPe is dependent upon this phosphatase undergoing phosphorylation at its C-terminal Tyr-638 by partially active Src. To understand how cyt-PTPe activates Src, we screened 73 Src homology 2 (SH2) domains for binding to Tyr(P)-638 of cyt-PTPe. The SH2 domain of GRB2 bound Tyr(P)-638 of cyt-PTPe most prominently, whereas the Src SH2 domain did not bind at all, suggesting that GRB2 may link PTPe with downstream molecules. Further studies indicated that GRB2 is required for activation of Src by cyt-PTPe in osteoclast-like cells (OCLs) in culture. Overexpression of GRB2 in OCLs increased activating phosphorylation of Src at Tyr-416 and of cyt-PTPe at Tyr-638; opposite results were obtained when GRB2 expression was reduced by shRNA or by gene inactivation. Phosphorylation of cyt-PTPe at Tyr-683 and its association with GRB2 are integrin-driven processes in OCLs, and cyt-PTPe undergoes autodephosphorylation at Tyr-683, thus limiting Src activation by integrins. Reduced GRB2 expression also reduced the ability of bone marrow precursors to differentiate into OCLs and reduced the fraction of OCLs in which podosomal adhesion structures assume organization typical of active, resorbing cells. We conclude that GRB2 physically links cyt-PTPe with Src and enables cyt-PTPe to activate Src downstream of activated integrins in OCLs. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway

    Science.gov (United States)

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-01-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. PMID:25399018

  14. Voltage-sensing phosphatase: its molecular relationship with PTEN.

    Science.gov (United States)

    Okamura, Yasushi; Dixon, Jack E

    2011-02-01

    Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

  15. Pterocarpans with inhibitory effects on protein tyrosine phosphatase 1B from Erythrina lysistemon Hutch

    DEFF Research Database (Denmark)

    Dao, Trong Tuan; Nguyen, Phi Hung; Thuong, Phuong Thien

    2009-01-01

    ',5':3,4]-2'',2''-dimethyldihydropyrano[6'',5'':9,10]pterocarpan (1), furano[5',4':3,4]-9-hydroxy-10-prenylpterocarpan (2), and 8-formyl-3,9-dihydroxy-4,10-diprenylpterocarpan (3), based on spectroscopic analyses. All the isolates, with the exception of 3, 6, and 11, strongly inhibited protein tyrosine phosphatase 1B (PTP1B) activity...

  16. The interaction between AMPKβ2 and the PP1-targeting subunit R6 is dynamically regulated by intracellular glycogen content.

    Science.gov (United States)

    Oligschlaeger, Yvonne; Miglianico, Marie; Dahlmans, Vivian; Rubio-Villena, Carla; Chanda, Dipanjan; Garcia-Gimeno, Maria Adelaida; Coumans, Will A; Liu, Yilin; Voncken, J Willem; Luiken, Joost J F P; Glatz, Jan F C; Sanz, Pascual; Neumann, Dietbert

    2016-04-01

    AMP-activated protein kinase (AMPK) is a metabolic stress-sensing kinase. We previously showed that glucose deprivation induces autophosphorylation of AMPKβ at Thr-148, which prevents the binding of AMPK to glycogen. Furthermore, in MIN6 cells, AMPKβ1 binds to R6 (PPP1R3D), a glycogen-targeting subunit of protein phosphatase type 1 (PP1), thereby regulating the glucose-induced inactivation of AMPK. In the present study, we further investigated the interaction of R6 with AMPKβ and the possible dependency on Thr-148 phosphorylation status. Yeast two-hybrid (Y2H) analyses and co-immunoprecipitation (IP) of the overexpressed proteins in human embryonic kidney (HEK) 293T) cells revealed that both AMPKβ1 and AMPK-β2 wild-type (WT) isoforms bind to R6. The AMPKβ-R6 interaction was stronger with the muscle-specific AMPKβ2-WT and required association with the substrate-binding motif of R6. When HEK293T cells or C2C12 myotubes were cultured in high-glucose medium, AMPKβ2-WT and R6 weakly interacted. In contrast, glycogen depletion significantly enhanced this protein interaction. Mutation of AMPKβ2 Thr-148 prevented the interaction with R6 irrespective of the intracellular glycogen content. Treatment with the AMPK activator oligomycin enhanced the AMPKβ2-R6 interaction in conjunction with increased Thr-148 phosphorylation in cells grown in low-glucose medium. These data are in accordance with R6 binding directly to AMPKβ2 when both proteins detach from the diminishing glycogen particle, which is simultaneous with increased AMPKβ2 Thr-148 autophosphorylation. Such a model points to a possible control of AMPK by PP1-R6 upon glycogen depletion in muscle. © 2016 Authors; published by Portland Press Limited.

  17. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae

    Directory of Open Access Journals (Sweden)

    Sun Weiwei

    2011-01-01

    Full Text Available Abstract Background Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2, one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. Results The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. Conclusions The repression in phloem-feeding activities of

  18. Harpin-induced expression and transgenic overexpression of the phloem protein gene AtPP2-A1 in Arabidopsis repress phloem feeding of the green peach aphid Myzus persicae.

    Science.gov (United States)

    Zhang, Chunling; Shi, Haojie; Chen, Lei; Wang, Xiaomeng; Lü, Beibei; Zhang, Shuping; Liang, Yuan; Liu, Ruoxue; Qian, Jun; Sun, Weiwei; You, Zhenzhen; Dong, Hansong

    2011-01-13

    Treatment of plants with HrpNEa, a protein of harpin group produced by Gram-negative plant pathogenic bacteria, induces plant resistance to insect herbivores, including the green peach aphid Myzus persicae, a generalist phloem-feeding insect. Under attacks by phloem-feeding insects, plants defend themselves using the phloem-based defense mechanism, which is supposed to involve the phloem protein 2 (PP2), one of the most abundant proteins in the phloem sap. The purpose of this study was to obtain genetic evidence for the function of the Arabidopsis thaliana (Arabidopsis) PP2-encoding gene AtPP2-A1 in resistance to M. persicae when the plant was treated with HrpNEa and after the plant was transformed with AtPP2-A1. The electrical penetration graph technique was used to visualize the phloem-feeding activities of apterous agamic M. persicae females on leaves of Arabidopsis plants treated with HrpNEa and an inactive protein control, respectively. A repression of phloem feeding was induced by HrpNEa in wild-type (WT) Arabidopsis but not in atpp2-a1/E/142, the plant mutant that had a defect in the AtPP2-A1 gene, the most HrpNEa-responsive of 30 AtPP2 genes. In WT rather than atpp2-a1/E/142, the deterrent effect of HrpNEa treatment on the phloem-feeding activity accompanied an enhancement of AtPP2-A1 expression. In PP2OETAt (AtPP2-A1-overexpression transgenic Arabidopsis thaliana) plants, abundant amounts of the AtPP2-A1 gene transcript were detected in different organs, including leaves, stems, calyces, and petals. All these organs had a deterrent effect on the phloem-feeding activity compared with the same organs of the transgenic control plant. When a large-scale aphid population was monitored for 24 hours, there was a significant decrease in the number of aphids that colonized leaves of HrpNEa-treated WT and PP2OETAt plants, respectively, compared with control plants. The repression in phloem-feeding activities of M. persicae as a result of AtPP2-A1 overexpression, and

  19. TGF-β1/Smad3 Pathway Targets PP2A-AMPK-FoxO1 Signaling to Regulate Hepatic Gluconeogenesis.

    Science.gov (United States)

    Yadav, Hariom; Devalaraja, Samir; Chung, Stephanie T; Rane, Sushil G

    2017-02-24

    Maintenance of glucose homeostasis is essential for normal physiology. Deviation from normal glucose levels, in either direction, increases susceptibility to serious medical complications such as hypoglycemia and diabetes. Maintenance of glucose homeostasis is achieved via functional interactions among various organs: liver, skeletal muscle, adipose tissue, brain, and the endocrine pancreas. The liver is the primary site of endogenous glucose production, especially during states of prolonged fasting. However, enhanced gluconeogenesis is also a signature feature of type 2 diabetes (T2D). Thus, elucidating the signaling pathways that regulate hepatic gluconeogenesis would allow better insight into the process of normal endogenous glucose production as well as how this process is impaired in T2D. Here we demonstrate that the TGF-β1/Smad3 signaling pathway promotes hepatic gluconeogenesis, both upon prolonged fasting and during T2D. In contrast, genetic and pharmacological inhibition of TGF-β1/Smad3 signals suppressed endogenous glucose production. TGF-β1 and Smad3 signals achieved this effect via the targeting of key regulators of hepatic gluconeogenesis, protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK), and FoxO1 proteins. Specifically, TGF-β1 signaling suppressed the LKB1-AMPK axis, thereby facilitating the nuclear translocation of FoxO1 and activation of key gluconeogenic genes, glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. These findings underscore an important role of TGF-β1/Smad3 signaling in hepatic gluconeogenesis, both in normal physiology and in the pathophysiology of metabolic diseases such as diabetes, and are thus of significant medical relevance. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  20. Mitogen-Activated Protein Kinase Phosphatase-2 Deletion Impairs Synaptic Plasticity and Hippocampal-Dependent Memory.

    Science.gov (United States)

    Abdul Rahman, Nor Zaihana; Greenwood, Sam M; Brett, Ros R; Tossell, Kyoko; Ungless, Mark A; Plevin, Robin; Bushell, Trevor J

    2016-02-24

    Mitogen-activated protein kinases (MAPKs) regulate brain function and their dysfunction is implicated in a number of brain disorders, including Alzheimer's disease. Thus, there is great interest in understanding the signaling systems that control MAPK function. One family of proteins that contribute to this process, the mitogen-activated protein kinase phosphatases (MKPs), directly inactivate MAPKs through dephosphorylation. Recent studies have identified novel functions of MKPs in development, the immune system, and cancer. However, a significant gap in our knowledge remains in relation to their role in brain functioning. Here, using transgenic mice where the Dusp4 gene encoding MKP-2 has been knocked out (MKP-2(-/-) mice), we show that long-term potentiation is impaired in MKP-2(-/-) mice compared with MKP-2(+/+) controls whereas neuronal excitability, evoked synaptic transmission, and paired-pulse facilitation remain unaltered. Furthermore, spontaneous EPSC (sEPSC) frequency was increased in acute slices and primary hippocampal cultures prepared from MKP-2(-/-) mice with no effect on EPSC amplitude observed. An increase in synapse number was evident in primary hippocampal cultures, which may account for the increase in sEPSC frequency. In addition, no change in ERK activity was detected in both brain tissue and primary hippocampal cultures, suggesting that the effects of MKP-2 deletion were MAPK independent. Consistent with these alterations in hippocampal function, MKP-2(-/-) mice show deficits in spatial reference and working memory when investigated using the Morris water maze. These data show that MKP-2 plays a role in regulating hippocampal function and that this effect may be independent of MAPK signaling. Copyright © 2016 Abdul Rahman et al.

  1. Genotype-Phenotype Associations of the CD-Associated Single Nucleotide Polymorphism within the Gene Locus Encoding Protein Tyrosine Phosphatase Non-Receptor Type 22 in Patients of the Swiss IBD Cohort.

    Directory of Open Access Journals (Sweden)

    Marianne R Spalinger

    Full Text Available Protein tyrosine phosphatase non-receptor type 22 (PTPN22 plays an important role in immune cell function and intestinal homeostasis. The single nucleotide polymorphism (SNP rs2476601 within the PTPN22 gene locus results in aberrant function of PTPN22 protein and protects from Crohn's disease (CD. Here, we investigated associations of PTPN22 SNP rs2476601 in inflammatory bowel disease (IBD patients in the Swiss IBD Cohort Study (SIBDCS.2'028 SIBDCS patients (1173 CD and 855 ulcerative colitis (UC patients were included. The clinical characteristics were analysed for an association with the presence of the PTPN22 SNP rs2476601 genotypes 'homozygous variant' (AA, 'heterozygous' (GA and 'homozygous wild-type' (GG.13 patients (0.6% were homozygous variant (AA for the PTPN22 polymorphism, 269 (13.3% heterozygous variant (GA and 1'746 (86.1% homozygous wild-type (GG. In CD, AA and GA genotypes were associated with less use of steroids and antibiotics, and reduced prevalence of vitamin D and calcium deficiency. In UC the AA and GA genotype was associated with increased use of azathioprine and anti-TNF antibodies, but significantly less patients with the PTPN22 variant featured malabsorption syndrome (p = 0.026.Our study for the first time addressed how presence of SNP rs2476601 within the PTPN22 gene affects clinical characteristics in IBD-patients. Several factors that correlate with more severe disease were found to be less common in CD patients carrying the A-allele, pointing towards a protective role for this variant in affected CD patients. In UC patients however, we found the opposite trend, suggesting a disease-promoting effect of the A-allele.

  2. Phosphorylation of inhibitor-2 and activation of MgATP-dependent protein phosphatase by rat skeletal muscle glycogen synthase kinase

    International Nuclear Information System (INIS)

    Hegazy, M.G.; Reimann, E.M.; Thysseril, T.J.; Schlender, K.K.

    1986-01-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca 2+ or cAMP. This kinase has an apparent Mr of 62,000 and uses ATP but not GTP as a phosphoryl donor. GSK-M phosphorylated glycogen synthase at sites 2 and 3. It phosphorylated ATP-citrate lyase and activated MgATP-dependent phosphatase in the presence of ATP but not GTP. As expected, the kinase also phosphorylated phosphatase inhibitor 2 (I-2). Phosphatase incorporation reached approximately 0.3 mol/mol of I-2. Phosphopeptide maps were obtained by digesting 32 P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32 P-labeled peaks were obtained when I-2 was phosphorylated with either GSK-M or glycogen synthase kinase 3 (GSK-3) and these peptides were different from those obtained when I-2 was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase (CSU) or casein kinase II (CK-II). When I-2 was phosphorylated with GSK-M or GSK-3 and cleaved by CNBr, a single radioactive peak was obtained. Phosphoamino acid analysis showed that I-2 was phosphorylated by GSK-M or GSK-3 predominately in Thr whereas CSU and CK-II phosphorylated I-2 exclusively in Ser. These results indicate that GSK-M is similar to GSK-3 and to ATP-citrate lyase kinase. However, it appears to differ in Mr from ATP-citrate lyase kinase and it differs from GSK-3 in that it phosphorylates glycogen synthase at site 2 and it does not use GTP as a phosphoryl donor

  3. Pion interferometry and resonances in pp and AA collisions

    Energy Technology Data Exchange (ETDEWEB)

    Padula, S.S. (UNESP, Inst. de Fisica Teorica, Sao Paulo (Brazil)); Gyulassy, M. (Lawrence Berkeley Lab., Nuclear Science Div., CA (United States))

    1992-07-20

    We study the sensitivity of pion interferometry in pp and anti pp collisions at ISR energies to the resonance abundance. We show that those data are not compatible with the full resonance fractions predicted by the Lund model. The preliminary S+S and O+Au data at 200 A GeV are, however, not incompatible with the Lund predictions, although their sensitivity to resonances is significantly weaker than in the pp/anti pp case. (orig.).

  4. Pion interferometry and resonances in pp and AA collisions

    International Nuclear Information System (INIS)

    Padula, S.S.; Gyulassy, M.

    1992-01-01

    We study the sensitivity of pion interferometry in pp and anti pp collisions at ISR energies to the resonance abundance. We show that those data are not compatible with the full resonance fractions predicted by the Lund model. The preliminary S+S and O+Au data at 200 A GeV are, however, not incompatible with the Lund predictions, although their sensitivity to resonances is significantly weaker than in the pp/anti pp case. (orig.)

  5. Pion interferometry and resonances in pp and AA collisions

    International Nuclear Information System (INIS)

    Padula, S.S.

    1991-01-01

    We study the sensitivity of pion interferometry in bar pp and bar pp collisions at ISR energies to the resonance abundance. We show that those data are not compatible with the full resonance fractions predicted by the Lund model. The preliminary S+S and O+Au data at 200 GeV are, however, not incompatible with the Lund predictions, although their sensitivity to resonances is significantly weaker than in the pp/bar pp case

  6. Avicin D: a protein reactive plant isoprenoid dephosphorylates Stat 3 by regulating both kinase and phosphatase activities.

    Directory of Open Access Journals (Sweden)

    Valsala Haridas

    Full Text Available Avicins, a class of electrophilic triterpenoids with pro-apoptotic, anti-inflammatory and antioxidant properties, have been shown to induce redox-dependant post-translational modification of cysteine residues to regulate protein function. Based on (a the cross-talk that occurs between redox and phosphorylation processes, and (b the role of Stat3 in the process of apoptosis and carcinogenesis, we chose to study the effects of avicins on the processes of phosphorylation/dephosphorylation in Stat3. Avicins dephosphorylate Stat3 in a variety of human tumor cell lines, leading to a decrease in the transcriptional activity of Stat3. The expression of Stat3-regulated proteins such as c-myc, cyclin D1, Bcl2, survivin and VEGF were reduced in response to avicin treatment. Underlying avicin-induced dephosphorylation of Stat3 was dephosphorylation of JAKs, as well as activation of protein phosphatase-1. Downregulation of both Stat3 activity and expression of Stat 3-controlled pro-survival proteins, contributes to the induction of apoptosis in avicin treated tumor cells. Based on the role of Stat3 in inflammation and wounding, and the in vivo inhibition of VEGF by avicins in a mouse skin carcinogenesis model, it is likely that avicin-induced inhibition of Stat3 activity results in the suppression of the pro-inflammatory and pro-oxidant stromal environment of tumors. Activation of PP-1, which also acts as a cellular economizer, combined with the redox regulation by avicins, can aid in redirecting metabolism from growth promoting anabolic to energy sparing pathways.

  7. Folic Acid Reduces Tau Phosphorylation by Regulating PP2A Methylation in Streptozotocin-Induced Diabetic Mice

    Science.gov (United States)

    Zheng, Miaoyan; Zou, Chen; Li, Mengyue; Huang, Guowei; Gao, Yuxia; Liu, Huan

    2017-01-01

    High incidence rate of Alzheimer’s disease (AD) is observed in patients with type 2 diabetes. Aggregated β-amyloid (Aβ) and hyperphosphorylated tau are the hallmarks of AD. Hyperphosphorylated tau has been detected in diabetic animals as well as in diabetic patients. Folates mediate the transfer of one carbon unit, required in various biochemical reactions. The effect of folate on tau phosphorylation in diabetic models still remains unknown. In this study, we investigated the effect and mechanism of folic acid on hyperphosphorylation of tau in streptozotocin (STZ)-induced diabetic mice. Diabetic mice induced by STZ, at the age of 10 weeks, were administered with three levels of folic acid: folic acid-deficient diet, diet with normal folic acid content, and 120 μg/kg folic acid diet for 8 weeks. Levels of serum folate and blood glucose were monitored. Tau phosphorylation, protein phosphatase 2A (PP2A) methylation, and Glycogen synthase kinase 3β (GSK-3β) phosphorylation were detected using Western blot. The S-adenosyl methionine:S-adenosyl homocysteine ratio (SAM:SAH) in brain tissues was also determined. DNA methyltransferase (DNMT) mRNA expression levels were detected using real-time PCR. Folic acid reduced tau hyperphosphorylation at Ser396 in the brain of diabetes mellitus (DM) mice. In addition, PP2A methylation and DNMT1 mRNA expression were significantly increased in DM mice post folic acid treatment. GSK-3β phosphorylation was not regulated by folic acid administration. Folic acid can reduce tau phosphorylation by regulating PP2A methylation in diabetic mice. These results support that folic acid can serve as a multitarget neuronal therapeutic agent for treating diabetes-associated cognitive dysfunction. PMID:28422052

  8. Structural stability of human protein tyrosine phosphatase ρ catalytic domain: effect of point mutations.

    Directory of Open Access Journals (Sweden)

    Alessandra Pasquo

    Full Text Available Protein tyrosine phosphatase ρ (PTPρ belongs to the classical receptor type IIB family of protein tyrosine phosphatase, the most frequently mutated tyrosine phosphatase in human cancer. There are evidences to suggest that PTPρ may act as a tumor suppressor gene and dysregulation of Tyr phosphorylation can be observed in diverse diseases, such as diabetes, immune deficiencies and cancer. PTPρ variants in the catalytic domain have been identified in cancer tissues. These natural variants are nonsynonymous single nucleotide polymorphisms, variations of a single nucleotide occurring in the coding region and leading to amino acid substitutions. In this study we investigated the effect of amino acid substitution on the structural stability and on the activity of the membrane-proximal catalytic domain of PTPρ. We expressed and purified as soluble recombinant proteins some of the mutants of the membrane-proximal catalytic domain of PTPρ identified in colorectal cancer and in the single nucleotide polymorphisms database. The mutants show a decreased thermal and thermodynamic stability and decreased activation energy relative to phosphatase activity, when compared to wild- type. All the variants show three-state equilibrium unfolding transitions similar to that of the wild- type, with the accumulation of a folding intermediate populated at ~4.0 M urea.

  9. Inheritance of Protein Patterns in a Synthetic Allopolyploid of Triticum Monococcum (AA) and Aegilops Ventricosa (DDMvMv)

    DEFF Research Database (Denmark)

    Siddiqui, K. A.; Ingversen, J.; Køie, B.

    1972-01-01

    Patterns of seed proteins in Triticum monococcum (2n = 2x = 14 = AA), Aegilops ventricosa (2n = 4x = 28 = DDMVMV), and their synthetic amphiploid were studied. The distribution of proteins in the individual Osborne protein fractions of the amphiploid was characterized by a 14 per cent increase...

  10. Water molecule network and active site flexibility of apo protein tyrosine phosphatase 1B

    DEFF Research Database (Denmark)

    Pedersen, A.K.; Peters, Günther H.J.; Møller, K.B.

    2004-01-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role as a negative regulator of insulin and leptin signalling and is therefore considered to be an important molecular target for the treatment of type 2 diabetes and obesity. Detailed structural information about the structure of PTP1B, including...

  11. A Drosophila protein-tyrosine phosphatase associates with an adapter protein required for axonal guidance.

    Science.gov (United States)

    Clemens, J C; Ursuliak, Z; Clemens, K K; Price, J V; Dixon, J E

    1996-07-19

    We have used the yeast two-hybrid system to isolate a novel Drosophila adapter protein, which interacts with the Drosophila protein-tyrosine phosphatase (PTP) dPTP61F. Absence of this protein in Drosophila causes the mutant photoreceptor axon phenotype dreadlocks (dock) (Garrity, P. A., Rao, Y., Salecker, I., and Zipursky, S. L.(1996) Cell 85, 639-650). Dock is similar to the mammalian oncoprotein Nck and contains three Src homology 3 (SH3) domains and one Src homology 2 (SH2) domain. The interaction of dPTP61F with Dock was confirmed in vivo by immune precipitation experiments. A sequence containing five PXXP motifs from the non-catalytic domain of the PTP is sufficient for interaction with Dock. This suggests that binding to the PTP is mediated by one or more of the SH3 domains of Dock. Immune precipitations of Dock also co-precipitate two tyrosine-phosphorylated proteins having molecular masses of 190 and 145 kDa. Interactions between Dock and these tyrosine-phosphorylated proteins are likely mediated by the Dock SH2 domain. These findings identify potential signal-transducing partners of Dock and propose a role for dPTP61F and the unidentified phosphoproteins in axonal guidance.

  12. Overexpression of the PP2A regulatory subunit Tap46 leads to enhanced plant growth through stimulation of the TOR signalling pathway.

    Science.gov (United States)

    Ahn, Chang Sook; Ahn, Hee-Kyung; Pai, Hyun-Sook

    2015-02-01

    Tap46, a regulatory subunit of protein phosphatase 2A (PP2A), plays an essential role in plant growth and development through a functional link with the Target of Rapamycin (TOR) signalling pathway. Here, we have characterized the molecular mechanisms behind a gain-of-function phenotype of Tap46 and its relationship with TOR to gain further insights into Tap46 function in plants. Constitutive overexpression of Tap46 in Arabidopsis resulted in overall growth stimulation with enlarged organs, such as leaves and siliques. Kinematic analysis of leaf growth revealed that increased cell size was mainly responsible for the leaf enlargement. Tap46 overexpression also enhanced seed size and viability under accelerated ageing conditions. Enhanced plant growth was also observed in dexamethasone (DEX)-inducible Tap46 overexpression Arabidopsis lines, accompanied by increased cellular activities of nitrate-assimilating enzymes. DEX-induced Tap46 overexpression and Tap46 RNAi resulted in increased and decreased phosphorylation of S6 kinase (S6K), respectively, which is a sensitive indicator of endogenous TOR activity, and Tap46 interacted with S6K in planta based on bimolecular fluorescence complementation and co-immunoprecipitation. Furthermore, inactivation of TOR by estradiol-inducible RNAi or rapamycin treatment decreased Tap46 protein levels, but increased PP2A catalytic subunit levels. Real-time quantitative PCR analysis revealed that Tap46 overexpression induced transcriptional modulation of genes involved in nitrogen metabolism, ribosome biogenesis, and lignin biosynthesis. These findings suggest that Tap46 modulates plant growth as a positive effector of the TOR signalling pathway and Tap46/PP2Ac protein abundance is regulated by TOR activity. © The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  13. AbetaPP/APLP2 family of Kunitz serine proteinase inhibitors regulate cerebral thrombosis.

    Science.gov (United States)

    Xu, Feng; Previti, Mary Lou; Nieman, Marvin T; Davis, Judianne; Schmaier, Alvin H; Van Nostrand, William E

    2009-04-29

    The amyloid beta-protein precursor (AbetaPP) is best recognized as the precursor to the Abeta peptide that accumulates in the brains of patients with Alzheimer's disease, but less is known about its physiological functions. Isoforms of AbetaPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AbetaPP regulates cerebral thrombosis in vivo (Xu et al., 2005, 2007). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AbetaPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here, we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AbetaPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model, both AbetaPP(-/-) and APLP2(-/-) mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a prothrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage, both AbetaPP(-/-) and APLP2(-/-) mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AbetaPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury.

  14. Phosphatase Activity of Microbial Populations in Different Milk Samples in Relation to Protein and Carbohydrate Content

    Directory of Open Access Journals (Sweden)

    Sosanka Protim SANDILYA

    2014-12-01

    Full Text Available Cattle milk is a rich source of protein, carbohydrate, vitamins, minerals and all other major and micro nutrients. At a moderate pH, milk is an excellent media for the growth of microbes and thus, intake of raw milk is precarious. In this study, attempt was made for a qualitative study of eight raw milk samples of different varieties of cow and goat milk, collected from Jorhat district of Assam, India, on the basis of nutritional value and microbial population. The highest microbial population was found in the milk collected from cross hybrid variety of cow, whereas microbial contamination was the least in Jersey cow milk. Samples of C1 (Jersey cow variety showed presence of the highest amount of protein and carbohydrate content as compared to the others. Almost all the milk samples showed positive acid and alkaline phosphatase activity. Maximum acid phosphatase activity was observed in cross hybrid cow milk, whereas local cow milk exhibited the highest alkaline phosphatase activity. Phosphatase activity did not show any co-relationship with microbial population of the milk samples. Similarly, the protein and carbohydrate content of the samples did not have any significant impact on both acid and alkaline phosphatase activity.

  15. Characterization of pneumococcal Ser/Thr protein phosphatase phpP mutant and identification of a novel PhpP substrate, putative RNA binding protein Jag

    Czech Academy of Sciences Publication Activity Database

    Ulrych, Aleš; Holečková, Nela; Goldová, Jana; Doubravová, Linda; Benada, Oldřich; Kofroňová, Olga; Halada, Petr; Branny, Pavel

    2016-01-01

    Roč. 16, OCT 24 (2016), s. 247 ISSN 1471-2180 R&D Projects: GA ČR GAP302/12/0256; GA ČR GAP207/12/1568; GA MŠk LH12055 Institutional support: RVO:61388971 Keywords : Signal transduction * Protein phosphatase * Protein kinase Subject RIV: EE - Microbiology, Virology Impact factor: 2.644, year: 2016

  16. Bt rice expressing Cry2Aa does not harm Cyrtorhinus lividipennis, a main predator of the nontarget herbivore Nilapavarta lugens.

    Directory of Open Access Journals (Sweden)

    Yu Han

    Full Text Available T2A-1 is a newly developed transgenic rice that expresses a synthesized cry2Aa gene driven by the maize ubiquitin promoter. T2A-1 exhibits high resistance against lepidopteran pests of rice. The brown planthopper, Nilapavarta lugens (Stål, is a main nontarget sap-sucking insect pest of rice, and Cyrtorhinus lividipennis (Reuter is the major predator of the eggs and young nymphs of planthoppers. As C. lividipennis may expose to the Cry2Aa protein via N. lugens, it is therefore essential to assess the potential effects of transgenic cry2Aa rice on this predator. In the present study, three experiments were conducted to evaluate the ecological risk of transgenic cry2Aa rice to C. lividipennis: (1 a direct feeding experiment in which C. lividipennis was fed an artificial diet containing Cry2Aa at the dose of 10-time higher than that it may encounter in the realistic field condition; (2 a tritrophic experiment in which the Cry2Aa protein was delivered to C. lividipennis indirectly through prey eggs or nymphs; (3 a realistic field experiment in which the population dynamics of C. lividipennis were investigated using vacuum-suction. Both direct exposure to elevated doses of the Cry2Aa protein and prey-mediated exposure to realistic doses of the protein did not result in significant detrimental effects on the development, survival, female ratio and body weight of C. lividipennis. No significant differences in population density and population dynamics were observed between C. lividipennis in transgenic cry2Aa and nontransgenic rice fields. It may be concluded that transgenic cry2Aa rice had no detrimental effects on C. lividipennis. This study represents the first report of an assessment continuum for the effects of transgenic cry2Aa rice on C. lividipennis.

  17. Lack of evidence for protein AA reactivity in amyloid deposits of lattice corneal dystrophy and amyloid corneal degeneration.

    Science.gov (United States)

    Gorevic, P D; Rodrigues, M M; Krachmer, J H; Green, C; Fujihara, S; Glenner, G G

    1984-08-15

    Amyloid fibrils occurring in primary and myeloma-associated (AL), secondary (AA), and certain neuropathic hereditary forms of systemic amyloidosis can be distinguished biochemically or immunohistologically as being composed of immunoglobulin light chain, protein AA, or prealbumin respectively. All types of systemic and several localized forms of amyloidosis contain amyloid P component (protein AP). We studied formalin-fixed tissue from eight cases of lattice corneal dystrophy by the immunoperoxidase method using antisera to proteins AA and AP, to normal serum prealbumin and prealbumin isolated from a case of hereditary amyloidosis, and to light-chain determinants; additional cases were examined by indirect immunofluorescence of fresh-frozen material. We found weak (1:10 dilution) staining with anti-AP, but no reactivity with other antisera. Congo red staining was resistant to pretreatment of sections with potassium permanganate, a characteristic of non-AA amyloid. Two-dimensional gels of solubilized proteins from frozen tissue from two cases of lattice corneal dystrophy resembled those obtained from normal human cornea. Western blots of two cases of polymorphous amyloid degeneration and solubilized protein from normal cornea did not react with radioactive iodine-labeled anti-AA or anti-AP with purified protein AP and unfixed protein AA amyloid tissue as controls. We were unable to corroborate the presence of protein AA in the amyloid deposits of lattice corneal dystrophy. Although staining with antiserum to protein AP was demonstrable, the molecular configuration of this protein in stromal deposits remains to be defined.

  18. Antiangiogenic effects of AA-PMe on HUVECs in vitro and zebrafish in vivo

    Directory of Open Access Journals (Sweden)

    Jing Y

    2018-04-01

    phosphatase assay. VEGFR2 expression in whole embryos following AA-PMe treatment was also determined. Results: We found AA-PMe decreased cell viability and inhibited migration and tube formation in a dose-dependent manner in HUVECs. Similarly, AA-PMe disrupted the formation of intersegmental vessels, the dorsal aorta, and the posterior cardinal vein in zebrafish embryos. Both in vitro and in vivo AA-PMe surpassed AA in its ability to block angiogenesis by suppressing VEGF-induced phosphorylation of VEGFR2 and disrupting downstream extracellular regulated protein kinase and AKT signaling. Conclusion: For the first time, this study reveals that AA-PMe acts as a potent VEGFR2 kinase inhibitor and exerts powerful antiangiogenic activity, suggesting it to be a promising therapeutic candidate for further research. Keywords: AA-PMe, angiogenesis inhibitor, zebrafish, HUVEC

  19. Phosphatase activity of Poa pratensis seeds. II. Purification and characterization of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Two acid phosphatases (Ia2, Ia3 have been isolated from Poa pratensis seeds and partially purified. Both enzymes showed maximal activity at pH 4,9. They exhibited high activity towards p-nitrophenyl phosphate, inorganic pyrophosphate and phenyl phosphate, much less activity towards glucose-6 phosphate, and mononucleotides. Phosphatases a2 and a3 differed in their activity towards ADP. Orthophosphate, fluoride and Zn2+ were effective inhibitors. EDTA, β-mercaptoethanol and Mg2+ activated phophatase a2 but had no effect on phosphatase a3. Zn2+ inhibited the activity of phosphatase a2 noncompetitively, whereas phosphatase a3 showed inhibition of mixed type. Trypsin, chymotrypsin and pronase had no effect on the enzyme activities of both molecular forms.

  20. Anxious moments for the protein tyrosine phosphatase PTP1B

    OpenAIRE

    Krishnan, Navasona; Tonks, Nicholas K.

    2015-01-01

    Chronic stress can lead to the development of anxiety and mood disorders. Thus, novel therapies for preventing adverse effects of stress are vitally important. Recently, the protein tyrosine phosphatase PTP1B was identified as a novel regulator of stress-induced anxiety. This opens up exciting opportunities to exploit PTP1B inhibitors as anxiolytics.

  1. Characterization of cDNA encoding human placental anticoagulant protein (PP4): Homology with the lipocortin family

    International Nuclear Information System (INIS)

    Grundmann, U.; Abel, K.J.; Bohn, H.; Loebermann, H.; Lottspeich, F.; Kuepper, H.

    1988-01-01

    A cDNA library prepared from human placenta was screened for sequences encoding the placental protein 4 (PP4). PP4 is an anticoagulant protein that acts as an indirect inhibitor of the thromboplastin-specific complex, which is involved in the blood coagulation cascade. Partial amino acid sequence information from PP4-derived cyanogen bromide fragments was used to design three oligonucleotide probes for screening the library. From 10 6 independent recombinants, 18 clones were identified that hybridized to all three probes. These 18 recombinants contained cDNA inserts encoding a protein of 320 amino acid residues. In addition to the PP4 cDNA the authors identified 9 other recombinants encoding a protein with considerable similarity (74%) to PP4, which was termed PP4-X. PP4 and PP4-X belong to the lipocortin family, as judged by their homology to lipocortin I and calpactin I

  2. Expression of protein-tyrosine phosphatases in the major insulin target tissues

    DEFF Research Database (Denmark)

    Norris, K; Norris, F; Kono, D H

    1997-01-01

    Protein-tyrosine phosphatases (PTPs) are key regulators of the insulin receptor signal transduction pathway. We have performed a detailed analysis of PTP expression in the major human insulin target tissues or cells (liver, adipose tissue, skeletal muscle and endothelial cells). To obtain a repre...

  3. Receptor-like protein-tyrosine phosphatase alpha specifically inhibits insulin-increased prolactin gene expression

    DEFF Research Database (Denmark)

    Jacob, K K; Sap, J; Stanley, F M

    1998-01-01

    A physiologically relevant response to insulin, stimulation of prolactin promoter activity in GH4 pituitary cells, was used as an assay to study the specificity of protein-tyrosine phosphatase function. Receptor-like protein-tyrosine phosphatase alpha (RPTPalpha) blocks the effect of insulin...... is specific by two criteria. A number of potential RPTPalpha targets were ruled out by finding (a) that they are not affected or (b) that they are not on the pathway to insulin-increased prolactin-CAT activity. The negative effect of RPTPalpha on insulin activation of the prolactin promoter is not due...... to reduced phosphorylation or kinase activity of the insulin receptor or to reduced phosphorylation of insulin receptor substrate-1 or Shc. Inhibitor studies suggest that insulin-increased prolactin gene expression is mediated by a Ras-like GTPase but is not mitogen-activated protein kinase dependent...

  4. Selection and characterization of resistance to the Vip3Aa20 protein from Bacillus thuringiensis in Spodoptera frugiperda.

    Science.gov (United States)

    Bernardi, Oderlei; Bernardi, Daniel; Horikoshi, Renato J; Okuma, Daniela M; Miraldo, Leonardo L; Fatoretto, Julio; Medeiros, Fernanda Cl; Burd, Tony; Omoto, Celso

    2016-09-01

    Spodoptera frugiperda is one the main target pests of maize events expressing Vip3Aa20 protein from Bacillus thuringiensis (Bt) in Brazil. In this study, we selected a resistant strain of S. frugiperda on Bt maize expressing Vip3Aa20 protein and characterized the inheritance and fitness costs of the resistance. The resistance ratio of the Vip3Aa20-resistant strain of S. frugiperda was >3200-fold. Neonates of the Vip3Aa20-resistant strain were able to survive and emerge as fertile adults on Vip3Aa20 maize, while larvae from susceptible and heterozygous strains did not survive. The inheritance of Vip3Aa20 resistance was autosomal recessive and monogenic. Life history studies to investigate fitness cost revealed an 11% reduction in the survival rate until adult stage and a ∼50% lower reproductive rate of the Vip3Aa20-resistant strain compared with susceptible and heterozygous strains. This is the first characterization of S. frugiperda resistance to Vip3Aa protein. Our results provide useful information for resistance management programs designed to prevent or delay resistance evolution to Vip3Aa proteins in S. frugiperda. © 2016 Society of Chemical Industry. © 2016 Society of Chemical Industry.

  5. A Nucleotide Phosphatase Activity in the Nucleotide Binding Domain of an Orphan Resistance Protein from Rice*

    Science.gov (United States)

    Fenyk, Stepan; de San Eustaquio Campillo, Alba; Pohl, Ehmke; Hussey, Patrick J.; Cann, Martin J.

    2012-01-01

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack. PMID:22157756

  6. A nucleotide phosphatase activity in the nucleotide binding domain of an orphan resistance protein from rice.

    Science.gov (United States)

    Fenyk, Stepan; Campillo, Alba de San Eustaquio; Pohl, Ehmke; Hussey, Patrick J; Cann, Martin J

    2012-02-03

    Plant resistance proteins (R-proteins) are key components of the plant immune system activated in response to a plethora of different pathogens. R-proteins are P-loop NTPase superfamily members, and current models describe their main function as ATPases in defense signaling pathways. Here we show that a subset of R-proteins have evolved a new function to combat pathogen infection. This subset of R-proteins possesses a nucleotide phosphatase activity in the nucleotide-binding domain. Related R-proteins that fall in the same phylogenetic clade all show the same nucleotide phosphatase activity indicating a conserved function within at least a subset of R-proteins. R-protein nucleotide phosphatases catalyze the production of nucleoside from nucleotide with the nucleotide monophosphate as the preferred substrate. Mutation of conserved catalytic residues substantially reduced activity consistent with the biochemistry of P-loop NTPases. Kinetic analysis, analytical gel filtration, and chemical cross-linking demonstrated that the nucleotide-binding domain was active as a multimer. Nuclear magnetic resonance and nucleotide analogues identified the terminal phosphate bond as the target of a reaction that utilized a metal-mediated nucleophilic attack by water on the phosphoester. In conclusion, we have identified a group of R-proteins with a unique function. This biochemical activity appears to have co-evolved with plants in signaling pathways designed to resist pathogen attack.

  7. Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) interacts with neurofilament L and inhibits its filament association

    Energy Technology Data Exchange (ETDEWEB)

    Ozaki, Hana [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Katoh, Tsuyoshi [Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510 (Japan); Nakagawa, Ryoko; Ishihara, Yasuhiro [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Sueyoshi, Noriyuki; Kameshita, Isamu [Department of Life Sciences, Faculty of Agriculture, Kagawa University, Kagawa, 761-0795 (Japan); Taniguchi, Takanobu [Department of Biochemistry, Asahikawa Medical University, Asahikawa, 078-8510 (Japan); Hirano, Tetsuo; Yamazaki, Takeshi [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan); Ishida, Atsuhiko, E-mail: aishida@hiroshima-u.ac.jp [Laboratory of Molecular Brain Science, Graduate School of Integrated Arts and Sciences, Hiroshima University, Higashi-Hiroshima, 739-8521 (Japan)

    2016-09-02

    Ca{sup 2+}/calmodulin-dependent protein kinase phosphatase (CaMKP/PPM1F) is a Ser/Thr phosphatase that belongs to the PPM family. Growing evidence suggests that PPM phosphatases including CaMKP act as a complex with other proteins to regulate cellular functions. In this study, using the two-dimensional far-western blotting technique with digoxigenin-labeled CaMKP as a probe, in conjunction with peptide mass fingerprinting analysis, we identified neurofilament L (NFL) as a CaMKP-binding protein in a Triton-insoluble fraction of rat brain. We confirmed binding of fluorescein-labeled CaMKP (F-CaMKP) to NFL in solution by fluorescence polarization. The analysis showed that the dissociation constant of F-CaMKP for NFL is 73 ± 17 nM (n = 3). Co-immunoprecipitation assay using a cytosolic fraction of NGF-differentiated PC12 cells showed that endogenous CaMKP and NFL form a complex in cells. Furthermore, the effect of CaMKP on self-assembly of NFL was examined. Electron microscopy revealed that CaMKP markedly prevented NFL from forming large filamentous aggregates, suggesting that CaMKP-binding to NFL inhibits its filament association. These findings may provide new insights into a novel mechanism for regulating network formation of neurofilaments during neuronal differentiation. - Highlights: • NFL was identified as a CaMKP-binding protein in an insoluble fraction of rat brain. • CaMKP bound to NFL in solution with a K{sub d} value of 73 ± 17 nM. • A CaMKP-NFL complex was found in NGF-differentiated PC12 cells. • CaMKP-binding to NFL inhibited its filament association. • CaMKP may regulate network formation of neurofilaments in neurons.

  8. HD-PTP is a catalytically inactive tyrosine phosphatase due to a conserved divergence in its phosphatase domain.

    Directory of Open Access Journals (Sweden)

    Marie-Claude Gingras

    Full Text Available The HD-PTP protein has been described as a tumor suppressor candidate and based on its amino acid sequence, categorized as a classical non-transmembrane protein tyrosine phosphatase (PTP. To date, no HD-PTP phosphorylated substrate has been identified and controversial results concerning its catalytic activity have been recently reported.Here we report a rigorous enzymatic analysis demonstrating that the HD-PTP protein does not harbor tyrosine phosphatase or lipid phosphatase activity using the highly sensitive DiFMUP substrate and a panel of different phosphatidylinositol phosphates. We found that HD-PTP tyrosine phosphatase inactivity is caused by an evolutionary conserved amino acid divergence of a key residue located in the HD-PTP phosphatase domain since its back mutation is sufficient to restore the HD-PTP tyrosine phosphatase activity. Moreover, in agreement with a tumor suppressor activity, HD-PTP expression leads to colony growth reduction in human cancer cell lines, independently of its catalytic PTP activity status.In summary, we demonstrate that HD-PTP is a catalytically inactive protein tyrosine phosphatase. As such, we identify one residue involved in its inactivation and show that its colony growth reduction activity is independent of its PTP activity status in human cancer cell lines.

  9. MCLR-induced PP2A inhibition and subsequent Rac1 inactivation and hyperphosphorylation of cytoskeleton-associated proteins are involved in cytoskeleton rearrangement in SMMC-7721 human liver cancer cell line.

    Science.gov (United States)

    Wang, Hao; Liu, Jinghui; Lin, Shuyan; Wang, Beilei; Xing, Mingluan; Guo, Zonglou; Xu, Lihong

    2014-10-01

    Cyanobacteria-derived toxin microcystin-LR (MCLR) has been widely investigated in its effects on normal cells, there is little information concerning its effects on cancer cells. In the present study, the SMMC-7721 human liver cancer cell line treated with MCLR was used to investigate the change of PP2A, cytoskeleton rearrangement, phosphorylation levels of PP2A substrates that related with cytoskeleton stability and explored underlying mechanisms. Here, we confirmed that MCLR entered into SMMC-7721 cells, bound to PP2A/C subunit and inhibited the activity of PP2A. The upregulation of phosphorylation of the PP2A/C subunit and PP2A regulation protein α4, as well as the change in the association of PP2A/C with α4, were responsible for the decrease in PP2A activity. Another novel finding is that the rearrangement of filamentous actin and microtubules led by MCLR may attribute to the increased phosphorylation of HSP27, VASP and cofilin due to PP2A inhibition. As a result of weakened interactions with PP2A and alterations in its subcellular localization, Rac1 may contribute to the cytoskeletal rearrangement induced by MCLR in SMMC-7721 cells. The current paper presents the first report demonstrating the characteristic of PP2A in MCLR exposed cancer cells, which were more susceptible to MCLR compared with the normal cell lines we previously found, which may be owing to the absence of some type of compensatory mechanisms. The hyperphosphorylation of cytoskeleton-associated proteins and Rac1 inactivation which were induced by inhibition of PP2A are shown to be involved in cytoskeleton rearrangement. Copyright © 2014 Elsevier Ltd. All rights reserved.

  10. Allosteric inhibition of SHP2 phosphatase inhibits cancers driven by receptor tyrosine kinases

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Ying-Nan P.; LaMarche, Matthew J.; Chan, Ho Man; Fekkes, Peter; Garcia-Fortanet, Jorge; Acker, Michael G.; Antonakos, Brandon; Chen, Christine Hiu-Tung; Chen, Zhouliang; Cooke, Vesselina G.; Dobson, Jason R.; Deng, Zhan; Fei, Feng; Firestone, Brant; Fodor, Michelle; Fridrich, Cary; Gao, Hui; Grunenfelder, Denise; Hao, Huai-Xiang; Jacob, Jaison; Ho, Samuel; Hsiao, Kathy; Kang, Zhao B.; Karki, Rajesh; Kato, Mitsunori; Larrow, Jay; La Bonte, Laura R.; Lenoir, Francois; Liu, Gang; Liu, Shumei; Majumdar, Dyuti; Meyer, Matthew J.; Palermo, Mark; Perez, Lawrence; Pu, Minying; Price, Edmund; Quinn, Christopher; Shakya, Subarna; Shultz, Michael D.; Slisz, Joanna; Venkatesan, Kavitha; Wang, Ping; Warmuth, Markus; Williams, Sarah; Yang, Guizhi; Yuan, Jing; Zhang, Ji-Hu; Zhu, Ping; Ramsey, Timothy; Keen, Nicholas J.; Sellers, William R.; Stams, Travis; Fortin , Pascal D. (Novartis)

    2016-06-29

    The non-receptor protein tyrosine phosphatase SHP2, encoded by PTPN11, has an important role in signal transduction downstream of growth factor receptor signalling and was the first reported oncogenic tyrosine phosphatase1. Activating mutations of SHP2 have been associated with developmental pathologies such as Noonan syndrome and are found in multiple cancer types, including leukaemia, lung and breast cancer and neuroblastoma1, 2, 3, 4, 5. SHP2 is ubiquitously expressed and regulates cell survival and proliferation primarily through activation of the RAS–ERK signalling pathway2, 3. It is also a key mediator of the programmed cell death 1 (PD-1) and B- and T-lymphocyte attenuator (BTLA) immune checkpoint pathways6, 7. Reduction of SHP2 activity suppresses tumour cell growth and is a potential target of cancer therapy8, 9. Here we report the discovery of a highly potent (IC50 = 0.071 μM), selective and orally bioavailable small-molecule SHP2 inhibitor, SHP099, that stabilizes SHP2 in an auto-inhibited conformation. SHP099 concurrently binds to the interface of the N-terminal SH2, C-terminal SH2, and protein tyrosine phosphatase domains, thus inhibiting SHP2 activity through an allosteric mechanism. SHP099 suppresses RAS–ERK signalling to inhibit the proliferation of receptor-tyrosine-kinase-driven human cancer cells in vitro and is efficacious in mouse tumour xenograft models. Together, these data demonstrate that pharmacological inhibition of SHP2 is a valid therapeutic approach for the treatment of cancers.

  11. Cell surface expression of channel catfish leukocyte immune-type receptors (IpLITRs) and recruitment of both Src homology 2 domain-containing protein tyrosine phosphatase (SHP)-1 and SHP-2.

    Science.gov (United States)

    Montgomery, Benjamin C S; Mewes, Jacqueline; Davidson, Chelsea; Burshtyn, Deborah N; Stafford, James L

    2009-04-01

    Channel catfish leukocyte immune-type receptors (IpLITRs) are immunoglobulin superfamily (IgSF) members believed to play a role in the control and coordination of cellular immune responses in teleost. Putative stimulatory and inhibitory IpLITRs are co-expressed by different types of catfish immune cells (e.g. NK cells, T cells, B cells, and macrophages) but their signaling potential has not been determined. Following cationic polymer-mediated transfections into human cell lines we examined the surface expression, tyrosine phosphorylation, and phosphatase recruitment potential of two types of putative inhibitory IpLITRs using 'chimeric' expression constructs and an epitope-tagged 'native' IpLITR. We also cloned and expressed the teleost Src homology 2 domain-containing protein tyrosine phosphatases (SHP)-1 and SHP-2 and examined their expression in adult tissues and developing zebrafish embryos. Co-immunoprecipitation experiments support the inhibitory signaling potential of distinct IpLITR-types that bound both SHP-1 and SHP-2 following the phosphorylation of tyrosine residues within their cytoplasmic tail (CYT) regions. Phosphatase recruitment by IpLITRs represents an important first step in understanding their influence on immune cell effector functions and suggests that certain inhibitory signaling pathways are conserved among vertebrates.

  12. A PP2C-1 Allele Underlying a Quantitative Trait Locus Enhances Soybean 100-Seed Weight

    Institute of Scientific and Technical Information of China (English)

    Xiang Lu; Yong-Cai Lai; Wei-Guang Du; Wei-Qun Man; Shou-Yi Chen; Jin-Song Zhang; Qing Xiong; Tong Cheng; Qing-Tian Li; Xin-Lei Liu; Ying-Dong Bi; Wei Li; Wan-Ke Zhang; Biao Ma

    2017-01-01

    Cultivated soybeans may lose some useful genetic loci during domestication.Introgression of genes from wild soybeans could broaden the genetic background and improve soybean agronomic traits.In this study,through whole-genome sequencing of a recombinant inbred line population derived from a cross between a wild soybean ZYD7 and a cultivated soybean HN44,and mapping of quantitative trait loci for seed weight,we discovered that a phosphatase 2C-1 (PP2C-1) allele from wild soybean ZYD7 contributes to the increase in seed weight/size.PP2C-1 may achieve this function by enhancing cell size of integument and activating a subset of seed trait-related genes.We found that PP2C-1 is associated with GmBZR1,a soybean ortholog of Arabidopsis BZR1,one of key transcription factors in brassinosteroid (BR) signaling,and facilitate accumulation of dephosphorylated GmBZR1.In contrast,the PP2C-2 allele with variations of a few amino acids at the N-terminus did not exhibit this function.Moreover,we showed that GmBZR1 could promote seed weight/size in transgenic plants.Through analysis of cultivated soybean accessions,we found that 40% of the examined accessions do not have the PP2C-1 allele,suggesting that these accessions can be improved by introduction of this allele.Taken together,our study identifies an elite allele PP2C-1,which can enhance seed weight and/or size in soybean,and pinpoints that manipulation of this allele by molecular-assisted breeding may increase production in soybean and other legumes/crops.

  13. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David  S.; Poulin, Benoit; Ramaprasad, Abhinay; Wall, Richard  J.; Ferguson, David  J.P.; Brady, Declan; Patzewitz, Eva-Maria; Whipple, Sarah; Straschil, Ursula; Wright, Megan  H.; Mohamed, Alyaa  M.A.H.; Radhakrishnan, Anand; Arold, Stefan T.; Tate, Edward  W.; Holder, Anthony  A.; Wickstead, Bill; Pain, Arnab; Tewari, Rita

    2014-01-01

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  14. Genome-wide functional analysis of plasmodium protein phosphatases reveals key regulators of parasite development and differentiation

    KAUST Repository

    Guttery, David S.

    2014-07-09

    Reversible protein phosphorylation regulated by kinases and phosphatases controls many cellular processes. Although essential functions for the malaria parasite kinome have been reported, the roles of most protein phosphatases (PPs) during Plasmodium development are unknown. We report a functional analysis of the Plasmodium berghei protein phosphatome, which exhibits high conservation with the P. falciparum phosphatome and comprises 30 predicted PPs with differential and distinct expression patterns during various stages of the life cycle. Gene disruption analysis of P. berghei PPs reveals that half of the genes are likely essential for asexual blood stage development, whereas six are required for sexual development/sporogony in mosquitoes. Phenotypic screening coupled with transcriptome sequencing unveiled morphological changes and altered gene expression in deletion mutants of two N-myristoylated PPs. These findings provide systematic functional analyses of PPs in Plasmodium, identify how phosphatases regulate parasite development and differentiation, and can inform the identification of drug targets for malaria. © 2014 The Authors.

  15. Bridging the Gap Between Protein Carboxyl Methylation and Phospholipid Methylation to Understand Glucose-Stimulated Insulin Secretion From the Pancreatic β Cell

    OpenAIRE

    Kowluru, Anjaneyulu

    2007-01-01

    Recent findings have implicated post-translational modifications at C-terminal cysteines [e.g., methylation] of specific proteins [e.g., G-proteins] in glucose-stimulated insulin secretion [GSIS]. Furthermore, methylation at the C-terminal leucine of the catalytic subunit of protein phosphatase 2A [PP2Ac] has also been shown to be relevant for GSIS. In addition to these two classes of protein methyl transferases, a novel class of glucose-activated phospholipid methyl transferases have also be...

  16. Role of Zinc and Magnesium Ions in the Modulation of Phosphoryl Transfer in Protein Tyrosine Phosphatase 1B.

    Science.gov (United States)

    Bellomo, Elisa; Abro, Asma; Hogstrand, Christer; Maret, Wolfgang; Domene, Carmen

    2018-03-28

    While the majority of phosphatases are metalloenzymes, the prevailing model for the reactions catalyzed by protein tyrosine phosphatases does not involve any metal ion, yet both metal cations and oxoanions affect their enzymatic activity. Mg 2+ and Zn 2+ activate and inhibit, respectively, protein tyrosine phosphatase 1B (PTP1B). Molecular dynamics simulations, metadynamics, and quantum chemical calculations in combination with experimental investigations demonstrate that Mg 2+ and Zn 2+ compete for the same binding site in the active site only in the closed conformation of the enzyme in its phosphorylated state. The two cations have different effects on the arrangements and activities of water molecules that are necessary for the hydrolysis of the phosphocysteine intermediate in the second catalytic step of the reaction. Remarkable differences between the established structural enzymology of PTP1B investigated ex vivo and the function of PTP1B in vivo become evident. Different reaction pathways are viable when the presence of metal ions and their cellular concentrations are considered. The findings suggest that the substrate delivers the inhibitory Zn 2+ ion to the active site. The inhibition and activation can be ascribed to the different coordination chemistries of Zn 2+ and Mg 2+ ions and the orientation of the metal-coordinated water molecules. Metallochemistry adds an additional dimension to the regulation of PTP1B and presumably other members of this enzyme family.

  17. Arabidopsis protein phosphatase DBP1 nucleates a protein network with a role in regulating plant defense.

    Directory of Open Access Journals (Sweden)

    José Luis Carrasco

    Full Text Available Arabidopsis thaliana DBP1 belongs to the plant-specific family of DNA-binding protein phosphatases. Although recently identified as a novel host factor mediating susceptibility to potyvirus, little is known about DBP1 targets and partners and the molecular mechanisms underlying its function. Analyzing changes in the phosphoproteome of a loss-of-function dbp1 mutant enabled the identification of 14-3-3λ isoform (GRF6, a previously reported DBP1 interactor, and MAP kinase (MAPK MPK11 as components of a small protein network nucleated by DBP1, in which GRF6 stability is modulated by MPK11 through phosphorylation, while DBP1 in turn negatively regulates MPK11 activity. Interestingly, grf6 and mpk11 loss-of-function mutants showed altered response to infection by the potyvirus Plum pox virus (PPV, and the described molecular mechanism controlling GRF6 stability was recapitulated upon PPV infection. These results not only contribute to a better knowledge of the biology of DBP factors, but also of MAPK signalling in plants, with the identification of GRF6 as a likely MPK11 substrate and of DBP1 as a protein phosphatase regulating MPK11 activity, and unveils the implication of this protein module in the response to PPV infection in Arabidopsis.

  18. In vitro characterization of the Bacillus subtilis protein tyrosine phosphatase YwqE

    DEFF Research Database (Denmark)

    Mijakovic, Ivan; Musumeci, Lucia; Tautz, Lutz

    2005-01-01

    Both gram-negative and gram-positive bacteria possess protein tyrosine phosphatases (PTPs) with a catalytic Cys residue. In addition, many gram-positive bacteria have acquired a new family of PTPs, whose first characterized member was CpsB from Streptococcus pneumoniae. Bacillus subtilis contains...

  19. Detection of AA-type amyloid protein in labial salivary glands.

    Science.gov (United States)

    Sacsaquispe, Sonia-Julia; Antúnez-de Mayolo, Eleazar-Antonio; Vicetti, Rodolfo; Delgado, Wilson-Alejandro

    2011-03-01

    Among the diverse forms of amyloidosis, secondary type is the most frequent one. Diagnosis of amyloid deposition is based on the identification of the fibrillary protein amyloid by means of Congo Red (CR) or crystal violet (CV) stains, but these techniques do not differentiate between the different types of amyloid fibrils. The aim of this study was to identify by immunofluorescence (IF) AA amyloid a pathological fibrillar low-molecular-weight protein formed by cleavage of serum amyloid A (SAA) protein in labial salivary gland (LSG) biopsies from patients with secondary amyloidosis. 98 LSG were studied, 65 were from patients with secondary amyloidosis and 33 from subjects with chronic inflammatory diseases without evidence of this anomaly. All sections were stained with hematoxylin and eosin (H &E), CV, CR and IF using anti-AA antibodies. Positive and negative controls were used for all techniques. CV and CR demonstrated that the amyloid substance was found mainly distributed periductally (93.8%), followed by periacinar and perivascular locations (p <0.001); however, the IF demonstrated that amyloid AA substance predominates in the periacinar area (73.8%), followed by periductal and perivascular locations (p <0.001). IF has a sensitivity of 83%, 100% of specificity, 100% of predictive positive value and 75% of predictive negative value. The results of this study confirm the efficacy of the LSG biopsy as a highly reliable method for diagnosis of secondary amyloidosis.

  20. Lanthanum Containing Polymer's Modification to PP

    Institute of Scientific and Technical Information of China (English)

    Dai Shaojun; Zhang Ming

    2004-01-01

    Polypropylene (PP)'s low impact strength limits its usages. Adding some a rare earth polymer can enhance PP's tensile strength and impact strength. Acrylic lanthanum was prepared by the reaction between lanthanum oxide and acrylic acid. The IR spectrum prove that and optimum reacting conditions are that the bulk ratio of La(AA) 3 and MMA is not less than one and temperature is about 80 ℃. Lanthanum containing Polymer were added into PP. When percent of addition only was 3%, strength were enhanced 10% , and impact strength 40%. SEM shows the compatibility of rare earth polymer and PP; lanthanum containing polymer can form physical crosslinking between PP's molecules, then every particle's surface connect with several PP molecules and the PP mechanical property were enhanced.

  1. PP/APLP2 Family of Kunitz Serine Proteinase Inhibitors Regulate Cerebral Thrombosis

    Science.gov (United States)

    Xu, Feng; Previti, Mary Lou; Nieman, Marvin T.; Davis, Judianne; Schmaier, Alvin H.; Van Nostrand, William E.

    2009-01-01

    The amyloid β-protein precursor (AβPP) is best recognized as the precursor to the Aβ peptide that accumulates in the brains of patients with Alzheimer’s disease, but less is known about its physiological functions. Isoforms of AβPP that contain a Kunitz-type serine proteinase inhibitor (KPI) domain are expressed in brain and, outside the CNS, in circulating blood platelets. Recently, we showed that KPI-containing forms of AβPP regulates cerebral thrombosis in vivo (Xu et al., 2005 Proc. Natl. Acad. Sci. USA 102:18135–18140; Xu et al. 2007 Stroke 38:2598–2601). Amyloid precursor like protein-2 (APLP2), a closely related homolog to AβPP, also possesses a highly conserved KPI domain. Virtually nothing is known of its function. Here we show that APLP2 also regulates cerebral thrombosis risk. Recombinant purified KPI domains of AβPP and APLP2 both inhibit the plasma clotting in vitro. In a carotid artery thrombosis model both AβPP−/− and APLP2−/− mice exhibit similar significantly shorter times to vessel occlusion compared with wild-type mice indicating a pro-thrombotic phenotype. Similarly, in an experimental model of intracerebral hemorrhage both AβPP−/− and APLP2−/− mice produce significantly smaller hematomas with reduced brain hemoglobin content compared with wild-type mice. Together, these results indicate that AβPP and APLP2 share overlapping anticoagulant functions with regard to regulating thrombosis after cerebral vascular injury. PMID:19403832

  2. Placental Protein 13 (PP13 – a placental immunoregulatory galectin protecting pregnancy

    Directory of Open Access Journals (Sweden)

    Nandor Gabor Than

    2014-08-01

    Full Text Available Galectins are glycan-binding proteins that regulate innate and adaptive immune responses, and some confer maternal-fetal immune tolerance in eutherian mammals. A chromosome 19 cluster of galectins has emerged in anthropoid primates, species with deep placentation and long gestation. Three of the five human cluster galectins are solely expressed in the placenta, where they may confer additional immunoregulatory functions to enable deep placentation. One of these is galectin-13, also known as Placental Protein 13 (PP13. It has a jelly-roll fold, carbohydrate-recognition domain and sugar-binding preference resembling to other mammalian galectins. PP13 is predominantly expressed by the syncytiotrophoblast and released from the placenta into the maternal circulation. Its ability to induce apoptosis of activated T cells in vitro, and to divert and kill T cells as well as macrophages in the maternal decidua in situ suggests important immune functions. Indeed, mutations in the promoter and an exon of LGALS13 presumably leading to altered or non-functional protein expression are associated with a higher frequency of preeclampsia and other obstetrical syndromes, which involve immune dysregulation. Moreover, decreased placental expression of PP13 and its low first trimester maternal serum concentrations are associated with elevated risk of preeclampsia. Indeed, PP13 turned to be a good early biomarker to assess maternal risk for the subsequent development of pregnancy complications caused by impaired placentation. Due to the ischemic placental stress in preterm preeclampsia, there is an increased trophoblastic shedding of PP13 immunopositive microvesicles starting in the second trimester, which leads to high maternal blood PP13 concentrations. Our meta-analysis suggests that this phenomenon may enable the potential use of PP13 in directing patient management near to or at the time of delivery. Recent findings on the beneficial effects of PP13 on decreasing

  3. Alkaline Phosphatase, an Unconventional Immune Protein

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    Bethany A. Rader

    2017-08-01

    Full Text Available Recent years have seen an increase in the number of studies focusing on alkaline phosphatases (APs, revealing an expanding complexity of function of these enzymes. Of the four human AP (hAP proteins, most is known about tissue non-specific AP (TNAP and intestinal AP (IAP. This review highlights current understanding of TNAP and IAP in relation to human health and disease. TNAP plays a role in multiple processes, including bone mineralization, vitamin B6 metabolism, and neurogenesis, is the genetic cause of hypophosphatasia, influences inflammation through regulation of purinergic signaling, and has been implicated in Alzheimer’s disease. IAP regulates fatty acid absorption and has been implicated in the regulation of diet-induced obesity and metabolic syndrome. IAP and TNAP can dephosphorylate bacterial-derived lipopolysaccharide, and IAP has been identified as a potential regulator of the composition of the intestinal microbiome, an evolutionarily conserved function. Endogenous and recombinant bovine APs and recombinant hAPs are currently being explored for their potential as pharmacological agents to treat AP-associated diseases and mitigate multiple sources of inflammation. Continued research on these versatile proteins will undoubtedly provide insight into human pathophysiology, biochemistry, and the human holobiont.

  4. GADD34 Function in Protein Trafficking Promotes Adaptation to Hyperosmotic Stress in Human Corneal Cells

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    Dawid Krokowski

    2017-12-01

    Full Text Available Summary: GADD34, a stress-induced regulatory subunit of the phosphatase PP1, is known to function in hyperosmotic stress through its well-known role in the integrated stress response (ISR pathway. Adaptation to hyperosmotic stress is important for the health of corneal epithelial cells exposed to changes in extracellular osmolarity, with maladaptation leading to dry eye syndrome. This adaptation includes induction of SNAT2, an endoplasmic reticulum (ER-Golgi-processed protein, which helps to reverse the stress-induced loss of cell volume and promote homeostasis through amino acid uptake. Here, we show that GADD34 promotes the processing of proteins synthesized on the ER during hyperosmotic stress independent of its action in the ISR. We show that GADD34/PP1 phosphatase activity reverses hyperosmotic-stress-induced Golgi fragmentation and is important for cis- to trans-Golgi trafficking of SNAT2, thereby promoting SNAT2 plasma membrane localization and function. These results suggest that GADD34 is a protective molecule for ocular diseases such as dry eye syndrome. : Here, Krokowski et al. show that GADD34/PP1 protects the microtubule network, prevents Golgi fragmentation, and preserves protein trafficking independent of its action in the integrated stress response (ISR. In osmoadaptation, GADD34 facilitates trans-Golgi-mediated processing of the endoplasmic reticulum (ER-synthesized amino acid transporter SNAT2, which in turn increases amino acid uptake. Keywords: SNAT2, GADD34, hyperosmotic stress, amino acid transport, Golgi fragmentation, ISR

  5. Measurement of long-range correlations in pp collisions characterized by presence of a Z-boson

    CERN Document Server

    Milov, Alexander; The ATLAS collaboration

    2017-01-01

    Recent measurements of correlations between two particles separated in pesudorapidity and azimuthal angles have shown striking similarities between results obtained in pp, and in p+A and A+A collision systems. In pp collision system, unlike in the p+A and A+A systems, the strength of the correlations quantified by the anisotropy parameter $v_2$ does not show any dependence on the charged-particle multiplicity. Recent theoretical models suggest that this can be due to lack of correlation between the charged-particle multiplicity and the impact parameter of the pp collision. An independent handle on the impact parameter can be obtained by requiring the presence of a hard-scattering process in the collision. This talk presents the first measurement of two-particle correlations in pp collisions with a Z boson identified via its dimuon decay channel. The analysis uses ATLAS data recorded under nominal pp luminosities, and a procedure to correct for contribution of the tracks coming from pileup vertices is used. Th...

  6. Application of ascorbic acid 2-phosphate as a new voltammetric substrate for alkaline phosphatase determination in human serum

    Directory of Open Access Journals (Sweden)

    Wei Sun

    2005-12-01

    Full Text Available An electrochemical assay of the enzyme alkaline phosphatase (ALP using ascorbic acid 2-phosphate (AAP as a new voltammetric substrate has been described in this paper. In the alkaline buffer solution the ALP enzymatic hydrolysis product of AAP was ascorbic acid (AA, which was an electro-active substance and had a sensitive differential pulse voltammetric (DPV oxidative response on glassy carbon electrode (GCE at +380 mV (versus Ag/AgCl, so the activity of ALP could be monitored voltammetrically of the oxidative peak current of AA. The electrochemical behaviours of AA were carefully studied and the AA standard solution could be measured by DPV method in the linear range from 10.0 to 1000.0 μmol/L with the detection limit of 8.0 μmol/L. The optimal conditions for ALP enzymatic reaction and the voltammetric detection were optimized. Under the optimal conditions the calibration curve for ALP assay exhibited a linear range from 0.4 to 2000.0 U/L with a detection limit of 0.3 U/L. This proposed method was further applied to determine the ALP content in healthy human serum and the results were in good agreement with the traditional p-nitrophenyl phosphate spectrophotometric method. The kinetic constants of enzymatic reaction were also investigated with the apparent kinetic constant Km as 2.77 mmol/L and the maximum velocity Vmax as 0.33 mol/min.

  7. Setting sail for glucose homeostasis with the AKAP150-PP2B-anchor.

    Science.gov (United States)

    Teo, Adrian Kee Keong; Kulkarni, Rohit N

    2012-10-17

    Glucose-stimulated insulin secretion, controlled by multiple protein phosphorylation events, is critical for the regulation of glucose homeostasis. Protein kinase A (PKA) is known to play a role in β cell physiology, but the role of its anchoring protein is not fully understood. Hinke et al (2012) illustrate the significance of A-kinase anchoring protein 150 in tethering protein phosphatase 2B to mediate nutrient-stimulated insulin secretion and thus modulate glucose homeostasis.

  8. A pp32-retinoblastoma protein complex modulates androgen receptor-mediated transcription and associates with components of the splicing machinery

    International Nuclear Information System (INIS)

    Adegbola, Onikepe; Pasternack, Gary R.

    2005-01-01

    We have previously shown pp32 and the retinoblastoma protein interact. pp32 and the retinoblastoma protein are nuclear receptor transcriptional coregulators: the retinoblastoma protein is a coactivator for androgen receptor, the major regulator of prostate cancer growth, while pp32, which is highly expressed in prostate cancer, is a corepressor of the estrogen receptor. We now show pp32 increases androgen receptor-mediated transcription and the retinoblastoma protein modulates this activity. Using affinity purification and mass spectrometry, we identify members of the pp32-retinoblastoma protein complex as PSF and nonO/p54nrb, proteins implicated in coordinate regulation of nuclear receptor-mediated transcription and splicing. We show that the pp32-retinoblastoma protein complex is modulated during TPA-induced K562 differentiation. Present evidence suggests that nuclear receptors assemble multiprotein complexes to coordinately regulate transcription and mRNA processing. Our results suggest that pp32 and the retinoblastoma protein may be part of a multiprotein complex that coordinately regulates nuclear receptor-mediated transcription and mRNA processing

  9. Cell density-dependent nuclear/cytoplasmic localization of NORPEG (RAI14) protein

    International Nuclear Information System (INIS)

    Kutty, R. Krishnan; Chen, Shanyi; Samuel, William; Vijayasarathy, Camasamudram; Duncan, Todd; Tsai, Jen-Yue; Fariss, Robert N.; Carper, Deborah; Jaworski, Cynthia; Wiggert, Barbara

    2006-01-01

    NORPEG (RAI14), a developmentally regulated gene induced by retinoic acid, encodes a 980 amino acid (aa) residue protein containing six ankyrin repeats and a long coiled-coil domain [Kutty et al., J. Biol. Chem. 276 (2001), pp. 2831-2840]. We have expressed aa residues 1-287 of NORPEG and used the recombinant protein to produce an anti-NORPEG polyclonal antibody. Confocal immunofluorescence analysis showed that the subcellular localization of NORPEG in retinal pigment epithelial (ARPE-19) cells varies with cell density, with predominantly nuclear localization in nonconfluent cells, but a cytoplasmic localization, reminiscent of cytoskeleton, in confluent cultures. Interestingly, an evolutionarily conserved putative monopartite nuclear localization signal (P 27 KKRKAP 276 ) was identified by analyzing the sequences of NORPEG and its orthologs. GFP-NORPEG (2-287 aa), a fusion protein containing this signal, was indeed localized to nuclei when expressed in ARPE-19 or COS-7 cells. Deletion and mutation analysis indicated that the identified nuclear localization sequence is indispensable for nuclear targeting

  10. A new monoclonal antibody detects downregulation of protein tyrosine phosphatase receptor type γ in chronic myeloid leukemia patients

    Directory of Open Access Journals (Sweden)

    Marzia Vezzalini

    2017-06-01

    Full Text Available Abstract Background Protein tyrosine phosphatase receptor gamma (PTPRG is a ubiquitously expressed member of the protein tyrosine phosphatase family known to act as a tumor suppressor gene in many different neoplasms with mechanisms of inactivation including mutations and methylation of CpG islands in the promoter region. Although a critical role in human hematopoiesis and an oncosuppressor role in chronic myeloid leukemia (CML have been reported, only one polyclonal antibody (named chPTPRG has been described as capable of recognizing the native antigen of this phosphatase by flow cytometry. Protein biomarkers of CML have not yet found applications in the clinic, and in this study, we have analyzed a group of newly diagnosed CML patients before and after treatment. The aim of this work was to characterize and exploit a newly developed murine monoclonal antibody specific for the PTPRG extracellular domain (named TPγ B9-2 to better define PTPRG protein downregulation in CML patients. Methods TPγ B9-2 specifically recognizes PTPRG (both human and murine by flow cytometry, western blotting, immunoprecipitation, and immunohistochemistry. Results Co-localization experiments performed with both anti-PTPRG antibodies identified the presence of isoforms and confirmed protein downregulation at diagnosis in the Philadelphia-positive myeloid lineage (including CD34+/CD38bright/dim cells. After effective tyrosine kinase inhibitor (TKI treatment, its expression recovered in tandem with the return of Philadelphia-negative hematopoiesis. Of note, PTPRG mRNA levels remain unchanged in tyrosine kinase inhibitors (TKI non-responder patients, confirming that downregulation selectively occurs in primary CML cells. Conclusions The availability of this unique antibody permits its evaluation for clinical application including the support for diagnosis and follow-up of these disorders. Evaluation of PTPRG as a potential therapeutic target is also facilitated by the

  11. Deletion of an Endoplasmic Reticulum Stress Response Element in a ZmPP2C-A Gene Facilitates Drought Tolerance of Maize Seedlings.

    Science.gov (United States)

    Xiang, Yanli; Sun, Xiaopeng; Gao, Shan; Qin, Feng; Dai, Mingqiu

    2017-03-06

    Drought is a major abiotic stress that causes the yearly yield loss of maize, a crop cultured worldwide. Breeding drought-tolerant maize cultivars is a priority requirement of world agriculture. Clade A PP2C phosphatases (PP2C-A), which are conserved in most plant species, play important roles in abscisic acid (ABA) signaling and plant drought response. However, natural variations of PP2C-A genes that are directly associated with drought tolerance remain to be elucidated. Here, we conducted a candidate gene association analysis of the ZmPP2C-A gene family in a maize panel consisting of 368 varieties collected worldwide, and identified a drought responsive gene ZmPP2C-A10 that is tightly associated with drought tolerance. We found that the degree of drought tolerance of maize cultivars negatively correlates with the expression levels of ZmPP2C-A10. ZmPP2C-A10, like its Arabidopsis orthologs, interacts with ZmPYL ABA receptors and ZmSnRK2 kinases, suggesting that ZmPP2C-A10 is involved in mediating ABA signaling in maize. Transgenic studies in maize and Arabidopsis confirmed that ZmPP2C-A10 functions as a negative regulator of drought tolerance. Further, a causal natural variation, deletion allele-338, which bears a deletion of ERSE (endoplasmic reticulum stress response element) in the 5'-UTR region of ZmPP2C-A10, was detected. This deletion causes the loss of endoplasmic reticulum (ER) stress-induced expression of ZmPP2C-A10, leading to increased plant drought tolerance. Our study provides direct evidence linking ER stress signaling with drought tolerance and genetic resources that can be used directly in breeding drought-tolerant maize cultivars. Copyright © 2016 Elsevier Ltd. All rights reserved.

  12. Striatal-enriched Tyrosine Protein Phosphatase (STEP) in the Mechanisms of Depressive Disorders.

    Science.gov (United States)

    Kulikova, Elizabeth; Kulikov, Alexander

    2017-08-30

    Striatal-enriched tyrosine protein phosphatase (STEP) is expressed mainly in the brain. Its dysregulation is associated with Alzheimer's and Huntington's diseases, schizophrenia, fragile X syndrome, drug abuse and stroke/ischemia. However, an association between STEP and depressive disorders is still obscure. The review discusses the theoretical foundations and experimental facts concerning possible relationship between STEP dysregulation and depression risk. STEP dephosphorylates and inactivates several key neuronal signaling proteins such as extracellular signal-regulating kinase 1 and 2 (ERK1/2), stress activated protein kinases p38, the Src family tyrosine kinases Fyn, Pyk2, NMDA and AMPA glutamate receptors. The inactivation of these proteins decreases the expression of brain derived neurotrophic factor (BDNF) necessary for neurogenesis and neuronal survival. The deficit of BDNF results in progressive degeneration of neurons in the hippocampus and cortex and increases depression risk. At the same time, a STEP inhibitor, 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153), increases BDNF expression in the hippocampus and attenuated the depressivelike behavior in mice. Thus, STEP is involved in the mechanism of depressive disorders and it is a promising molecular target for atypical antidepressant drugs of new generation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  13. Personalized Therapy Against Preeclampsia by Replenishing Placental Protein 13 (PP13 Targeted to Patients With Impaired PP13 Molecule or Function

    Directory of Open Access Journals (Sweden)

    Hamutal Meiri

    Full Text Available Hypertensive disorders affect about one third of all people aged 20 and above, and are treated with anti-hypertensive drugs. Preeclampsia (PE is one form of such disorders that only develops during pregnancy. It affects ten million pregnant women globally and additionally causes fetal loss and major newborn disabilities. The syndrome's origin is multifactorial, and anti-hypertensive drugs are ineffective in treating it. Biomarkers are helpful for predict its development. Generic drugs, such as low dose aspirin, were proven effective in preventing preterm PE. However, it does not cure the majority of cases and many studies are underway for fighting PE with extended use of additional generic drugs, or through new drug development programs.This review focuses on placental protein 13 (PP13. This protein is only expressed in the placenta. Impaired PP13 DNA structure and/or its reduced mRNA expression leads to lower blood PP13 level that predict a higher risk of developing PE. Two polymorphic PP13 variants have been identified: (1 The promoter PP13 variant with an “A/A” genotype in the -98 position (versus “A/C” or “C/C”. Having the “A/A” genotype is coupled to lower PP13 expression, mainly during placental syncytiotrophoblast differentiation and, if associated with obesity and history of previous preeclampsia, it accurately predicts higher risk for developing the disorder. (2 A thymidine deletion at position 221 causes a frame shift in the open reading frame, and the formation of an early stop codon resulting in the formation of DelT221, a truncated variant of PP13. In pregnant rodents, both short- and long- term replenishment of PP13 causes reversible hypotension and vasodilation of uterine vessels. Long-term exposure is also accompanied by the development of larger placentas and newborns. Also, only w/t PP13 is capable of inducing leukocyte apoptosis, providing maternal immune tolerance to pregnancy.Based on published data, we

  14. 2-D Difference in gel electrophoresis combined with Pro-Q Diamond staining: a successful approach for the identification of kinase/phosphatase targets.

    Science.gov (United States)

    Orsatti, Laura; Forte, Eleonora; Tomei, Licia; Caterino, Marianna; Pessi, Antonello; Talamo, Fabio

    2009-07-01

    The protein tyrosine phosphatase PRL-3 is an appealing therapeutic cancer target for its well described involvement in the metastasis progression. Nevertheless, very little is known about PRL-3 role in tumorigenesis. In the attempt to identify the protein target of this phosphatase we have devised a model system based on the use of highly invasive HCT116 colon cancer cells over-expressing PRL-3. We used 2-D difference gel electrophoresis combined with the fluorescence staining Pro-Q Diamond selective for phosphorylated proteins to monitor changes in the phosphorylation status of possible substrates. Proteins whose phosphorylation level was negatively affected by PRL-3 over-expression were identified by MS. Two proteins were found to be significantly dephosphorylated in this condition, the cytoskeletal protein ezrin and elongation factor 2. Ezrin has already been described as having a proactive role in cancer metastasis through control of its phosphorylation status, and the PRL-3-induced modulation of ezrin phosphorylation in HCT116 and human umblical vascular endothelial cells is the subject of a separate paper by Forte et al. [Biochim. Biophys. Acta 2008, 1783, 334-344]. The combination of 2-D difference in gel electrophoresis and Pro-Q Diamond was hence confirmed successful in analyzing changes of protein phosphorylation which enable the identification of kinase/phosphatase targets.

  15. Retinoic Acid Modulates Interferon-γ Production by Hepatic Natural Killer T Cells via Phosphatase 2A and the Extracellular Signal-Regulated Kinase Pathway

    Science.gov (United States)

    Chang, Heng-Kwei

    2015-01-01

    Retinoic acid (RA), an active metabolite converted from vitamin A, plays an active role in immune function, such as defending against infections and immune regulation. Although RA affects various types of immune cells, including antigen-presenting cells, B lymphocytes, and T lymphocytes, whether it affects natural killer T (NKT) cells remain unknown. In this study, we found that RA decreased interferon (IFN)-γ production by activated NKT cells through T-cell receptor (TCR) and CD28. We also found that RA reduced extracellular signal-regulated kinase (ERK) phosphorylation, but increased phosphatase 2A (PP2A) activity in TCR/CD28-stimulated NKT cells. The increased PP2A activity, at least partly, contributed to the reduction of ERK phosphorylation. Since inhibition of ERK activation decreases IFN-γ production by TCR/CD28-stimulated NKT cells, RA may downregulate IFN-γ production by TCR/CD28-stimulated NKT cells through the PP2A-ERK pathway. Our results demonstrated a novel function of RA in modulating the IFN-γ expression by activated NKT cells. PMID:25343668

  16. Strangeness production in AA and pp collisions

    Energy Technology Data Exchange (ETDEWEB)

    Castorina, Paolo [Universita di Catania, Dipartimento di Fisica ed Astronomia, Catania (Italy); INFN, Catania (Italy); Satz, Helmut [Universitaet Bielefeld, Fakultaet fuer Physik, Bielefeld (Germany)

    2016-07-15

    Boost-invariant hadron production in high-energy collisions occurs in causally disconnected regions of finite space-time size. As a result, globally conserved quantum numbers (charge, strangeness, baryon number) are conserved locally in spatially restricted correlation clusters. Their size is determined by two time scales: the equilibration time specifying the formation of a quark-gluon plasma, and the hadronization time, specifying the onset of confinement. The expected values for these scales provide the theoretical basis for the suppression observed for strangeness production in elementary interactions (pp, e{sup +}e{sup -}) below LHC energies. In contrast, the space-time superposition of individual collisions in high-energy heavy-ion interactions leads to higher energy densities, resulting in much later hadronization and hence much larger hadronization volumes. This largely removes the causality constraints and results in an ideal hadronic resonance gas in full chemical equilibrium. In the present paper, we determine the collision energies needed for that; we also estimate when pp collisions reach comparable hadronization volumes and thus determine when strangeness suppression should disappear there as well. (orig.)

  17. Regulatory role of kinases and phosphatases on the internalisation of caveolae in HepG2 cells.

    Science.gov (United States)

    Botos, Erzsébet; Turi, Agnes; Müllner, Nándor; Kovalszky, Ilona; Tátrai, Péter; Kiss, Anna L

    2007-01-01

    The caveolar cycle is thought to be regulated by synchronised function of kinases and phosphatases. Using ocadaic acid--a serine/threonine protein phosphatase inhibitor--and an inhibitor of tyrosine phosphatase (sodium orthovanadate) we have followed the internalisation of caveolae. Since albumin binding to its receptor (gp60) can induce pinching off of caveolae from the plasma membrane, we also used this physiological ligand to induce the internalisation. Our confocal microscopic results show that both ocadaic acid and vanadate treatments have significantly decreased caveolin (caveolin-1 and -2) labelling on the cell surface, while the cytoplasmic labelling became much stronger. Quite often large, strongly labelled "granules" appear at the perinuclear region. Very strong caveolin labelling was detected along the actin-cytoskeleton suggesting that caveolae might move along these filaments. Our electron microscopic results also show an intensive caveolae pinching off from the plasma membrane. After ocadaic acid and vanadate treatments the number of surface connected vesicles (caveolae) decreases. At the same time, large multivesicular bodies (termed caveosomes) appear in the perinuclear area of the cytoplasm. By immunoprecipitation and Western blot analysis we detect an increased tyrosine phosphorylation of a approximately 29kDa protein in ocadaic acid and vanadate treated samples. This protein was identified as caveolin-2. No significant change in the tyrosine phosphorylation of caveolin-1 was found. From these data we can conclude that caveolae internalisation is regulated by phosphorylation of caveolin-2.

  18. Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

    Science.gov (United States)

    Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

    1998-11-01

    Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

  19. Regulation of Brain-Derived Neurotrophic Factor and Growth Factor Signaling Pathways by Tyrosine Phosphatase Shp2 in the Retina: A Brief Review

    Directory of Open Access Journals (Sweden)

    Mojdeh Abbasi

    2018-03-01

    Full Text Available SH2 domain-containing tyrosine phosphatase-2 (PTPN11 or Shp2 is a ubiquitously expressed protein that plays a key regulatory role in cell proliferation, differentiation and growth factor (GF signaling. This enzyme is well expressed in various retinal neurons and has emerged as an important player in regulating survival signaling networks in the neuronal tissues. The non-receptor phosphatase can translocate to lipid rafts in the membrane and has been implicated to regulate several signaling modules including PI3K/Akt, JAK-STAT and Mitogen Activated Protein Kinase (MAPK pathways in a wide range of biochemical processes in healthy and diseased states. This review focuses on the roles of Shp2 phosphatase in regulating brain-derived neurotrophic factor (BDNF neurotrophin signaling pathways and discusses its cross-talk with various GF and downstream signaling pathways in the retina.

  20. The Rts1 regulatory subunit of protein phosphatase 2A is required for control of G1 cyclin transcription and nutrient modulation of cell size.

    Directory of Open Access Journals (Sweden)

    Karen Artiles

    2009-11-01

    Full Text Available The key molecular event that marks entry into the cell cycle is transcription of G1 cyclins, which bind and activate cyclin-dependent kinases. In yeast cells, initiation of G1 cyclin transcription is linked to achievement of a critical cell size, which contributes to cell-size homeostasis. The critical cell size is modulated by nutrients, such that cells growing in poor nutrients are smaller than cells growing in rich nutrients. Nutrient modulation of cell size does not work through known critical regulators of G1 cyclin transcription and is therefore thought to work through a distinct pathway. Here, we report that Rts1, a highly conserved regulatory subunit of protein phosphatase 2A (PP2A, is required for normal control of G1 cyclin transcription. Loss of Rts1 caused delayed initiation of bud growth and delayed and reduced accumulation of G1 cyclins. Expression of the G1 cyclin CLN2 from an inducible promoter rescued the delayed bud growth in rts1Delta cells, indicating that Rts1 acts at the level of transcription. Moreover, loss of Rts1 caused altered regulation of Swi6, a key component of the SBF transcription factor that controls G1 cyclin transcription. Epistasis analysis revealed that Rts1 does not work solely through several known critical upstream regulators of G1 cyclin transcription. Cells lacking Rts1 failed to undergo nutrient modulation of cell size. Together, these observations demonstrate that Rts1 is a key player in pathways that link nutrient availability, cell size, and G1 cyclin transcription. Since Rts1 is highly conserved, it may function in similar pathways in vertebrates.

  1. p-p minimum-bias dijets and nonjet quadrupole in relation to conjectured collectivity (flows in high-energy nuclear collisions

    Directory of Open Access Journals (Sweden)

    Trainor Thomas A.

    2016-01-01

    Full Text Available Recent observations of ridge-like structure in p-p and p-A angular correlations at the RHIC and LHC have been interpreted to imply collective motion in smaller collision systems. It is argued that if correlation structures accepted as manifestations of flow in A-A collisions appear in smaller systems collectivity (flow must extend to the smaller systems. But the argument could be reversed to conclude that such structures appearing in A-A collisions may not imply hydrodynamic flow. I present spectrum, correlation and fluctuation data from RHIC p-p and Au-Au collisions and p-p, p-Pb and Pb-Pb results from the LHC described accurately by a two-component (soft+dijet model of hadron production. I also present evidence for a significant p-p nonjet (NJ quadrupole (v2 component with nch systematics directly related to A-A NJ quadrupole systematics. The combination suggests that soft, dijet and NJ quadrupole com- ponents are distinct phenomena in all cases, inconsistent with hadron production from a common bulk medium exhibiting collective motion (flow.

  2. Src homology domain 2-containing protein-tyrosine phosphatase-1 (SHP-1) binds and dephosphorylates G(alpha)-interacting, vesicle-associated protein (GIV)/Girdin and attenuates the GIV-phosphatidylinositol 3-kinase (PI3K)-Akt signaling pathway.

    Science.gov (United States)

    Mittal, Yash; Pavlova, Yelena; Garcia-Marcos, Mikel; Ghosh, Pradipta

    2011-09-16

    GIV (Gα-interacting vesicle-associated protein, also known as Girdin) is a bona fide enhancer of PI3K-Akt signals during a diverse set of biological processes, e.g. wound healing, macrophage chemotaxis, tumor angiogenesis, and cancer invasion/metastasis. We recently demonstrated that tyrosine phosphorylation of GIV by receptor and non-receptor-tyrosine kinases is a key step that is required for GIV to directly bind and enhance PI3K activity. Here we report the discovery that Src homology 2-containing phosphatase-1 (SHP-1) is the major protein-tyrosine phosphatase that targets two critical phosphotyrosines within GIV and antagonizes phospho-GIV-dependent PI3K enhancement in mammalian cells. Using phosphorylation-dephosphorylation assays, we demonstrate that SHP-1 is the major and specific protein-tyrosine phosphatase that catalyzes the dephosphorylation of tyrosine-phosphorylated GIV in vitro and inhibits ligand-dependent tyrosine phosphorylation of GIV downstream of both growth factor receptors and GPCRs in cells. In vitro binding and co-immunoprecipitation assays demonstrate that SHP-1 and GIV interact directly and constitutively and that this interaction occurs between the SH2 domain of SHP-1 and the C terminus of GIV. Overexpression of SHP-1 inhibits tyrosine phosphorylation of GIV and formation of phospho-GIV-PI3K complexes, and specifically suppresses GIV-dependent activation of Akt. Consistently, depletion of SHP-1 enhances peak tyrosine phosphorylation of GIV, which coincides with an increase in peak Akt activity. We conclude that SHP-1 antagonizes the action of receptor and non-receptor-tyrosine kinases on GIV and down-regulates the phospho-GIV-PI3K-Akt axis of signaling.

  3. Platelet-derived growth factor induces phosphorylation of a 64-kDa nuclear protein

    International Nuclear Information System (INIS)

    Shawver, L.K.; Pierce, G.F.; Kawahara, R.S.; Deuel, T.F.

    1989-01-01

    The platelet-derived growth factor (PDGF) stimulated the phosphorylation of a nuclear protein of 64 kDa (pp64) in nuclei of nontransformed normal rat kidney (NRK) cells. Low levels of phosphorylation of pp64 were observed in nuclei of serum-starved NRK cells. Fetal calf serum (FCS), PDGF, and homodimeric v-sis and PDGF A-chain protein enhanced the incorporation of 32P into pp64 over 4-fold within 30 min and over 8-fold within 2 h of exposure of NRK cells to the growth factors. In contrast, constitutive phosphorylation of 32P-labeled pp64 in nuclei of NRK cells transformed by the simian sarcoma virus (SSV) was high and only minimally stimulated by PDGF and FCS. 32P-Labeled pp64 was isolated from nuclei of PDGF-stimulated nontransformed NRK cells; the 32P of pp64 was labile in 1 M KOH, and pp64 was not significantly recognized by anti-phosphotyrosine antisera, suggesting that the PDGF-induced phosphorylation of pp64 occurred on serine or on threonine residues. However, pp64 from SSV-transformed NRK cell nuclei was significantly stable to base hydrolysis and was immunoprecipitated with anti-phosphotyrosine antisera, suggesting that pp64 from SSV-transformed cell nuclei is phosphorylated also on tyrosine. FCS, PDGF, and PDGF A- and B-chain homodimers thus stimulate the rapid time-dependent phosphorylation of a 64-kDa nuclear protein shortly after stimulation of responsive cells. The growth factor-stimulated phosphorylation of pp64 and the constitutive high levels of pp64 phosphorylation in cells transformed by SSV suggest important roles for pp64 and perhaps regulated nuclear protein kinases and phosphatases in cell division and proliferation

  4. Protection against gamma-radiation injury by protein tyrosine phosphatase 1B

    Directory of Open Access Journals (Sweden)

    Marina Mojena

    2018-07-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is widely expressed in mammalian tissues, in particular in immune cells, and plays a pleiotropic role in dephosphorylating many substrates. Moreover, PTP1B expression is enhanced in response to pro-inflammatory stimuli and to different cell stressors. Taking advantage of the use of mice deficient in PTP1B we have investigated the effect of γ-radiation in these animals and found enhanced lethality and decreased respiratory exchange ratio vs. the corresponding wild type animals. Using bone-marrow derived macrophages and mouse embryonic fibroblasts (MEFs from wild-type and PTP1B-deficient mice, we observed a differential response to various cell stressors. PTP1B-deficient macrophages exhibited an enhanced response to γ-radiation, UV-light, LPS and S-nitroso-glutathione. Macrophages exposed to γ-radiation show DNA damage and fragmentation, increased ROS production, a lack in GSH elevation and enhanced acidic β-galactosidase activity. Interestingly, these differences were not observed in MEFs. Differential gene expression analysis of WT and KO macrophages revealed that the main pathways affected after irradiation were an up-regulation of protein secretion, TGF-β signaling and angiogenesis among other, and downregulation of Myc targets and Hedgehog signaling. These results demonstrate a key role for PTP1B in the protection against the cytotoxicity of irradiation in intact animal and in macrophages, which might be therapeutically relevant. Keywords: Protein tyrosine phosphatase, Cell viability, Irradiation sensitivity, Lethality, p53

  5. Vanillic acid derivatives from the green algae Cladophora socialis as potent protein tyrosine phosphatase 1B inhibitors.

    Science.gov (United States)

    Feng, Yunjiang; Carroll, Anthony R; Addepalli, Rama; Fechner, Gregory A; Avery, Vicky M; Quinn, Ronald J

    2007-11-01

    A novel vanillic acid derivative (1) and its sulfate adduct (2) were isolated from a green algae, Cladophora socialis. The structures of 1 and 2 were elucidated from NMR and HRESIMS experiments. Both compounds showed potent inhibitory activity against protein tyrosine phosphatase 1B (PTP1B), an enzyme involved in the regulation of insulin cell signaling. Compounds 1 and 2 had IC50 values of 3.7 and 1.7 microM, respectively.

  6. pp32 (ANP32A expression inhibits pancreatic cancer cell growth and induces gemcitabine resistance by disrupting HuR binding to mRNAs.

    Directory of Open Access Journals (Sweden)

    Timothy K Williams

    Full Text Available The expression of protein phosphatase 32 (PP32, ANP32A is low in poorly differentiated pancreatic cancers and is linked to the levels of HuR (ELAV1, a predictive marker for gemcitabine response. In pancreatic cancer cells, exogenous overexpression of pp32 inhibited cell growth, supporting its long-recognized role as a tumor suppressor in pancreatic cancer. In chemotherapeutic sensitivity screening assays, cells overexpressing pp32 were selectively resistant to the nucleoside analogs gemcitabine and cytarabine (ARA-C, but were sensitized to 5-fluorouracil; conversely, silencing pp32 in pancreatic cancer cells enhanced gemcitabine sensitivity. The cytoplasmic levels of pp32 increased after cancer cells are treated with certain stressors, including gemcitabine. pp32 overexpression reduced the association of HuR with the mRNA encoding the gemcitabine-metabolizing enzyme deoxycytidine kinase (dCK, causing a significant reduction in dCK protein levels. Similarly, ectopic pp32 expression caused a reduction in HuR binding of mRNAs encoding tumor-promoting proteins (e.g., VEGF and HuR, while silencing pp32 dramatically enhanced the binding of these mRNA targets. Low pp32 nuclear expression correlated with high-grade tumors and the presence of lymph node metastasis, as compared to patients' tumors with high nuclear pp32 expression. Although pp32 expression levels did not enhance the predictive power of cytoplasmic HuR status, nuclear pp32 levels and cytoplasmic HuR levels associated significantly in patient samples. Thus, we provide novel evidence that the tumor suppressor function of pp32 can be attributed to its ability to disrupt HuR binding to target mRNAs encoding key proteins for cancer cell survival and drug efficacy.

  7. Susceptibility of Spodoptera frugiperda and S. exigua to Bacillus thuringiensis Vip3Aa insecticidal protein.

    Science.gov (United States)

    Chakroun, Maissa; Bel, Yolanda; Caccia, Silvia; Abdelkefi-Mesrati, Lobna; Escriche, Baltasar; Ferré, Juan

    2012-07-01

    The Vip3Aa protein is an insecticidal protein secreted by Bacillus thuringiensis during the vegetative stage of growth. The activity of this protein has been tested after different steps/protocols of purification using Spodoptera frugiperda as a control insect. The results showed that the Vip3Aa protoxin was stable and retained full toxicity after being subjected to common biochemical steps used in protein purification. Bioassays with the protoxin in S. frugiperda and S. exigua showed pronounced differences in LC(50) values when mortality was measured at 7 vs. 10d. At 7d most live larvae were arrested in their development. LC(50) values of "functional mortality" (dead larvae plus larvae remaining in the first instar), measured at 7d, were similar or even lower than the LC(50) values of mortality at 10d. This strong growth inhibition was not observed when testing the trypsin-activated protein (62 kDa) in either species. S. exigua was less susceptible than S. frugiperda to the protoxin form, with LC(50) values around 10-fold higher. However, both species were equally susceptible to the trypsin-activated form. Processing of Vip3Aa protoxin to the activated form was faster with S. frugiperda midgut juice than with S. exigua midgut juice. The results strongly suggest that the differences in the rate of activation of the Vip3Aa protoxin between both species are the basis for the differences in susceptibility towards the protoxin form. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    Science.gov (United States)

    Leis, J F; Kaplan, N O

    1982-11-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid phosphatases tested from potato, wheat germ, milk, and bovine prostate did not show this degree of specificity. The plasma membrane activity also dephosphorylated phosphotyrosine histone at a much greater rate than did the other acid phosphatases. pH profiles for free O-phosphotyrosine and phosphotyrosine histone showed a shift toward physiological pH, indicating possible physiological significance. Phosphotyrosine histone dephosphorylation activity was nearly 10 times greater than that seen for phosphoserine histone dephosphorylation, and Km values were much lower for phosphotyrosine histone dephosphorylation (0.5 microM vs. 10 microM). Fluoride and zinc significantly inhibited phosphoserine histone dephosphorylation. Vanadate, on the other hand, was a potent inhibitor of phosphotyrosine histone dephosphorylation (50% inhibition at 0.5 microM) but not of phosphoserine histone. ATP stimulated phosphotyrosine histone dephosphorylation (160-250%) but inhibited phosphoserine histone dephosphorylation (95%). These results suggest the existence of a highly specific phosphotyrosine protein phosphatase activity associated with the plasma membrane of human astrocytoma.

  9. Three Pseudomonas putida FNR Family Proteins with Different Sensitivities to O2.

    Science.gov (United States)

    Ibrahim, Susan A; Crack, Jason C; Rolfe, Matthew D; Borrero-de Acuña, José Manuel; Thomson, Andrew J; Le Brun, Nick E; Schobert, Max; Stapleton, Melanie R; Green, Jeffrey

    2015-07-03

    The Escherichia coli fumarate-nitrate reduction regulator (FNR) protein is the paradigm for bacterial O2-sensing transcription factors. However, unlike E. coli, some bacterial species possess multiple FNR proteins that presumably have evolved to fulfill distinct roles. Here, three FNR proteins (ANR, PP_3233, and PP_3287) from a single bacterial species, Pseudomonas putida KT2440, have been analyzed. Under anaerobic conditions, all three proteins had spectral properties resembling those of [4Fe-4S] proteins. The reactivity of the ANR [4Fe-4S] cluster with O2 was similar to that of E. coli FNR, and during conversion to the apo-protein, via a [2Fe-2S] intermediate, cluster sulfur was retained. Like ANR, reconstituted PP_3233 and PP_3287 were converted to [2Fe-2S] forms when exposed to O2, but their [4Fe-4S] clusters reacted more slowly. Transcription from an FNR-dependent promoter with a consensus FNR-binding site in P. putida and E. coli strains expressing only one FNR protein was consistent with the in vitro responses to O2. Taken together, the experimental results suggest that the local environments of the iron-sulfur clusters in the different P. putida FNR proteins influence their reactivity with O2, such that ANR resembles E. coli FNR and is highly responsive to low concentrations of O2, whereas PP_3233 and PP_3287 have evolved to be less sensitive to O2. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  10. Pathogen exploitation of an abscisic acid- and jasmonate-inducible MAPK phosphatase and its interception by Arabidopsis immunity.

    Science.gov (United States)

    Mine, Akira; Berens, Matthias L; Nobori, Tatsuya; Anver, Shajahan; Fukumoto, Kaori; Winkelmüller, Thomas M; Takeda, Atsushi; Becker, Dieter; Tsuda, Kenichi

    2017-07-11

    Phytopathogens promote virulence by, for example, exploiting signaling pathways mediated by phytohormones such as abscisic acid (ABA) and jasmonate (JA). Some plants can counteract pathogen virulence by invoking a potent form of immunity called effector-triggered immunity (ETI). Here, we report that ABA and JA mediate inactivation of the immune-associated MAP kinases (MAPKs), MPK3 and MPK6, in Arabidopsis thaliana ABA induced expression of genes encoding the protein phosphatases 2C (PP2Cs), HAI1 , HAI2 , and HAI3 through ABF/AREB transcription factors. These three HAI PP2Cs interacted with MPK3 and MPK6 and were required for ABA-mediated MPK3/MPK6 inactivation and immune suppression. The bacterial pathogen Pseudomonas syringae pv. tomato ( Pto ) DC3000 activates ABA signaling and produces a JA-mimicking phytotoxin, coronatine (COR), that promotes virulence. We found that Pto DC3000 induces HAI1 through COR-mediated activation of MYC2, a master transcription factor in JA signaling. HAI1 dephosphorylated MPK3 and MPK6 in vitro and was necessary for COR-mediated suppression of MPK3/MPK6 activation and immunity. Intriguingly, upon ETI activation, A. thaliana plants overcame the HAI1-dependent virulence of COR by blocking JA signaling. Finally, we showed conservation of induction of HAI PP2Cs by ABA and JA in other Brassicaceae species. Taken together, these results suggest that ABA and JA signaling pathways, which are hijacked by the bacterial pathogen, converge on the HAI PP2Cs that suppress activation of the immune-associated MAPKs. Also, our data unveil interception of JA-signaling activation as a host counterstrategy against the bacterial suppression of MAPKs during ETI.

  11. Fluorescence turn-on detection of alkaline phosphatase activity based on controlled release of PEI-capped Cu nanoclusters from MnO2 nanosheets.

    Science.gov (United States)

    Zhang, Yunyi; Li, Yongxin; Zhang, Cuiyun; Zhang, Qingfeng; Huang, Xinan; Yang, Meiding; Shahzad, Sohail Anjum; Lo, Kenneth Kam-Wing; Yu, Cong; Jiang, Shichun

    2017-08-01

    A fluorescence turn-on assay for alkaline phosphatase (ALP) activity is developed through the controlled release of polyethyleneimine-capped copper nanoclusters (PEI-capped CuNCs) from the MnO 2 nanosheets. In an aqueous solution, the positively charged PEI-capped CuNCs could be adsorbed onto the surface of the negatively charged MnO 2 nanosheets. Such adsorption through favorable electrostatic interactions could efficiently quench the nanocluster fluorescence emission via resonance energy transfer from the PEI-capped CuNCs to the MnO 2 nanosheets. 2-Phospho-L-ascorbic acid (AAP) could be hydrolyzed to L-ascorbic acid (AA) in the presence of ALP. AA could reduce MnO 2 into Mn 2+ and trigger the disintegration of the MnO 2 nanosheets. As a result, the CuNCs were released and the quenched fluorescence was recovered efficiently. The detection strategy is simple, inexpensive, sensitive, selective, with low toxicity, and has better biocompatibility. The newly fabricated biosensor for ALP activity will potentially make it a robust candidate for numerous biological and biomedical applications.

  12. A paralogue of the phosphomutase-like gene family in Candida glabrata, CgPmu2, gained broad-range phosphatase activity due to a small number of clustered substitutions.

    Science.gov (United States)

    Orlando, Kelly A; Iosue, Christine L; Leone, Sarah G; Davies, Danielle L; Wykoff, Dennis D

    2015-10-15

    Inorganic phosphate is required for a range of cellular processes, such as DNA/RNA synthesis and intracellular signalling. The phosphate starvation-inducible phosphatase activity of Candida glabrata is encoded by the gene CgPMU2 (C. glabrata phosphomutase-like protein). CgPMU2 is part of a three-gene family (∼75% identical) created through gene duplication in the C. glabrata clade; only CgPmu2 is a PHO-regulated broad range acid phosphatase. We identified amino acids that confer broad range phosphatase activity on CgPmu2 by creating fusions of sections of CgPMU2 with CgPMU1, a paralogue with little broad range phosphatase activity. We used site-directed mutagenesis on various fusions to sequentially convert CgPmu1 to CgPmu2. Based on molecular modelling of the Pmu proteins on to a histidine phosphatase crystal structure, clusters of amino acids were found in two distinct regions that were able to confer phosphatase activity. Substitutions in these two regions together conferred broad phosphatase activity on CgPmu1. Interestingly, one change is a histidine adjacent to the active site histidine of CgPmu2 and it exhibits a novel ability to partially replace the conserved active site histidine in CgPmu2. Additionally, a second amino acid change was able to confer nt phosphatase activity to CgPmu1, suggesting single amino acid changes neofunctionalize CgPmu2. © 2015 Authors; published by Portland Press Limited.

  13. Overexpression, purification, characterization and preliminary crystallographic study of phosphoglycolate phosphatase from Shigella flexneri 2a strain 301

    International Nuclear Information System (INIS)

    Liu, Heli; Zhou, Huina; Zhu, Deyu; Bi, Ruchang

    2008-01-01

    Recombinant phosphoglycolate phosphatase from S. flexneri was overexpressed, purified, characterized and crystallized using the hanging-drop vapour-diffusion method. SeMet-labelled protein was also prepared and was crystallized for phase determination using the MAD technique. Phosphoglycolate phosphatase has a salvage function in the metabolism of the 2-phosphoglycolate formed during bacterial DNA repair. In order to better understand its dimerization behaviour, the influence of metal ions on its activity and its catalytic mechanism at the molecular level, recombinant phosphoglycolate phosphatase from Shigella flexneri was overexpressed, purified, characterized and crystallized by the hanging-drop vapour-diffusion method at 291 K using polyethylene glycol 3500 as a precipitant and zinc acetate as an additive. The crystals belonged to space group R3, with unit-cell parameters a = 88.1, b = 88.1, c = 259.2 Å, corresponding to the presence of two molecules in the asymmetric unit. SeMet-labelled protein was also prepared and crystallized for use in phase determination. Initial structure determination using the multiwavelength anomalous dispersion (MAD) method clearly revealed that SfPGPase bears an α-helical cap domain that differs from that of a previously reported orthologue

  14. PLATINUM SENSITIVE 2 LIKE impacts growth, root morphology, seed set, and stress responses.

    Directory of Open Access Journals (Sweden)

    Amr R A Kataya

    Full Text Available Eukaryotic protein phosphatase 4 (PP4 is a PP2A-type protein phosphatase that is part of a conserved complex with regulatory factors PSY2 and PP4R2. Various lines of Arabidopsis thaliana with mutated PP4 subunit genes were constructed to study the so far completely unknown functions of PP4 in plants. Mutants with knocked out putative functional homolog of the PSY2 LIKE (PSY2L gene were dwarf and bushy, while plants with knocked out PP4R2 LIKE (PP4R2L looked very similar to WT. The psy2l seedlings had short roots with disorganized morphology and impaired meristem. Seedling growth was sensitive to the genotoxin cisplatin. Global transcript analysis (RNA-seq of seedlings and rosette leaves revealed several groups of genes, shared between both types of tissues, strongly influenced by knocked out PSY2L. Receptor kinases, CRINKLY3 and WAG1, important for growth and development, were down-regulated 3-7 times. EUKARYOTIC ELONGATION FACTOR5A1 was down-regulated 4-6 fold. Analysis of hormone sensitive genes indicated that abscisic acid levels were high, while auxin, cytokinin and gibberellic acid levels were low in psy2l. Expression of specific transcription factors involved in regulation of anthocyanin synthesis were strongly elevated, e.g. the master regulator PAP1, and intriguingly TT8, which is otherwise mainly expressed in seeds. The psy2l mutants accumulated anthocyanins under conditions where WT did not, pointing to PSY2L as a possible upstream negative regulator of PAP1 and TT8. Expression of the sugar-phosphate transporter GPT2, important for cellular sugar and phosphate homeostasis, was enhanced 7-8 times. Several DNA damage response genes, including the cell cycle inhibitor gene WEE1, were up-regulated in psy2l. The activation of DNA repair signaling genes, in combination with phenotypic traits showing aberrant root meristem and sensitivity to the genotoxic cisplatin, substantiate the involvement of Arabidopsis PSY2L in maintenance of genome

  15. Modulation of Spc1 stress-activated protein kinase activity by methylglyoxal through inhibition of protein phosphatase in the fission yeast Schizosaccharomyces pombe

    International Nuclear Information System (INIS)

    Takatsume, Yoshifumi; Izawa, Shingo; Inoue, Yoshiharu

    2007-01-01

    Methylglyoxal, a ubiquitous metabolite derived from glycolysis has diverse physiological functions in yeast cells. Previously, we have reported that extracellularly added methylglyoxal activates Spc1, a stress-activated protein kinase (SAPK), in the fission yeast Schizosaccharomyces pombe [Y. Takatsume, S. Izawa, Y. Inoue, J. Biol. Chem. 281 (2006) 9086-9092]. Phosphorylation of Spc1 by treatment with methylglyoxal in S. pombe cells defective in glyoxalase I, an enzyme crucial for the metabolism of methylglyoxal, continues for a longer period than in wild-type cells. Here we show that methylglyoxal inhibits the activity of the protein phosphatase responsible for the dephosphorylation of Spc1 in vitro. In addition, we found that methylglyoxal inhibits human protein tyrosine phosphatase 1B (PTP1B) also. We propose a model for the regulation of the activity of the Spc1-SAPK signaling pathway by methylglyoxal in S. pombe

  16. Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

    Science.gov (United States)

    Galic, Sandra; Hauser, Christine; Kahn, Barbara B.; Haj, Fawaz G.; Neel, Benjamin G.; Tonks, Nicholas K.; Tiganis, Tony

    2005-01-01

    The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell. PMID:15632081

  17. The function of Shp2 tyrosine phosphatase in the dispersal of acetylcholine receptor clusters

    Directory of Open Access Journals (Sweden)

    Madhavan Raghavan

    2008-07-01

    Full Text Available Abstract Background A crucial event in the development of the vertebrate neuromuscular junction (NMJ is the postsynaptic enrichment of muscle acetylcholine (ACh receptors (AChRs. This process involves two distinct steps: the local clustering of AChRs at synapses, which depends on the activation of the muscle-specific receptor tyrosine kinase MuSK by neural agrin, and the global dispersal of aneural or "pre-patterned" AChR aggregates, which is triggered by ACh or by synaptogenic stimuli. We and others have previously shown that tyrosine phosphatases, such as the SH2 domain-containing phosphatase Shp2, regulate AChR cluster formation in muscle cells, and that tyrosine phosphatases also mediate the dispersal of pre-patterned AChR clusters by synaptogenic stimuli, although the specific phosphatases involved in this latter step remain unknown. Results Using an assay system that allows AChR cluster assembly and disassembly to be studied separately and quantitatively, we describe a previously unrecognized role of the tyrosine phosphatase Shp2 in AChR cluster disassembly. Shp2 was robustly expressed in embryonic Xenopus muscle in vivo and in cultured myotomal muscle cells, and treatment of the muscle cultures with an inhibitor of Shp2 (NSC-87877 blocked the dispersal of pre-patterned AChR clusters by synaptogenic stimuli. In contrast, over-expression in muscle cells of either wild-type or constitutively active Shp2 accelerated cluster dispersal. Significantly, forced expression in muscle of the Shp2-activator SIRPα1 (signal regulatory protein α1 also enhanced the disassembly of AChR clusters, whereas the expression of a truncated SIRPα1 mutant that suppresses Shp2 signaling inhibited cluster disassembly. Conclusion Our results suggest that Shp2 activation by synaptogenic stimuli, through signaling intermediates such as SIRPα1, promotes the dispersal of pre-patterned AChR clusters to facilitate the selective accumulation of AChRs at developing NMJs.

  18. Measurement of long-range correlations in pp collisions characterized by presence of a Z boson with the ATLAS detector

    CERN Document Server

    Cole, Brian; The ATLAS collaboration

    2018-01-01

    Recent measurements of correlations between two particles separated in pseudorapidity and azimuthal angles have shown striking similarities between results obtained in pp, p+A and A+A collision systems. In the pp collision system, unlike in p+A and A+A collisions, the strength of the correlations, quantified by the anisotropy parameter v2, shows little dependence on the observed charged-particle multiplicity. Recent theoretical models suggest that this can result from an intrinsically weak correlation between the charged-particle multiplicity and the impact parameter of the pp collision. An independent handle on the impact parameter can be obtained in principle by requiring the presence of a hard-scattering process in the collision. This talk presents the first measurement of two-particle correlations in pp collisions with a presence of Z boson identified via its μμ decay channel. The analysis uses ATLAS data recorded with nominal pp luminosity with high pileup. A new procedure is used to correct for the co...

  19. Experimental transmission of AA amyloidosis by injecting the AA amyloid protein into interleukin-1 receptor antagonist knockout (IL-1raKO) mice.

    Science.gov (United States)

    Watanabe, K; Uchida, K; Chambers, J K; Tei, M; Shoji, A; Ushio, N; Nakayama, H

    2015-05-01

    The incidence of AA amyloidosis is high in humans with rheumatoid arthritis and several animal species, including cats and cattle with prolonged inflammation. AA amyloidosis can be experimentally induced in mice using severe inflammatory stimuli and a coinjection of AA amyloid; however, difficulties have been associated with transmitting AA amyloidosis to a different animal species, and this has been attributed to the "species barrier." The interleukin-1 receptor antagonist knockout (IL-1raKO) mouse, a rodent model of human rheumatoid arthritis, has been used in the transmission of AA amyloid. When IL-1raKO and BALB/c mice were intraperitoneally injected with mouse AA amyloid together with a subcutaneous pretreatment of 2% AgNO3, all mice from both strains that were injected with crude or purified murine AA amyloid developed AA amyloidosis. However, the amyloid index, which was determined by the intensity of AA amyloid deposition, was significantly higher in IL-1raKO mice than in BALB/c mice. When IL-1raKO and BALB/c mice were injected with crude or purified bovine AA amyloid together with the pretreatment, 83% (5/6 cases) and 38% (3/8 cases) of IL-1raKO mice and 17% (1/6 cases) and 0% (0/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. Similarly, when IL-1raKO and BALB/c mice were injected with crude or purified feline AA amyloid, 33% (2/6 cases) and 88% (7/8 cases) of IL-1raKO mice and 0% (0/6 cases) and 29% (2/6 cases) of BALB/c mice, respectively, developed AA amyloidosis. These results indicated that IL-1raKO mice are a useful animal model for investigating AA amyloidogenesis. © The Author(s) 2014.

  20. Comparative proteomic analysis of Salmonella enterica serovar Typhimurium ppGpp-deficient mutant to identify a novel virulence protein required for intracellular survival in macrophages

    Directory of Open Access Journals (Sweden)

    Kumagai Yoshinori

    2010-12-01

    Full Text Available Abstract Background The global ppGpp-mediated stringent response in pathogenic bacteria plays an important role in the pathogenesis of bacterial infections. In Salmonella enterica serovar Typhimurium (S. Typhimurium, several genes, including virulence genes, are regulated by ppGpp when bacteria are under the stringent response. To understand the control of virulence genes by ppGpp in S. Typhimurium, agarose 2-dimensional electrophoresis (2-DE combined with mass spectrometry was used and a comprehensive 2-DE reference map of amino acid-starved S. Typhimurium strain SH100, a derivative of ATCC 14028, was established. Results Of the 366 examined spots, 269 proteins were successfully identified. The comparative analysis of the wild-type and ppGpp0 mutant strains revealed 55 proteins, the expression patterns of which were affected by ppGpp. Using a mouse infection model, we further identified a novel virulence-associated factor, STM3169, from the ppGpp-regulated and Salmonella-specific proteins. In addition, Salmonella strains carrying mutations in the gene encoding STM3169 showed growth defects and impaired growth within macrophage-like RAW264.7 cells. Furthermore, we found that expression of stm3169 was controlled by ppGpp and SsrB, a response regulator of the two-component system located on Salmonella pathogenicity island 2. Conclusions A proteomic approach using a 2-DE reference map can prove a powerful tool for analyzing virulence factors and the regulatory network involved in Salmonella pathogenesis. Our results also provide evidence of a global response mediated by ppGpp in S. enterica.

  1. Phosphoglycolate phosphatase and 2,3-diphosphoglycerate in red cells of normal and anemic subjects.

    Science.gov (United States)

    Somoza, R; Beutler, E

    1983-10-01

    Red cell phosphoglycolate phosphatase (PGP) and 2,3-diphosphoglycerate (2,3-DPG) were investigated in normal and anemic patients and rabbits. In hemolytic anemia and blood-loss anemia, characterized by a young red cell population, there was an increase in both phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. In aplastic anemia, the phosphoglycolate phosphatase activity was normal, but the 2,3-diphosphoglycerate values were nonetheless increased. Thus, no relationship was found between phosphoglycolate phosphatase activity and 2,3-diphosphoglycerate levels. The lack of correlation between the activity of phosphoglycolate phosphatase and 2,3-DPG levels suggests that modulation of phosphoglycolate phosphatase activity does not control the level of 2,3-DPG in erythrocytes.

  2. Tyrosine phosphorylation in T cells is regulated by phosphatase activity: studies with phenylarsine oxide.

    OpenAIRE

    Garcia-Morales, P; Minami, Y; Luong, E; Klausner, R D; Samelson, L E

    1990-01-01

    Activation of T cells induces rapid tyrosine phosphorylation on the T-cell receptor zeta chain and other substrates. These phosphorylations can be regulated by a number of protein-tyrosine kinases (ATP: protein-tyrosine O-phosphotransferase, EC 2.7.1.112) and protein-tyrosine-phosphatases (protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48). In this study, we demonstrate that phenylarsine oxide can inhibit tyrosine phosphatases while leaving tyrosine kinase function intact. We use this ...

  3. Phytotoxicity associated to microcystins: a review

    Directory of Open Access Journals (Sweden)

    MC Bittencourt-Oliveira

    Full Text Available Microcystins (MC are the most studied toxins of cyanobacteria since they are widely distributed and account for several cases of human and animal poisoning, being potent inhibitors of the serine/threonine protein phosphatases 1 (PP1 and 2A (PP2A. The phosphatases PP1 and PP2A are also present in plants, which may also suffer adverse effects due to the inhibition of these enzymes. In aquatic plants, biomass reduction is usually observed after absorption of cyanotoxins, which can bioaccumulate in its tissues. In terrestrial plants, the effects caused by microcystins vary from inhibition to stimulation as the individuals develop from seedling to adult, and include reduction of protein phosphatases 1 and 2A, oxidative stress, decreased photosynthetic activity and even cell apoptosis, as well as bioaccumulation in plant tissues. Thus, the irrigation of crop plants by water contaminated with microcystins is not only an economic problem but becomes a public health issue because of the possibility of food contamination, and this route of exposure requires careful monitoring by the responsible authorities.

  4. Tailor-Made Protein Tyrosine Phosphatases: In Vitro Site-Directed Mutagenesis of PTEN and PTPRZ-B

    NARCIS (Netherlands)

    Luna, S.; Mingo, J.; Aurtenetxe, O.; Blanco, L.; Amo, L.; Schepens, J.; Hendriks, W.J.A.J.; Pulido, R.

    2016-01-01

    In vitro site-directed mutagenesis (SDM) of protein tyrosine phosphatases (PTPs) is a commonly used approach to experimentally analyze PTP functions at the molecular and cellular level and to establish functional correlations with PTP alterations found in human disease. Here, using the

  5. Mutational analysis of the pumpkin (Cucurbita maxima) phloem exudate lectin, PP2 reveals Ser-104 is crucial for carbohydrate binding.

    Science.gov (United States)

    Bobbili, Kishore Babu; Bandari, Shyam; Grobe, Kay; Swamy, Musti J

    2014-07-18

    The pumpkin phloem lectin (PP2) is an RNA-binding, defense-related, chitooligosaccharide-specific, homodimeric lectin of Mr 48 kDa expressed at high concentrations in the sieve elements and companion cells of pumpkin (Cucurbita maxima). In the present study, PP2 was expressed in the methylotrophic yeast Pichia pastoris with the Saccharomyces α-factor sequence to direct the recombinant protein into the secretory pathway as a prerequisite for unimpaired folding and posttranslational glycosylation of recombinant PP2. Previous computational modeling and ligand docking studies predicted a putative chitooligosaccharide-binding site on the PP2 surface, which was divided into three subsites, with two amino acid residues in each subsite identified as possible candidates for interaction with chitooligosaccharides (CHOs). In this work, mutational analysis and hemagglutination assays were employed to verify the role of the predicted residues in the carbohydrate binding activity of the protein. The results obtained revealed that mutation of Ser-104 to Ala (S104A) at subsite-2 resulted in about 90% loss of agglutination activity of the protein, indicating that Ser-104 is crucial for the binding of CHOs to PP2. Also, L100A (at subsite-1) and K200A (at subsite-3) independently decreased the lectin activity by about 40%, indicating that these two residues also contribute significantly to sugar binding by PP2. Together, these findings confirm that all the three subsites contribute to varying degrees toward PP2-carbohydrate interaction, and confirm the validity of the computational model, as proposed earlier. Copyright © 2014 Elsevier Inc. All rights reserved.

  6. Cloning and characterization of R-PTP-kappa, a new member of the receptor protein tyrosine phosphatase family with a proteolytically cleaved cellular adhesion molecule-like extracellular region

    DEFF Research Database (Denmark)

    Jiang, Y P; Wang, H; D'Eustachio, P

    1993-01-01

    We describe a new member of the receptor protein tyrosine phosphatase family, R-PTP-kappa, cDNA cloning predicts that R-PTP-kappa is synthesized from a precursor protein of 1,457 amino acids. Its intracellular domain displays the classical tandemly repeated protein tyrosine phosphatase homology, ...

  7. Phosphorylation of stress protein pp80 is related to promotion of transformation

    International Nuclear Information System (INIS)

    Smith, B.M.; Gindhart, T.D.; Hirano, K.; Colburn, N.H.

    1986-01-01

    The JB6 mouse epidermal cell system is an in vitro model of late stage promotion, and includes cell lines sensitive (P+) or resistant (P-) to phorbol ester-induced anchorage independent transformation, and transformed (T/sub x/) lines. Certain promoter-induced changes in phosphoproteins, identified by gel electrophoresis, are unique to cells of one phenotype, and occur only with specific promoters. An 80Kd protein is inversely correlated with phenotype: P- cells have a constitutively higher level (p 35 S-methionine. pp80 shares properties with the 80Kd heat stress protein: molecular weight relative abundance, and isoelectric point (4.5). Pharmacological analogs of calcium, the lanthanides, promote transformation of JB6 cells, but have no effect on phosphorylation of the 80Kd protein. If pp80 is on the promotion pathway, it is limited to a specific subset of transformation promoters

  8. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    DEFF Research Database (Denmark)

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.

    2000-01-01

    Molecular dynamics simulations of protein tyrosine phosphatase 1B (PTP1B) complexed with the phosphorylated peptide substrate DADEpYL and the free substrate have been conducted to investigate 1) the physical forces involved in substrate-protein interactions, 2) the importance of enzyme...... to substrate binding. Based on essential dynamics analysis of the PTP1B/DADEpYL trajectory, it is shown that internal motions in the binding pocket occur in a subspace of only a few degrees of freedom. in particular, relatively large flexibilities are observed along several eigenvectors in the segments: Arg(24...... for catalysis. Analysis of the individual enzyme-substrate interaction energies revealed that mainly electrostatic forces contribute to binding. Indeed, calculation of the electrostatic field of the enzyme reveals that only the field surrounding the binding pocket is positive, while the remaining protein...

  9. Identification of okadaic acid-induced phosphorylation events by a mass spectrometry approach

    International Nuclear Information System (INIS)

    Hill, Jennifer J.; Callaghan, Deborah A.; Ding Wen; Kelly, John F.; Chakravarthy, Balu R.

    2006-01-01

    Okadaic acid (OA) is a widely used small-molecule phosphatase inhibitor that is thought to selectively inhibit protein phosphatase 2A (PP2A). Multiple studies have demonstrated that PP2A activity is compromised in Brains of Alzheimer's disease patients. Thus, we set out to determine changes in phosphorylation that occur upon OA treatment of neuronal cells. Utilizing isotope-coded affinity tags and mass spectrometry analysis, we determined the relative abundance of proteins in a phosphoprotein enriched fraction from control and OA-treated primary cortical neurons. We identified many proteins whose phosphorylation state is regulated by OA, including glycogen synthase kinase 3β, collapsin-response mediator proteins (DRP-2, DPYSL-5, and CRMP-4), and the B subunit of PP2A itself. Most interestingly, we have found that complexin 2, an important regulator of neurotransmitter release and synaptic plasticity, is phosphorylated at serine 93 upon OA treatment of neurons. This is First report of a phosphorylation site on complexin 2

  10. Heterogenous expression of Pyrus pyrifolia PpCAD2 and PpEXP2 in tobacco impacts lignin accumulation in transgenic plants.

    Science.gov (United States)

    Wang, Yuling; Zhang, Xinfu; Yang, Shaolan; Wang, Caihong; Lu, Guilong; Wang, Ran; Yang, Yingjie; Li, Dingli

    2017-12-30

    Lignin, a natural macromolecular compound, plays an important role in the texture and taste of fruit. Hard end is a physiological disorder of pear fruit, in which the level of lignification in fruit tissues is dramatically elevated. Cinnamyl alcohol dehydrogenase and expansin genes (PpCAD2 and PpEXP2, respectively) exhibit higher levels of expression in 'Whangkeumbae' (Pyrus pyrifolia) pear fruit exhibiting this physiological disorder, relative to control fruit without symptoms. These genes were isolated from pear fruit and subsequently expressed in tobacco (Nicotiana tabacum) to investigate their function. Histochemical staining for lignin revealed that the degree of lignification in leaf veins and stem tissues increased in plants transformed with sense constructs and decreased in plants transformed with antisense constructs of PpCAD2. The expression of native NtCADs was also inhibited in the antisense PpCAD2 transgenic tobacco. Sense and antisense PpCAD2 transgenic tobacco exhibited an 86.7% increase and a 60% decrease in CAD activity, respectively, accompanied by a complementary response in lignin content in root tissues. The basal portion of the stem in PpEXP2 transgenic tobacco was bent and highly lignified. Additionally, the level of cellulose also increased in the stem of PpEXP2 transgenic tobacco. Collectively, these results suggested that PpCAD2 and PpEXP2 genes play a significant role in lignin accumulation in transgenic tobacco plants, and it is inferred that these two genes may also participate in the increased lignification observed in hard end pear fruit. Copyright © 2017. Published by Elsevier B.V.

  11. Bone Morphogenic Protein 4-Smad-Induced Upregulation of Platelet-Derived Growth Factor AA Impairs Endothelial Function.

    Science.gov (United States)

    Hu, Weining; Zhang, Yang; Wang, Li; Lau, Chi Wai; Xu, Jian; Luo, Jiang-Yun; Gou, Lingshan; Yao, Xiaoqiang; Chen, Zhen-Yu; Ma, Ronald Ching Wan; Tian, Xiao Yu; Huang, Yu

    2016-03-01

    Bone morphogenic protein 4 (BMP4) is an important mediator of endothelial dysfunction in cardio-metabolic diseases, whereas platelet-derived growth factors (PDGFs) are major angiogenic and proinflammatory mediator, although the functional link between these 2 factors is unknown. The present study investigated whether PDGF mediates BMP4-induced endothelial dysfunction in diabetes mellitus. We generated Ad-Bmp4 to overexpress Bmp4 and Ad-Pdgfa-shRNA to knockdown Pdgfa in mice through tail intravenous injection. SMAD4-shRNA lentivirus, SMAD1-shRNA, and SMAD5 shRNA adenovirus were used for knockdown in human and mouse endothelial cells. We found that PDGF-AA impaired endothelium-dependent vasodilation in aortas and mesenteric resistance arteries. BMP4 upregulated PDGF-AA in human and mouse endothelial cells, which was abolished by BMP4 antagonist noggin or knockdown of SMAD1/5 or SMAD4. BMP4-impared relaxation in mouse aorta was also ameliorated by PDGF-AA neutralizing antibody. Tail injection of Ad-Pdgfa-shRNA ameliorates endothelial dysfunction induced by Bmp4 overexpression (Ad-Bmp4) in vivo. Serum PDGF-AA was elevated in both diabetic patients and diabetic db/db mice compared with nondiabetic controls. Pdgfa-shRNA or Bmp4-shRNA adenovirus reduced serum PDGF-AA concentration in db/db mice. PDGF-AA neutralizing antibody or tail injection with Pdgfa-shRNA adenovirus improved endothelial function in aortas and mesenteric resistance arteries from db/db mice. The effect of PDGF-AA on endothelial function in mouse aorta was also inhibited by Ad-Pdgfra-shRNA to inhibit PDGFRα. The present study provides novel evidences to show that PDGF-AA impairs endothelium-dependent vasodilation and PDGF-AA mediates BMP4-induced adverse effect on endothelial cell function through SMAD1/5- and SMAD4-dependent mechanisms. Inhibition of PGDF-AA ameliorates vascular dysfunction in diabetic mice. © 2016 American Heart Association, Inc.

  12. Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus

    Directory of Open Access Journals (Sweden)

    Gilbert Christophe

    2008-04-01

    Full Text Available Abstract Background Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS biosynthesis loci of numerous lactic acid bacteria genomes. Results The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP, with an optimal activity in presence of bovine serum albumin (BSA 1% at pH 7.3 and a temperature of 75°C. At 50°C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S showed close to 20% increase in phosphatase activity. Conclusion These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.

  13. Insights into the Structure of the Vip3Aa Insecticidal Protein by Protease Digestion Analysis

    Directory of Open Access Journals (Sweden)

    Yolanda Bel

    2017-04-01

    Full Text Available Vip3 proteins are secretable proteins from Bacillus thuringiensis whose mode of action is still poorly understood. In this study, the activation process for Vip3 proteins was closely examined in order to better understand the Vip3Aa protein stability and to shed light on its structure. The Vip3Aa protoxin (of 89 kDa was treated with trypsin at concentrations from 1:100 to 120:100 (trypsin:Vip3A, w:w. If the action of trypsin was not properly neutralized, the results of SDS-PAGE analysis (as well as those with Agrotis ipsilon midgut juice equivocally indicated that the protoxin could be completely processed. However, when the proteolytic reaction was efficiently stopped, it was revealed that the protoxin was only cleaved at a primary cleavage site, regardless of the amount of trypsin used. The 66 kDa and the 19 kDa peptides generated by the proteases co-eluted after gel filtration chromatography, indicating that they remain together after cleavage. The 66 kDa fragment was found to be extremely resistant to proteases. The trypsin treatment of the protoxin in the presence of SDS revealed the presence of secondary cleavage sites at S-509, and presumably at T-466 and V-372, rendering C-terminal fragments of approximately 29, 32, and 42 kDa, respectively. The fact that the predicted secondary structure of the Vip3Aa protein shows a cluster of beta sheets in the C-terminal region of the protein might be the reason behind the higher stability to proteases compared to the rest of the protein, which is mainly composed of alpha helices.

  14. Asperentin B, a New Inhibitor of the Protein Tyrosine Phosphatase 1B.

    Science.gov (United States)

    Wiese, Jutta; Aldemir, Hülya; Schmaljohann, Rolf; Gulder, Tobias A M; Imhoff, Johannes F

    2017-06-21

    In the frame of studies on secondary metabolites produced by fungi from deep-sea environments we have investigated inhibitors of enzymes playing key roles in signaling cascades of biochemical pathways relevant for the treatment of diseases. Here we report on a new inhibitor of the human protein tyrosine phosphatase 1B (PTP1B), a target in the signaling pathway of insulin. A new asperentin analog is produced by an Aspergillus sydowii strain isolated from the sediment of the deep Mediterranean Sea. Asperentin B ( 1 ) contains an additional phenolic hydroxy function at C-6 and exhibits an IC 50 value against PTP1B of 2 μM in vitro, which is six times stronger than the positive control, suramin. Interestingly, asperentin ( 2 ) did not show any inhibition of this enzymatic activity. Asperentin B ( 1 ) is discussed as possible therapeutic agents for type 2 diabetes and sleeping sickness.

  15. Finding the smoking gun: protein tyrosine phosphatases as tools and targets of unicellular microorganisms and viruses.

    Science.gov (United States)

    Heneberg, P

    2012-01-01

    Protein tyrosine phosphatases (PTPs) are increasingly recognized as important effectors of host-pathogen interactions. Since Guan and Dixon reported in 1990 that phosphatase YopH serves as an essential virulence determinant of Yersinia, the field shifted significantly forward, and dozens of PTPs were identified in various microorganisms and even in viruses. The discovery of extensive tyrosine signaling networks in non-metazoan organisms refuted the moth-eaten paradigm claiming that these organisms rely exclusively on phosphoserine/phosphothreonine signaling. Similarly to humans, phosphotyrosine signaling is thought to comprise a small fraction of total protein phosphorylation, but plays a disproportionately important role in cell-cycle control, differentiation, and invasiveness. Here we summarize the state-of-art knowledge on PTPs of important non-metazoan pathogens (Listeria monocytogenes, Staphylococcus aureus, Porphyromonas gingivalis, Caulobacter crescentus, Yersinia, Synechocystis, Leishmania, Plasmodium falciparum, Entamoeba histolytica, etc.), and focus also at the microbial proteins affecting directly or indirectly the PTPs of the host (Mycobacterium tuberculosis MTSA-10, Bacillus anthracis anthrax toxin, streptococcal β protein, Helicobacter pylori CagA and VacA, Leishmania GP63 and EF-1α, Plasmodium hemozoin, etc.). This is the first review summarizing the knowledge on biological activity and pharmacological inhibition of non-metazoan PTPs, with the emphasis of those important in host-pathogen interactions. Targeting of numerous non-metazoan PTPs is simplified by the fact that they act either as ectophosphatases or are secreted outside of the pathogen. Interfering with tyrosine phosphorylation represents a powerful pharmacologic approach, and even though the PTP inhibitors are difficult to develop, lifting the fog of phosphatase inhibition is of the great market potential and further clinical impact.

  16. Natural compounds as a source of protein tyrosine phosphatase inhibitors : Application to the rational design of small-molecule derivatives

    NARCIS (Netherlands)

    Ferreira, Carmen V.; Justo, Giselle Z.; Souza, Ana C. S.; Queiroz, Karla C. S.; Zambuzzi, William F.; Aoyama, Hiroshi; Peppelenbosch, Maikel P.

    2006-01-01

    Reversible phosphorylation of tyrosine residues is a key regulatory mechanism for numerous cellular events. Protein tyrosine kinases and protein tyrosine phosphatases (PTPs) have a pivotal role in regulating both normal cell physiology and pathophysiology. Accordingly, deregulated activity of both

  17. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    DEFF Research Database (Denmark)

    Cook, Naomi L.; Moeke, Cassidy H.; Fantoni, Luca I.

    2016-01-01

    Phosphorylation of protein tyrosine residues is critical to cellular processes, and is regulated by kinases and phosphatases (PTPs). PTPs contain a redox-sensitive active site Cys residue, which is readily oxidized. Myeloperoxidase, released from activated leukocytes, catalyzes thiocyanate ion (SCN...

  18. Phosphatase activity of Poa pratensis seeds. III. Effect of fluoride, citrate, urea and other substances on the activity of acid phosphatase Ia2 and Ia3

    Directory of Open Access Journals (Sweden)

    Irena Lorenc-Kubis

    2015-01-01

    Full Text Available Effects of fluoride, citrate, urea and other substances on the activity of acid phosphatase a2 and a3 toward p-nitrophenylphosphate and phenylphosphate were investigated. Both enzymes were inhibited by fluoride, p-chloromercuribenzoate and oxalate. Fluoride inhibited acid phosphatase a2 noncorapetitively with p-mitrophenylphosphate, whereas acid phosphatase a3 showed inhibition of mixed type. Hydrolysis of phenylphosphate by both acid phosphatases was activated by citrate. Cytosine and uridine inhibited the activity of phosphatase a2 toward p-nitrophenylphosphate and phenylphosphate, but no effect was observed in case of acid phosphatase a3. After 30 min. incubation with 4 M urea both enzymes lost about 30% of activity.

  19. Protein phosphatase PPM1G regulates protein translation and cell growth by dephosphorylating 4E binding protein 1 (4E-BP1).

    Science.gov (United States)

    Liu, Jianyu; Stevens, Payton D; Eshleman, Nichole E; Gao, Tianyan

    2013-08-09

    Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.

  20. FTY720 inhibits mesothelioma growth in vitro and in a syngeneic mouse model.

    Science.gov (United States)

    Szymiczek, Agata; Pastorino, Sandra; Larson, David; Tanji, Mika; Pellegrini, Laura; Xue, Jiaming; Li, Shuangjing; Giorgi, Carlotta; Pinton, Paolo; Takinishi, Yasutaka; Pass, Harvey I; Furuya, Hideki; Gaudino, Giovanni; Napolitano, Andrea; Carbone, Michele; Yang, Haining

    2017-03-15

    Malignant mesothelioma (MM) is a very aggressive type of cancer, with a dismal prognosis and inherent resistance to chemotherapeutics. Development and evaluation of new therapeutic approaches is highly needed. Immunosuppressant FTY720, approved for multiple sclerosis treatment, has recently raised attention for its anti-tumor activity in a variety of cancers. However, its therapeutic potential in MM has not been evaluated yet. Cell viability and anchorage-independent growth were evaluated in a panel of MM cell lines and human mesothelial cells (HM) upon FTY720 treatment to assess in vitro anti-tumor efficacy. The mechanism of action of FTY720 in MM was assessed by measuring the activity of phosphatase protein 2A (PP2A)-a major target of FTY720. The binding of the endogenous inhibitor SET to PP2A in presence of FTY720 was evaluated by immunoblotting and immunoprecipitation. Signaling and activation of programmed cell death were evaluated by immunoblotting and flow cytometry. A syngeneic mouse model was used to evaluate anti-tumor efficacy and toxicity profile of FTY720 in vivo. We show that FTY720 significantly suppressed MM cell viability and anchorage-independent growth without affecting normal HM cells. FTY720 inhibited the phosphatase activity of PP2A by displacement of SET protein, which appeared overexpressed in MM, as compared to HM cells. FTY720 promoted AKT dephosphorylation and Bcl-2 degradation, leading to induction of programmed cell death, as demonstrated by caspase-3 and PARP activation, as well as by cytochrome c and AIF intracellular translocation. Moreover, FTY720 administration in vivo effectively reduced tumor burden in mice without apparent toxicity. Our preclinical data indicate that FTY720 is a potentially promising therapeutic agent for MM treatment.

  1. SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

    Science.gov (United States)

    Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

    2013-08-15

    The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

  2. Loss of Function Studies in Mice and Genetic Association Link Receptor Protein Tyrosine Phosphatase a to Schizophrenia

    DEFF Research Database (Denmark)

    Takahashi, Nagahide; Nielsen, Karin Sandager; Aleksic, Branko

    2011-01-01

    Solid evidence links schizophrenia (SZ) susceptibility to neurodevelopmental processes involving tyrosine phosphorylation-mediated signaling. Mouse studies implicate the Ptpra gene, encoding protein tyrosine phosphatase RPTPa, in the control of radial neuronal migration, cortical cytoarchitecture...

  3. Cloning and expression of a widely expressed receptor tyrosine phosphatase

    DEFF Research Database (Denmark)

    Sap, J; D'Eustachio, P; Givol, D

    1990-01-01

    We describe the identification of a widely expressed receptor-type (transmembrane) protein tyrosine phosphatase (PTPase; EC 3.1.3.48). Screening of a mouse brain cDNA library under low-stringency conditions with a probe encompassing the intracellular (phosphatase) domain of the CD45 lymphocyte...... antigen yielded cDNA clones coding for a 794-amino acid transmembrane protein [hereafter referred to as receptor protein tyrosine phosphatase alpha (R-PTP-alpha)] with an intracellular domain displaying clear homology to the catalytic domains of CD45 and LAR (45% and 53%, respectively). The 142-amino acid...

  4. TCTEX1D4, a novel protein phosphatase 1 interactor: connecting the phosphatase to the microtubule network

    Science.gov (United States)

    Korrodi-Gregório, Luís; Vieira, Sandra I.; Esteves, Sara L. C.; Silva, Joana V.; Freitas, Maria João; Brauns, Ann-Kristin; Luers, Georg; Abrantes, Joana; Esteves, Pedro J.; da Cruz e Silva, Odete A. B.; Fardilha, Margarida; da Cruz e Silva, Edgar F.

    2013-01-01

    Summary Reversible phosphorylation plays an important role as a mechanism of intracellular control in eukaryotes. PPP1, a major eukaryotic Ser/Thr-protein phosphatase, acquires its specificity by interacting with different protein regulators, also known as PPP1 interacting proteins (PIPs). In the present work we characterized a physiologically relevant PIP in testis. Using a yeast two-hybrid screen with a human testis cDNA library, we identified a novel PIP of PPP1CC2 isoform, the T-complex testis expressed protein 1 domain containing 4 (TCTEX1D4) that has recently been described as a Tctex1 dynein light chain family member. The overlay assays confirm that TCTEX1D4 interacts with the different spliced isoforms of PPP1CC. Also, the binding domain occurs in the N-terminus, where a consensus PPP1 binding motif (PPP1BM) RVSF is present. The distribution of TCTEX1D4 in testis suggests its involvement in distinct functions, such as TGFβ signaling at the blood–testis barrier and acrosome cap formation. Immunofluorescence in human ejaculated sperm shows that TCTEX1D4 is present in the flagellum and in the acrosome region of the head. Moreover, TCTEX1D4 and PPP1 co-localize in the microtubule organizing center (MTOC) and microtubules in cell cultures. Importantly, the TCTEX1D4 PPP1BM seems to be relevant for complex formation, for PPP1 retention in the MTOC and movement along microtubules. These novel results open new avenues to possible roles of this dynein, together with PPP1. In essence TCTEX1D4/PPP1C complex appears to be involved in microtubule dynamics, sperm motility, acrosome reaction and in the regulation of the blood–testis barrier. PMID:23789093

  5. Potential role of voltage-sensing phosphatases in regulation of cell structure through the production of PI(3,4)P2.

    Science.gov (United States)

    Yamaguchi, Shinji; Kurokawa, Tatsuki; Taira, Ikuko; Aoki, Naoya; Sakata, Souhei; Okamura, Yasushi; Homma, Koichi J

    2014-04-01

    Voltage-sensing phosphatase, VSP, consists of the transmembrane domain, operating as the voltage sensor, and the cytoplasmic domain with phosphoinositide-phosphatase activities. The voltage sensor tightly couples with the cytoplasmic phosphatase and membrane depolarization induces dephosphorylation of several species of phosphoinositides. VSP gene is conserved from urochordate to human. There are some diversities among VSP ortholog proteins; range of voltage of voltage sensor motions as well as substrate selectivity. In contrast with recent understandings of biophysical mechanisms of VSPs, little is known about its physiological roles. Here we report that chick ortholog of VSP (designated as Gg-VSP) induces morphological feature of cell process outgrowths with round cell body in DF-1 fibroblasts upon its forced expression. Expression of the voltage sensor mutant, Gg-VSPR153Q with shifted voltage dependence to a lower voltage led to more frequent changes of cell morphology than the wild-type protein. Coexpression of PTEN that dephosphorylates PI(3,4)P2 suppressed this effect by Gg-VSP, indicating that the increase of PI(3,4)P2 leads to changes of cell shape. In addition, visualization of PI(3,4)P2 with the fluorescent protein fused with the TAPP1-derived pleckstrin homology (PH) domain suggested that Gg-VSP influenced the distribution of PI(3,4)P2 . These findings raise a possibility that one of the VSP's functions could be to regulate cell morphology through voltage-sensitive tuning of phosphoinositide profile. © 2013 Wiley Periodicals, Inc.

  6. Dragmacidins: new protein phosphatase inhibitors from a southern australian deep-water marine sponge, spongosorites sp

    Science.gov (United States)

    Capon; Rooney; Murray; Collins; Sim; Rostas; Butler; Carroll

    1998-05-01

    A Spongosorites sp. collected during trawling operations off the southern coast of Australia returned the new alkaloid dragmacidin E (3), the structure of which was secured by detailed spectroscopic analysis. Dragmacidin E (3), and its co-metabolite dragmacidin D (1) have been identified as potent inhibitors of serine-threonine protein phosphatases.

  7. Transgenic plants expressing the AaIT/GNA fusion protein show increased resistance and toxicity to both chewing and sucking pests.

    Science.gov (United States)

    Liu, Shu-Min; Li, Jie; Zhu, Jin-Qi; Wang, Xiao-Wei; Wang, Cheng-Shu; Liu, Shu-Sheng; Chen, Xue-Xin; Li, Sheng

    2016-04-01

    The adoption of pest-resistant transgenic plants to reduce yield losses and decrease pesticide use has been successful. To achieve the goal of controlling both chewing and sucking pests in a given transgenic plant, we generated transgenic tobacco, Arabidopsis, and rice plants expressing the fusion protein, AaIT/GNA, in which an insecticidal scorpion venom neurotoxin (Androctonus australis toxin, AaIT) is fused to snowdrop lectin (Galanthus nivalis agglutinin, GNA). Compared with transgenic tobacco and Arabidopsis plants expressing AaIT or GNA, transgenic plants expressing AaIT/GNA exhibited increased resistance and toxicity to one chewing pest, the cotton bollworm, Helicoverpa armigera. Transgenic tobacco and rice plants expressing AaIT/GNA showed increased resistance and toxicity to two sucking pests, the whitefly, Bemisia tabaci, and the rice brown planthopper, Nilaparvata lugens, respectively. Moreover, in the field, transgenic rice plants expressing AaIT/GNA exhibited a significant improvement in grain yield when infested with N. lugens. This study shows that expressing the AaIT/GNA fusion protein in transgenic plants can be a useful approach for controlling pests, particularly sucking pests which are not susceptible to the toxin in Bt crops. © 2015 Institute of Zoology, Chinese Academy of Sciences.

  8. Protein tyrosine phosphatase receptor type R deficient mice exhibit increased exploration in a new environment and impaired novel object recognition memory

    NARCIS (Netherlands)

    Erkens, M.; Bakker, B.; Duijn, L.M. van; Hendriks, W.J.A.J.; Zee, C.E.E.M. van der

    2014-01-01

    Mouse gene Ptprr encodes multiple protein tyrosine phosphatase receptor type R (PTPRR) isoforms that negatively regulate mitogen-activated protein kinase (MAPK) signaling pathways. In the mouse brain, PTPRR proteins are expressed in cerebellum, olfactory bulb, hippocampus, amygdala and perirhinal

  9. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    LENUS (Irish Health Repository)

    Meehan, Maria

    2012-02-05

    Abstract Background Protein tyrosine phosphatase receptor delta (PTPRD) is a member of a large family of protein tyrosine phosphatases which negatively regulate tyrosine phosphorylation. Neuroblastoma is a major childhood cancer arising from precursor cells of the sympathetic nervous system which is known to acquire deletions and alterations in the expression patterns of PTPRD, indicating a potential tumor suppressor function for this gene. The molecular mechanism, however, by which PTPRD renders a tumor suppressor effect in neuroblastoma is unknown. Results As a molecular mechanism, we demonstrate that PTPRD interacts with aurora kinase A (AURKA), an oncogenic protein that is over-expressed in multiple forms of cancer, including neuroblastoma. Ectopic up-regulation of PTPRD in neuroblastoma dephosphorylates tyrosine residues in AURKA resulting in a destabilization of this protein culminating in interfering with one of AURKA\\'s primary functions in neuroblastoma, the stabilization of MYCN protein, the gene of which is amplified in approximately 25 to 30% of high risk neuroblastoma. Conclusions PTPRD has a tumor suppressor function in neuroblastoma through AURKA dephosphorylation and destabilization and a downstream destabilization of MYCN protein, representing a novel mechanism for the function of PTPRD in neuroblastoma.

  10. Deficiency in Protein Tyrosine Phosphatase PTP1B Shortens Lifespan and Leads to Development of Acute Leukemia.

    Science.gov (United States)

    Le Sommer, Samantha; Morrice, Nicola; Pesaresi, Martina; Thompson, Dawn; Vickers, Mark A; Murray, Graeme I; Mody, Nimesh; Neel, Benjamin G; Bence, Kendra K; Wilson, Heather M; Delibegović, Mirela

    2018-01-01

    Protein tyrosine phosphatase PTP1B is a critical regulator of signaling pathways controlling metabolic homeostasis, cell proliferation, and immunity. In this study, we report that global or myeloid-specific deficiency of PTP1B in mice decreases lifespan. We demonstrate that myeloid-specific deficiency of PTP1B is sufficient to promote the development of acute myeloid leukemia. LysM-PTP1B -/- mice lacking PTP1B in the innate myeloid cell lineage displayed a dysregulation of bone marrow cells with a rapid decline in population at midlife and a concomitant increase in peripheral blood blast cells. This phenotype manifested further with extramedullary tumors, hepatic macrophage infiltration, and metabolic reprogramming, suggesting increased hepatic lipid metabolism prior to overt tumor development. Mechanistic investigations revealed an increase in anti-inflammatory M2 macrophage responses in liver and spleen, as associated with increased expression of arginase I and the cytokines IL10 and IL4. We also documented STAT3 hypersphosphorylation and signaling along with JAK-dependent upregulation of antiapoptotic proteins Bcl2 and BclXL. Our results establish a tumor suppressor role for PTP1B in the myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Significance: This study defines a tumor suppressor function for the protein tyrosine phosphatase PTP1B in myeloid lineage cells, with evidence that its genetic inactivation in mice is sufficient to drive acute myeloid leukemia. Cancer Res; 78(1); 75-87. ©2017 AACR . ©2017 American Association for Cancer Research.

  11. In-silico determination of insecticidal potential of Vip3Aa-Cry1Ac fusion protein against Lepidopteran targets using molecular docking

    Directory of Open Access Journals (Sweden)

    Aftab eAhmad

    2015-12-01

    Full Text Available Study and research of Bt (Bacillus thuringiensis transgenic plants have opened new ways to combat insect pests. Over the decades, however, insect pests, especially the Lepidopteran, have developed tolerance against Bt delta-endotoxins. Such issues can be addressed through the development of novel toxins with greater toxicity and affinity against a broad range of insect receptors. In this computational study, functional domains of Bacillus thuringiensis crystal delta-endotoxin (Cry1Ac insecticidal protein and vegetative insecticidal protein (Vip3Aa have been fused to develop a broad-range Vip3Aa-Cry1Ac fusion protein. Cry1Ac and Vip3Aa are non-homologous insecticidal proteins possessing receptors against different targets within the midgut of insects. The insecticidal proteins were fused to broaden the insecticidal activity. Molecular docking analysis of the fusion protein against aminopeptidase-N (APN and cadherin receptors of five Lepidopteran insects (Agrotis ipsilon, Helicoverpa armigera, Pectinophora gossypiella, Spodoptera exigua and Spodoptera litura revealed that the Ser290, Ser293, Leu337, Thr340 and Arg437 residues of the fusion protein are involved in the interaction with insect receptors. The Helicoverpa armigera cadherin receptor, however, showed no interaction, which might be due to either loss or burial of interactive residues inside the fusion protein. These findings revealed that the Vip3Aa-Cry1Ac fusion protein has a strong affinity against Lepidopteran insect receptors and hence has a potential to be an efficient broad-range insecticidal protein.

  12. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

  13. Loss of Oca2 disrupts the unfolded protein response and increases resistance to endoplasmic reticulum stress in melanocytes.

    Science.gov (United States)

    Cheng, Tsing; Orlow, Seth J; Manga, Prashiela

    2013-11-01

    Accumulation of proteins in the endoplasmic reticulum (ER) typically induces stress and initiates the unfolded protein response (UPR) to facilitate recovery. If homeostasis is not restored, apoptosis is induced. However, adaptation to chronic UPR activation can increase resistance to subsequent acute ER stress. We therefore investigated adaptive mechanisms in Oculocutaneous albinism type 2 (Oca2)-null melanocytes where UPR signaling is arrested despite continued tyrosinase accumulation leading to resistance to the chemical ER stressor thapsigargin. Although thapsigargin triggers UPR activation, instead of Perk-mediated phosphorylation of eIF2α, in Oca2-null melanocytes, eIF2α was rapidly dephosphorylated upon treatment. Dephosphorylation was mediated by the Gadd34-PPphosphatase complex. Gadd34-complex inhibition blocked eIF2α dephosphorylation and significantly increased Oca2-null melanocyte sensitivity to thapsigargin. Thus, Oca2-null melanocytes adapt to acute ER stress by disruption of pro-apoptotic Perk signaling, which promotes cell survival. This is the first study to demonstrate rapid eIF2α dephosphorylation as an adaptive mechanism to ER stress. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  14. Identification and bioinformatics comparison of two novel phosphatases in monoecious and gynoecious cucumber lines

    Science.gov (United States)

    Pawełkowicz, Magdalena E.; Wojcieszek, Michał; Osipowski, Paweł; Krzywkowski, Tomasz; PlÄ der, Wojciech; Przybecki, Zbigniew

    2016-09-01

    Two Arabidopsis thaliana genes from the PP2C family of protein phosphatases (AtABI1 and AtABI2) were used to find orthologous genes in the Cucumis sativus L. cv. Borszczagowski (cucumber) genome. Cucumber has been used as a model plant for sex expression studies because although it has been defined as a monoecious species, numerous genotypes are known to produce only female, only male, or hermaphroditic flowers. We identified two new orthologous genes of AtABI1 and AtABI2 in the cucumber genome and named them CsABI1 and CsABI2. To determine the relationships between the regulation of CsABI1 and CsABI2 and flower morphogenesis in cucumber, we performed various computational analyses to define the structure of the genes, and to predict regulatory elements and protein motifs in their sequences. We also performed an expression analysis to identify differences in the expression levels of CsABI1 and CsABI2 in vegetative and generative tissues (leaf, shoot apex, and flower buds) of monoecious (B10) and gynoecious (2gg) cucumber lines. We found that the expressions of CsABI1 and CsABI2 differed in male and female floral buds, and correlated these findings with the abscisic acid signaling pathways in male and female flowers.

  15. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    Science.gov (United States)

    Epidemiological studies have implicated zinc in the toxicity of ambient particulate matter (PM) inhalation. We previously showed that exposure to metal-laden PM inhibits protein tyrosine phosphatase (PTP) activity in human primary bronchial epithelial cells (HAEC) and leads t...

  16. Uncoupling protein 2 G(-866A polymorphism: a new gene polymorphism associated with C-reactive protein in type 2 diabetic patients C-reactive protein in type 2 diabetic patients

    Directory of Open Access Journals (Sweden)

    Cocozza Sergio

    2010-10-01

    Full Text Available Abstract Background This study evaluated the relationship between the G(-866A polymorphism of the uncoupling protein 2 (UCP2 gene and high-sensitivity C reactive protein (hs-CRP plasma levels in diabetic patients. Methods We studied 383 unrelated people with type 2 diabetes aged 40-70 years. Anthropometry, fasting lipids, glucose, HbA1c, and hs-CRP were measured. Participants were genotyped for the G (-866A polymorphism of the uncoupling protein 2 gene. Results Hs-CRP (mg/L increased progressively across the three genotype groups AA, AG, or GG, being respectively 3.0 ± 3.2, 3.6 ± 5.0, and 4.8 ± 5.3 (p for trend = 0.03. Since hs-CRP values were not significantly different between AA and AG genotype, these two groups were pooled for further analyses. Compared to participants with the AA/AG genotypes, homozygotes for the G allele (GG genotype had significantly higher hs-CRP levels (4.8 ± 5.3 vs 3.5 ± 4.7 mg/L, p = 0.01 and a larger proportion (53.9% vs 46.1%, p = 0.013 of elevated hs-CRP (> 2 mg/L. This was not explained by major confounders such as age, gender, BMI, waist circumference, HbA1c, smoking, or medications use which were comparable in the two genotype groups. Conclusions The study shows for the first time, in type 2 diabetic patients, a significant association of hs-CRP levels with the G(-866A polymorphism of UCP2 beyond the effect of major confounders.

  17. High-Pressure NMR and SAXS Reveals How Capping Modulates Folding Cooperativity of the pp32 Leucine-rich Repeat Protein.

    Science.gov (United States)

    Zhang, Yi; Berghaus, Melanie; Klein, Sean; Jenkins, Kelly; Zhang, Siwen; McCallum, Scott A; Morgan, Joel E; Winter, Roland; Barrick, Doug; Royer, Catherine A

    2018-04-27

    Many repeat proteins contain capping motifs, which serve to shield the hydrophobic core from solvent and maintain structural integrity. While the role of capping motifs in enhancing the stability and structural integrity of repeat proteins is well documented, their contribution to folding cooperativity is not. Here we examined the role of capping motifs in defining the folding cooperativity of the leucine-rich repeat protein, pp32, by monitoring the pressure- and urea-induced unfolding of an N-terminal capping motif (N-cap) deletion mutant, pp32-∆N-cap, and a C-terminal capping motif destabilization mutant pp32-Y131F/D146L, using residue-specific NMR and small-angle X-ray scattering. Destabilization of the C-terminal capping motif resulted in higher cooperativity for the unfolding transition compared to wild-type pp32, as these mutations render the stability of the C-terminus similar to that of the rest of the protein. In contrast, deletion of the N-cap led to strong deviation from two-state unfolding. In both urea- and pressure-induced unfolding, residues in repeats 1-3 of pp32-ΔN-cap lost their native structure first, while the C-terminal half was more stable. The residue-specific free energy changes in all regions of pp32-ΔN-cap were larger in urea compared to high pressure, indicating a less cooperative destabilization by pressure. Moreover, in contrast to complete structural disruption of pp32-ΔN-cap at high urea concentration, its pressure unfolded state remained compact. The contrasting effects of the capping motifs on folding cooperativity arise from the differential local stabilities of pp32, whereas the contrasting effects of pressure and urea on the pp32-ΔN-cap variant arise from their distinct mechanisms of action. Copyright © 2018 Elsevier Ltd. All rights reserved.

  18. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

    Science.gov (United States)

    Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

    2015-01-01

    The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  19. Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK Complex.

    Directory of Open Access Journals (Sweden)

    Stefan Frey

    Full Text Available The striatin-interacting phosphatase and kinase (STRIPAK complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP. In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

  20. Bacillus thuringiensis Cry1Ia10 and Vip3Aa protein interactions and their toxicity in Spodoptera spp. (Lepidoptera).

    Science.gov (United States)

    Bergamasco, V B; Mendes, D R P; Fernandes, O A; Desidério, J A; Lemos, M V F

    2013-02-01

    The polyphagous pests belonging to the genus Spodoptera are considered to be among the most important causes of damage and are widely distributed throughout the Americas'. Due to the extensive use of genetically modified plants containing Bacillus thuringiensis genes that code for insecticidal proteins, resistant insects may arise. To prevent the development of resistance, pyramided plants, which express multiple insecticidal proteins that act through distinct mode of actions, can be used. This study analyzed the mechanisms of action for the proteins Cry1Ia10 and Vip3Aa on neonatal Spodoptera frugiperda, Spodoptera albula, Spodoptera eridania and Spodoptera cosmioides larvae. The interactions of these toxins with receptors on the intestinal epithelial membrane were also analyzed by binding biotinylated toxins to brush border membrane vesicles (BBMVs) from the intestines of these insects. A putative receptor of approximately 65 kDa was found by ligand blotting in all of these species. In vitro competition assays using biotinylated proteins have indicated that Vip3Aa and Cry1Ia10 do not compete for the same receptor for S. frugiperda, S. albula and S. cosmioides and that Vip3Aa was more efficient than Cry1Ia10 when tested individually, by bioassays. A synergistic effect of the toxins in S. frugiperda, S. albula and S. cosmioides was observed when they were combined. However, in S. eridania, Cry1Ia10 and Vip3Aa might compete for the same receptor and through bioassays Cry1Ia10 was more efficient than Vip3Aa and showed an antagonistic effect when the proteins were combined. These results suggest that using these genes to develop pyramided plants may not prove effective in preventing the development of resistance in S. eridiana. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Large magnetoresistance in (AA')2FeReO6 double perovskites

    International Nuclear Information System (INIS)

    Teresa, J.M. de; Serrate, D.; Blasco, J.; Ibarra, M.R.; Morellon, L.

    2005-01-01

    We review the main structural, magnetic and magnetotransport properties of the intriguing (AA') 2 FeReO 6 magnetic double perovskites. As the average cation size decreases, the crystallographic structure at room temperature evolves from cubic [(AA') 2 =Ba 2 , Ba 1.5 Sr 0.5 , BaSr, Ba 0.5 Sr 1.5 ] to tetragonal [(AA') 2 =Sr 2 ] and monoclinic [(AA') 2 =Ca 0.5 Sr 1.5 , CaSr, Ca 1.5 Sr 0.5 , Ca 2 ]. The Curie temperature increases anomalously from ∼303K for Ba 2 to ∼522K for Ca 2 in sharp contrast with the observed behaviour in the isostructural compounds (AA') 2 FeMoO 6 . Other anomalous features in the (AA') 2 FeReO 6 series are: the large magnetic anisotropy, the large magnetoelastic coupling and the semiconducting behaviour of the monoclinic compounds. The monoclinic compounds undergo a structural/magnetic transition at T S below 125K. Three different magnetoresistance mechanisms have been identified: the intergrain negative magnetoresistance effect, which is present across the whole series of compounds, and in the case of the monoclinic compounds below T S a negative magnetoresistance effect associated to the melting of the low-temperature phase and a positive magnetoresistance effect only present in (AA') 2 =Ca 2 below T∼50K

  2. Geranylated 2-arylbenzofurans from Morus alba var. tatarica and their α-glucosidase and protein tyrosine phosphatase 1B inhibitory activities.

    Science.gov (United States)

    Zhang, Ya-Long; Luo, Jian-Guang; Wan, Chuan-Xing; Zhou, Zhong-Bo; Kong, Ling-Yi

    2014-01-01

    Ten new geranylated 2-arylbenzofuran derivatives, including two monoterpenoid 2-arylbenzofurans (1 and 2), two geranylated 2-arylbenzofuran enantiomers (3a and 3b), and six geranylated 2-arylbenzofurans (4-9), along with four known 2-arylbenzofurans (10-13) were isolated from the root bark of Morus alba var. tatarica. Their structures and relative configurations were established on the basis of spectroscopic data analysis. Compounds 3-7 with one asymmetric carbon at C-7″ were supposed to be enantiomeric mixtures confirmed by chiral HPLC analysis, and the absolute configurations of each enantiomer in 3-7 were determined by Rh2(OCOCF3)4-induced CD and Snatzke's method. The enantiomers with the substituting group at C-2' exhibited better resolutions on a Chiralpak AD-H column than those with the substituting group at C-4'. Compounds 1-7, 10, 11 and 13, showed α-glucosidase inhibitory activities with IC50 values of 11.9-131.9 μM, and compounds 1 and 9-13 inhibited protein tyrosine phosphatase 1B (PTP1B) with IC50 values of 7.9-38.1 μM. Copyright © 2013 Elsevier B.V. All rights reserved.

  3. Penostatin Derivatives, a Novel Kind of Protein Phosphatase 1B Inhibitors Isolated from Solid Cultures of the Entomogenous Fungus Isaria tenuipes

    Directory of Open Access Journals (Sweden)

    Yu-Peng Chen

    2014-01-01

    Full Text Available Protein tyrosine phosphatase 1B (PTP1B is implicated as a negative regulator of insulin receptor (IR signaling and a potential drug target for the treatment of type II diabetes and other associated metabolic syndromes. Therefore, small molecular inhibitors of PTP1B can be considered as an attractive approach for the design of new therapeutic agents of type II diabetes diseases. In a continuing search for new protein phosphatase inhibitors from fungi, we have isolated a new compound, named penostatin J (1, together with three known ones, penostatin C (2, penostatin A (3, and penostatin B (4, from cultures of the entomogenous fungus Isaria tenuipes. The structure of penostatin J (1 was elucidated by extensive spectroscopic analysis. We also demonstrate for the first time that penostatin derivatives exhibit the best PTP1B inhibitory action. These findings suggest that penostatin derivatives are a potential novel kind of PTP1B inhibitors.

  4. Characterization of the Functional Domains of a Mammalian Voltage-Sensitive Phosphatase.

    Science.gov (United States)

    Rosasco, Mario G; Gordon, Sharona E; Bajjalieh, Sandra M

    2015-12-15

    Voltage-sensitive phosphatases (VSPs) are proteins that directly couple changes in membrane electrical potential to inositol lipid phosphatase activity. VSPs thus couple two signaling pathways that are critical for cellular functioning. Although a number of nonmammalian VSPs have been characterized biophysically, mammalian VSPs are less well understood at both the physiological and biophysical levels. In this study, we aimed to address this gap in knowledge by determining whether the VSP from mouse, Mm-VSP, is expressed in the brain and contains a functional voltage-sensing domain (VSD) and a phosphatase domain. We report that Mm-VSP is expressed in neurons and is developmentally regulated. To address whether the functions of the VSD and phosphatase domain are retained in Mm-VSP, we took advantage of the modular nature of these domains and expressed each independently as a chimeric protein in a heterologous expression system. We found that the Mm-VSP VSD, fused to a viral potassium channel, was able to drive voltage-dependent gating of the channel pore. The Mm-VSP phosphatase domain, fused to the VSD of a nonmammalian VSP, was also functional: activation resulted in PI(4,5)P2 depletion that was sufficient to inhibit the PI(4,5)P2-regulated KCNQ2/3 channels. While testing the functionality of the VSD and phosphatase domain, we observed slight differences between the activities of Mm-VSP-based chimeras and those of nonmammalian VSPs. Although the properties of VSP chimeras may not completely reflect the properties of native VSPs, the differences we observed in voltage-sensing and phosphatase activity provide a starting point for future experiments to investigate the function of Mm-VSP and other mammalian VSPs. In conclusion, our data reveal that both the VSD and the lipid phosphatase domain of Mm-VSP are functional, indicating that Mm-VSP likely plays an important role in mouse neurophysiology. Copyright © 2015 Biophysical Society. Published by Elsevier Inc. All

  5. Molecular dynamics simulations of protein-tyrosine phosphatase 1B. I. Ligand-induced changes in the protein motions

    DEFF Research Database (Denmark)

    Peters, Günther H. J.; Frimurer, T.M.; Andersen, J.N.

    1999-01-01

    Activity of enzymes, such as protein tyrosine phosphatases (PTPs), is often associated with structural changes in the enzyme, resulting in selective and stereospecific reactions with the substrate. To investigate the effect of a substrate on the motions occurring in PTPs, we have performed...... molecular dynamics simulations of PTP1B and PTP1B complexed with a high-affinity peptide DADEpYL, where pY stands for phosphorylated tyrosine. The peptide sequence is derived from the epidermal growth factor receptor (EGFR(988-993)). Simulations were performed in water for 1 ns, and the concerted motions...... in the protein were analyzed using the essential dynamics technique. Our results indicate that the predominately internal motions in PTP1B occur in a subspace of only a few degrees of freedom. Upon substrate binding, the flexibility of the protein is reduced by similar to 10%. The largest effect is found...

  6. Osteocalcin and bone-specific alkaline phosphatase in Sickle cell ...

    African Journals Online (AJOL)

    specific alkaline phosphatase (b-AP) total protein levels were evaluated as indicators of bone turnover in twenty patients with sickle cell haemoglobinopathies and in twenty normal healthy individuals. The serum bonespecific alkaline phosphatase ...

  7. Redox Regulation of Receptor Protein-Tyrosine Phosphatases

    NARCIS (Netherlands)

    Groen, A.J.

    2006-01-01

    Phosphorylation is of major importance in cell signalling processes like cell migration, cell proliferation and cell differentiation within higher eukaryotic organisms. Therefore, the balance between phosphorylation, mediated by kinases, and dephosphorylation, mediated by phosphatases, must be

  8. Domain-to-domain coupling in voltage-sensing phosphatase.

    Science.gov (United States)

    Sakata, Souhei; Matsuda, Makoto; Kawanabe, Akira; Okamura, Yasushi

    2017-01-01

    Voltage-sensing phosphatase (VSP) consists of a transmembrane voltage sensor and a cytoplasmic enzyme region. The enzyme region contains the phosphatase and C2 domains, is structurally similar to the tumor suppressor phosphatase PTEN, and catalyzes the dephosphorylation of phosphoinositides. The transmembrane voltage sensor is connected to the phosphatase through a short linker region, and phosphatase activity is induced upon membrane depolarization. Although the detailed molecular characteristics of the voltage sensor domain and the enzyme region have been revealed, little is known how these two regions are coupled. In addition, it is important to know whether mechanism for coupling between the voltage sensor domain and downstream effector function is shared among other voltage sensor domain-containing proteins. Recent studies in which specific amino acid sites were genetically labeled using a fluorescent unnatural amino acid have enabled detection of the local structural changes in the cytoplasmic region of Ciona intestinalis VSP that occur with a change in membrane potential. The results of those studies provide novel insight into how the enzyme activity of the cytoplasmic region of VSP is regulated by the voltage sensor domain.

  9. Bombyx mori ABC transporter C2 structures responsible for the receptor function of Bacillus thuringiensis Cry1Aa toxin.

    Science.gov (United States)

    Tanaka, Shiho; Endo, Haruka; Adegawa, Satomi; Iizuka, Ami; Imamura, Kazuhiro; Kikuta, Shingo; Sato, Ryoichi

    2017-12-01

    Because Bombyx mori ABC transporter C2 (BmABCC2) has 1000-fold higher potential than B. mori cadherin-like protein as a receptor for Bacillus thuringiensis Cry1Aa toxin (Tanaka et al., 2013), the gate-opening ability of the latent pore under six extracellular loops (ECLs) of BmABCC2 was expected to be the reason for its higher potential (Heckel, 2012). In this study, cell swelling assays in Sf9 cells showed that BmABCC2 mutants lacking substrate-excreting activity retained receptor activity, indicating that the gate-opening activity of BmABCC2 is not responsible for Cry1Aa toxicity. The analysis of 29 BmABCC2 mutants demonstrated that 770 DYWL 773 of ECL 4 comprise a putative binding site to Cry1Aa. This suggests that specific toxicity of Cry1Aa toxin to a restricted range of lepidopteran insects is dependent on conservation and variation in the amino acid residues around 770 DYWL 773 of ECL 4 in the ABCC2. Copyright © 2017 Elsevier Ltd. All rights reserved.

  10. A rapid lateral flow immunoassay for the detection of tyrosine phosphatase-like protein IA-2 autoantibodies in human serum.

    Directory of Open Access Journals (Sweden)

    Ingrid Kikkas

    Full Text Available Type 1 diabetes (T1D results from the destruction of pancreatic insulin-producing beta cells and is strongly associated with the presence of islet autoantibodies. Autoantibodies to tyrosine phosphatase-like protein IA-2 (IA-2As are considered to be highly predictive markers of T1D. We developed a novel lateral flow immunoassay (LFIA based on a bridging format for the rapid detection of IA-2As in human serum samples. In this assay, one site of the IA-2As is bound to HA-tagged-IA-2, which is subsequently captured on the anti-HA-Tag antibody-coated test line on the strip. The other site of the IA-2As is bound to biotinylated IA-2, allowing the complex to be visualized using colloidal gold nanoparticle-conjugated streptavidin. For this study, 35 serum samples from T1D patients and 44 control sera from non-diabetic individuals were analyzed with our novel assay and the results were correlated with two IA-2A ELISAs. Among the 35 serum samples from T1D patients, the IA-2A LFIA, the in-house IA-2A ELISA and the commercial IA-2A ELISA identified as positive 21, 29 and 30 IA-2A-positive sera, respectively. The major advantages of the IA-2A LFIA are its rapidity and simplicity.

  11. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    Science.gov (United States)

    Liu, Yingying; Li, Fengna; Kong, Xiangfeng; Tan, Bie; Li, Yinghui; Duan, Yehui; Blachier, François; Hu, Chien-An A; Yin, Yulong

    2015-01-01

    Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA) pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet)- or higher/NRC (National Research Council)-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I) and longissimus dorsi muscle (LDM, type II) were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR) signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (Prelated AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05) than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K), and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05). There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05) the levels for mTOR and p70S6K in Bama mini-pigs, but repressed (P<0.05) the level for p70S6K in Landrace pigs. The higher protein-NRC diet increased ratio of p-mTOR/mTOR in

  12. The structural insights of stem cell factor receptor (c-Kit interaction with tyrosine phosphatase-2 (Shp-2: An in silico analysis

    Directory of Open Access Journals (Sweden)

    Gurudutta Gangenahalli U

    2010-01-01

    Full Text Available Abstract Background Stem cell factor (SCF receptor c-Kit is recognized as a key signaling molecule, which transduces signals for the proliferation, differentiation and survival of stem cells. Binding of SCF to its receptor triggers transactivation, leading to the recruitment of kinases and phosphatases to the docking platforms of c-Kit catalytic domain. Tyrosine phosphatase-1 (Shp-1 deactivates/attenuates 'Kit' kinase activity. Whereas, Asp816Val mutation in the Kit activation loop transforms kinase domain to a constitutively activated state (switch off-to-on state, in a ligand-independent manner. This phenomenon completely abrogates negative regulation of Shp-1. To predict the possible molecular basis of interaction between c-Kit and Shp-1, we have performed an in silico protein-protein docking study between crystal structure of activated c-Kit (phosphorylated c-Kit and full length crystal structure of Shp-2, a close structural counterpart of Shp-1. Findings Study revealed a stretch of conserved amino acids (Lys818 to Ser821 in the Kit activation domain, which makes decisive H-bonds with N-sh2 and phosphotyrosine binding pocket residues of the phosphatase. These H-bonds may impose an inhibitory steric hindrance to the catalytic domain of c-Kit, there by blocking further interaction of the activation loop molecules with incoming kinases. We have also predicted a phosphotyrosine binding pocket in SH2 domains of Shp-1, which is found to be predominantly closer to a catalytic groove like structure in c-Kit kinase domain. Conclusions This study predicts that crucial hydrogen bonding between N-sh2 domain of Shp-1 and Kit activation loop can modulate the negative regulation of c-Kit kinase by Shp-1. Thus, this finding is expected to play a significant role in designing suitable gain-of-function c-Kit mutants for inducing conditional proliferation of hematopoietic stem cells.

  13. Dopamine D2L receptor-interacting proteins regulate dopaminergic signaling

    Directory of Open Access Journals (Sweden)

    Norifumi Shioda

    2017-10-01

    Full Text Available Dopamine receptor family proteins include seven transmembrane and trimeric GTP-binding protein-coupled receptors (GPCRs. Among them, the dopamine D2 receptor (D2R is most extensively studied. All clinically used antipsychotic drugs serve as D2R antagonists in the mesolimbic dopamine system, and their ability to block D2R signaling is positively correlated with antipsychotic efficiency. Human genetic studies also show a significant association of DRD2 polymorphisms with disorders including schizophrenia and Parkinson's disease. D2R exists as two alternatively spliced isoforms, the long isoform (D2LR and the short isoform (D2SR, which differ in a 29-amino acid (AA insert in the third cytoplasmic loop. Importantly, previous reports demonstrate functional diversity between the two isoforms in humans. In this review, we focus on binding proteins that specifically interact with the D2LR 29AA insert. We discuss how D2R activities are mediated not only by heterotrimeric G proteins but by D2LR-interacting proteins, which in part regulate diverse D2R activities. Keywords: Dopamine D2L receptor, Antipsychotic drugs, DRD2 polymorphisms, Alternatively spliced isoforms, D2LR-interacting proteins

  14. Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium.

    Science.gov (United States)

    Murata, Yoji; Kotani, Takenori; Supriatna, Yana; Kitamura, Yasuaki; Imada, Shinya; Kawahara, Kohichi; Nishio, Miki; Daniwijaya, Edwin Widyanto; Sadakata, Hisanobu; Kusakari, Shinya; Mori, Munemasa; Kanazawa, Yoshitake; Saito, Yasuyuki; Okawa, Katsuya; Takeda-Morishita, Mariko; Okazawa, Hideki; Ohnishi, Hiroshi; Azuma, Takeshi; Suzuki, Akira; Matozaki, Takashi

    2015-08-04

    Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer-associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10-deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1-deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.

  15. Presence of ecto-protein tyrosine phosphatase activity is vital for survival of Setaria cervi, a bovine filarial parasite.

    Science.gov (United States)

    Singh, Neetu; Heneberg, Petr; Rathaur, Sushma

    2014-10-01

    The ecto protein tyrosine phosphatases (PTP) are known to play a crucial role in the pathogenesis and survival of the intracellular parasites. However, their presence and role in filarial parasites is still unknown. We found a significant amount of tyrosine phosphatase activity in the surface antigen fraction extracted from Setaria cervi (S. cervi), a bovine filarial parasite. An antibody designed against the conserved catalytic core of human protein tyrosine phosphatases, PTP1B cross reacted with a 63 kDa band in the surface antigen. We detected a significant amount of PTP activity in the intact S. cervi adult parasites as well as microfilariae in this study for the first time. This PTP may be localized on the surface of the parasite with an exposed active site available for the external substrates. The PTP activity was also inhibited by sodium orthovanadate and phenyl arsine oxide, specific inhibitors of PTP in both the life stages. The Km and Vmax for PTP in the adult parasites and microfilariae were determined to be 2.574 ± 0.14 mM; 206.3 ± 2.75 μM Pi/h/two parasites and 5.510 ± 0.59 mM; 62.27 ± 2.27 μM Pi/h/10(6) parasites respectively using O-P-L-Tyrosine as substrate. Interestingly, a positive correlation was observed between the inhibition in PTP activity and reduction in the motility/ viability of the parasites when they were subjected to the specific PTP inhibitors (Orthovanadate and Phenyl arsine oxide) for 4 h in the KRB maintenance medium. The activity was also significantly inhibited in the parasites exposed to antifilarial drug/compounds for e.g. Diethylcarbamazine, Acetylsalicylic Acid and SK7, a methyl chalcone. Therefore suggesting a possible role played by PTP in the survival of the parasite, its interaction with the host as well as in the screening of newly synthesized antifilarials/drugs.

  16. Regulation of cardiac C-protein phosphorylation

    International Nuclear Information System (INIS)

    Titus, F.L.

    1985-01-01

    Molecular mechanisms of cardiac sympathetic and parasympathetic responses were addressed by studying subcellular changes in protein phosphorylation, cAMP-dependent protein kinase activity and protein phosphatase activity in frog hearts. B-adrenergic agonists increased and muscarinic cholinergic agonists decreased [ 32 P]phosphate incorporation into C-protein, a thick filament component. Regulation of protein phosphatase activity by Iso and methacholine (MCh) was assayed using extracts of drug treated frog hearts and [ 32 P]phospho-C-protein as substrate. Total phosphatase activity decreased 21% in extracts from hearts perfused with 0.1 μM Iso and 17% in hearts exposed to Iso plus 1 μM methacholine. This decrease reflected decreased phosphatase-2A activity. No changes in total phosphatase activity were measurable in broken cells treated with Iso or MCh. The results suggest adrenergic stimulation changes contractile activity in frog hearts by activating cAMP-dependent protein kinase associated with particulate cellular elements and inactivating soluble protein phosphatase-2A. This is the first demonstration of coordinated regulation of these enzymes by B-adrenergic agonists favoring phosphorylation of effector proteins. Coordinated regulation by methacholine in the presence of Iso was not observed

  17. Isothiazolidinone (IZD) as a phosphoryl mimetic in inhibitors of the Yersinia pestis protein tyrosine phosphatase YopH

    International Nuclear Information System (INIS)

    Kim, Sung-Eun; Bahta, Medhanit; Lountos, George T.; Ulrich, Robert G.; Burke, Terrence R. Jr; Waugh, David S.

    2011-01-01

    The first X-ray crystal structure of the Y. pestis protein tyrosine phosphatase YopH in complex with an isothiazolidinone-based lead-fragment compound is reported. Isothiazolidinone (IZD) heterocycles can act as effective components of protein tyrosine phosphatase (PTP) inhibitors by simultaneously replicating the binding interactions of both a phosphoryl group and a highly conserved water molecule, as exemplified by the structures of several PTP1B–inhibitor complexes. In the first unambiguous demonstration of IZD interactions with a PTP other than PTP1B, it is shown by X-ray crystallography that the IZD motif binds within the catalytic site of the Yersinia pestis PTP YopH by similarly displacing a highly conserved water molecule. It is also shown that IZD-based bidentate ligands can inhibit YopH in a nonpromiscuous fashion at low micromolar concentrations. Hence, the IZD moiety may represent a useful starting point for the development of YopH inhibitors

  18. An Ancient Protein Phosphatase, SHLP1, Is Critical to Microneme Development in Plasmodium Ookinetes and Parasite Transmission

    Directory of Open Access Journals (Sweden)

    Eva-Maria Patzewitz

    2013-03-01

    Full Text Available Signaling pathways controlled by reversible protein phosphorylation (catalyzed by kinases and phosphatases in the malaria parasite Plasmodium are of great interest, for both increased understanding of parasite biology and identification of novel drug targets. Here, we report a functional analysis in Plasmodium of an ancient bacterial Shewanella-like protein phosphatase (SHLP1 found only in bacteria, fungi, protists, and plants. SHLP1 is abundant in asexual blood stages and expressed at all stages of the parasite life cycle. shlp1 deletion results in a reduction in ookinete (zygote development, microneme formation, and complete ablation of oocyst formation, thereby blocking parasite transmission. This defect is carried by the female gamete and can be rescued by direct injection of mutant ookinetes into the mosquito hemocoel, where oocysts develop. This study emphasizes the varied functions of SHLP1 in Plasmodium ookinete biology and suggests that it could be a novel drug target for blocking parasite transmission.

  19. Use of Okadaic Acid to Identify Relevant Phosphoepitopes in Pathology: A Focus on Neurodegeneration

    Directory of Open Access Journals (Sweden)

    Jesús Avila

    2013-05-01

    Full Text Available Protein phosphorylation is involved in the regulation of a wide variety of physiological processes and is the result of a balance between protein kinase and phosphatase activities. Biologically active marine derived compounds have been shown to represent an interesting source of novel compounds that could modify that balance. Among them, the marine toxin and tumor promoter, okadaic acid (OA, has been shown as an inhibitor of two of the main cytosolic, broad-specificity protein phosphatases, PP1 and PP2A, thus providing an excellent cell-permeable probe for examining the role of protein phosphorylation, and PP1 and PP2A in particular, in any physiological or pathological process. In the present work, we review the use of okadaic acid to identify specific phosphoepitopes mainly in proteins relevant for neurodegeneration. We will specifically highlight those cases of highly dynamic phosphorylation-dephosphorylation events and the ability of OA to block the high turnover phosphorylation, thus allowing the detection of modified residues that could be otherwise difficult to identify. Finally, its effect on tau hyperhosphorylation and its relevance in neurodegenerative pathologies such as Alzheimer’s disease and related dementia will be discussed.

  20. Vanadate monomers and dimers both inhibit the human prostatic acid phosphatase.

    Science.gov (United States)

    Crans, D C; Simone, C M; Saha, A K; Glew, R H

    1989-11-30

    A combination of enzyme kinetics and 51V NMR spectroscopy was used to identify the species of vanadate that inhibits acid phosphatases. Monomeric vanadate was shown to inhibit wheat germ and potato acid phosphatases. At pH 5.5, the vanadate dimer inhibits the human prostatic acid phosphatase whereas at pH 7.0 it is the vanadate monomer that inhibits this enzyme. The pH-dependent shift in the affinity of the prostatic phosphatase for vanadate is presumably due to deprotonation of an amino acid side chain in or near the binding site resulting in a conformational change in the protein. pH may be a subtle effector of the insulin-like vanadate activity in biological systems and may explain some of the differences in selectivity observed with the protein phosphatases.

  1. Structural and biochemical analysis of a unique phosphatase from Bdellovibrio bacteriovorus reveals its structural and functional relationship with the protein tyrosine phosphatase class of phytase.

    Directory of Open Access Journals (Sweden)

    Robert J Gruninger

    Full Text Available Bdellovibrio bacteriovorus is an unusual δ-proteobacterium that invades and preys on other Gram-negative bacteria and is of potential interest as a whole cell therapeutic against pathogens of man, animals and crops. PTPs (protein tyrosine phosphatases are an important class of enzyme involved in desphosphorylating a variety of substrates, often with implications in cell signaling. The B. bacteriovorus open reading frame Bd1204 is predicted to encode a PTP of unknown function. Bd1204 is both structurally and mechanistically related to the PTP-like phytase (PTPLP class of enzymes and possesses a number of unique properties not observed in any other PTPLPs characterized to date. Bd1204 does not display catalytic activity against some common protein tyrosine phosphatase substrates but is highly specific for hydrolysis of phosphomonoester bonds of inositol hexakisphosphate. The structure reveals that Bd1204 has the smallest and least electropositive active site of all characterized PTPLPs to date yet possesses a unique substrate specificity characterized by a strict preference for inositol hexakisphosphate. These two active site features are believed to be the most significant contributors to the specificity of phytate degrading enzymes. We speculate that Bd1204 may be involved in phosphate acquisition outside of prey.

  2. Vip3Aa induces apoptosis in cultured Spodoptera frugiperda (Sf9) cells.

    Science.gov (United States)

    Jiang, Kun; Mei, Si-Qi; Wang, Ting-Ting; Pan, Jin-Hua; Chen, Yue-Hua; Cai, Jun

    2016-09-15

    The vegetative insecticidal proteins (Vip) secreted by many Bacillus thuringiensis strains during their vegetative growth stage are regarded as second generation insecticidal proteins, as they share no sequence or structural homology with known crystal insecticidal proteins (Cry) and have a broad insecticidal spectrum. Compared with insecticidal crystal proteins (ICPs), the insecticidal mechanisms of Vips have been little studied. Here we investigated the mechanism responsible for Vip3Aa toxicity in cultured insect cells. Using, flow cytometry analyzes, TUNEL staining and DNA fragmentation assays, we show that Vip3Aa can induce apoptosis in Spodoptera frugiperda (Sf9) cells and cause cells to arrest at the G2/M phase. We also show that Vip3Aa can disrupt mitochondrial membrane potential (ΔΨm), leading to the activation of Sf-caspase-1, suggesting that a mitochondrial mediated and caspase dependent pathway may be involved in Vip3Aa-induced apoptosis in Sf9 cells. Copyright © 2016 Elsevier Ltd. All rights reserved.

  3. Recruitment of SHP-1 protein tyrosine phosphatase and signalling by a chimeric T-cell receptor-killer inhibitory receptor

    DEFF Research Database (Denmark)

    Christensen, M D; Geisler, C

    2000-01-01

    Receptors expressing the immunoreceptor tyrosine-based inhibitory motif (ITIM) in their cytoplasmic tail play an important role in the negative regulation of natural killer and B-cell activation. A subpopulation of T cells expresses the ITIM containing killer cell inhibitory receptor (KIR), which...... recognize MHC class I molecules. Following coligation of KIR with an activating receptor, the tyrosine in the ITIM is phosphorylated and the cytoplasmic protein tyrosine phosphatase SHP-1 is recruited to the ITIM via its SH2 domains. It is still not clear how SHP-1 affects T-cell receptor (TCR) signalling...... regarding total protein tyrosine phosphorylation, TCR down-regulation, mobilization of intracellular free calcium, or induction of the activation markers CD69 and CD25....

  4. Activation of Src kinase by protein-tyrosine phosphatase-PEST in osteoclasts: comparative analysis of the effects of bisphosphonate and protein-tyrosine phosphatase inhibitor on Src activation in vitro.

    Science.gov (United States)

    Chellaiah, Meenakshi A; Schaller, Michael D

    2009-08-01

    PTP-PEST is involved in the regulation of sealing ring formation in osteoclasts. In this article, we have shown a regulatory role for PTP-PEST on dephosphorylation of c-Src at Y527 and phosphorylation at Y418 in the catalytic site. Activation of Src in osteoclasts by over-expression of PTP-PEST resulted in the phosphorylation of cortactin at Y421 and WASP at Y294. Also enhanced as a result, is the interaction of Src, cortactin, and Arp2 with WASP. Moreover, the number of osteoclasts displaying sealing ring and bone resorbing activity was increased in response to PTP-PEST over-expression as compared with control osteoclasts. Cells expressing constitutively active-Src (527YDeltaF) simulate the effects mediated by PTP-PEST. Treatment of osteoclasts with a bisphosphonate alendronate or a potent PTP inhibitor PAO decreased the activity and phosphorylation of Src at Y418 due to reduced dephosphorylation state at Y527. Therefore, Src-mediated phosphorylation of cortactin and WASP as well as the formation of WASP.cortactin.Arp2 complex and sealing ring were reduced in these osteoclasts. Similar effects were observed in osteoclasts treated with an Src inhibitor PP2. We have shown that bisphosphonates could modulate the function of osteoclasts by inhibiting downstream signaling mediated by PTP-PEST/Src, in addition to its effect on the inhibition of the post-translational modification of small GTP-binding proteins such as Rab, Rho, and Rac as shown by others. The promising effects of the inhibitors PP2 and PAO on osteoclast function suggest a therapeutic approach for patients with bone metastases and osteoporosis as an alternative to bisphosphonates.

  5. A family of metal-dependent phosphatases implicated in metabolite damage-control

    Energy Technology Data Exchange (ETDEWEB)

    Huang, Lili; Khusnutdinova, Anna; Nocek, Boguslaw; Brown, Greg; Xu, Xiaohui; Cui, Hong; Petit, Pierre; Flick, Robert; Zallot, Rémi; Balmant, Kelly; Ziemak, Michael J.; Shanklin, John; de Crécy-Lagard, Valérie; Fiehn, Oliver; Gregory, Jesse F.; Joachimiak, Andrzej; Savchenko, Alexei; Yakunin, Alexander F.; Hanson, Andrew D.

    2016-06-20

    DUF89 family proteins occur widely in both prokaryotes and eukaryotes, but their functions are unknown. Here we define three DUF89 subfamilies (I, II, and III), with subfamily II being split into stand-alone proteins and proteins fused to pantothenate kinase (PanK). We demonstrated that DUF89 proteins have metal-dependent phosphatase activity against reactive phosphoesters or their damaged forms, notably sugar phosphates (subfamilies II and III), phosphopantetheine and its S-sulfonate or sulfonate (subfamily II-PanK fusions), and nucleotides (subfamily I). Genetic and comparative genomic data strongly associated DUF89 genes with phosphoester metabolism. The crystal structure of the yeast (Saccharomyces cerevisiae) subfamily III protein YMR027W revealed a novel phosphatase active site with fructose 6-phosphate and Mg2+ bound near conserved signature residues Asp254 and Asn255 that are critical for activity. These findings indicate that DUF89 proteins are previously unrecognized hydrolases whose characteristic in vivo function is to limit potentially harmful buildups of normal or damaged phosphometabolites.

  6. Pharmacological inhibition of protein tyrosine phosphatase 1B: a promising strategy for the treatment of obesity and type 2 diabetes mellitus.

    Science.gov (United States)

    Panzhinskiy, E; Ren, J; Nair, S

    2013-01-01

    Obesity and metabolic syndrome represent major public health problems, and are the biggest preventable causes of death worldwide. Obesity is the leading risk factor for type 2 diabetes mellitus (T2DM), cardiovascular diseases and non-alcoholic fatty liver disease. Obesity-associated insulin resistance, which is characterized by reduced uptake and utilization of glucose in muscle, adipose and liver tissues, is a key predictor of metabolic syndrome and T2DM. With increasing prevalence of obesity in adults and children, the need to identify and characterize potential targets for treating metabolic syndrome is imminent. Emerging evidence from animal models, clinical studies and cell lines studies suggest that protein tyrosine phosphatase 1B (PTP1B), an enzyme that negatively regulates insulin signaling, is likely to be involved in the pathways leading to insulin resistance. PTP1B is tethered to the cytosolic surface of endoplasmic reticulum (ER), an organelle that is responsible for folding, modification, and trafficking of proteins. Recent evidence links the disruption of ER homeostasis, referred to as ER stress, to the pathogenesis of obesity and T2DM. PTP1B has been recognized as an important player linking ER stress and insulin resistance in obese subjects. This review highlights recent advances in the research related to the role of PTP1B in signal transduction processes implicated in pathophysiology of obesity and type 2 diabetes, and focuses on the potential therapeutic exploitation of PTP1B inhibitors for the management of these conditions.

  7. Host cell subversion by Toxoplasma GRA16, an exported dense granule protein that targets the host cell nucleus and alters gene expression.

    Science.gov (United States)

    Bougdour, Alexandre; Durandau, Eric; Brenier-Pinchart, Marie-Pierre; Ortet, Philippe; Barakat, Mohamed; Kieffer, Sylvie; Curt-Varesano, Aurélie; Curt-Bertini, Rose-Laurence; Bastien, Olivier; Coute, Yohann; Pelloux, Hervé; Hakimi, Mohamed-Ali

    2013-04-17

    After invading host cells, Toxoplasma gondii multiplies within a parasitophorous vacuole (PV) that is maintained by parasite proteins secreted from organelles called dense granules. Most dense granule proteins remain within the PV, and few are known to access the host cell cytosol. We identify GRA16 as a dense granule protein that is exported through the PV membrane and reaches the host cell nucleus, where it positively modulates genes involved in cell-cycle progression and the p53 tumor suppressor pathway. GRA16 binds two host enzymes, the deubiquitinase HAUSP and PP2A phosphatase, which exert several functions, including regulation of p53 and the cell cycle. GRA16 alters p53 levels in a HAUSP-dependent manner and induces nuclear translocation of the PP2A holoenzyme. Additionally, certain GRA16-deficient strains exhibit attenuated virulence, indicating the importance of these host alterations in pathogenesis. Therefore, GRA16 represents a potentially emerging subfamily of exported dense granule proteins that modulate host function. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Characterizing the interactions between prolyl isomerase pin1 and phosphatase inhibitor-2 in living cells with FRET and FCS

    Science.gov (United States)

    Sun, Yuansheng; Wang, Lifu; Jyothikumar, Vinod; Brautigan, David L.; Periasamy, Ammasi

    2012-03-01

    Phosphatase inhibitor-2 (I2) was discovered as a regulator of protein Ser/Thr phosphatase-1 and is conserved from yeast to human. Binding between purified recombinant I2 from different species and the prolyl isomerase Pin1 has been demonstrated with pull-down assays, size exclusion chromatography and nuclear magnetic resonance spectroscopy. Despite this, questions persist as to whether these proteins associate together in living cells. In this study, we prepared fluorescent protein (FP) fusions of I2 and Pin1 and employed both Förster Resonance Energy Transfer (FRET) and Fluorescence Correlation Spectroscopy (FCS) imaging techniques to characterize their interactions in living cells. In both intensity-based and time-resolved FRET studies, we observed FRET uniformly across whole cells co-expressing I2-Cerulean and Pin1-Venus that was significantly higher than in negative controls expressing Cerulean FP (without fusing to I2) as the FRET donor and Pin1-Venus, showing a specific interaction between I2-Cerulean and Pin1-Venus in living cells. We also observed the co-diffusion of I2-Cerulean and Pin1-mCherry in Fluorescence Cross Correlation Spectroscopy (FCCS) measurements. We further showed that I2 itself as well as I2-Pin1 formed complexes in living cells (predicted from in vitro studies) via a quantitative FRET assay, and demonstrated from FCS measurements that both I2 and Pin1 (fused to Cerulean) are highly mobile in living cells.

  9. Phosphorylation of Mycobacterium tuberculosis Ser/Thr phosphatase by PknA and PknB.

    Directory of Open Access Journals (Sweden)

    Andaleeb Sajid

    2011-03-01

    Full Text Available The integrated functions of 11 Ser/Thr protein kinases (STPKs and one phosphatase manipulate the phosphorylation levels of critical proteins in Mycobacterium tuberculosis. In this study, we show that the lone Ser/Thr phosphatase (PstP is regulated through phosphorylation by STPKs.PstP is phosphorylated by PknA and PknB and phosphorylation is influenced by the presence of Zn(2+-ions and inorganic phosphate (Pi. PstP is differentially phosphorylated on the cytosolic domain with Thr(137, Thr(141, Thr(174 and Thr(290 being the target residues of PknB while Thr(137 and Thr(174 are phosphorylated by PknA. The Mn(2+-ion binding residues Asp(38 and Asp(229 are critical for the optimal activity of PstP and substitution of these residues affects its phosphorylation status. Native PstP and its phosphatase deficient mutant PstP(c (D38G are phosphorylated by PknA and PknB in E. coli and addition of Zn(2+/Pi in the culture conditions affect the phosphorylation level of PstP. Interestingly, the phosphorylated phosphatase is more active than its unphosphorylated equivalent.This study establishes the novel mechanisms for regulation of mycobacterial Ser/Thr phosphatase. The results indicate that STPKs and PstP may regulate the signaling through mutually dependent mechanisms. Consequently, PstP phosphorylation may play a critical role in regulating its own activity. Since, the equilibrium between phosphorylated and non-phosphorylated states of mycobacterial proteins is still unexplained, understanding the regulation of PstP may help in deciphering the signal transduction pathways mediated by STPKs and the reversibility of the phenomena.

  10. Discovery and study of novel protein tyrosine phosphatase 1B inhibitors

    Science.gov (United States)

    Zhang, Qian; Chen, Xi; Feng, Changgen

    2017-10-01

    Protein tyrosine phosphatase 1B (PTP1B) is considered to be a target for therapy of type II diabetes and obesity. So it is of great significance to take advantage of a computer aided drug design protocol involving the structured-based virtual screening with docking simulations for fast searching small molecule PTP1B inhibitors. Based on optimized complex structure of PTP1B bound with specific inhibitor of IX1, structured-based virtual screening against a library of natural products containing 35308 molecules, which was constructed based on Traditional Chinese Medicine database@ Taiwan (TCM database@ Taiwan), was conducted to determine the occurrence of PTP1B inhibitors using the Lubbock module and CDOCKER module from Discovery Studio 3.1 software package. The results were further filtered by predictive ADME simulation and predictive toxic simulation. As a result, 2 good drug-like molecules, namely para-benzoquinone compound 1 and Clavepictine analogue 2 were identified ultimately with the dock score of original inhibitor (IX1) and the receptor as a threshold. Binding model analyses revealed that these two candidate compounds have good interactions with PTP1B. The PTP1B inhibitory activity of compound 2 hasn't been reported before. The optimized compound 2 has higher scores and deserves further study.

  11. The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

    KAUST Repository

    Cui, Peng; Chen, Tao; Qin, Tao; Ding, Feng; Wang, Zhenyu; Chen, Hao; Xiong, Liming

    2016-01-01

    © 2016 American Society of Plant Biologists. All rights reserved. Nonsense-mediated decay (NMD) is a posttranscriptional surveillance mechanism in eukaryotes that recognizes and degrades transcripts with premature translation-termination codons. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2 (FRY2; also known as C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 [CPL1]) plays multiple roles in RNA processing in Arabidopsis thaliana. Here, we found that FRY2/CPL1 interacts with two NMD factors, eIF4AIII and UPF3, and is involved in the dephosphorylation of eIF4AIII. This dephosphorylation retains eIF4AIII in the nucleus and limits its accumulation in the cytoplasm. By analyzing RNA-seq data combined with quantitative RT-PCR validation, we found that a subset of alternatively spliced transcripts and 59-extended mRNAs with NMD-eliciting features accumulated in the fry2-1 mutant, cycloheximidetreated wild type, and upf3 mutant plants, indicating that FRY2 is essential for the degradation of these NMD transcripts.

  12. The RNA Polymerase II C-Terminal Domain Phosphatase-Like Protein FIERY2/CPL1 Interacts with eIF4AIII and Is Essential for Nonsense-Mediated mRNA Decay in Arabidopsis

    KAUST Repository

    Cui, Peng

    2016-02-18

    © 2016 American Society of Plant Biologists. All rights reserved. Nonsense-mediated decay (NMD) is a posttranscriptional surveillance mechanism in eukaryotes that recognizes and degrades transcripts with premature translation-termination codons. The RNA polymerase II C-terminal domain phosphatase-like protein FIERY2 (FRY2; also known as C-TERMINAL DOMAIN PHOSPHATASE-LIKE1 [CPL1]) plays multiple roles in RNA processing in Arabidopsis thaliana. Here, we found that FRY2/CPL1 interacts with two NMD factors, eIF4AIII and UPF3, and is involved in the dephosphorylation of eIF4AIII. This dephosphorylation retains eIF4AIII in the nucleus and limits its accumulation in the cytoplasm. By analyzing RNA-seq data combined with quantitative RT-PCR validation, we found that a subset of alternatively spliced transcripts and 59-extended mRNAs with NMD-eliciting features accumulated in the fry2-1 mutant, cycloheximidetreated wild type, and upf3 mutant plants, indicating that FRY2 is essential for the degradation of these NMD transcripts.

  13. WatAA: Atlas of Protein Hydration. Exploring synergies between data mining and ab initio calculations.

    Science.gov (United States)

    Černý, Jiří; Schneider, Bohdan; Biedermannová, Lada

    2017-07-14

    Water molecules represent an integral part of proteins and a key determinant of protein structure, dynamics and function. WatAA is a newly developed, web-based atlas of amino-acid hydration in proteins. The atlas provides information about the ordered first hydration shell of the most populated amino-acid conformers in proteins. The data presented in the atlas are drawn from two sources: experimental data and ab initio quantum-mechanics calculations. The experimental part is based on a data-mining study of a large set of high-resolution protein crystal structures. The crystal-derived data include 3D maps of water distribution around amino-acids and probability of occurrence of each of the identified hydration sites. The quantum mechanics calculations validate and extend this primary description by optimizing the water position for each hydration site, by providing hydrogen atom positions and by quantifying the interaction energy that stabilizes the water molecule at the particular hydration site position. The calculations show that the majority of experimentally derived hydration sites are positioned near local energy minima for water, and the calculated interaction energies help to assess the preference of water for the individual hydration sites. We propose that the atlas can be used to validate water placement in electron density maps in crystallographic refinement, to locate water molecules mediating protein-ligand interactions in drug design, and to prepare and evaluate molecular dynamics simulations. WatAA: Atlas of Protein Hydration is freely available without login at .

  14. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    Energy Technology Data Exchange (ETDEWEB)

    Felts, Richard L. [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Reilly, Thomas J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Veterinary Medical Diagnostic Laboratory, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Calcutt, Michael J. [Department of Veterinary Pathobiology, College of Veterinary Medicine, University of Missouri-Columbia, Columbia, Missouri 65212 (United States); Tanner, John J., E-mail: tannerjj@missouri.edu [Department of Chemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States); Department of Biochemistry, University of Missouri-Columbia, Columbia, Missouri 65211 (United States)

    2006-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative.

  15. Crystallization of a newly discovered histidine acid phosphatase from Francisella tularensis

    International Nuclear Information System (INIS)

    Felts, Richard L.; Reilly, Thomas J.; Calcutt, Michael J.; Tanner, John J.

    2005-01-01

    A histidine acid phosphatase from the CDC Category A pathogen F. tularensis has been crystallized in space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. A 1.75 Å resolution data set was collected at Advanced Light Source beamline 4.2.2. Francisella tularensis is a highly infectious bacterial pathogen that is considered by the Centers for Disease Control and Prevention to be a potential bioterrorism weapon. Here, the crystallization of a 37.2 kDa phosphatase encoded by the genome of F. tularensis subsp. holarctica live vaccine strain is reported. This enzyme shares 41% amino-acid sequence identity with Legionella pneumophila major acid phosphatase and contains the RHGXRXP motif that is characteristic of the histidine acid phosphatase family. Large diffraction-quality crystals were grown in the presence of Tacsimate, HEPES and PEG 3350. The crystals belong to space group P4 1 2 1 2, with unit-cell parameters a = 61.96, c = 210.78 Å. The asymmetric unit is predicted to contain one protein molecule, with a solvent content of 53%. A 1.75 Å resolution native data set was recorded at beamline 4.2.2 of the Lawrence Berkeley National Laboratory Advanced Light Source. Molecular-replacement trials using the human prostatic acid phosphatase structure as the search model (28% amino-acid sequence identity) did not produce a satisfactory solution. Therefore, the structure of F. tularensis histidine acid phosphatase will be determined by multiwavelength anomalous dispersion phasing using a selenomethionyl derivative

  16. Overexpression of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Karr, Laurel; Malone, Christine, C.; Rose, M. Franklin (Technical Monitor)

    2000-01-01

    The Pichiapastoris expression system was utilized to produce functionally active human bone alkaline phosphatase in gram quantities. Bone alkaline phosphatase is a key enzyme in bone formation and biomineralization, yet important questions about its structural chemistry and interactions with other cellular enzymes in mineralizing tissues remain unanswered. A soluble form of human bone alkaline phosphatase was constructed by deletion of the 25 amino acid hydrophobic C-terminal region of the encoding cDNA and inserted into the X-33 Pichiapastoris strain. An overexpression system was developed in shake flasks and converted to large-scale fermentation. Alkaline phosphatase was secreted into the medium to a level of 32mgAL when cultured in shake flasks. Enzyme activity was 12U/mg measured by a spectrophotometric assay. Fermentation yielded 880mgAL with enzymatic activity of 968U/mg. Gel electrophoresis analysis indicates that greater than 50% of the total protein in the fermentation is alkaline phosphatase. A purification scheme has been developed using ammonium sulfate precipitation followed by hydrophobic interaction chromatography. We are currently screening crystallization conditions of the purified recombinant protein for subsequent X-ray diffraction analyses. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  17. Acid phosphatase turnover during repressed and derepressed cultivation of Aspergillus niger

    International Nuclear Information System (INIS)

    Komano, Teruya

    1975-01-01

    Enhancement of the activity of acid phosphatase (EC 3.1.3.2) by phosphate starvation in growing Aspergillus niger mycelia was prevented by cycloheximide. This indicates that the enhancement was due to de novo protein synthesis caused by derepression. Radioactive acid phosphatase extracted from mycelia labeled with 14 C-amino acid was separated into at least four fractions. Experiments on pulse labeling and the chasing of the four acid phosphatases revealed the synthesis and degradation of each fraction occurred at different rates; showing a different rate of turnover of the enzyme molecules. The results of similar experiments performed during culture in the presence of phosphate (partially repressed condition) suggested that the marked change in the activity ratios of the four acid phosphatases during cultivation was the result of the active turnover of enzyme molecules. In contrast, the slight changes in the ratios observed during derepressed cultivation seemed to be the result of similar of synthesis and degradation of each phosphatase fraction. (auth.)

  18. EST Table: FS873739 [KAIKOcDNA[Archive

    Lifescience Database Archive (English)

    Full Text Available FS873739 E_FL_fner_51P23_R_0 10/09/28 100 %/172 aa ref|NP_001037572.1| translationa...pp0225303|DvirGJ10886-PA 10/08/29 44 %/180 aa F25H2.11#CE09656#WBGene00009122#locus:tct-1#TCTP protein#statu...s:Confirmed#UniProt:Q93573#protein_id:CAB02 099.1 10/09/10 73 %/172 aa AGAP002667

  19. Maximal COX-2 and ppRb expression in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia in Alzheimer's disease

    Directory of Open Access Journals (Sweden)

    Arendt Thomas

    2005-11-01

    Full Text Available Abstract Neuronal expression of cyclooxygenase-2 (COX-2 and cell cycle proteins is suggested to contribute to neurodegeneration during Alzheimer's disease (AD. The stimulus that induces COX-2 and cell cycle protein expression in AD is still elusive. Activated glia cells are shown to secrete substances that can induce expression of COX-2 and cell cycle proteins in vitro. Using post mortem brain tissue we have investigated whether activation of microglia and astrocytes in AD brain can be correlated with the expression of COX-2 and phosphorylated retinoblastoma protein (ppRb. The highest levels of neuronal COX-2 and ppRb immunoreactivity are observed in the first stages of AD pathology (Braak 0–II, Braak A. No significant difference in COX-2 or ppRb neuronal immunoreactivity is observed between Braak stage 0 and later Braak stages for neurofibrillary changes or amyloid plaques. The mean number of COX-2 or ppRb immunoreactive neurons is significantly decreased in Braak stage C compared to Braak stage A for amyloid deposits. Immunoreactivity for glial markers KP1, CR3/43 and GFAP appears in the later Braak stages and is significantly increased in Braak stage V-VI compared to Braak stage 0 for neurofibrillary changes. In addition, a significant negative correlation is observed between the presence of KP1, CR3/43 and GFAP immunoreactivity and the presence of neuronal immunoreactivity for COX-2 and ppRb. These data show that maximal COX-2 and ppRb immunoreactivity in neurons occurs during early Braak stages prior to the maximal activation of astrocytes and microglia. In contrast to in vitro studies, post mortem data do not support a causal relation between the activation of microglia and astrocytes and the expression of neuronal COX-2 and ppRb in the pathological cascade of AD.

  20. Phosphorylation-mediated regulation of the Staphylococcus aureus secreted tyrosine phosphatase PtpA.

    Science.gov (United States)

    Brelle, Solène; Baronian, Grégory; Huc-Brandt, Sylvaine; Zaki, Laila Gannoun; Cohen-Gonsaud, Martin; Bischoff, Markus; Molle, Virginie

    2016-01-15

    Due to the emergence of methicillin-resistant strains, Staphylococcus aureus has become as major public-health threat. Studies aimed at deciphering the molecular mechanism of virulence are thus required to identify new targets and develop efficient therapeutic agents. Protein phosphorylations are known to play key regulatory functions and their roles in pathogenesis are under intense scrutiny. Here we analyzed the protein tyrosine phosphatase PtpA of S. aureus, a member of the family of low molecular weight protein tyrosine phosphatases that are often secreted by pathogenic bacteria. We report for the first time that PtpA is phosphorylated in vitro by the S. aureus tyrosine kinase CapA1B2. A mass spectrometry approach allowed determining that Tyr122 and Tyr123 were the only two residues phosphorylated by this kinase. This result was confirmed by analysis of a double PtpA_Y122A/Y123A mutant that showed no phosphorylation by CapA1B2. Interestingly, PtpA phosphatase activity was abrogated in this mutant, suggesting a key regulatory function for these two tyrosine residues. This was further reinforced by the observation that CapA1B2-mediated phosphorylation significantly increased PtpA phosphatase activity. Moreover, we provide evidence that PtpA is secreted during growth of S. aureus. Together our results suggest that PtpA is an exported S. aureus signaling molecule controlled by tyrosine phosphorylation which may interfere with host cell signaling. Copyright © 2015 Elsevier Inc. All rights reserved.

  1. Regulation of Akt/Protein Kinase B Signaling by a Novel Protein Phosphatase in Breast Cancer Cells

    National Research Council Canada - National Science Library

    Brognard, John; Newton, Alexandra

    2008-01-01

    ...: cell proliferation, growth, and apoptosis. Finally, since this phosphatase resides in a location of frequent loss of heterozygosity in breast cancer, we sought to determine if this phosphatase played a role in breast tumorigenesis...

  2. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    International Nuclear Information System (INIS)

    Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

    2008-01-01

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

  3. Receptor protein tyrosine phosphatase alpha activates Src-family kinases and controls integrin-mediated responses in fibroblasts

    DEFF Research Database (Denmark)

    Su, J; Muranjan, M; Sap, J

    1999-01-01

    of tyrosine kinases, the activity of which is tightly controlled by inhibitory phosphorylation of a carboxyterminal tyrosine residue (Tyr527 in chicken c-Src); this phosphorylation induces the kinases to form an inactive conformation. Whereas the identity of such inhibitory Tyr527 kinases has been well...... established, no corresponding phosphatases have been identified that, under physiological conditions, function as positive regulators of c-Src and Fyn in fibroblasts. RESULTS: Receptor protein tyrosine phosphatase alpha (RPTPalpha) was inactivated by homologous recombination. Fibroblasts derived from...... these RPTPalpha-/- mice had impaired tyrosine kinase activity of both c-Src and Fyn, and this was accompanied by a concomitant increase in c-Src Tyr527 phosphorylation. RPTPalpha-/- fibroblasts also showed a reduction in the rate of spreading on fibronectin substrates, a trait that is a phenocopy of the effect...

  4. Microvillus-Specific Protein Tyrosine Phosphatase SAP-1 Plays a Role in Regulating the Intestinal Paracellular Transport of Macromolecules.

    Science.gov (United States)

    Mori, Shingo; Kamei, Noriyasu; Murata, Yoji; Takayama, Kozo; Matozaki, Takashi; Takeda-Morishita, Mariko

    2017-09-01

    The stomach cancer-associated protein tyrosine phosphatase 1 (SAP-1) is a receptor-type protein tyrosine phosphatase that is specifically expressed on the apical membrane of the intestinal epithelium. SAP-1 is known to maintain the balance of phosphorylation of proteins together with protein kinases; however, its biological function and impact on pharmacokinetics in the intestine remain unclear. The present study, therefore, aimed at clarifying the relationship between SAP-1 and the intestinal absorption behaviors of typical transporter substrates and macromolecules. The endogenous levels of glucose and total cholesterol in the blood were similar between wild-type and SAP-1-deficient mice (Sap1 -/- ), suggesting no contribution of SAP-1 to biogenic influx. Moreover, in vitro transport study with everted ileal sacs demonstrated that there was no difference in the absorption of breast cancer resistance protein, P-glycoprotein, and peptide transporter substrates between both mice. However, absorptive clearance of macromolecular model dextrans (FD-4 and FD-10) in Sap1 -/- mice was significantly higher than that in wild-type mice, and this was confirmed by the trend of increased FD-4 absorption from colonic loops of Sap1 -/- mice. Therefore, the results of this study suggest the partial contribution of SAP-1 to the regulated transport of hydrophilic macromolecules through paracellular tight junctions. Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.

  5. Molecular mechanism of ERK dephosphorylation by striatal-enriched protein tyrosine phosphatase (STEP)

    Science.gov (United States)

    Li, Hui; Li, Kang-shuai; Su, Jing; Chen, Lai-Zhong; Xu, Yun-Fei; Wang, Hong-Mei; Gong, Zheng; Cui, Guo-Ying; Yu, Xiao; Wang, Kai; Yao, Wei; Xin, Tao; Li, Min-Yong; Xiao, Kun-Hong; An, Xiao-fei; Huo, Yuqing; Xu, Zhi-gang; Sun, Jin-Peng; Pang, Qi

    2013-01-01

    Striatal-enriched tyrosine phosphatase (STEP) is an important regulator of neuronal synaptic plasticity, and its abnormal level or activity contributes to cognitive disorders. One crucial downstream effector and direct substrate of STEP is extracellular signal-regulated protein kinase (ERK), which has important functions in spine stabilisation and action potential transmission. The inhibition of STEP activity toward phospho-ERK has the potential to treat neuronal diseases, but the detailed mechanism underlying the dephosphorylation of phospho-ERK by STEP is not known. Therefore, we examined STEP activity toward pNPP, phospho-tyrosine-containing peptides, and the full-length phospho-ERK protein using STEP mutants with different structural features. STEP was found to be a highly efficient ERK tyrosine phosphatase that required both its N-terminal regulatory region and key residues in its active site. Specifically, both KIM and KIS of STEP were required for ERK interaction. In addition to the N-terminal KIS region, S245, hydrophobic residues L249/L251, and basic residues R242/R243 located in the KIM region were important in controlling STEP activity toward phospho-ERK. Further kinetic experiments revealed subtle structural differences between STEP and HePTP that affected the interactions of their KIMs with ERK. Moreover, STEP recognised specific positions of a phospho-ERK peptide sequence through its active site, and the contact of STEP F311 with phospho-ERK V205 and T207 were crucial interactions. Taken together, our results not only provide the information for interactions between ERK and STEP, but will also help in the development of specific strategies to target STEP-ERK recognition, which could serve as a potential therapy for neurological disorders. PMID:24117863

  6. Toward the identification of a reliable 3D-QSAR model for the protein tyrosine phosphatase 1B inhibitors

    Science.gov (United States)

    Wang, Fangfang; Zhou, Bo

    2018-04-01

    Protein tyrosine phosphatase 1B (PTP1B) is an intracellular non-receptor phosphatase that is implicated in signal transduction of insulin and leptin pathways, thus PTP1B is considered as potential target for treating type II diabetes and obesity. The present article is an attempt to formulate the three-dimensional quantitative structure-activity relationship (3D-QSAR) modeling of a series of compounds possessing PTP1B inhibitory activities using comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) techniques. The optimum template ligand-based models are statistically significant with great CoMFA (R2cv = 0.600, R2pred = 0.6760) and CoMSIA (R2cv = 0.624, R2pred = 0.8068) values. Molecular docking was employed to elucidate the inhibitory mechanisms of this series of compounds against PTP1B. In addition, the CoMFA and CoMSIA field contour maps agree well with the structural characteristics of the binding pocket of PTP1B active site. The knowledge of structure-activity relationship and ligand-receptor interactions from 3D-QSAR model and molecular docking will be useful for better understanding the mechanism of ligand-receptor interaction and facilitating development of novel compounds as potent PTP1B inhibitors.

  7. 2,3-diphosphoglycerate phosphatase activity of phosphoglycerate mutase: stimulation by vanadate and phosphate

    International Nuclear Information System (INIS)

    Stankiewicz, P.J.; Gresser, M.J.; Tracey, A.S.; Hass, L.F.

    1987-01-01

    The binding of inorganic vanadate (V/sub i/) to rabbit muscle phosphoglycerate mutase (PGM), studied by using 51 V nuclear magnetic resonance spectroscopy, shows a sigmoidal dependence on vanadate concentration with a stoichiometry of four vanadium atoms per PGM molecule at saturating [V/sub i/]. The data are consistent with binding of one divanadate ion to each of the two subunits of PGM in a noncooperative manner with an intrinsic dissociation constant of 4 x 10 -6 M. The relevance of this result to other studies which have shown that the V/sub i/-stimulated 2,3-diphosphoglycerate (2,3-DPG) phosphatase activity of PGM has a sigmoidal dependence on [V/sub i/] with a Hill coefficient of 2.0 is discussed. At pH 7.0, inorganic phosphate has little effect on the 2,3-DPG phosphatase activity of PGM, even at concentrations as high as 50 mM. Similarly, 25 μM V/sub i/ has little effect on the phosphatase activity. However, in the presence of 25 μM V/sub i/, a phosphate concentration of 20 mM increases the phosphatase activity by more than 3-fold. This behavior is rationalized in terms of activation of the phosphatase activity by a phosphate/vanadate mixed anhydride. This interpretation is supported by the observation of strong activation of the phosphatase activity by inorganic pyrophosphate. A molecular mechanism for the observed effects of vanadate is proposed, and the relevance of this study to the possible use of vanadate as a therapeutic agent for the treatment of sickle cell anemia is discussed

  8. SH2-inositol phosphatase 1 negatively influences early megakaryocyte progenitors.

    Directory of Open Access Journals (Sweden)

    Lia E Perez

    Full Text Available The SH2-containing-5'inositol phosphatase-1 (SHIP influences signals downstream of cytokine/chemokine receptors that play a role in megakaryocytopoiesis, including thrombopoietin, stromal-cell-derived-Factor-1/CXCL-12 and interleukin-3. We hypothesize that SHIP might control megakaryocytopoiesis through effects on proliferation of megakaryocyte progenitors (MKP and megakaryocytes (MK.Herein, we report the megakaryocytic phenotype and MK functional assays of hematopoietic organs of two strains of SHIP deficient mice with deletion of the SHIP promoter/first exon or the inositol phosphatase domain. Both SHIP deficient strains exhibit a profound increase in MKP numbers in bone marrow (BM, spleen and blood as analyzed by flow cytometry (Lin(-c-Kit+CD41+ and functional assays (CFU-MK. SHIP deficient MKP display increased phosphorylation of Signal Transducers and Activators of Transcription 3 (STAT-3, protein kinase B (PKB/AKT and extracellular signal-regulated kinases (ERKs. Despite increased MKP content, total body number of mature MK (Lin(-c-kit(-CD41+ are not significantly changed as SHIP deficient BM contains reduced MK while spleen MK numbers are increased. Reduction of CXCR-4 expression in SHIP deficient MK may influence MK localization to the spleen instead of the BM. Endomitosis, process involved in MK maturation, was preserved in SHIP deficient MK. Circulating platelets and red blood cells are also reduced in SHIP deficient mice.SHIP may play an important role in regulation of essential signaling pathways that control early megakaryocytopoiesis in vivo.

  9. Bardoxolone methyl (BARD) ameliorates aristolochic acid (AA)-induced acute kidney injury through Nrf2 pathway.

    Science.gov (United States)

    Wu, Juan; Liu, Xinhui; Fan, Jinjin; Chen, Wenfang; Wang, Juan; Zeng, Youjia; Feng, Xiaorang; Yu, Xueqing; Yang, Xiao

    2014-04-06

    Bardoxolone methyl (BARD) is an antioxidant modulator that acts through induction of the nuclear factor erythroid 2-related factor 2 (Nrf2) signaling pathway. This study aimed to investigate the role of BARD in protecting kidneys from aristolochic acid (AA)-induced acute kidney injury (AKI). Male C57BL/6 mice received intraperitoneal (i.p.) injections of aristolochic acid I (AAI) (5mg/kg/day) for 5 days to produce acute AA nephropathy (AAN) model. BARD (10mg/kg/day, i.p.) was applied for 7 consecutive days, starting 2 days prior to AAI administration. The mice in the AA group showed AKI as evidenced by worsening kidney function evaluated by blood urea nitrogen (BUN) and serum creatinine (SCr) levels, and severe tubulointerstitial injury marked by massive tubule necrosis in kidney tissues. BARD significantly reduced BUN and SCr levels which were elevated by AAI. Additionally, AAI-induced histopathological renal damage was ameliorated by BARD. Furthermore, the expression of Nrf2 was reduced, and its repressor Kelch-like ECH-associated protein 1 (Keap1) was increased significantly, whereas heme oxygenase-1 (HO-1) was upregulated and NAD(P)H quinone oxidoreductase-1 (NQO1) was barely increased in the cytoplasm of tubules in kidneys after treatment with AAI. BARD significantly upregulated renal Nrf2, NQO1 and HO-1 expression and downregulated Keap1 expression compared with those in the AA group. Moreover, it was found that Nrf2 was expressed both in the cytoplasm and nuclear of glomeruli and tubules, whereas NQO1 and HO-1 were localized in the cytoplasm of tubules only. In conclusion, AA-induced acute renal injury was associated with impaired Nrf2 activation and expression of its downstream target genes in renal tissues. BARD prevented renal damage induced by AAI, and this renoprotective effect may be exerted by activating the Nrf2 signaling pathway and increasing expression of the downstream target genes. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  10. Efeito do citrato e taurina em meio CR2aa no desenvolvimento de embriões bovinos fecundados in vitro Effect of citrate and taurine added to CR2aa medium on the development of in vitro-fertilized bovine embryos

    Directory of Open Access Journals (Sweden)

    L.S.A. Camargo

    2009-02-01

    Full Text Available Avaliou-se o efeito do citrato em meio CR2aa suplementado com soro fetal bovino (SFB ou livre de proteínas séricas e sua associação com taurina no desenvolvimento de embriões bovinos fecundados in vitro. Embriões foram cultivados em CR2aa contendo 0, 0,5, 1,0 e 3,0mM citrato, suplementado com 10% SFB (experimento 1 ou com álcool polivinil (PVA; experimento 2. No terceiro experimento, embriões foram cultivados em meio com 0,5mM citrato, ou 7mM taurina, ou com a associação de ambos, suplementado com SFB. Os cultivos foram realizados com células do cumulus em ambiente a 38,8ºC com 5% de CO2 em ar atmosférico. Melhora no desenvolvimento embrionário foi observado no cultivo de embriões em CR2aa com 0,5 e 1,0mM citrato na ausência de SFB (P0,05 a produção de embriões ou o número de células. Citrato em meio CR2aa pode ser uma alternativa para cultivo embrionário em condições atmosféricas com 5% de CO2 em ar na ausência de proteína sérica.The effect of citrate added to CR2aa medium supplemented with fetal calf serum (FCS or serum-proteinfree and its association with taurine on the development of in vitro-fertilized bovine embryos was evaluated. Embryos were cultured with 0, 0.5, 1.0, and 3.0mM citrate, in CR2aa supplemented with 10% FCS (experiment 1, or polyvinyl alcohol (PVA; experiment 2. In experiment 3, embryos were cultured with 0.5mM citrate, 7.0mM taurine or with association of both, in medium supplemented with FCS. Embryo culture was performed with cumulus cells at 38.8ºC in 5% CO2 under air for all experiments. Positive effect on embryo development was only observed with 0.5 and 1.0mM citrate in FCS-free CR2aa (P0.05 embryo rate nor total cell number. Citrate in CR2aa medium can be an alternative for serumfree embryo culture under 5% CO2 in air, absence of serum protein.

  11. Stimulation of Slack K(+) Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex.

    Science.gov (United States)

    Fleming, Matthew R; Brown, Maile R; Kronengold, Jack; Zhang, Yalan; Jenkins, David P; Barcia, Gulia; Nabbout, Rima; Bausch, Anne E; Ruth, Peter; Lukowski, Robert; Navaratnam, Dhasakumar S; Kaczmarek, Leonard K

    2016-08-30

    Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  12. Stimulation of Slack K+ channels alters mass at the plasma membrane by triggering dissociation of a phosphatase-regulatory complex

    Science.gov (United States)

    Fleming, Matthew R.; Brown, Maile R.; Kronengold, Jack; Zhang, Yalan; Jenkins, David P.; Barcia, Gulia; Nabbout, Rima; Bausch, Anne E.; Ruth, Peter; Lukowski, Robert; Navaratnam, Dhasakumar S.; Kaczmarek, Leonard K.

    2016-01-01

    Summary Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa) channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1) targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways, and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies. PMID:27545877

  13. Stimulation of Slack K+ Channels Alters Mass at the Plasma Membrane by Triggering Dissociation of a Phosphatase-Regulatory Complex

    Directory of Open Access Journals (Sweden)

    Matthew R. Fleming

    2016-08-01

    Full Text Available Human mutations in the cytoplasmic C-terminal domain of Slack sodium-activated potassium (KNa channels result in childhood epilepsy with severe intellectual disability. Slack currents can be increased by pharmacological activators or by phosphorylation of a Slack C-terminal residue by protein kinase C. Using an optical biosensor assay, we find that Slack channel stimulation in neurons or transfected cells produces loss of mass near the plasma membrane. Slack mutants associated with intellectual disability fail to trigger any change in mass. The loss of mass results from the dissociation of the protein phosphatase 1 (PP1 targeting protein, Phactr-1, from the channel. Phactr1 dissociation is specific to wild-type Slack channels and is not observed when related potassium channels are stimulated. Our findings suggest that Slack channels are coupled to cytoplasmic signaling pathways and that dysregulation of this coupling may trigger the aberrant intellectual development associated with specific childhood epilepsies.

  14. MID1 and MID2 homo- and heterodimerise to tether the rapamycin-sensitive PP2A regulatory subunit, Alpha 4, to microtubules: implications for the clinical variability of X-linked Opitz GBBB syndrome and other developmental disorders

    Directory of Open Access Journals (Sweden)

    Cox Timothy C

    2002-01-01

    Full Text Available Abstract Background Patients with Opitz GBBB syndrome present with a variable array of developmental defects including craniofacial, cardiac, and genital anomalies. Mutations in the X-linked MID1 gene, which encodes a microtubule-binding protein, have been found in ~50% of Opitz GBBB syndrome patients consistent with the genetically heterogeneous nature of the disorder. A protein highly related to MID1, called MID2, has also been described that similarly associates with microtubules. Results To identify protein partners of MID1 and MID2 we undertook two separate yeast two-hybrid screens. Using this system we identified Alpha 4, a regulatory subunit of PP2-type phosphatases and a key component of the rapamycin-sensitive signaling pathway, as a strong interactor of both proteins. Analysis of domain-specific deletions has shown that the B-boxes of both MID1 and MID2 mediate the interaction with Alpha 4, the first demonstration in an RBCC protein of a specific role for the B-box region. In addition, we show that the MID1/2 coiled-coil motifs mediate both homo- and hetero-dimerisation, and that dimerisation is a prerequisite for association of the MID-Alpha 4 complex with microtubules. Conclusions Our findings not only implicate Alpha 4 in the pathogenesis of Opitz GBBB syndrome but also support our earlier hypothesis that MID2 is a modifier of the X-linked phenotype. Of further note is the observation that Alpha 4 maps to Xq13 within the region showing linkage to FG (Opitz-Kaveggia syndrome. Overlap in the clinical features of FG and Opitz GBBB syndromes warrants investigation of Alpha 4 as a candidate for causing FG syndrome.

  15. Detection of endogenous alkaline phosphatase activity in intact cells by flow cytometry using the fluorogenic ELF-97 phosphatase substrate

    Science.gov (United States)

    Telford, W. G.; Cox, W. G.; Stiner, D.; Singer, V. L.; Doty, S. B.

    1999-01-01

    BACKGROUND: The alkaline phosphatase (AP) substrate 2-(5'-chloro-2'-phosphoryloxyphenyl)-6-chloro-4-(3H)-quinazolinone (ELF((R))-97 for enzyme-labeled fluorescence) has been found useful for the histochemical detection of endogenous AP activity and AP-tagged proteins and oligonucleotide probes. In this study, we evaluated its effectiveness at detecting endogenous AP activity by flow cytometry. METHODS: The ELF-97 phosphatase substrate was used to detect endogenous AP activity in UMR-106 rat osteosarcoma cells and primary cultures of chick chondrocytes. Cells were labeled with the ELF-97 reagent and analyzed by flow cytometry using an argon ultraviolet (UV) laser. For comparison purposes, cells were also assayed for AP using a Fast Red Violet LB azo dye assay previously described for use in detecting AP activity by flow cytometry. RESULTS: The ELF-97 phosphatase substrate effectively detected endogenous AP activity in UMR-106 cells, with over 95% of the resulting fluorescent signal resulting from AP-specific activity (as determined by levamisole inhibition of AP activity). In contrast, less than 70% of the fluorescent signal from the Fast Red Violet LB (FRV) assay was AP-dependent, reflecting the high intrinsic fluorescence of the unreacted components. The ELF-97 phosphatase assay was also able to detect very low AP activity in chick chondrocytes that was undetectable by the azo dye method. CONCLUSIONS: The ELF-97 phosphatase assay was able to detect endogenous AP activity in fixed mammalian and avian cells by flow cytometry with superior sensitivity to previously described assays. This work also shows the applicability of ELF-97 to flow cytometry, supplementing its previously demonstrated histochemical applications. Copyright 1999 Wiley-Liss, Inc.

  16. Signaling Pathways Related to Protein Synthesis and Amino Acid Concentration in Pig Skeletal Muscles Depend on the Dietary Protein Level, Genotype and Developmental Stages.

    Directory of Open Access Journals (Sweden)

    Yingying Liu

    Full Text Available Muscle growth is regulated by the homeostatic balance of the biosynthesis and degradation of muscle proteins. To elucidate the molecular interactions among diet, pig genotype, and physiological stage, we examined the effect of dietary protein concentration, pig genotype, and physiological stages on amino acid (AA pools, protein deposition, and related signaling pathways in different types of skeletal muscles. The study used 48 Landrace pigs and 48 pure-bred Bama mini-pigs assigned to each of 2 dietary treatments: lower/GB (Chinese conventional diet- or higher/NRC (National Research Council-protein diet. Diets were fed from 5 weeks of age to respective market weights of each genotype. Samples of biceps femoris muscle (BFM, type I and longissimus dorsi muscle (LDM, type II were collected at nursery, growing, and finishing phases according to the physiological stage of each genotype, to determine the AA concentrations, mRNA levels for growth-related genes in muscles, and protein abundances of mechanistic target of rapamycin (mTOR signaling pathway. Our data showed that the concentrations of most AAs in LDM and BFM of pigs increased (P<0.05 gradually with increasing age. Bama mini-pigs had generally higher (P<0.05 muscle concentrations of flavor-related AA, including Met, Phe, Tyr, Pro, and Ser, compared with Landrace pigs. The mRNA levels for myogenic determining factor, myogenin, myocyte-specific enhancer binding factor 2 A, and myostatin of Bama mini-pigs were higher (P<0.05 than those of Landrace pigs, while total and phosphorylated protein levels for protein kinase B, mTOR, and p70 ribosomal protein S6 kinases (p70S6K, and ratios of p-mTOR/mTOR, p-AKT/AKT, and p-p70S6K/p70S6K were lower (P<0.05. There was a significant pig genotype-dependent effect of dietary protein on the levels for mTOR and p70S6K. When compared with the higher protein-NRC diet, the lower protein-GB diet increased (P<0.05 the levels for mTOR and p70S6K in Bama mini-pigs, but

  17. Growth, extracellular alkaline phosphatase activity, and kinetic characteristic responses of the bloom-forming toxic cyanobacterium, Microcystis aeruginosa, to atmospheric particulate matter (PM2.5, PM2.5-10, and PM>10).

    Science.gov (United States)

    Xu, Ziran; Wang, Shoubing; Wang, Yuanan; Zhang, Jie

    2018-03-01

    Atmospheric particulate matter (APM), commonly seen and widely excited in environment, appears great enough to influence the biochemical processes in aquatic microorganisms and phytoplankton. Understanding the response of cyanobacteria to various factors is fundamental for eutrophication control. To clarify the response of cyanobacteria to APM, the effects of PM 2.5 , PM 2.5-10 , and PM >10 on Microcystis aeruginosa were researched. Variabilities in cell density, chlorophyll a, soluble protein, malondialdehyde, extracellular activity, and kinetic parameters of alkaline phosphatase were evaluated by lab-cultured experiments. Results showed that the PM 2.5 had a slight stimulation impact on the growth and enhanced both of the 48- and 72-h extracellular alkaline phosphatase activity (APA), the affinity of alkaline phosphatase for substrate, and the 72-h maximum enzymatic reaction velocity (V max ). Moreover, the stimulations in extracellular APA and V max enhanced with the increasing exposure concentrations. We also found there were no obvious distinctions on the effects of growth and alkaline phosphatase in M. aeruginosa between PM 2.5-10 and PM >10 exposure groups. Obviously, inhibitory effects on growth existed in 4.0 and 8.0 mg/L PM 2.5-10 and 8.0 mg/L PM >10 at 120 h. Furthermore, PM 2.5-10 and PM >10 exerted inhibitory effects on the extracellular APA during the 72-h exposure. Simultaneously, the V max was notably inhibited and the affinity of alkaline phosphatase for substrate was more inseparable compared with control in PM 2.5-10 and PM >10 treatments. Nevertheless, the inhibitors in extracellular APA and kinetic parameters were unrelated to PM 2.5-10 and PM >10 exposure concentrations. Two-way ANOVA results revealed that there were significant interactions between exposure concentration and diameter of APM on the 120-h cell density, soluble protein content, APA, and 72 h APA of M. aeruginosa. These results in our study would be meaningful to further

  18. Overexpression of the pumpkin (Cucurbita maxima) 16 kDa phloem protein CmPP16 increases tolerance to water deficit.

    Science.gov (United States)

    Ramírez-Ortega, Francisco Arturo; Herrera-Pola, Paul Starsky; Toscano-Morales, Roberto; Xoconostle-Cázares, Beatriz; Ruiz-Medrano, Roberto

    2014-01-01

    The phloem plays an important role in the delivery of nutrients and signals between photosynthetic to heterotrophic tissues. Proteins and RNAs in the phloem translocation stream may have an important role in maintaining the integrity of the sieve tube system, as well as in long-distance signaling. CmPP16 is a pumpkin phloem protein, which has been shown to bind RNA in a non-sequence specific manner, and move it cell-to-cell and conceivably, long-distance. The protein and RNA are found in both companion cell (CC) and sieve elements (SE). However, a more precise function for this protein is not known. In this work we report the overexpression of CmPP16 fused to GFP via transformation of pumpkin (Cucurbita maxima cv. Big Max) plants in the cotyledonary stage by direct inoculation of Agrobacterium tumefaciens and Agrobacterium rhizogenes. Plants overexpressing CmPP16 did not show an obvious phenotype. However, these plants displayed higher photosynthetic capacity during drought than wild-type (WT) pumpkin or transformed with another construct. These results suggest that CmPP16 may be involved in the response to stress through long-distance signaling.

  19. Microsolvation of the acetanilide cation (AA(+)) in a nonpolar solvent: IR spectra of AA(+)-L(n) clusters (L = He, Ar, N2; n ≤ 10).

    Science.gov (United States)

    Schmies, Matthias; Patzer, Alexander; Schütz, Markus; Miyazaki, Mitsuhiko; Fujii, Masaaki; Dopfer, Otto

    2014-05-07

    Infrared photodissociation (IRPD) spectra of mass-selected cluster ions of acetanilide (N-phenylacetamide), AA(+)-Ln, with the ligands L = He (n = 1-2), Ar (n = 1-7), and N2 (n = 1-10) are recorded in the hydride stretch (amide A, νNH, νCH) and fingerprint (amide I-III) ranges of AA(+) in its (2)A'' ground electronic state. Cold AA(+)-Ln clusters are generated in an electron impact ion source, which predominantly produces the most stable isomer of a given cluster ion. Systematic vibrational frequency shifts of the N-H stretch fundamentals (νNH) provide detailed information about the sequential microsolvation process of AA(+) in a nonpolar (L = He and Ar) and quadrupolar (L = N2) solvent. In the most stable AA(+)-Ln clusters, the first ligand forms a hydrogen bond (H-bond) with the N-H proton of trans-AA(+) (t-AA(+)), whereas further ligands bind weakly to the aromatic ring (π-stacking). There is no experimental evidence for complexes with the less stable cis-AA(+) isomer. Quantum chemical calculations at the M06-2X/aug-cc-pVTZ level confirm the cluster growth sequence derived from the IR spectra. The calculated binding energies of De(H) = 720 and 1227 cm(-1) for H-bonded and De(π) = 585 and 715 cm(-1) for π-bonded Ar and N2 ligands in t-AA(+)-L are consistent with the observed photofragmentation branching ratios of AA(+)-Ln. Comparison between charged and neutral AA((+))-L dimers indicates that ionization switches the preferred ion-ligand binding motif from π-stacking to H-bonding. Electron removal from the HOMO of AA(+) delocalized over both the aromatic ring and the amide group significantly strengthens the C[double bond, length as m-dash]O bond and weakens the N-H bond of the amide group.

  20. Dynamic substrate enhancement for the identification of specific, second-site-binding fragments targeting a set of protein tyrosine phosphatases

    NARCIS (Netherlands)

    Schmidt, Marco F; Groves, Matthew R; Rademann, Jörg

    2011-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators in living systems and thus are attractive drug targets. The development of potent, selective PTP inhibitors has been a difficult challenge mainly due to the high homology of the phosphotyrosine substrate pockets. Here, a strategy of dynamic

  1. Regulation of gap junction conductance by calcineurin through Cx43 phosphorylation: implications for action potential conduction.

    Science.gov (United States)

    Jabr, Rita I; Hatch, Fiona S; Salvage, Samantha C; Orlowski, Alejandro; Lampe, Paul D; Fry, Christopher H

    2016-11-01

    Cardiac arrhythmias are associated with raised intracellular [Ca 2+ ] and slowed action potential conduction caused by reduced gap junction (GJ) electrical conductance (Gj). Ventricular GJs are composed of connexin proteins (Cx43), with Gj determined by Cx43 phosphorylation status. Connexin phosphorylation is an interplay between protein kinases and phosphatases but the precise pathways are unknown. We aimed to identify key Ca 2+ -dependent phosphorylation sites on Cx43 that regulate cardiac gap junction conductance and action potential conduction velocity. We investigated the role of the Ca 2+ -dependent phosphatase, calcineurin. Intracellular [Ca 2+ ] was raised in guinea-pig myocardium by a low-Na solution or increased stimulation. Conduction velocity and Gj were measured in multicellular strips. Phosphorylation of Cx43 serine residues (S365 and S368) and of the intermediary regulator I1 at threonine35 was measured by Western blot. Measurements were made in the presence and absence of inhibitors to calcineurin, I1 or protein phosphatase-1 and phosphatase-2.Raised [Ca 2 + ] i decreased Gj, reduced Cx43 phosphorylation at S365 and increased it at S368; these changes were reversed by calcineurin inhibitors. Cx43-S368 phosphorylation was reversed by the protein kinase C inhibitor chelerythrine. Raised [Ca 2+ ] i also decreased I1 phosphorylation, also prevented by calcineurin inhibitors, to increase activity of the Ca 2+ -independent phosphatase, PPI. The PP1 inhibitor, tautomycin, prevented Cx43-365 dephosphorylation, Cx43-S368 phosphorylation and Gj reduction in raised [Ca 2+ ] i . PP2A had no role. Conduction velocity was reduced by raised [Ca 2+ ] i and reversed by calcineurin inhibitors. Reduced action potential conduction and Gj in raised [Ca 2+ ] are regulated by calcineurin-dependent Cx43-S365 phosphorylation, leading to Cx43-S368 dephosphorylation. The calcineurin action is indirect, via I1 dephosphorylation and subsequent activation of PP1.

  2. Structural and biochemical analysis of atypically low dephosphorylating activity of human dual-specificity phosphatase 28.

    Directory of Open Access Journals (Sweden)

    Bonsu Ku

    Full Text Available Dual-specificity phosphatases (DUSPs constitute a subfamily of protein tyrosine phosphatases, and are intimately involved in the regulation of diverse parameters of cellular signaling and essential biological processes. DUSP28 is one of the DUSP subfamily members that is known to be implicated in the progression of hepatocellular and pancreatic cancers, and its biological functions and enzymatic characteristics are mostly unknown. Herein, we present the crystal structure of human DUSP28 determined to 2.1 Å resolution. DUSP28 adopts a typical DUSP fold, which is composed of a central β-sheet covered by α-helices on both sides and contains a well-ordered activation loop, as do other enzymatically active DUSP proteins. The catalytic pocket of DUSP28, however, appears hardly accessible to a substrate because of the presence of nonconserved bulky residues in the protein tyrosine phosphatase signature motif. Accordingly, DUSP28 showed an atypically low phosphatase activity in the biochemical assay, which was remarkably improved by mutations of two nonconserved residues in the activation loop. Overall, this work reports the structural and biochemical basis for understanding a putative oncological therapeutic target, DUSP28, and also provides a unique mechanism for the regulation of enzymatic activity in the DUSP subfamily proteins.

  3. Enzyme domain affects the movement of the voltage sensor in ascidian and zebrafish voltage-sensing phosphatases.

    Science.gov (United States)

    Hossain, Md Israil; Iwasaki, Hirohide; Okochi, Yoshifumi; Chahine, Mohamed; Higashijima, Shinichi; Nagayama, Kuniaki; Okamura, Yasushi

    2008-06-27

    The ascidian voltage-sensing phosphatase (Ci-VSP) consists of the voltage sensor domain (VSD) and a cytoplasmic phosphatase region that has significant homology to the phosphatase and tensin homolog deleted on chromosome TEN (PTEN). The phosphatase activity of Ci-VSP is modified by the conformational change of the VSD. In many proteins, two protein modules are bidirectionally coupled, but it is unknown whether the phosphatase domain could affect the movement of the VSD in VSP. We addressed this issue by whole-cell patch recording of gating currents from a teleost VSP (Dr-VSP) cloned from Danio rerio expressed in tsA201 cells. Replacement of a critical cysteine residue, in the phosphatase active center of Dr-VSP, by serine sharpened both ON- and OFF-gating currents. Similar changes were produced by treatment with phosphatase inhibitors, pervanadate and orthovanadate, that constitutively bind to cysteine in the active catalytic center of phosphatases. The distinct kinetics of gating currents dependent on enzyme activity were not because of altered phosphatidylinositol 4,5-bisphosphate levels, because the kinetics of gating current did not change by depletion of phosphatidylinositol 4,5-bisphosphate, as reported by coexpressed KCNQ2/3 channels. These results indicate that the movement of the VSD is influenced by the enzymatic state of the cytoplasmic domain, providing an important clue for understanding mechanisms of coupling between the VSD and its effector.

  4. Direct determination of phosphatase activity from physiological substrates in cells.

    Directory of Open Access Journals (Sweden)

    Zhongyuan Ren

    Full Text Available A direct and continuous approach to determine simultaneously protein and phosphate concentrations in cells and kinetics of phosphate release from physiological substrates by cells without any labeling has been developed. Among the enzymes having a phosphatase activity, tissue non-specific alkaline phosphatase (TNAP performs indispensable, multiple functions in humans. It is expressed in numerous tissues with high levels detected in bones, liver and neurons. It is absolutely required for bone mineralization and also necessary for neurotransmitter synthesis. We provided the proof of concept that infrared spectroscopy is a reliable assay to determine a phosphatase activity in the osteoblasts. For the first time, an overall specific phosphatase activity in cells was determined in a single step by measuring simultaneously protein and substrate concentrations. We found specific activities in osteoblast like cells amounting to 116 ± 13 nmol min(-1 mg(-1 for PPi, to 56 ± 11 nmol min(-1 mg(-1 for AMP, to 79 ± 23 nmol min(-1 mg(-1 for beta-glycerophosphate and to 73 ± 15 nmol min(-1 mg(-1 for 1-alpha-D glucose phosphate. The assay was also effective to monitor phosphatase activity in primary osteoblasts and in matrix vesicles. The use of levamisole--a TNAP inhibitor--served to demonstrate that a part of the phosphatase activity originated from this enzyme. An IC50 value of 1.16 ± 0.03 mM was obtained for the inhibition of phosphatase activity of levamisole in osteoblast like cells. The infrared assay could be extended to determine any type of phosphatase activity in other cells. It may serve as a metabolomic tool to monitor an overall phosphatase activity including acid phosphatases or other related enzymes.

  5. Mitogen-activated protein kinase and abscisic acid signal transduction

    NARCIS (Netherlands)

    Heimovaara-Dijkstra, S.; Testerink, C.; Wang, M.

    1998-01-01

    The phytohormone abscisic acid (ABA) is a classical plant hormone, responsible for regulation of abscission, diverse aspects of plant and seed development, stress responses and germination. It was found that ABA signal transduction in plants can involve the activity of type 2C-phosphatases (PP2C),

  6. PHF20 regulates NF-κB signalling by disrupting recruitment of PP2A to p65

    Science.gov (United States)

    Zhang, Tiejun; Park, Kyeong Ah; Li, Yuwen; Byun, Hee Sun; Jeon, Juhee; Lee, Yoonjung; Hong, Jang Hee; Kim, Jin Man; Huang, Song-Mei; Choi, Seung-Won; Kim, Sun-Hwan; Sohn, Kyung-Cheol; Ro, Hyunju; Lee, Ji Hoon; Lu, Tao; Stark, George R.; Shen, Han-Ming; Liu, Zheng-gang; Park, Jongsun; Hur, Gang Min

    2014-01-01

    Constitutive NF-κB activation in cancer cells is caused by defects in the signalling network responsible for terminating the NF-κB response. Here we report that plant homeodomain finger protein 20 maintains NF-κB in an active state in the nucleus by inhibiting the interaction between PP2A and p65. We show that plant homeodomain finger protein 20 induces canonical NF-κB signalling by increasing the DNA-binding activity of NF-κB subunit p65. In plant homeodomain finger protein 20-overexpressing cells, the termination of tumour necrosis factor-induced p65 phosphorylation is impaired whereas upstream signalling events triggered by tumour necrosis factor are unaffected. This effect strictly depends on the interaction between plant homeodomain finger protein 20 and methylated lysine residues of p65, which hinders recruitment of PP2A to p65, thereby maintaining p65 in a phosphorylated state. We further show that plant homeodomain finger protein 20 levels correlate with p65 phosphorylation levels in human glioma specimens. Our work identifies plant homeodomain finger protein 20 as a novel regulator of NF-κB activation and suggests that elevated expression of plant homeodomain finger protein 20 may drive constitutive NF-κB activation in some cancers. PMID:23797602

  7. Structural basis for basal activity and autoactivation of abscisic acid (ABA) signaling SnRK2 kinases

    OpenAIRE

    Ng, Ley-Moy; Soon, Fen-Fen; Zhou, X. Edward; West, Graham M.; Kovach, Amanda; Suino-Powell, Kelly M.; Chalmers, Michael J.; Li, Jun; Yong, Eu-Leong; Zhu, Jian-Kang; Griffin, Patrick R.; Melcher, Karsten; Xu, H. Eric

    2011-01-01

    Abscisic acid (ABA) is an essential hormone that controls plant growth, development, and responses to abiotic stresses. Central for ABA signaling is the ABA-mediated autoactivation of three monomeric Snf1-related kinases (SnRK2.2, -2.3, and -2.6). In the absence of ABA, SnRK2s are kept in an inactive state by forming physical complexes with type 2C protein phosphatases (PP2Cs). Upon relief of this inhibition, SnRK2 kinases can autoactivate through unknown mechanisms. Here, we report the cryst...

  8. Hematopoietic cell phosphatase is recruited to CD22 following B cell antigen receptor ligation

    NARCIS (Netherlands)

    Lankester, A. C.; van Schijndel, G. M.; van Lier, R. A.

    1995-01-01

    Hematopoietic cell phosphatase is a nonreceptor protein tyrosine phosphatase that is preferentially expressed in hematopoietic cell lineages. Motheaten mice, which are devoid of (functional) hematopoietic cell phosphatase, have severe disturbances in the regulation of B cell activation and

  9. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

    Science.gov (United States)

    Zheng, Yanhua; Lu, Zhimin

    2013-01-01

    Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

  10. Combining affinity proteomics and network context to identify new phosphatase substrates and adapters in growth pathways.

    Directory of Open Access Journals (Sweden)

    Francesca eSacco

    2014-05-01

    Full Text Available Protein phosphorylation homoeostasis is tightly controlled and pathological conditions are caused by subtle alterations of the cell phosphorylation profile. Altered levels of kinase activities have already been associated to specific diseases. Less is known about the impact of phosphatases, the enzymes that down-regulate phosphorylation by removing the phosphate groups. This is partly due to our poor understanding of the phosphatase-substrate network. Much of phosphatase substrate specificity is not based on intrinsic enzyme specificity with the catalytic pocket recognizing the sequence/structure context of the phosphorylated residue. In addition many phosphatase catalytic subunits do not form a stable complex with their substrates. This makes the inference and validation of phosphatase substrates a non-trivial task. Here, we present a novel approach that builds on the observation that much of phosphatase substrate selection is based on the network of physical interactions linking the phosphatase to the substrate. We first used affinity proteomics coupled to quantitative mass spectrometry to saturate the interactome of eight phosphatases whose down regulations was shown to affect the activation of the RAS-PI#K pathway. By integrating information from functional siRNA with protein interaction information, we develop a strategy that aims at inferring phosphatase physiological substrates. Graph analysis is used to identify protein scaffolds that may link the catalytic subunits to their substrates. By this approach we rediscover several previously described phosphatase substrate interactions and characterize two new protein scaffolds that promote the dephosphorylation of PTPN11 and ERK by DUSP18 and DUSP26 respectively.

  11. Controlled release of bioactive PDGF-AA from a hydrogel/nanoparticle composite.

    Science.gov (United States)

    Elliott Donaghue, Irja; Shoichet, Molly S

    2015-10-01

    Polymer excipients, such as low molar mass poly(ethylene glycol) (PEG), have shown contradictory effects on protein stability when co-encapsulated in polymeric nanoparticles. To gain further insight into these effects, platelet-derived growth factor (PDGF-AA) was encapsulated in polymeric nanoparticles with vs. without PEG. PDGF-AA is a particularly compelling protein, as it has been demonstrated to promote cell survival and induce the oligodendrocyte differentiation of neural stem/progenitor cells (NSPCs) both in vitro and in vivo. Here we show, for the first time, the controlled release of bioactive PDGF-AA from an injectable nanoparticle/hydrogel drug delivery system (DDS). PDGF-AA was encapsulated, with high efficiency, in poly(lactide-co-glycolide) nanoparticles, and its release from the drug delivery system was followed over 21 d. Interestingly, the co-encapsulation of low molecular weight poly(ethylene glycol) increased the PDGF-AA loading but, unexpectedly, accelerated the aggregation of PDGF-AA, resulting in reduced activity and detection by enzyme-linked immunosorbent assay (ELISA). In the absence of PEG, released PDGF-AA remained bioactive as demonstrated with NSPC oligodendrocyte differentiation, similar to positive controls, and significantly different from untreated controls. This work presents a novel delivery method for differentiation factors, such as PDGF-AA, and provides insights into the contradictory effects reported in the literature of excipients, such as PEG, on the loading and release of proteins from polymeric nanoparticles. Previously, the polymer poly(ethylene glycol) (PEG) has been used in many biomaterials applications, from surface coatings to the encapsulation of proteins. In this work, we demonstrate that, unexpectedly, low molecular weight PEG has a deleterious effect on the release of the encapsulated protein platelet-derived growth factor AA (PDGF-AA). We also demonstrate release of bioactive PDGF-AA (in the absence of PEG

  12. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    International Nuclear Information System (INIS)

    Blanquart, Christophe; Karouri, Salah-Eddine; Issad, Tarik

    2009-01-01

    The protein tyrosine phosphatase 1B (PTP1B) and the T-cell protein tyrosine phosphatase (TC-PTP) were initially thought to be mainly anti-oncogenic. However, overexpression of PTP1B and TC-PTP has been observed in human tumors, and recent studies have demonstrated that PTP1B contributes to the appearance of breast tumors by modulating ERK pathway. In the present work, we observed that decreasing the expression of TC-PTP or PTP1B in MCF-7 cells using siRNA reduced cell proliferation without affecting cell death. This reduction in proliferation was associated with decreased ERK phosphorylation. Moreover, selection of tamoxifen-resistant MCF-7 cells, by long-term culture in presence of 4-OH tamoxifen, resulted in cells that display overexpression of PTP1B and TC-PTP, and concomitant increase in ERK and STAT3 phosphorylation. siRNA experiments showed that PTP1B, but not TC-PTP, is necessary for resistance to 4-OH tamoxifen. Therefore, our work indicates that PTP1B could be a relevant therapeutic target for treatment of tamoxifen-resistant breast cancers.

  13. A P387L variant in protein tyrosine phosphatase-1B (PTP-1B) is associated with type 2 diabetes and impaired serine phosphorylation of PTP-1B in vitro

    DEFF Research Database (Denmark)

    Echwald, Søren M; Riis, Helle Bach; Vestergaard, Henrik

    2002-01-01

    In the present study, we tested the hypothesis that variability in the protein tyrosine phosphatase-1B (PTP-1B) gene is associated with type 2 diabetes. Using single-strand conformational polymorphism analysis, we examined cDNA of PTP-1B from 56 insulin-resistant patients with type 2 diabetes.......0012). In summary, a rare P387L variant of the PTP-1B gene is associated with a 3.7 (CI 1.26-10.93, P = 0.02) genotype relative risk of type 2 diabetes in the examined population of Danish Caucasian subjects and results in impaired in vitro serine phosphorylation of the PTP-1B peptide....

  14. BKR 26(2) pp. 36-42 (Igunnu)

    African Journals Online (AJOL)

    Femi Olorunniji

    2014-06-30

    Jun 30, 2014 ... (monoesterase reaction) by calf intestinal alkaline phosphatase (CIAP). The results showed ... phosphatase (AP) is a metalloenzyme that catalyses phosphoryl ..... required for both catalysis and thermodynamic stability while.

  15. Measurement of nuclear modification factor $R_\\mathrm{AA}$ in Pb+Pb collisions at $\\sqrt{s_{NN}}=5.02$TeV with the ATLAS detector at the LHC

    CERN Document Server

    The ATLAS collaboration

    2017-01-01

    Measurements of charged hadron spectra and the nuclear modification factor in Pb+Pb collisions at $\\sqrt{s_{NN}}=5.02$TeV with the ATLAS detector at the LHC are presented. The measurements are performed using data with an integrated luminosity of 0.49nb${}^{-1}$ in Pb+Pb collisions and 25pb${}^{-1}$ in pp collisions, obtained in the 2015 LHC physics run. Charged hadron Pb+Pb spectra measured in several centrality intervals are divided by pp spectrum to form the nuclear modification factor, $R_\\mathrm{AA}$, which provides insight into the effects of jet quenching in nuclear collisions. The behavior of $R_\\mathrm{AA}$ is studied as a function of transverse momentum up to 300GeV in different centralities. The values of $R_\\mathrm{AA}$ measured at $\\sqrt{s_{NN}}=5.02$TeV are found to be consistent with the previous measurements at $\\sqrt{s_{NN}}=2.76$TeV published by the ATLAS experiment.

  16. Expression and clinical significance of tyrosine phosphatase SHP-2 in colon cancer.

    Science.gov (United States)

    Cai, Peifen; Guo, Wenjie; Yuan, Huaqin; Li, Qian; Wang, Weicheng; Sun, Yang; Li, Xiaomin; Gu, Yanhong

    2014-04-01

    Protein-tyrosine phosphatase SHP-2, encoded by gene PTPN11, has been identified as a tumor-promoting factor in several types of leukemia and is hyper-activated by other mechanisms in some solid tumors including gastric cancer, breast cancer, non-small cell lung cancer (NSCLC), etc. But few were reported on the expression and significances of SHP-2 in colon cancer. Here, we detect SHP-2 expression in colon cancer cells, colon cancer-induced by AOM+DSS in mice and 232 human colon cancer specimens, including 58 groups of self-matched adjacent peritumor tissues and normal tissues. We found that compared to the normal colon tissues, SHP-2 significantly decreased in tumor tissues (Pcolon tumor cells as well as mice colon tumors. And in humans samples, low SHP-2 expression showed a significantly correlation with poor tumor differentiation (P<0.05), late TNM stage (P=0.1666) and lymph node metastasis (P<0.05). Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  17. Functional diversity of voltage-sensing phosphatases in two urodele amphibians.

    Science.gov (United States)

    Mutua, Joshua; Jinno, Yuka; Sakata, Souhei; Okochi, Yoshifumi; Ueno, Shuichi; Tsutsui, Hidekazu; Kawai, Takafumi; Iwao, Yasuhiro; Okamura, Yasushi

    2014-07-16

    Voltage-sensing phosphatases (VSPs) share the molecular architecture of the voltage sensor domain (VSD) with voltage-gated ion channels and the phosphoinositide phosphatase region with the phosphatase and tensin homolog (PTEN), respectively. VSPs enzymatic activities are regulated by the motions of VSD upon depolarization. The physiological role of these proteins has remained elusive, and insights may be gained by investigating biological variations in different animal species. Urodele amphibians are vertebrates with potent activities of regeneration and also show diverse mechanisms of polyspermy prevention. We cloned cDNAs of VSPs from the testes of two urodeles; Hynobius nebulosus and Cynops pyrrhogaster, and compared their expression and voltage-dependent activation. Their molecular architecture is highly conserved in both Hynobius VSP (Hn-VSP) and Cynops VSP (Cp-VSP), including the positively-charged arginine residues in the S4 segment of the VSD and the enzymatic active site for substrate binding, yet the C-terminal C2 domain of Hn-VSP is significantly shorter than that of Cp-VSP and other VSP orthologs. RT-PCR analysis showed that gene expression pattern was distinct between two VSPs. The voltage sensor motions and voltage-dependent phosphatase activities were investigated electrophysiologically by expression in Xenopus oocytes. Both VSPs showed "sensing" currents, indicating that their voltage sensor domains are functional. The phosphatase activity of Cp-VSP was found to be voltage dependent, as shown by its ability to regulate the conductance of coexpressed GIRK2 channels, but Hn-VSP lacked such phosphatase activity due to the truncation of its C2 domain. © 2014 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of the American Physiological Society and The Physiological Society.

  18. Zinc-ion-dependent acid phosphatase exhibits magnesium-ion-dependent myo-inositol-1-phosphatase activity.

    Science.gov (United States)

    Fujimoto, S; Okano, I; Tanaka, Y; Sumida, Y; Tsuda, J; Kawakami, N; Shimohama, S

    1996-06-01

    We have purified bovine brain Zn(2+)-dependent acid phosphatase (Zn(2+)-APase), which requires Zn2+ ions to hydrolyze the substrate p-nitrophenyl phosphate (pNPP) in an acidic environment. The substrate specificity and metal requirement of Zn(2+)-APase at a physiological pH was also studied. The enzyme exhibited hydrolytic activity on myo-inositol-1- and -2-monophosphates, 2'-adenosine monophosphate, 2'-guanosine monophosphate, and the alpha- and beta-glycerophosphates, glucose-1-phosphate, and fructose-6-phosphate in 50 mM Tris-HCl buffer (pH 7.4) in the presence of Mg2+ ions, but not on pNPP and phosphotyrosine. Zn2+, Mn2+ and Co2+ ions were less effective for activation. Among the above substrates, myo-inositol-1-phosphate was the most susceptible to hydrolysis by the enzyme in the presence of 3 mM Mg2+ ions. The enzyme exhibited an optimum pH at around 8 for myo-inositol-1-phosphate in the presence of 3 mM Mg2+ ions. The Mg(2+)-dependent myo-inositol-1-phosphatase activity of the enzyme was significantly inhibited by Li+ ions. The Zn(2+)-dependent p-nitrophenyl phosphatase activity and Mg(2+)-dependent myo-inositol-1-phosphatase activity of the purified enzyme fraction exhibited similar behavior on Sephadex G-100 and Mono Q colomns. These findings suggest that Zn(2+)-APase also exhibits Mg(2+)-dependent myo-inositol-1-phosphatase activity under physiological conditions.

  19. Coupling between the voltage-sensing and phosphatase domains of Ci-VSP.

    Science.gov (United States)

    Villalba-Galea, Carlos A; Miceli, Francesco; Taglialatela, Maurizio; Bezanilla, Francisco

    2009-07-01

    The Ciona intestinalis voltage sensor-containing phosphatase (Ci-VSP) shares high homology with the phosphatidylinositol phosphatase enzyme known as PTEN (phosphatase and tensin homologue deleted on chromosome 10). We have taken advantage of the similarity between these proteins to inquire about the coupling between the voltage sensing and the phosphatase domains in Ci-VSP. Recently, it was shown that four basic residues (R11, K13, R14, and R15) in PTEN are critical for its binding onto the membrane, required for its catalytic activity. Ci-VSP has three of the basic residues of PTEN. Here, we show that when R253 and R254 (which are the homologues of R14 and R15 in PTEN) are mutated to alanines in Ci-VSP, phosphatase activity is disrupted, as revealed by a lack of effect on the ionic currents of KCNQ2/3, where current decrease is a measure of phosphatase activity. The enzymatic activity was not rescued by the introduction of lysines, indicating that the binding is an arginine-specific interaction between the phosphatase binding domain and the membrane, presumably through the phosphate groups of the phospholipids. We also found that the kinetics and steady-state voltage dependence of the S4 segment movement are affected when the arginines are not present, indicating that the interaction of R253 and R254 with the membrane, required for the catalytic action of the phosphatase, restricts the movement of the voltage sensor.

  20. Ethanol and Other Short-Chain Alcohols Inhibit NLRP3 Inflammasome Activation through Protein Tyrosine Phosphatase Stimulation

    Science.gov (United States)

    Hoyt, Laura R.; Ather, Jennifer L.; Randall, Matthew J.; DePuccio, Daniel P.; Landry, Christopher C.; Wewers, Mark D.; Gavrilin, Mikhail A.; Poynter, Matthew E.

    2016-01-01

    Immunosuppression is a major complication of alcoholism that contributes to increased rates of opportunistic infections and sepsis in alcoholics. The NLRP3 inflammasome, a multi-protein intracellular pattern recognition receptor complex that facilitates the cleavage and secretion of the pro-inflammatory cytokines IL-1β and IL-18, can be inhibited by ethanol and we sought to better understand the mechanism through which this occurs and whether chemically similar molecules exert comparable effects. We show that ethanol can specifically inhibit activation of the NLRP3 inflammasome, resulting in attenuated IL-1β and caspase-1 cleavage and secretion, as well as diminished ASC speck formation, without affecting potassium efflux, in a mouse macrophage cell line (J774), mouse bone marrow derived dendritic cells, mouse neutrophils, and human PBMCs. The inhibitory effects on the Nlrp3 inflammasome were independent of GABAA receptor activation or NMDA receptor inhibition, but was associated with decreased oxidant production. Ethanol treatment markedly decreased cellular tyrosine phosphorylation, while administration of the tyrosine phosphatase inhibitor sodium orthovanadate prior to ethanol restored tyrosine phosphorylation and IL-1β secretion subsequent to ATP stimulation. Furthermore, sodium orthovanadate-induced phosphorylation of ASC Y144, necessary and sufficient for Nlrp3 inflammasome activation, and secretion of phosphorylated ASC, were inhibited by ethanol. Finally, multiple alcohol-containing organic compounds exerted inhibitory effects on the Nlrp3 inflammasome, whereas 2-methylbutane (isopentane), the analogous alkane of the potent inhibitor isoamyl alcohol (isopentanol), did not. Our results demonstrate that ethanol antagonizes the NLRP3 inflammasome at an apical event in its activation through the stimulation of protein tyrosine phosphatases, an effect shared by other short-chain alcohols. PMID:27421477

  1. Identification of the interaction and interaction domains of chicken anemia virus VP2 and VP3 proteins.

    Science.gov (United States)

    Sun, Fenfen; Pan, Wei; Gao, Honglei; Qi, Xiaole; Qin, Liting; Wang, Yongqiang; Gao, Yulong; Wang, Xiaomei

    2018-01-01

    Chicken anemia virus (CAV) is a small, single-stranded DNA virus of Anelloviridae family. Its genome segments encode three proteins, VP1, VP2, and VP3. This study identified an interaction between VP2 and VP3 and mapped the interaction domains. Through the yeast two-hybrid (Y2H) system, VP2 was found to interact with VP3. The presence of the VP2-VP3 complex in CAV-infected chicken cells was confirmed by co-immunoprecipitation. Confocal microscopy showed that VP2 and VP3 were expressed in the cytoplasm in cotransfected Vero cells. In the Y2H system, the interaction domains were identified as being within the N-terminal aa 1-30 and C-terminal aa 17-60 for VP2 and the N-terminal aa 46-60 and C-terminal aa 1-7 for VP3. This study showed the interaction between VP2 and VP3 of CAV and identified multiple independent interactive domains within the two proteins. This provides novel information for investigating the biological functions of these proteins. Copyright © 2017. Published by Elsevier Inc.

  2. Differential protein expression profile in the hypothalamic GT1-7 cell line after exposure to anabolic androgenic steroids.

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    Freddyson J Martínez-Rivera

    Full Text Available The abuse of anabolic androgenic steroids (AAS has been considered a major public health problem during decades. Supraphysiological doses of AAS may lead to a variety of neuroendocrine problems. Precisely, the hypothalamic-pituitary-gonadal (HPG axis is one of the body systems that is mainly influenced by steroidal hormones. Fluctuations of the hormonal milieu result in alterations of reproductive function, which are made through changes in hypothalamic neurons expressing gonadotropin-releasing hormone (GnRH. In fact, previous studies have shown that AAS modulate the activity of these neurons through steroid-sensitive afferents. To increase knowledge about the cellular mechanisms induced by AAS in GnRH neurons, we performed proteomic analyses of the murine hypothalamic GT1-7 cell line after exposure to 17α-methyltestosterone (17α-meT; 1 μM. These cells represent a good model for studying regulatory processes because they exhibit the typical characteristics of GnRH neurons, and respond to compounds that modulate GnRH in vivo. Two-dimensional difference in gel electrophoresis (2D-DIGE and mass spectrometry analyses identified a total of 17 different proteins that were significantly affected by supraphysiological levels of AAS. Furthermore, pathway analyses showed that modulated proteins were mainly associated to glucose metabolism, drug detoxification, stress response and cell cycle. Validation of many of these proteins, such as GSTM1, ERH, GAPDH, PEBP1 and PDIA6, were confirmed by western blotting. We further demonstrated that AAS exposure decreased expression of estrogen receptors and GnRH, while two important signaling pathway proteins p-ERK, and p-p38, were modulated. Our results suggest that steroids have the capacity to directly affect the neuroendocrine system by modulating key cellular processes for the control of reproductive function.

  3. Coronavirus gene 7 counteracts host defenses and modulates virus virulence.

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    Jazmina L G Cruz

    2011-06-01

    Full Text Available Transmissible gastroenteritis virus (TGEV genome contains three accessory genes: 3a, 3b and 7. Gene 7 is only present in members of coronavirus genus a1, and encodes a hydrophobic protein of 78 aa. To study gene 7 function, a recombinant TGEV virus lacking gene 7 was engineered (rTGEV-Δ7. Both the mutant and the parental (rTGEV-wt viruses showed the same growth and viral RNA accumulation kinetics in tissue cultures. Nevertheless, cells infected with rTGEV-Δ7 virus showed an increased cytopathic effect caused by an enhanced apoptosis mediated by caspase activation. Macromolecular synthesis analysis showed that rTGEV-Δ7 virus infection led to host translational shut-off and increased cellular RNA degradation compared with rTGEV-wt infection. An increase of eukaryotic translation initiation factor 2 (eIF2α phosphorylation and an enhanced nuclease, most likely RNase L, activity were observed in rTGEV-Δ7 virus infected cells. These results suggested that the removal of gene 7 promoted an intensified dsRNA-activated host antiviral response. In protein 7 a conserved sequence motif that potentially mediates binding to protein phosphatase 1 catalytic subunit (PP1c, a key regulator of the cell antiviral defenses, was identified. We postulated that TGEV protein 7 may counteract host antiviral response by its association with PP1c. In fact, pull-down assays demonstrated the interaction between TGEV protein 7, but not a protein 7 mutant lacking PP1c binding motif, with PP1. Moreover, the interaction between protein 7 and PP1 was required, during the infection, for eIF2α dephosphorylation and inhibition of cell RNA degradation. Inoculation of newborn piglets with rTGEV-Δ7 and rTGEV-wt viruses showed that rTGEV-Δ7 virus presented accelerated growth kinetics and pathology compared with the parental virus. Overall, the results indicated that gene 7 counteracted host cell defenses, and modified TGEV persistence increasing TGEV survival. Therefore, the

  4. The IBO germination quantitative trait locus encodes a phosphatase 2C-related variant with a nonsynonymous amino acid change that interferes with abscisic acid signaling.

    Science.gov (United States)

    Amiguet-Vercher, Amélia; Santuari, Luca; Gonzalez-Guzman, Miguel; Depuydt, Stephen; Rodriguez, Pedro L; Hardtke, Christian S

    2015-02-01

    Natural genetic variation is crucial for adaptability of plants to different environments. Seed dormancy prevents precocious germination in unsuitable conditions and is an adaptation to a major macro-environmental parameter, the seasonal variation in temperature and day length. Here we report the isolation of IBO, a quantitative trait locus (QTL) that governs c. 30% of germination rate variance in an Arabidopsis recombinant inbred line (RIL) population derived from the parental accessions Eilenburg-0 (Eil-0) and Loch Ness-0 (Lc-0). IBO encodes an uncharacterized phosphatase 2C-related protein, but neither the Eil-0 nor the Lc-0 variant, which differ in a single amino acid, have any appreciable phosphatase activity in in vitro assays. However, we found that the amino acid change in the Lc-0 variant of the IBO protein confers reduced germination rate. Moreover, unlike the Eil-0 variant of the protein, the Lc-0 variant can interfere with the activity of the phosphatase 2C ABSCISIC ACID INSENSITIVE 1 in vitro. This suggests that the Lc-0 variant possibly interferes with abscisic acid signaling, a notion that is supported by physiological assays. Thus, we isolated an example of a QTL allele with a nonsynonymous amino acid change that might mediate local adaptation of seed germination timing. © 2014 The Authors. New Phytologist © 2014 New Phytologist Trust.

  5. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    International Nuclear Information System (INIS)

    Rehman, Kanwal; Chen, Zhe; Wang, Wen Wen; Wang, Yan Wei; Sakamoto, Akira; Zhang, Yan Fang; Naranmandura, Hua; Suzuki, Noriyuki

    2012-01-01

    Arsenic binding to biomolecules is considered one of the major toxic mechanisms, which may also be related to the carcinogenic risks of arsenic in humans. At the same time, arsenic is also known to activate the phosphorylation-dependent signaling pathways including the epidermal growth factor receptor, the mitogen-activated protein kinase and insulin/insulin-like growth factor-1 pathways. These signaling pathways originate at the level of receptor tyrosine kinases whose phosphorylation status is regulated by opposing protein tyrosine phosphatase (PTP) activity. Reversible tyrosine phosphorylation, which is governed by the balanced action of protein tyrosine kinases and phosphatases, regulates important signaling pathways that are involved in the control of cell proliferation, adhesion and migration. In the present study, we have focused on the interaction of cellular PTPs with toxic trivalent arsenite (iAs III ) and its intermediate metabolites such as monomethylarsonous acid (MMA III ) and dimethylarsinous acid (DMA III ) in vitro, and then determined the arsenic binding site in PTP by the use of recombinant PTPs (e.g., PTP1B and CD45). Interestingly, the activities of PTP1B (cytoplasm-form) or CD45 (receptor-linked form) were observed to be strongly inhibited by both methylated metabolites (i.e., MMA III and DMA III ) but not by iAs III . Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMA III directly bound to the active site cysteine residue of PTP1B (e.g., Cys215), resulting in inhibition of enzyme activity. These results suggest that arsenic exposure may disturb the cellular signaling pathways through PTP inactivation. Highlights: ► This study focused on the interaction of PTPs with trivalent arsenicals in vitro. ► We for the first time confirmed that DMA III strongly inhibited activity of PTP1B. ► DMA III directly bound to PTP1B, resulting in inhibition of

  6. Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction

    Directory of Open Access Journals (Sweden)

    Andrew C. Hiday

    2017-01-01

    Full Text Available Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1, are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.

  7. Catalytic and glycan-binding abilities of ppGalNAc-T2 are regulated by acetylation

    DEFF Research Database (Denmark)

    Zlocowski, Natacha; Sendra, Victor G; Lorenz, Virginia

    2011-01-01

    Post-translational acetylation is an important molecular regulatory mechanism affecting the biological activity of proteins. Polypeptide GalNAc transferases (ppGalNAc-Ts) are a family of enzymes that catalyze initiation of mucin-type O-glycosylation. All ppGalNAc-Ts in mammals are type II...

  8. 200 kDa and 160 kDa neurofilament protein phosphatase resistance following in vivo aluminum chloride exposure.

    Science.gov (United States)

    Strong, M J; Jakowec, D M

    1994-01-01

    We have used time-course dephosphorylation experiments and two dimensional isoelectric focusing to assess the phosphorylation state of neurofilament (NF) proteins following the intracisternal inoculation of AlCl3. Littermates of New Zealand white rabbits, age 5-6 weeks, were inoculated with either 1000, 750, 500, 250 or 100 micrograms AlCl3 in 0.9% NaCl or 0.9% NaCl alone, killed 48 hours later and the NF-enriched cytoskeletal fraction isolated from the spinal cord. Neurofilamentous inclusions did not occur following inoculums of 100 or 250 micrograms AlCl3, but thereafter developed in increasing quantities in a dosage-dependent manner. Incubation of the NF-enriched fraction with E. Coli. alkaline phosphatase (enzyme: substrate 1:50) induced a replacement of the highly phosphorylated 200 kDa isoform of NFH with a more poorly phosphorylated 170 kDa isoform, confirmed by immunoblot analysis. This reaction was complete within 20 minutes with NF derived from NaCl, 100 or 250 micrograms AlCl3 inoculated rabbits and within 30 minutes for 500 micrograms AlCl3 inoculums. However, residual highly phosphorylated NFH isoforms persisted at 60 minutes for 750 micrograms inoculums and 90 minutes for that derived from 1000 micrograms AlCl3 inoculums. A similar inhibition of phosphatase activity was observed for NFM. Following two dimensional electrophoresis of the NF-enriched isolate, no alteration in the net phosphorylation state of individual NF subunit proteins was observed--regardless of the inoculum. These results demonstrate a dose-dependent induction of neurofilamentous inclusions in spinal motor neurons following intracisternal AlCl3 inoculation accompanied by increasing phosphatase resistance without a demonstrable alteration in NF net phosphorylation state.

  9. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  10. Functional Analysis of Mouse G6pc1 Mutations Using a Novel In Situ Assay for Glucose-6-Phosphatase Activity and the Effect of Mutations in Conserved Human G6PC1/G6PC2 Amino Acids on G6PC2 Protein Expression.

    Directory of Open Access Journals (Sweden)

    Kayla A Boortz

    Full Text Available Elevated fasting blood glucose (FBG has been associated with increased risk for development of type 2 diabetes. Single nucleotide polymorphisms (SNPs in G6PC2 are the most important common determinants of variations in FBG in humans. Studies using G6pc2 knockout mice suggest that G6pc2 regulates the glucose sensitivity of insulin secretion. G6PC2 and the related G6PC1 and G6PC3 genes encode glucose-6-phosphatase catalytic subunits. This study describes a functional analysis of 22 non-synonymous G6PC2 SNPs, that alter amino acids that are conserved in human G6PC1, mouse G6pc1 and mouse G6pc2, with the goal of identifying variants that potentially affect G6PC2 activity/expression. Published data suggest strong conservation of catalytically important amino acids between all four proteins and the related G6PC3 isoform. Because human G6PC2 has very low glucose-6-phosphatase activity we used an indirect approach, examining the effect of these SNPs on mouse G6pc1 activity. Using a novel in situ functional assay for glucose-6-phosphatase activity we demonstrate that the amino acid changes associated with the human G6PC2 rs144254880 (Arg79Gln, rs149663725 (Gly114Arg and rs2232326 (Ser324Pro SNPs reduce mouse G6pc1 enzyme activity without affecting protein expression. The Arg79Gln variant alters an amino acid mutation of which, in G6PC1, has previously been shown to cause glycogen storage disease type 1a. We also demonstrate that the rs368382511 (Gly8Glu, rs138726309 (His177Tyr, rs2232323 (Tyr207Ser rs374055555 (Arg293Trp, rs2232326 (Ser324Pro, rs137857125 (Pro313Leu and rs2232327 (Pro340Leu SNPs confer decreased G6PC2 protein expression. In summary, these studies identify multiple G6PC2 variants that have the potential to be associated with altered FBG in humans.

  11. Electro-chemical coupling in the voltage-dependent phosphatase Ci-VSP

    Science.gov (United States)

    Kohout, Susy C.; Bell, Sarah C.; Liu, Lijun; Xu, Qiang; Minor, Daniel L.; Isacoff, Ehud Y.

    2010-01-01

    In the voltage sensing phosphatase, Ci-VSP, a voltage sensing domain (VSD) controls a lipid phosphatase domain (PD). The mechanism by which the domains are allosterically coupled is not well understood. Using an in vivo assay, we find that the inter-domain linker that connects the VSD to the PD is essential for coupling the full-length protein. Biochemical assays show that the linker is also needed for activity in the isolated PD. We identify a late step of VSD motion in the full-length protein that depends on the linker. Strikingly, this VSD motion is found to require PI(4,5)P2, a substrate of Ci-VSP. These results suggest that the voltage-driven motion of the VSD turns the enzyme on by rearranging the linker into an activated conformation, and that this activated conformation is stabilized by PI(4,5)P2. We propose that Ci-VSP activity is self-limited because its decrease of PI(4,5)P2 levels decouples the VSD from the enzyme. PMID:20364128

  12. G protein-coupled receptors (GPCRs) That Signal via Protein Kinase A (PKA) Cross-talk at Insulin Receptor Substrate 1 (IRS1) to Activate the phosphatidylinositol 3-kinase (PI3K)/AKT Pathway.

    Science.gov (United States)

    Law, Nathan C; White, Morris F; Hunzicker-Dunn, Mary E

    2016-12-30

    G protein-coupled receptors (GPCRs) activate PI3K/v-AKT thymoma viral oncoprotein (AKT) to regulate many cellular functions that promote cell survival, proliferation, and growth. However, the mechanism by which GPCRs activate PI3K/AKT remains poorly understood. We used ovarian preantral granulosa cells (GCs) to elucidate the mechanism by which the GPCR agonist FSH via PKA activates the PI3K/AKT cascade. Insulin-like growth factor 1 (IGF1) is secreted in an autocrine/paracrine manner by GCs and activates the IGF1 receptor (IGF1R) but, in the absence of FSH, fails to stimulate YXXM phosphorylation of IRS1 (insulin receptor substrate 1) required for PI3K/AKT activation. We show that PKA directly phosphorylates the protein phosphatase 1 (PP1) regulatory subunit myosin phosphatase targeting subunit 1 (MYPT1) to activate PP1 associated with the IGF1R-IRS1 complex. Activated PP1 is sufficient to dephosphorylate at least four IRS1 Ser residues, Ser 318 , Ser 346 , Ser 612 , and Ser 789 , and promotes IRS1 YXXM phosphorylation by the IGF1R to activate the PI3K/AKT cascade. Additional experiments indicate that this mechanism also occurs in breast cancer, thyroid, and preovulatory granulosa cells, suggesting that the PKA-dependent dephosphorylation of IRS1 Ser/Thr residues is a conserved mechanism by which GPCRs signal to activate the PI3K/AKT pathway downstream of the IGF1R. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  13. Protein-tyrosine phosphatase SHP2 contributes to GDNF neurotrophic activity through direct binding to phospho-Tyr687 in the RET receptor tyrosine kinase.

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F

    2010-10-08

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr(687) in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr(687) and association with components of the Tyr(1062) signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser(696), a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr(687) as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions.

  14. Tumor Suppressor RARRES1 Regulates DLG2, PP2A, VCP, EB1, and Ankrd26

    Directory of Open Access Journals (Sweden)

    Ziad J. Sahab, Michael D. Hall, Lihua Zhang, Amrita K. Cheema, Stephen W. Byers

    2010-01-01

    Full Text Available Retinoic Acid Receptor Responder (RARRES1 initially identified as a novel retinoic acid receptor regulated gene in the skin is a putative tumor suppressor of unknown function. RARRES1 was knocked down in immortalized human prostatic epithelial cell line PWR-1E cells and differential protein expression was identified using differential in-gel electrophoresis (DIGE followed by matrix-assisted laser desorption ionization (MALDI mass spectrometry and western Blot analysis excluding highly abundant proteins routinely identified in almost all proteomics projects. Knock-down of RARRES1: 1- down-regulates PP2A, an enzyme involved in the negative regulation of the growth hormone-stimulated signal transduction pathways; 2- down-regulates Valosin-containing protein causing impaired autophagy; 3- up-regulates the tumor suppressor disks large 2; 4- up-regulates Ankrd26 that belongs to the POTE family of genes that are highly expressed in cancer patients with poor outcome; and 5- down-regulates EB1, a protein that is involved in spindle dynamics and chromosome alignment during mitosis.

  15. Therapeutic Benefits of Tocilizumab Vary in Different Organs of a Patient with AA Amyloidosis

    Directory of Open Access Journals (Sweden)

    Masaru Matsui

    2014-01-01

    Full Text Available Systemic reactive AA amyloidosis is a life-threatening complication of chronic inflammatory diseases. Anti-interleukin-6 receptor, tocilizumab (TCZ, has been shown to improve clinical symptoms of patients with AA amyloidosis, accompanied with regression of the amyloid deposition. We report a case of AA amyloidosis evaluated by histology of multiple organs before and after TCZ treatment. A woman in her 60s with rheumatoid arthritis was referred to our hospital because of cardiac and renal dysfunction. A gastric and renal biopsy revealed the deposition of AA amyloid, and echocardiography revealed concentric left ventricular hypertrophy. Her estimated glomerular filtration rate was decreased to 8.6 mL/min/1.73 m2, and B-type natriuretic peptide, C-reactive protein, and serum amyloid A protein were significantly elevated. TCZ treatments markedly decreased her serum amyloid A protein and C-reactive protein levels, but hemodialysis was required 1 year later. Endoscopic gastric rebiopsy 3 years after initiation of TCZ treatments revealed the regression of amyloid deposition and echocardiography revealed improvement of her left ventricular hypertrophy. However, a renal rebiopsy revealed that the amyloid deposition had not regressed. In conclusion, these observations suggest that the therapeutic effects of TCZ can vary among organs in patients with AA amyloidosis.

  16. The adsorption features between insecticidal crystal protein and nano-Mg(OH)2.

    Science.gov (United States)

    Pan, Xiaohong; Xu, Zhangyan; Zheng, Yilin; Huang, Tengzhou; Li, Lan; Chen, Zhi; Rao, Wenhua; Chen, Saili; Hong, Xianxian; Guan, Xiong

    2017-12-01

    Nano-Mg(OH) 2 , with low biological toxicity, is an ideal nano-carrier for insecticidal protein to improve the bioactivity. In this work, the adsorption features of insecticidal protein by nano-Mg(OH) 2 have been studied. The adsorption capacity could reach as high as 136 mg g -1 , and the adsorption isotherm had been fitted with Langmuir and Freundlich models. Moreover, the adsorption kinetics followed a pseudo-first or -second order rate model, and the adsorption was spontaneous and an exothermic process. However, high temperatures are not suitable for adsorption, which implies that the temperature would be a critical factor during the adsorption process. In addition, FT-IR confirmed that the protein was adsorbed on the nano-Mg(OH) 2 , zeta potential analysis suggested that insecticidal protein was loaded onto the nano-Mg(OH) 2 not by electrostatic adsorption but maybe by intermolecular forces, and circular dichroism spectroscopy of Cry11Aa protein before and after loading with nano-Mg(OH) 2 was changed. The study applied the adsorption information between Cry11Aa and nano-Mg(OH) 2 , which would be useful in the practical application of nano-Mg(OH) 2 as a nano-carrier.

  17. Assay format as a critical success factor for identification of novel inhibitor chemotypes of tissue-nonspecific alkaline phosphatase from high-throughput screening.

    Science.gov (United States)

    Chung, Thomas D Y; Sergienko, Eduard; Millán, José Luis

    2010-04-27

    The tissue-nonspecific alkaline phosphatase (TNAP) isozyme is centrally involved in the control of normal skeletal mineralization and pathophysiological abnormalities that lead to disease states such as hypophosphatasia, osteoarthritis, ankylosis and vascular calcification. TNAP acts in concert with the nucleoside triphosphate pyrophosphohydrolase-1 (NPP1) and the Ankylosis protein to regulate the extracellular concentrations of inorganic pyrophosphate (PP(i)), a potent inhibitor of mineralization. In this review we describe the serial development of two miniaturized high-throughput screens (HTS) for TNAP inhibitors that differ in both signal generation and detection formats, but more critically in the concentrations of a terminal alcohol acceptor used. These assay improvements allowed the rescue of the initially unsuccessful screening campaign against a large small molecule chemical library, but moreover enabled the discovery of several unique classes of molecules with distinct mechanisms of action and selectivity against the related placental (PLAP) and intestinal (IAP) alkaline phosphatase isozymes. This illustrates the underappreciated impact of the underlying fundamental assay configuration on screening success, beyond mere signal generation and detection formats.

  18. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

    Science.gov (United States)

    Cross, Megan; Biberacher, Sonja; Park, Suk-Youl; Rajan, Siji; Korhonen, Pasi; Gasser, Robin B; Kim, Jeong-Sun; Coster, Mark J; Hofmann, Andreas

    2018-04-24

    The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

  19. Sequence Classification: 889834 [

    Lifescience Database Archive (English)

    Full Text Available ritance; deletion reveals defects in precursor tRNA splicing, sporulation and cell separation; Ptc1p || http://www.ncbi.nlm.nih.gov/protein/6320198 ... ...C protein phosphatase (PP2C); inactivates the osmosensing MAPK cascade by dephosphorylating Hog1p; mutation delays mitochondrial inhe

  20. Neutral pion production at midrapidity in pp and Pb-Pb collisions at $\\sqrt{s_{NN}}$ = 2.76 TeV

    CERN Document Server

    Abelev, Betty Bezverkhny; Adamova, Dagmar; Aggarwal, Madan Mohan; Aglieri Rinella, Gianluca; Agnello, Michelangelo; Agostinelli, Andrea; Agrawal, Neelima; Ahammed, Zubayer; Ahmad, Nazeer; Ahmed, Ijaz; Ahn, Sang Un; Ahn, Sul-Ah; Aimo, Ilaria; Aiola, Salvatore; Ajaz, Muhammad; Akindinov, Alexander; Alam, Sk Noor; Aleksandrov, Dmitry; Alessandro, Bruno; Alexandre, Didier; Alici, Andrea; Alkin, Anton; Alme, Johan; Alt, Torsten; Altinpinar, Sedat; Altsybeev, Igor; Alves Garcia Prado, Caio; Andrei, Cristian; Andronic, Anton; Anguelov, Venelin; Anielski, Jonas; Anticic, Tome; Antinori, Federico; Antonioli, Pietro; Aphecetche, Laurent Bernard; Appelshaeuser, Harald; Arcelli, Silvia; Armesto Perez, Nestor; Arnaldi, Roberta; Aronsson, Tomas; Arsene, Ionut Cristian; Arslandok, Mesut; Augustinus, Andre; Averbeck, Ralf Peter; Awes, Terry; Azmi, Mohd Danish; Bach, Matthias Jakob; Badala, Angela; Baek, Yong Wook; Bagnasco, Stefano; Bailhache, Raphaelle Marie; Bala, Renu; Baldisseri, Alberto; Baltasar Dos Santos Pedrosa, Fernando; Baral, Rama Chandra; Barbera, Roberto; Barile, Francesco; Barnafoldi, Gergely Gabor; Barnby, Lee Stuart; Ramillien Barret, Valerie; Bartke, Jerzy Gustaw; Basile, Maurizio; Bastid, Nicole; Basu, Sumit; Bathen, Bastian; Batigne, Guillaume; Batyunya, Boris; Batzing, Paul Christoph; Baumann, Christoph Heinrich; Bearden, Ian Gardner; Beck, Hans; Bedda, Cristina; Behera, Nirbhay Kumar; Belikov, Iouri; Bellini, Francesca; Bellwied, Rene; Belmont Moreno, Ernesto; Belmont Iii, Ronald John; Belyaev, Vladimir; Bencedi, Gyula; Beole, Stefania; Berceanu, Ionela; Bercuci, Alexandru; Berdnikov, Yaroslav; Berenyi, Daniel; Berger, Martin Emanuel; Bertens, Redmer Alexander; Berzano, Dario; Betev, Latchezar; Bhasin, Anju; Bhat, Inayat Rasool; Bhati, Ashok Kumar; Bhattacharjee, Buddhadeb; Bhom, Jihyun; Bianchi, Livio; Bianchi, Nicola; Bianchin, Chiara; Bielcik, Jaroslav; Bielcikova, Jana; Bilandzic, Ante; Bjelogrlic, Sandro; Blanco, Fernando; Blau, Dmitry; Blume, Christoph; Bock, Friederike; Bogdanov, Alexey; Boggild, Hans; Bogolyubskiy, Mikhail; Boehmer, Felix Valentin; Boldizsar, Laszlo; Bombara, Marek; Book, Julian Heinz; Borel, Herve; Borissov, Alexander; Bossu, Francesco; Botje, Michiel; Botta, Elena; Boettger, Stefan; Braun-Munzinger, Peter; Bregant, Marco; Breitner, Timo Gunther; Broker, Theo Alexander; Browning, Tyler Allen; Broz, Michal; Bruna, Elena; Bruno, Giuseppe Eugenio; Budnikov, Dmitry; Buesching, Henner; Bufalino, Stefania; Buncic, Predrag; Busch, Oliver; Buthelezi, Edith Zinhle; Caffarri, Davide; Cai, Xu; Caines, Helen Louise; Calero Diaz, Liliet; Caliva, Alberto; Calvo Villar, Ernesto; Camerini, Paolo; Carena, Francesco; Carena, Wisla; Castillo Castellanos, Javier Ernesto; Casula, Ester Anna Rita; Catanescu, Vasile Ioan; Cavicchioli, Costanza; Ceballos Sanchez, Cesar; Cepila, Jan; Cerello, Piergiorgio; Chang, Beomsu; Chapeland, Sylvain; Charvet, Jean-Luc Fernand; Chattopadhyay, Subhasis; Chattopadhyay, Sukalyan; Chelnokov, Volodymyr; Cherney, Michael Gerard; Cheshkov, Cvetan Valeriev; Cheynis, Brigitte; Chibante Barroso, Vasco Miguel; Dobrigkeit Chinellato, David; Chochula, Peter; Chojnacki, Marek; Choudhury, Subikash; Christakoglou, Panagiotis; Christensen, Christian Holm; Christiansen, Peter; Chujo, Tatsuya; Chung, Suh-Urk; Cicalo, Corrado; Cifarelli, Luisa; Cindolo, Federico; Cleymans, Jean Willy Andre; Colamaria, Fabio Filippo; Colella, Domenico; Collu, Alberto; Colocci, Manuel; Conesa Balbastre, Gustavo; Conesa Del Valle, Zaida; Connors, Megan Elizabeth; Contreras Nuno, Jesus Guillermo; Cormier, Thomas Michael; Corrales Morales, Yasser; Cortese, Pietro; Cortes Maldonado, Ismael; Cosentino, Mauro Rogerio; Costa, Filippo; Crochet, Philippe; Cruz Albino, Rigoberto; Cuautle Flores, Eleazar; Cunqueiro Mendez, Leticia; Dainese, Andrea; Dang, Ruina; Danu, Andrea; Das, Debasish; Das, Indranil; Das, Kushal; Das, Supriya; Dash, Ajay Kumar; Dash, Sadhana; De, Sudipan; Delagrange, Hugues; Deloff, Andrzej; Denes, Ervin Sandor; D'Erasmo, Ginevra; De Caro, Annalisa; De Cataldo, Giacinto; De Cuveland, Jan; De Falco, Alessandro; De Gruttola, Daniele; De Marco, Nora; De Pasquale, Salvatore; De Rooij, Raoul Stefan; Diaz Corchero, Miguel Angel; Dietel, Thomas; Dillenseger, Pascal; Divia, Roberto; Di Bari, Domenico; Di Liberto, Sergio; Di Mauro, Antonio; Di Nezza, Pasquale; Djuvsland, Oeystein; Dobrin, Alexandru Florin; Dobrowolski, Tadeusz Antoni; Domenicis Gimenez, Diogenes; Donigus, Benjamin; Dordic, Olja; Dorheim, Sverre; Dubey, Anand Kumar; Dubla, Andrea; Ducroux, Laurent; Dupieux, Pascal; Dutt Mazumder, Abhee Kanti; Hilden, Timo Eero; Ehlers Iii, Raymond James; Elia, Domenico; Engel, Heiko; Erazmus, Barbara Ewa; Erdal, Hege Austrheim; Eschweiler, Dominic; Espagnon, Bruno; Esposito, Marco; Estienne, Magali Danielle; Esumi, Shinichi; Evans, David; Evdokimov, Sergey; Fabris, Daniela; Faivre, Julien; Falchieri, Davide; Fantoni, Alessandra; Fasel, Markus; Fehlker, Dominik; Feldkamp, Linus; Felea, Daniel; Feliciello, Alessandro; Feofilov, Grigory; Ferencei, Jozef; Fernandez Tellez, Arturo; Gonzalez Ferreiro, Elena; Ferretti, Alessandro; Festanti, Andrea; Figiel, Jan; Araujo Silva Figueredo, Marcel; Filchagin, Sergey; Finogeev, Dmitry; Fionda, Fiorella; Fiore, Enrichetta Maria; Floratos, Emmanouil; Floris, Michele; Foertsch, Siegfried Valentin; Foka, Panagiota; Fokin, Sergey; Fragiacomo, Enrico; Francescon, Andrea; Frankenfeld, Ulrich Michael; Fuchs, Ulrich; Furget, Christophe; Fusco Girard, Mario; Gaardhoeje, Jens Joergen; Gagliardi, Martino; Gago Medina, Alberto Martin; Gallio, Mauro; Gangadharan, Dhevan Raja; Ganoti, Paraskevi; Garabatos Cuadrado, Jose; Garcia-Solis, Edmundo Javier; Gargiulo, Corrado; Garishvili, Irakli; Gerhard, Jochen; Germain, Marie; Gheata, Andrei George; Gheata, Mihaela; Ghidini, Bruno; Ghosh, Premomoy; Ghosh, Sanjay Kumar; Gianotti, Paola; Giubellino, Paolo; Gladysz-Dziadus, Ewa; Glassel, Peter; Gomez Ramirez, Andres; Gonzalez Zamora, Pedro; Gorbunov, Sergey; Gorlich, Lidia Maria; Gotovac, Sven; Graczykowski, Lukasz Kamil; Grelli, Alessandro; Grigoras, Alina Gabriela; Grigoras, Costin; Grigoryev, Vladislav; Grigoryan, Ara; Grigoryan, Smbat; Grynyov, Borys; Grion, Nevio; Grosse-Oetringhaus, Jan Fiete; Grossiord, Jean-Yves; Grosso, Raffaele; Guber, Fedor; Guernane, Rachid; Guerzoni, Barbara; Guilbaud, Maxime Rene Joseph; Gulbrandsen, Kristjan Herlache; Gulkanyan, Hrant; Gumbo, Mervyn; Gunji, Taku; Gupta, Anik; Gupta, Ramni; Khan, Kamal; Haake, Rudiger; Haaland, Oystein Senneset; Hadjidakis, Cynthia Marie; Haiduc, Maria; Hamagaki, Hideki; Hamar, Gergoe; Hanratty, Luke David; Hansen, Alexander; Harris, John William; Hartmann, Helvi; Harton, Austin Vincent; Hatzifotiadou, Despina; Hayashi, Shinichi; Heckel, Stefan Thomas; Heide, Markus Ansgar; Helstrup, Haavard; Herghelegiu, Andrei Ionut; Herrera Corral, Gerardo Antonio; Hess, Benjamin Andreas; Hetland, Kristin Fanebust; Hippolyte, Boris; Hladky, Jan; Hristov, Peter Zahariev; Huang, Meidana; Humanic, Thomas; Hussain, Nur; Hutter, Dirk; Hwang, Dae Sung; Ilkaev, Radiy; Ilkiv, Iryna; Inaba, Motoi; Innocenti, Gian Michele; Ionita, Costin; Ippolitov, Mikhail; Irfan, Muhammad; Ivanov, Marian; Ivanov, Vladimir; Jacholkowski, Adam Wlodzimierz; Jacobs, Peter Martin; Jahnke, Cristiane; Jang, Haeng Jin; Janik, Malgorzata Anna; Pahula Hewage, Sandun; Jena, Chitrasen; Jena, Satyajit; Jimenez Bustamante, Raul Tonatiuh; Jones, Peter Graham; Jung, Hyungtaik; Jusko, Anton; Kadyshevskiy, Vladimir; Kalcher, Sebastian; Kalinak, Peter; Kalweit, Alexander Philipp; Kamin, Jason Adrian; Kang, Ju Hwan; Kaplin, Vladimir; Kar, Somnath; Karasu Uysal, Ayben; Karavichev, Oleg; Karavicheva, Tatiana; Karpechev, Evgeny; Kebschull, Udo Wolfgang; Keidel, Ralf; Keijdener, Darius Laurens; Khan, Mohammed Mohisin; Khan, Palash; Khan, Shuaib Ahmad; Khanzadeev, Alexei; Kharlov, Yury; Kileng, Bjarte; Kim, Beomkyu; Kim, Do Won; Kim, Dong Jo; Kim, Jinsook; Kim, Mimae; Kim, Minwoo; Kim, Se Yong; Kim, Taesoo; Kirsch, Stefan; Kisel, Ivan; Kiselev, Sergey; Kisiel, Adam Ryszard; Kiss, Gabor; Klay, Jennifer Lynn; Klein, Jochen; Klein-Boesing, Christian; Kluge, Alexander; Knichel, Michael Linus; Knospe, Anders Garritt; Kobdaj, Chinorat; Kofarago, Monika; Kohler, Markus Konrad; Kollegger, Thorsten; Kolozhvari, Anatoly; Kondratev, Valerii; Kondratyeva, Natalia; Konevskikh, Artem; Kovalenko, Vladimir; Kowalski, Marek; Kox, Serge; Koyithatta Meethaleveedu, Greeshma; Kral, Jiri; Kralik, Ivan; Kravcakova, Adela; Krelina, Michal; Kretz, Matthias; Krivda, Marian; Krizek, Filip; Kryshen, Evgeny; Krzewicki, Mikolaj; Kucera, Vit; Kucheryaev, Yury; Kugathasan, Thanushan; Kuhn, Christian Claude; Kuijer, Paulus Gerardus; Kulakov, Igor; Kumar, Jitendra; Kurashvili, Podist; Kurepin, Alexander; Kurepin, Alexey; Kuryakin, Alexey; Kushpil, Svetlana; Kweon, Min Jung; Kwon, Youngil; Ladron De Guevara, Pedro; Lagana Fernandes, Caio; Lakomov, Igor; Langoy, Rune; Lara Martinez, Camilo Ernesto; Lardeux, Antoine Xavier; Lattuca, Alessandra; La Pointe, Sarah Louise; La Rocca, Paola; Lea, Ramona; Leardini, Lucia; Lee, Graham Richard; Legrand, Iosif; Lehnert, Joerg Walter; Lemmon, Roy Crawford; Lenti, Vito; Leogrande, Emilia; Leoncino, Marco; Leon Monzon, Ildefonso; Levai, Peter; Li, Shuang; Lien, Jorgen Andre; Lietava, Roman; Lindal, Svein; Lindenstruth, Volker; Lippmann, Christian; Lisa, Michael Annan; Ljunggren, Hans Martin; Lodato, Davide Francesco; Lonne, Per-Ivar; Loggins, Vera Renee; Loginov, Vitaly; Lohner, Daniel; Loizides, Constantinos; Lopez, Xavier Bernard; Lopez Torres, Ernesto; Lu, Xianguo; Luettig, Philipp Johannes; Lunardon, Marcello; Luparello, Grazia; Luzzi, Cinzia; Ma, Rongrong; Maevskaya, Alla; Mager, Magnus; Mahapatra, Durga Prasad; Mahmood, Sohail Musa; Maire, Antonin; Majka, Richard Daniel; Malaev, Mikhail; Maldonado Cervantes, Ivonne Alicia; Malinina, Liudmila; Mal'Kevich, Dmitry; Malzacher, Peter; Mamonov, Alexander; Manceau, Loic Henri Antoine; Manko, Vladislav; Manso, Franck; Manzari, Vito; Marchisone, Massimiliano; Mares, Jiri; Margagliotti, Giacomo Vito; Margotti, Anselmo; Marin, Ana Maria; Markert, Christina; Marquard, Marco; Martashvili, Irakli; Martin, Nicole Alice; Martinengo, Paolo; Martinez Hernandez, Mario Ivan; Martinez-Garcia, Gines; Martin Blanco, Javier; Martynov, Yevgen; Mas, Alexis Jean-Michel; Masciocchi, Silvia; Masera, Massimo; Masoni, Alberto; Massacrier, Laure Marie; Mastroserio, Annalisa; Matyja, Adam Tomasz; Mayer, Christoph; Mazer, Joel Anthony; Mazzoni, Alessandra Maria; Meddi, Franco; Menchaca-Rocha, Arturo Alejandro; Meninno, Elisa; Mercado-Perez, Jorge; Meres, Michal; Miake, Yasuo; Mikhaylov, Konstantin; Milano, Leonardo; Milosevic, Jovan; Mischke, Andre; Mishra, Aditya Nath; Miskowiec, Dariusz Czeslaw; Mitra, Jubin; Mitu, Ciprian Mihai; Mlynarz, Jocelyn; Mohammadi, Naghmeh; Mohanty, Bedangadas; Molnar, Levente; Montano Zetina, Luis Manuel; Montes Prado, Esther; Morando, Maurizio; Moreira De Godoy, Denise Aparecida; Moretto, Sandra; Morreale, Astrid; Morsch, Andreas; Muccifora, Valeria; Mudnic, Eugen; Muhlheim, Daniel Michael; Muhuri, Sanjib; Mukherjee, Maitreyee; Muller, Hans; Gameiro Munhoz, Marcelo; Murray, Sean; Musa, Luciano; Musinsky, Jan; Nandi, Basanta Kumar; Nania, Rosario; Nappi, Eugenio; Nattrass, Christine; Nayak, Kishora; Nayak, Tapan Kumar; Nazarenko, Sergey; Nedosekin, Alexander; Nicassio, Maria; Niculescu, Mihai; Nielsen, Borge Svane; Nikolaev, Sergey; Nikulin, Sergey; Nikulin, Vladimir; Nilsen, Bjorn Steven; Noferini, Francesco; Nomokonov, Petr; Nooren, Gerardus; Norman, Jaime; Nyanin, Alexander; Nystrand, Joakim Ingemar; Oeschler, Helmut Oskar; Oh, Saehanseul; Oh, Sun Kun; Okatan, Ali; Olah, Laszlo; Oleniacz, Janusz; Oliveira Da Silva, Antonio Carlos; Onderwaater, Jacobus; Oppedisano, Chiara; Ortiz Velasquez, Antonio; Oskarsson, Anders Nils Erik; Otwinowski, Jacek Tomasz; Oyama, Ken; Ozdemir, Mahmut; Sahoo, Pragati; Pachmayer, Yvonne Chiara; Pachr, Milos; Pagano, Paola; Paic, Guy; Painke, Florian; Pajares Vales, Carlos; Pal, Susanta Kumar; Palmeri, Armando; Pant, Divyash; Papikyan, Vardanush; Pappalardo, Giuseppe; Pareek, Pooja; Park, Woojin; Parmar, Sonia; Passfeld, Annika; Patalakha, Dmitry; Paticchio, Vincenzo; Paul, Biswarup; Pawlak, Tomasz Jan; Peitzmann, Thomas; Pereira Da Costa, Hugo Denis Antonio; Pereira De Oliveira Filho, Elienos; Peresunko, Dmitry Yurevich; Perez Lara, Carlos Eugenio; Pesci, Alessandro; Peskov, Vladimir; Pestov, Yury; Petracek, Vojtech; Petran, Michal; Petris, Mariana; Petrovici, Mihai; Petta, Catia; Piano, Stefano; Pikna, Miroslav; Pillot, Philippe; Pinazza, Ombretta; Pinsky, Lawrence; Piyarathna, Danthasinghe; Ploskon, Mateusz Andrzej; Planinic, Mirko; Pluta, Jan Marian; Pochybova, Sona; Podesta Lerma, Pedro Luis Manuel; Poghosyan, Martin; Pohjoisaho, Esko Heikki Oskari; Polishchuk, Boris; Poljak, Nikola; Pop, Amalia; Porteboeuf, Sarah Julie; Porter, R Jefferson; Potukuchi, Baba; Prasad, Sidharth Kumar; Preghenella, Roberto; Prino, Francesco; Pruneau, Claude Andre; Pshenichnov, Igor; Puddu, Giovanna; Pujahari, Prabhat Ranjan; Punin, Valery; Putschke, Jorn Henning; Qvigstad, Henrik; Rachevski, Alexandre; Raha, Sibaji; Rak, Jan; Rakotozafindrabe, Andry Malala; Ramello, Luciano; Raniwala, Rashmi; Raniwala, Sudhir; Rasanen, Sami Sakari; Rascanu, Bogdan Theodor; Rathee, Deepika; Rauf, Aamer Wali; Razazi, Vahedeh; Read, Kenneth Francis; Real, Jean-Sebastien; Redlich, Krzysztof; Reed, Rosi Jan; Rehman, Attiq Ur; Reichelt, Patrick Simon; Reicher, Martijn; Reidt, Felix; Renfordt, Rainer Arno Ernst; Reolon, Anna Rita; Reshetin, Andrey; Rettig, Felix Vincenz; Revol, Jean-Pierre; Reygers, Klaus Johannes; Riabov, Viktor; Ricci, Renato Angelo; Richert, Tuva Ora Herenui; Richter, Matthias Rudolph; Riedler, Petra; Riegler, Werner; Riggi, Francesco; Rivetti, Angelo; Rocco, Elena; Rodriguez Cahuantzi, Mario; Rodriguez Manso, Alis; Roeed, Ketil; Rogochaya, Elena; Sharma, Rohni; Rohr, David Michael; Roehrich, Dieter; Romita, Rosa; Ronchetti, Federico; Ronflette, Lucile; Rosnet, Philippe; Rossi, Andrea; Roukoutakis, Filimon; Roy, Ankhi; Roy, Christelle Sophie; Roy, Pradip Kumar; Rubio Montero, Antonio Juan; Rui, Rinaldo; Russo, Riccardo; Ryabinkin, Evgeny; Ryabov, Yury; Rybicki, Andrzej; Sadovskiy, Sergey; Safarik, Karel; Sahlmuller, Baldo; Sahoo, Raghunath; Sahu, Pradip Kumar; Saini, Jogender; Sakai, Shingo; Salgado Lopez, Carlos Alberto; Salzwedel, Jai Samuel Nielsen; Sambyal, Sanjeev Singh; Samsonov, Vladimir; Sanchez Castro, Xitzel; Sanchez Rodriguez, Fernando Javier; Sandor, Ladislav; Sandoval, Andres; Sano, Masato; Santagati, Gianluca; Sarkar, Debojit; Scapparone, Eugenio; Scarlassara, Fernando; Scharenberg, Rolf Paul; Schiaua, Claudiu Cornel; Schicker, Rainer Martin; Schmidt, Christian Joachim; Schmidt, Hans Rudolf; Schuchmann, Simone; Schukraft, Jurgen; Schulc, Martin; Schuster, Tim Robin; Schutz, Yves Roland; Schwarz, Kilian Eberhard; Schweda, Kai Oliver; Scioli, Gilda; Scomparin, Enrico; Scott, Rebecca Michelle; Segato, Gianfranco; Seger, Janet Elizabeth; Sekiguchi, Yuko; Selyuzhenkov, Ilya; Seo, Jeewon; Serradilla Rodriguez, Eulogio; Sevcenco, Adrian; Shabetai, Alexandre; Shabratova, Galina; Shahoyan, Ruben; Shangaraev, Artem; Sharma, Natasha; Sharma, Satish; Shigaki, Kenta; Shtejer Diaz, Katherin; Sibiryak, Yury; Siddhanta, Sabyasachi; Siemiarczuk, Teodor; Silvermyr, David Olle Rickard; Silvestre, Catherine Micaela; Simatovic, Goran; Singaraju, Rama Narayana; Singh, Ranbir; Singha, Subhash; Singhal, Vikas; Sinha, Bikash; Sarkar - Sinha, Tinku; Sitar, Branislav; Sitta, Mario; Skaali, Bernhard; Skjerdal, Kyrre; Slupecki, Maciej; Smirnov, Nikolai; Snellings, Raimond; Soegaard, Carsten; Soltz, Ron Ariel; Song, Jihye; Song, Myunggeun; Soramel, Francesca; Sorensen, Soren Pontoppidan; Spacek, Michal; Spiriti, Eleuterio; Sputowska, Iwona Anna; Spyropoulou-Stassinaki, Martha; Srivastava, Brijesh Kumar; Stachel, Johanna; Stan, Ionel; Stefanek, Grzegorz; Steinpreis, Matthew Donald; Stenlund, Evert Anders; Steyn, Gideon Francois; Stiller, Johannes Hendrik; Stocco, Diego; Stolpovskiy, Mikhail; Strmen, Peter; Alarcon Do Passo Suaide, Alexandre; Sugitate, Toru; Suire, Christophe Pierre; Suleymanov, Mais Kazim Oglu; Sultanov, Rishat; Sumbera, Michal; Susa, Tatjana; Symons, Timothy; Szabo, Alexander; Szanto De Toledo, Alejandro; Szarka, Imrich; Szczepankiewicz, Adam; Szymanski, Maciej Pawel; Takahashi, Jun; Tangaro, Marco-Antonio; Tapia Takaki, Daniel Jesus; Tarantola Peloni, Attilio; Tarazona Martinez, Alfonso; Tarzila, Madalina-Gabriela; Tauro, Arturo; Tejeda Munoz, Guillermo; Telesca, Adriana; Terrevoli, Cristina; Thaeder, Jochen Mathias; Thomas, Deepa; Tieulent, Raphael Noel; Timmins, Anthony Robert; Toia, Alberica; Trubnikov, Victor; Trzaska, Wladyslaw Henryk; Tsuji, Tomoya; Tumkin, Alexandr; Turrisi, Rosario; Tveter, Trine Spedstad; Ullaland, Kjetil; Uras, Antonio; Usai, Gianluca; Vajzer, Michal; Vala, Martin; Valencia Palomo, Lizardo; Vallero, Sara; Vande Vyvre, Pierre; Van Der Maarel, Jasper; Van Hoorne, Jacobus Willem; Van Leeuwen, Marco; Diozcora Vargas Trevino, Aurora; Vargyas, Marton; Varma, Raghava; Vasileiou, Maria; Vasiliev, Andrey; Vechernin, Vladimir; Veldhoen, Misha; Velure, Arild; Venaruzzo, Massimo; Vercellin, Ermanno; Vergara Limon, Sergio; Vernet, Renaud; Verweij, Marta; Vickovic, Linda; Viesti, Giuseppe; Viinikainen, Jussi Samuli; Vilakazi, Zabulon; Villalobos Baillie, Orlando; Vinogradov, Alexander; Vinogradov, Leonid; Vinogradov, Yury; Virgili, Tiziano; Viyogi, Yogendra; Vodopyanov, Alexander; Volkl, Martin Andreas; Voloshin, Kirill; Voloshin, Sergey; Volpe, Giacomo; Von Haller, Barthelemy; Vorobyev, Ivan; Vranic, Danilo; Vrlakova, Janka; Vulpescu, Bogdan; Vyushin, Alexey; Wagner, Boris; Wagner, Jan; Wagner, Vladimir; Wang, Mengliang; Wang, Yifei; Watanabe, Daisuke; Weber, Michael; Wessels, Johannes Peter; Westerhoff, Uwe; Wiechula, Jens; Wikne, Jon; Wilde, Martin Rudolf; Wilk, Grzegorz Andrzej; Wilkinson, Jeremy John; Williams, Crispin; Windelband, Bernd Stefan; Winn, Michael Andreas; Yaldo, Chris G; Yamaguchi, Yorito; Yang, Hongyan; Yang, Ping; Yang, Shiming; Yano, Satoshi; Yasnopolskiy, Stanislav; Yi, Jungyu; Yin, Zhongbao; Yoo, In-Kwon; Yushmanov, Igor; Zaccolo, Valentina; Zach, Cenek; Zaman, Ali; Zampolli, Chiara; Zaporozhets, Sergey; Zarochentsev, Andrey; Zavada, Petr; Zavyalov, Nikolay; Zbroszczyk, Hanna Paulina; Zgura, Sorin Ion; Zhalov, Mikhail; Zhang, Haitao; Zhang, Xiaoming; Zhang, Yonghong; Zhao, Chengxin; Zhigareva, Natalia; Zhou, Daicui; Zhou, Fengchu; Zhou, You; Zhou, Zhuo; Zhu, Hongsheng; Zhu, Jianhui; Zhu, Xiangrong; Zichichi, Antonino; Zimmermann, Alice; Zimmermann, Markus Bernhard; Zinovjev, Gennady; Zoccarato, Yannick Denis; Zyzak, Maksym

    2014-10-16

    Invariant yields of neutral pions at midrapidity in the transverse momentum range $0.6 < p_{T} < 12$ GeV/c measured in Pb-Pb collisions at $\\sqrt{s_{NN}}$ = 2.76 TeV are presented for six centrality classes. The pp reference spectrum was measured in the range $0.4 < p_{T} < 10$ GeV/c at the same center-of-mass energy. The nuclear modification factor, $R_{AA}$, shows a suppression of neutral pions in central Pb-Pb collisions by a factor of up to about $8-10$ for $5 \\lesssim p_{T} \\lesssim 7$ GeV/c. The presented measurements are compared with results at lower center-of-mass energies and with theoretical calculations.

  1. Transcriptomic and Functional Analyses Reveal That PpGLK1 Regulates Chloroplast Development in Peach (Prunus persica

    Directory of Open Access Journals (Sweden)

    Min Chen

    2018-01-01

    Full Text Available Peach is an ideal species for fruit tree research because of its small, fully sequenced genome. Chloroplast development is dependent on the tight cooperation between the nuclear and plastid genomes, and is regulated by GLK transcription factors. In this work, the pigment content was monitored and the chloroplast-to-chromoplast conversion during the fruit ripening was visualized by transmission electron microscopy. Localization and expression analyses showed that PpGLK1 was located in the nucleus and expressed mainly in the leaves and fruit skin. A transcriptome analysis showed that PpGLK1 and its target genes were significantly differentially expressed in ripening peach fruit skin. PpGLK1 silencing affected chlorophyll accumulation in peach leaves and fruits. Overexpression of PpGLK1 rescued the phenotypes of the Arabidopsis Atglk1Atglk2 double mutant and the tomato uniform ripening mutant. The results of a yeast two-hybrid analysis showed that PpGLK1 is autoactivated and that PpGLK1 (301-542 a.a. interacted with PpARF5. Together, our results indicate that PpGLK1 regulates chloroplast development in green tissues in peach. Therefore, it may be a promising target gene for improving the production and quality of peach by genetic engineering and breeding approaches.

  2. Peripheral administration of antisense oligonucleotides targeting the amyloid-β protein precursor reverses AβPP and LRP-1 overexpression in the aged SAMP8 mouse brain.

    Science.gov (United States)

    Erickson, Michelle A; Niehoff, Michael L; Farr, Susan A; Morley, John E; Dillman, Lucy A; Lynch, Kristin M; Banks, William A

    2012-01-01

    The senescence accelerated mouse-prone 8 (SAMP8) mouse model of Alzheimer's disease has a natural mutation leading to age-related increases in the amyloid-β protein precursor (AβPP) and amyloid-β (Aβ) in the brain, memory impairment, and deficits in Aβ removal from the brain. Previous studies show that centrally administered antisense oligonucleotide directed against AβPP can decrease AβPP expression and Aβ production in the brains of aged SAMP8 mice, and improve memory. The same antisense crosses the blood-brain barrier and reverses memory deficits when injected intravenously. Here, we give 6 μg of AβPP or control antisense 3 times over 2 week intervals to 12 month old SAMP8 mice. Object recognition test was done 48 hours later, followed by removal of whole brains for immunoblot analysis of AβPP, low-density lipoprotein-related protein-1 (LRP-1), p-glycoprotein (Pgp), receptor for advanced glycation endproducts (RAGE), or ELISA of soluble Aβ(40). Our results show that AβPP antisense completely reverses a 30% age-associated increase in AβPP signal (p < 0.05 versus untreated 4 month old SAMP8). Soluble Aβ(40) increased with age, but was not reversed by antisense. LRP-1 large and small subunits increased significantly with age (147.7%, p < 0.01 and 123.7%, p < 0.05 respectively), and AβPP antisense completely reversed these increases (p < 0.05). Pgp and RAGE were not significantly altered with age or antisense. Antisense also caused improvements in memory (p < 0.001). Together, these data support the therapeutic potential of AβPP antisense and show a unique association between AβPP and LRP-1 expression in the SAMP8 mouse.

  3. Rectification induced in N2AA-doped armchair graphene nanoribbon device

    International Nuclear Information System (INIS)

    Chen, Tong; Wang, Ling-Ling; Luo, Kai-Wu; Xu, Liang; Li, Xiao-Fei

    2014-01-01

    By using non-equilibrium Green function formalism in combination with density functional theory, we investigated the electronic transport properties of armchair graphene nanoribbon devices in which one lead is undoped and the other is N 2 AA -doped with two quasi-adjacent substitutional nitrogen atoms incorporating pairs of neighboring carbon atoms in the same sublattice A. Two kinds of N 2 AA -doped style are considered, for N dopants substitute the center or the edge carbon atoms. Our results show that the rectification behavior with a large rectifying ratio can be found in these devices and the rectifying characteristics can be modulated by changing the width of graphene nanoribbons or the position of the N 2 AA dopant. The mechanisms are revealed to explain the rectifying behaviors.

  4. Crystal structure and putative substrate identification for the Entamoeba histolytica low molecular weight tyrosine phosphatase.

    Science.gov (United States)

    Linford, Alicia S; Jiang, Nona M; Edwards, Thomas E; Sherman, Nicholas E; Van Voorhis, Wesley C; Stewart, Lance J; Myler, Peter J; Staker, Bart L; Petri, William A

    2014-01-01

    Entamoeba histolytica is a eukaryotic intestinal parasite of humans, and is endemic in developing countries. We have characterized the E. histolytica putative low molecular weight protein tyrosine phosphatase (LMW-PTP). The structure for this amebic tyrosine phosphatase was solved, showing the ligand-induced conformational changes necessary for binding of substrate. In amebae, it was expressed at low but detectable levels as detected by immunoprecipitation followed by immunoblotting. A mutant LMW-PTP protein in which the catalytic cysteine in the active site was replaced with a serine lacked phosphatase activity, and was used to identify a number of trapped putative substrate proteins via mass spectrometry analysis. Seven of these putative substrate protein genes were cloned with an epitope tag and overexpressed in amebae. Five of these seven putative substrate proteins were demonstrated to interact specifically with the mutant LMW-PTP. This is the first biochemical study of a small tyrosine phosphatase in Entamoeba, and sets the stage for understanding its role in amebic biology and pathogenesis. Copyright © 2014 Elsevier B.V. All rights reserved.

  5. Macrophage fusion is controlled by the cytoplasmic protein tyrosine phosphatase PTP-PEST/PTPN12.

    Science.gov (United States)

    Rhee, Inmoo; Davidson, Dominique; Souza, Cleiton Martins; Vacher, Jean; Veillette, André

    2013-06-01

    Macrophages can undergo cell-cell fusion, leading to the formation of multinucleated giant cells and osteoclasts. This process is believed to promote the proteolytic activity of macrophages toward pathogens, foreign bodies, and extracellular matrices. Here, we examined the role of PTP-PEST (PTPN12), a cytoplasmic protein tyrosine phosphatase, in macrophage fusion. Using a macrophage-targeted PTP-PEST-deficient mouse, we determined that PTP-PEST was not needed for macrophage differentiation or cytokine production. However, it was necessary for interleukin-4-induced macrophage fusion into multinucleated giant cells in vitro. It was also needed for macrophage fusion following implantation of a foreign body in vivo. Moreover, in the RAW264.7 macrophage cell line, PTP-PEST was required for receptor activator of nuclear factor kappa-B ligand (RANKL)-triggered macrophage fusion into osteoclasts. PTP-PEST had no impact on expression of fusion mediators such as β-integrins, E-cadherin, and CD47, which enable macrophages to become fusion competent. However, it was needed for polarization of macrophages, migration induced by the chemokine CC chemokine ligand 2 (CCL2), and integrin-induced spreading, three key events in the fusion process. PTP-PEST deficiency resulted in specific hyperphosphorylation of the protein tyrosine kinase Pyk2 and the adaptor paxillin. Moreover, a fusion defect was induced upon treatment of normal macrophages with a Pyk2 inhibitor. Together, these data argue that macrophage fusion is critically dependent on PTP-PEST. This function is seemingly due to the ability of PTP-PEST to control phosphorylation of Pyk2 and paxillin, thereby regulating cell polarization, migration, and spreading.

  6. Identification of amino acids essential for the human parainfluenza type 2 virus V protein to lower the intracellular levels of the STAT2

    International Nuclear Information System (INIS)

    Kozuka, Yuji; Yamashita, Yasufumi; Kawano, Mitsuo; Tsurudome, Masato; Ito, Morihiro; Nishio, Machiko; Komada, Hiroshi; Ito, Yasuhiko

    2003-01-01

    The V protein of SV41 targets STAT1, while a specific loss of STAT2 is induced by the hPIV2 V protein. We established HeLa cells constitutively expressing various chimeric proteins between the hPIV2 and SV41 V proteins, and which STAT (STAT1 or 2) was expressed in these cells was analyzed. Both the P-V common domain and the V specific domain of hPIV2 V protein are necessary for STAT2 lowering. The internal domain (aa145-173) containing a large number of nonidentical amino acids between hPIV2 and SV41 does not direct STAT tropism, and the regions necessary for STAT2 lowering are discontinuous. The N-terminal domain (aa1-104) and the internal domain (aa126-196) of the hPIV2 V protein do not determine STAT tropism. HeLa cells expressing A105E or H108P show distinct expression of STAT2, but do show low expression or a loss of STAT1, indicating that the amino acid residues 105 and 108 of the hPIV2 V protein are essential for STAT2 lowering. Interestingly, there is an important amino acid(s) in the region (aa121-125) for STAT2 lowering, and the presence of either amino acid residue 123 or 125 of the hPIV2 V protein is necessary for lowering of STAT2. In addition, HeLa cells expressing S216D or 1217R expressed STAT2, but no STAT1, indicating that the amino acid residues 216 and 217 of the hPIV2 V protein are indispensable for STAT2 lowering. HeLa/hPIV2V cells and HeLa/S104/P are resistant to IFN-β, while they are sensitive to IFN-γ. On the other hand, HeLa/SV41V, HeLa/S216D, and HeLa1217R cells are resistant to both IFNs. Intriguingly, HeLa/A105E and HeLa/H108P cells were found to be sensitive to IFN-γ

  7. Reversal of DDK-Mediated MCM Phosphorylation by Rif1-PP1 Regulates Replication Initiation and Replisome Stability Independently of ATR/Chk1.

    Science.gov (United States)

    Alver, Robert C; Chadha, Gaganmeet Singh; Gillespie, Peter J; Blow, J Julian

    2017-03-07

    Dbf4-dependent kinases (DDKs) are required for the initiation of DNA replication, their essential targets being the MCM2-7 proteins. We show that, in Xenopus laevis egg extracts and human cells, hyper-phosphorylation of DNA-bound Mcm4, but not phosphorylation of Mcm2, correlates with DNA replication. These phosphorylations are differentially affected by the DDK inhibitors PHA-767491 and XL413. We show that DDK-dependent MCM phosphorylation is reversed by protein phosphatase 1 (PP1) targeted to chromatin by Rif1. Loss of Rif1 increased MCM phosphorylation and the rate of replication initiation and also compromised the ability of cells to block initiation when challenged with replication inhibitors. We also provide evidence that Rif1 can mediate MCM dephosphorylation at replication forks and that the stability of dephosphorylated replisomes strongly depends on Chk1 activity. We propose that both replication initiation and replisome stability depend on MCM phosphorylation, which is maintained by a balance of DDK-dependent phosphorylation and Rif1-mediated dephosphorylation. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  8. [Effect of elevated atmospheric CO2 on soil urease and phosphatase activities].

    Science.gov (United States)

    Chen, Lijun; Wu, Zhijie; Huang, Guohong; Zhou, Likai

    2002-10-01

    The response of soil urease and phosphatase activities at different rice growth stages to free air CO2 enrichment (FACE) was studied. The results showed that comparing with the ambient atmospheric CO2 concentration (370 mumol.mol-1), FACE (570 mumol.mol-1) significantly increased the urease activity of 0-5 cm soil layer at the vigorous growth stage of rice, whole that of 5-10 cm layer had no significant change during the whole growing season. Phosphatase activity of 0-5 cm and 5-10 cm soil layers significantly increased, and the peak increment was at the vigorous growth stage of rice.

  9. PpHB22, a member of HD-Zip proteins, activates PpDAM1 to regulate bud dormancy transition in 'Suli' pear (Pyrus pyrifolia White Pear Group).

    Science.gov (United States)

    Yang, Qinsong; Niu, Qingfeng; Li, Jianzhao; Zheng, Xiaoyan; Ma, Yunjing; Bai, Songling; Teng, Yuanwen

    2018-06-01

    Homeodomain-leucine zipper (HD-Zip) proteins, which form one of the largest and most diverse families, regulate many biological processes in plants, including differentiation, flowering, vascular development, and stress signaling. Abscisic acid (ABA) has been proved to be one of the key regulators of bud dormancy and to influence several HD-Zip genes expression. However, the role of HD-Zip genes in regulating bud dormancy remains unclear. We identified 47 pear (P. pyrifolia White Pear Group) HD-Zip genes, which were classified into four subfamilies (HD-Zip I-IV). We further revealed that gene expression levels of some HD-Zip members were closely related to ABA concentrations in flower buds during dormancy transition. Exogenous ABA treatment confirmed that PpHB22 and several other HD-Zip genes responded to ABA. Yeast one-hybrid and dual luciferase assay results combining subcellular localization showed that PpHB22 was present in nucleus and directly induced PpDAM1 (dormancy associated MADS-box 1) expression. Thus, PpHB22 is a negative regulator of plant growth associated with the ABA response pathway and functions upstream of PpDAM1. These findings enrich our understanding of the function of HD-Zip genes related to the bud dormancy transition. Copyright © 2018 Elsevier Masson SAS. All rights reserved.

  10. PP32 and SET/TAF-Iβ proteins regulate the acetylation of newly synthesized histone H4.

    Science.gov (United States)

    Saavedra, Francisco; Rivera, Carlos; Rivas, Elizabeth; Merino, Paola; Garrido, Daniel; Hernández, Sergio; Forné, Ignasi; Vassias, Isabelle; Gurard-Levin, Zachary A; Alfaro, Iván E; Imhof, Axel; Almouzni, Geneviève; Loyola, Alejandra

    2017-11-16

    Newly synthesized histones H3 and H4 undergo a cascade of maturation steps to achieve proper folding and to establish post-translational modifications prior to chromatin deposition. Acetylation of H4 on lysines 5 and 12 by the HAT1 acetyltransferase is observed late in the histone maturation cascade. A key question is to understand how to establish and regulate the distinct timing of sequential modifications and their biological significance. Here, we perform proteomic analysis of the newly synthesized histone H4 complex at the earliest time point in the cascade. In addition to known binding partners Hsp90 and Hsp70, we also identify for the first time two subunits of the histone acetyltransferase inhibitor complex (INHAT): PP32 and SET/TAF-Iβ. We show that both proteins function to prevent HAT1-mediated H4 acetylation in vitro. When PP32 and SET/TAF-Iβ protein levels are down-regulated in vivo, we detect hyperacetylation on lysines 5 and 12 and other H4 lysine residues. Notably, aberrantly acetylated H4 is less stable and this reduces the interaction with Hsp90. As a consequence, PP32 and SET/TAF-Iβ depleted cells show an S-phase arrest. Our data demonstrate a novel function of PP32 and SET/TAF-Iβ and provide new insight into the mechanisms regulating acetylation of newly synthesized histone H4. © The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.

  11. 5-hydroxy-2-methyl-1,4-naphthoquinone, a vitamin K3 analogue, suppresses STAT3 activation pathway through induction of protein tyrosine phosphatase, SHP-1: potential role in chemosensitization.

    Science.gov (United States)

    Sandur, Santosh K; Pandey, Manoj K; Sung, Bokyung; Aggarwal, Bharat B

    2010-01-01

    The activation of signal transducers and activators of transcription 3 (STAT3) has been linked with carcinogenesis through survival, proliferation, and angiogenesis of tumor cells. Agents that can suppress STAT3 activation have potential not only for prevention but also for treatment of cancer. In the present report, we investigated whether 5-hydroxy-2-methyl-1,4-naphthoquinone (plumbagin), an analogue of vitamin K, and isolated from chitrak (Plumbago zeylanica), an Ayurvedic medicinal plant, can modulate the STAT3 pathway. We found that plumbagin inhibited both constitutive and interleukin 6-inducible STAT3 phosphorylation in multiple myeloma (MM) cells and this correlated with the inhibition of c-Src, Janus-activated kinase (JAK)1, and JAK2 activation. Vanadate, however, reversed the plumbagin-induced downregulation of STAT3 activation, suggesting the involvement of a protein tyrosine phosphatase. Indeed, we found that plumbagin induced the expression of the protein tyrosine phosphatase, SHP-1, and silencing of the SHP-1 abolished the effect of plumbagin. This agent also downregulated the expression of STAT3-regulated cyclin D1, Bcl-xL, and vascular endothelial growth factor; activated caspase-3; induced poly (ADP ribose) polymerase cleavage; and increased the sub-G(1) population of MM cells. Consistent with these results, overexpression of constitutive active STAT3 significantly reduced the plumbagin-induced apoptosis. When compared with AG490, a rationally designed STAT3/JAK2 inhibitor, plumbagin was found more potent in suppressing the proliferation of cells. Plumbagin also significantly potentiated the apoptotic effects of thalidomide and bortezomib in MM cells. Overall, these results suggest that the plumbagin inhibits STAT3 activation pathway through the induction of SHP-1 and this may mediate the sensitization of STAT3 overexpressing cancers to chemotherapeutic agents.

  12. Dual roles for CoAA and its counterbalancing isoform CoAM in human kidney cell tumorigenesis

    Science.gov (United States)

    Kang, Yun Kyoung; Schiff, Rachel; Ko, Lan; Wang, Tao; Tsai, Sophia Y.; Tsai, Ming-Jer; W. O’Malley, Bert

    2008-01-01

    Co-Activator Activator (CoAA) has been reported to be a coactivator that regulates steroid receptor-mediated transcription and alternative RNA splicing. Herein we show that CoAA is a dual-function coregulator that inhibits G1/S transition in human kidney cells and suppresses anchorage independent growth and xenograft tumor formation. Suppression occurs in part by downregulating c-myc and its downstream effectors ccnd1 and skp2, and causing accumulation of p27/Kip1 protein. In this cellular setting, CoAA directly represses the proto-oncogene, c-myc by recruiting HDAC3 protein and decreasing both the acetylation of histone H3 and the presence of RNA polymerase II on the c-myc promoter. Interestingly, a splicing isoform of CoAA, Coactivator Modulator (CoAM), antagonizes CoAA-induced G1/S transition and growth inhibition by negatively regulating the mRNA levels of the endogenous CoAA isoform. In addition, we found that expression of CoAA protein is significantly decreased in human renal cell carcinoma as compared to normal kidney. Our study presents evidence that CoAA is a potential tumor suppressor in renal carcinoma and that CoAM is a counterbalancing splice-isoform. This is so far the only example of a nuclear receptor coregulator involved in suppression of kidney cancer, and suggests potentially significant new roles for coregulators in renal cancer biology. PMID:18829545

  13. Mitogen-activated protein kinase phosphatase-3 (MKP-3 in the surgical wound is necessary for the resolution of postoperative pain in mice

    Directory of Open Access Journals (Sweden)

    Skopelja-Gardner S

    2017-03-01

    Full Text Available Sladjana Skopelja-Gardner,1,* Madhurima Saha,1,* Perla Abigail Alvarado-Vazquez,2 Brenna S Liponis,1 Elena Martinez,1 E Alfonso Romero-Sandoval2 1Department of Anesthesiology, Geisel School of Medicine at Dartmouth, Lebanon, NH, 2Department of Pharmaceutical and Administrative Sciences, Presbyterian College School of Pharmacy, Clinton, SC, USA *These authors contributed equally to this work. Abstract: Mitogen-activated protein kinase (MAPK phosphatase-3 (MKP-3 and its substrates (extracellular signal-regulated kinase [ERK] and p38 play an important role in pathophysiological mechanisms of acute postoperative and chronic neuropathic pain in the spinal cord. This study aimed to understand the role of MKP-3 and its target MAPKs at the site of surgical incision in nociceptive behavior. Wild-type (WT and MKP-3 knockout (KO mice underwent unilateral plantar hind paw incision. Mechanical allodynia was assessed by using von Frey filaments. Peripheral ERK-1/2 and p38 phosphorylation were measured by Western blot. Cell infiltration was determined using hematoxylin and eosin histological staining. Peripheral phosphorylated ERK-1/2 (p-ERK-1/2 inhibition was performed in MKP-3 KO mice. In WT mice, mechanical hypersensitivity was observed on postoperative day 1 (0.69±0.17 g baseline vs 0.13±0.08 g day 1, which resolved normally by postoperative day 12 (0.46±0.08 g, N=6. In MKP-3 KO mice, this hypersensitivity persisted at least 12 days after surgery (0.19±0.06 g; N=6. KO mice displayed higher numbers of infiltrating cells (51.4±6 cells/0.1 mm2 than WT mice (8.7±1.2 cells/0.1 mm2 on postoperative day 1 (vs 5–6 cells/0.1 mm2 at baseline that returned to baseline 12 days after surgery (10–12 cells/0.1 mm2. In WT mice, peripheral p-p38 and p-ERK-1/2 expression increased (5- and 3-fold, respectively on postoperative days 1 and 5, and returned to basal levels 7–12 days after surgery (N=3 per group. Peripheral p-p38 levels in MKP-3 KO mice followed

  14. The Cytokinin Requirement for Cell Division in Cultured Nicotiana plumbaginifolia Cells Can Be Satisfied by Yeast Cdc25 Protein Tyrosine Phosphatase. Implications for Mechanisms of Cytokinin Response and Plant Development

    Science.gov (United States)

    Zhang, Kerong; Diederich, Ludger; John, Peter C.L.

    2005-01-01

    Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67Cdc25 protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr. PMID:15618425

  15. The cytokinin requirement for cell division in cultured Nicotiana plumbaginifolia cells can be satisfied by yeast Cdc25 protein tyrosine phosphatase: implications for mechanisms of cytokinin response and plant development.

    Science.gov (United States)

    Zhang, Kerong; Diederich, Ludger; John, Peter C L

    2005-01-01

    Cultured cells of Nicotiana plumbaginifolia, when deprived of exogenous cytokinin, arrest in G2 phase prior to mitosis and then contain cyclin-dependent protein kinase (CDK) that is inactive because phosphorylated on tyrosine (Tyr). The action of cytokinin in stimulating the activation of CDK by removal of inhibitory phosphorylation from Tyr is not a secondary downstream consequence of other hormone actions but is the key primary effect of the hormone in its stimulation of cell proliferation, since cytokinin could be replaced by expression of cdc25, which encodes the main Cdc2 (CDK)-Tyr dephosphorylating enzyme of yeast (Saccharomyces cerevisiae). The cdc25 gene, under control of a steroid-inducible promoter, induced a rise in cdc25 mRNA, accumulation of p67(Cdc25) protein, and increase in Cdc25 phosphatase activity that was measured in vitro with Tyr-phosphorylated Cdc2 as substrate. Cdc25 phosphatase activity peaked during mitotic prophase at the time CDK activation was most rapid. Mitosis that was induced by cytokinin also involved increase in endogenous plant CDK Tyr phosphatase activity during prophase, therefore indicating that this is a normal part of plant mitosis. These results suggest a biochemical mechanism for several previously described transgene phenotypes in whole plants and suggest that a primary signal from cytokinin leading to progression through mitosis is the activation of CDK by dephosphorylation of Tyr.

  16. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis

    Directory of Open Access Journals (Sweden)

    Bennett Hayley J

    2010-08-01

    Full Text Available Abstract Background Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. Results We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. Conclusion This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases

  17. A new family of phosphoinositide phosphatases in microorganisms: identification and biochemical analysis.

    Science.gov (United States)

    Beresford, Nicola J; Saville, Charis; Bennett, Hayley J; Roberts, Ian S; Tabernero, Lydia

    2010-08-02

    Phosphoinositide metabolism is essential to membrane dynamics and impinges on many cellular processes, including phagocytosis. Modulation of phosphoinositide metabolism is important for pathogenicity and virulence of many human pathogens, allowing them to survive and replicate in the host cells. Phosphoinositide phosphatases from bacterial pathogens are therefore key players in this modulation and constitute attractive targets for chemotherapy. MptpB, a virulence factor from Mycobacterium tuberculosis, has phosphoinositide phosphatase activity and a distinct active site P-loop signature HCXXGKDR that shares characteristics with eukaryotic lipid phosphatases and protein tyrosine phosphatases. We used this P-loop signature as a "diagnostic motif" to identify related putative phosphatases with phosphoinositide activity in other organisms. We found more than 200 uncharacterised putative phosphatase sequences with the conserved signature in bacteria, with some related examples in fungi and protozoa. Many of the sequences identified belong to recognised human pathogens. Interestingly, no homologues were found in any other organisms including Archaea, plants, or animals. Phylogenetic analysis revealed that these proteins are unrelated to classic eukaryotic lipid phosphatases. However, biochemical characterisation of those from Listeria monocytogenes and Leishmania major, demonstrated that, like MptpB, they have phosphatase activity towards phosphoinositides. Mutagenesis studies established that the conserved Asp and Lys in the P-loop signature (HCXXGKDR) are important in catalysis and substrate binding respectively. Furthermore, we provide experimental evidence that the number of basic residues in the P-loop is critical in determining activity towards poly-phosphoinositides. This new family of enzymes in microorganisms shows distinct sequence and biochemical characteristics to classic eukaryotic lipid phosphatases and they have no homologues in humans. This study provides

  18. Purification, crystallization and preliminary X-ray analysis of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli

    International Nuclear Information System (INIS)

    Zheng, Jimin; Lee, Daniel C.; Jia, Zongchao

    2009-01-01

    Isocitrate dehydrogenase kinase/phosphatase has been crystallized in three different crystal forms. Data were collected from each crystal form for structure determination. The Escherichia coli aceK gene encodes isocitrate dehydrogenase kinase/phosphatase (EC 2.7.11.5), a bifunctional protein that phosphorylates and dephosphorylates isocitrate dehydrogenase (IDH), resulting in its inactivation and activation, respectively. This reversible (de)phosphorylation directs isocitrate, an intermediate of the citric acid cycle, to either go through the full cycle or to enter the glyoxylate bypass. In the present study, the AceK protein from E. coli has been purified and crystallized. Three crystal forms were obtained from very similar crystallization conditions. The crystals belong to space groups P4 1 2 1 2, P3 2 21 and P2 1 2 1 2 1 and diffracted X-rays to resolutions of 2.9, 3.0 and 2.7 Å, respectively

  19. Phosphatase activity of Poa pratensis seeds. I. Preliminary studies on acid phosphatase II

    Directory of Open Access Journals (Sweden)

    I. Lorenc-Kubis

    2015-01-01

    Full Text Available Acid phosphatase (EC 3.1.3.2 was extracted with 0.1 M sodium acetate buffer pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction Il-b (acid phosphatase II. The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward β-naphtyl phosphate and phenyl phosphate, very low activity towards β-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca2+ and fluoride, but activated by Mg2+. EDTA had no influence on the activity of the enzyme.

  20. Phosphatase activity of Poa pratensis seeds. l. Preliminary studies on acid phosphatase II

    Energy Technology Data Exchange (ETDEWEB)

    Lorenc-Kubis, I.; Morawiecka, B.

    1973-01-01

    Acid phosphatase (EC 3.1.3.2) was extracted from 0.1 M sodium acetate buffer, pH 5.1 from Poa pratensis seeds, and separated into three fractions by chromatography on DEAE cellulose. The highest activity was found in fraction II-b (acid phosphatase II). The activity of the enzyme was optimal at pH 4.9. It hydrolyzed p-nitrophenyl phosphate most readily among the various phosphomonoesters examined. Acid phosphatase II showed also a high activity toward ..beta..-naphtyl phosphate and phenyl phosphate, very low activity towards ..beta..-glycero phosphate, 5'-GMP and no activity with glucose-1 phosphate. The enzyme was inhibited by Ca/sup 2 +/ and fluoride, but activated by Mg/sup 2 +/. EDTA had no influence on the activity of the enzyme. 12 references, 3 figures, 4 tables.

  1. Factors Affecting Pro- and Anti-Oxidant Properties of Fragments of the b-Protein Precursor (bPP): Implication for Alzheimer's Disease.

    Science.gov (United States)

    Andorn, Anne C.; Kalaria, Rajesh N.

    2000-06-01

    Oxidative stress may have a key pathogenetic role in neurodegenerative diseases including Alzheimer's disease (AD). While there is evidence that some amyloid-b (Ab) peptides can initiate oxidative stress at micromolar doses, there is also some evidence that oxidative stress increases the concentration of the b-protein precursor (bPP) and the potential for increased formation of the Ab peptides. The following studies were performed to test the hypothesis that fragments of bPP could be antioxidants and hence that oxidative stress might be an early event in AD. We found that several fragments of bPP, including the Ab peptides, inhibit ascorbate-stimulated lipid peroxidation (ASLP) in membrane fragment preparations of postmortem human brain. In contrast, other fragments of bPP enhance ASLP. These data indicate that bPP or fragments of bPP could play a key role in the redox status of cells and that alterations in bPP processing could have profound effects on the cellular response to oxidative stress.

  2. Protein-tyrosine Phosphatase SHP2 Contributes to GDNF Neurotrophic Activity through Direct Binding to Phospho-Tyr687 in the RET Receptor Tyrosine Kinase*

    Science.gov (United States)

    Perrinjaquet, Maurice; Vilar, Marçal; Ibáñez, Carlos F.

    2010-01-01

    The signaling mechanisms by which neurotrophic receptors regulate neuronal survival and axonal growth are still incompletely understood. In the receptor tyrosine kinase RET, a receptor for GDNF (glial cell line-derived neurotrophic factor), the functions of the majority of tyrosine residues that become phosphorylated are still unknown. Here we have identified the protein-tyrosine phosphatase SHP2 as a novel direct interactor of RET and the first effector known to bind to phosphorylated Tyr687 in the juxtamembrane region of the receptor. We show that SHP2 is recruited to RET upon ligand binding in a cooperative fashion, such that both interaction with Tyr687 and association with components of the Tyr1062 signaling complex are required for stable recruitment of SHP2 to the receptor. SHP2 recruitment contributes to the ability of RET to activate the PI3K/AKT pathway and promote survival and neurite outgrowth in primary neurons. Furthermore, we find that activation of protein kinase A (PKA) by forskolin reduces the recruitment of SHP2 to RET and negatively affects ligand-mediated neurite outgrowth. In agreement with this, mutation of Ser696, a known PKA phosphorylation site in RET, enhances SHP2 binding to the receptor and eliminates the effect of forskolin on ligand-induced outgrowth. Together, these findings establish SHP2 as a novel positive regulator of the neurotrophic activities of RET and reveal Tyr687 as a critical platform for integration of RET and PKA signals. We anticipate that several other phosphotyrosines of unknown function in neuronal receptor tyrosine kinases will also support similar regulatory functions. PMID:20682772

  3. [AA amyloidosis: a little-known complication of chronic leg ulcer].

    Science.gov (United States)

    Waton, J; Fays-Michel, S; Chandeclerc, M L; Corby, S; Cuny, J F; Barbaud, A; Schmutz, J-L

    2008-02-01

    AA amyloidosis, secondary to inflammatory chronic diseases like rheumatoid arthritis, is often complicated by renal failure. Chronic inflammatory dermatoses constitute rare causes of AA amyloidosis. We describe two cases of AA amyloidosis discovered after renal failure in patients presenting leg ulcers for several years. AL amyloidosis was suspected in both cases because of a history of monoclonal gammopathy in one patient and of plasmocytoma in the other. The diagnosis of AA amyloidosis was confirmed on renal histology through the detection of AA antibodies in amyloid deposits. No extrarenal amyloidosis was seen in either patient and there were no inflammatory diseases other than chronic leg ulcers. AA amyloidosis is caused by serum amyloid protein A (SAA), a reactive inflammatory protein. AA amyloidosis is thus caused by chronic inflammatory diseases, but only rarely by cutaneous inflammatory diseases. To our knowledge, the literature contains only seven other published cases of AA amyloidosis secondary to chronic leg ulcers. A review of the literature does not indicate whether cure of ulcers has any effect on the accompanying renal failure. We imagine that AA amyloidosis secondary to leg ulcer is in fact under-diagnosed. However, since the first specific treatment for AA amyloidosis is currently being evaluated by the Food and Drug Administration, it is essential that this serious complication of chronic leg ulcers be widely recognised.

  4. Our interests in protein-protein interactions

    Indian Academy of Sciences (India)

    protein interactions. Evolution of P-P partnerships. Evolution of P-P structures. Evolutionary dynamics of P-P interactions. Dynamics of P-P interaction network. Host-pathogen interactions. CryoEM mapping of gigantic protein assemblies.

  5. Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis

    DEFF Research Database (Denmark)

    Hein, Jamin B; Hertz, Emil P T; Garvanska, Dimitriya H

    2017-01-01

    Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit. Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains ...

  6. Modulation of fatty acid synthase degradation by concerted action of p38 MAP kinase, E3 ligase COP1, and SH2-tyrosine phosphatase Shp2.

    Science.gov (United States)

    Yu, Jianxiu; Deng, Rong; Zhu, Helen H; Zhang, Sharon S; Zhu, Changhong; Montminy, Marc; Davis, Roger; Feng, Gen-Sheng

    2013-02-08

    The Src-homology 2 (SH2) domain-containing tyrosine phosphatase Shp2 has been known to regulate various signaling pathways triggered by receptor and cytoplasmic tyrosine kinases. Here we describe a novel function of Shp2 in control of lipid metabolism by mediating degradation of fatty acid synthase (FASN). p38-phosphorylated COP1 accumulates in the cytoplasm and subsequently binds FASN through Shp2 here as an adapter, leading to FASN-Shp2-COP1 complex formation and FASN degradation mediated by ubiquitination pathway. By fasting p38 is activated and stimulates FASN protein degradation in mice. Consistently, the FASN protein levels are dramatically elevated in mouse liver and pancreas in which Shp2/Ptpn11 is selectively deleted. Thus, this study identifies a new activity for Shp2 in lipid metabolism.

  7. Expression, prognostic significance and mutational analysis of protein tyrosine phosphatase SHP-1 in chronic myeloid leukemia.

    Science.gov (United States)

    Papadopoulou, Vasiliki; Kontandreopoulou, Elina; Panayiotidis, Panayiotis; Roumelioti, Maria; Angelopoulou, Maria; Kyriazopoulou, Lydia; Diamantopoulos, Panagiotis T; Vaiopoulos, George; Variami, Eleni; Kotsianidis, Ioannis; Athina Viniou, Nora

    2016-05-01

    The protein tyrosine phosphatase SHP-1 dephosphorylates BCR-ABL1, thereby serving as a potential control mechanism of BCR-ABL1 kinase activity. Pathways regulating SHP-1 expression, which could be exploited in the therapeutics of TKI-resistant chronic myeloid leukemia (CML), remain unknown. Moreover, the questions of whether there is any kind of SHP-1 deregulation in CML, contributing to disease initiation or evolution, as well as the question of prognostic significance of SHP-1, have not been definitively answered. This study shows moderately lower SHP-1 mRNA expression in chronic phase CML patients in comparison to healthy individuals and no change in SHP-1 mRNA levels after successful TKI treatment. Mutational analysis of the aminoterminal and phosphatase domains of SHP-1 in patients did not reveal genetic lesions. This study also found no correlation of SHP-1 expression at diagnosis with response to treatment, although a trend for lower SHP-1 expression was noted in the very small non-responders' group of the 3-month therapeutic milestone.

  8. Irradiation effect on PP/PMMA and PP/PP-g-PMMA matrices

    International Nuclear Information System (INIS)

    Marsongko; Soebianto, Yanti S.

    1998-01-01

    The effects of PMMA and PP-g-PMMA on the oxidation of polypropylene (PP) have been studied. The mixing was done in Laboplastomill at the temperature of 200 o C, and screw speed of 20 rpm, for 5 minutes. The PMMA concentrations were 1, 2, 5, and 10% by weight, and PP-g-PMMA (12% grafting) 5, 10, and 20% by weight. Mechanical properties (tensile strength (Tb) and elongation at break (Eb)( of the mixture decreased with the increase of PMMA content over 5%. The addition of PMMA over 3% produced non-transparent film. Electron beam irradiation at the dose of 5, 10, 30, and 50 kGy was carried out to accelerate the matrix oxidation is accelerated. The optimum properties of PP/PMMA blends can be achieved by addition of maximum 2% PMMA either direct as PMMA or as compatibilizer (PP-g-PMMA). (authors)

  9. Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

    DEFF Research Database (Denmark)

    Whiteford, James R; Xian, Xiaojie; Chaussade, Claire

    2011-01-01

    Syndecan-2 is a heparan sulfate proteoglycan that has a cell adhesion regulatory domain contained within its extracellular core protein. Cell adhesion to the syndecan-2 extracellular domain (S2ED) is ß1 integrin dependent; however, syndecan-2 is not an integrin ligand. Here the protein tyrosine...

  10. Caged xanthones displaying protein tyrosine phosphatase 1B (PTP1B) inhibition from Cratoxylum cochinchinense.

    Science.gov (United States)

    Li, Zuo Peng; Lee, Hyeong-Hwan; Uddin, Zia; Song, Yeong Hun; Park, Ki Hun

    2018-08-01

    Four new caged xanthones (1-4) and two known compounds (5, 6) were isolated from the roots of Cratoxylum cochinchinense, a polyphenol rich plant, collected in China. The structures of the isolated compounds (1-6) were characterized by obtaining their detailed spectroscopic data. In particular, compounds 1 and 6 were fully identified by X-ray crystallographic data. The isolated compounds (1-6) were evaluated against protein tyrosine phosphatase 1B (PTP1B), which plays an important role in diabetes, obesity, and cancer. Among these compounds, 3, 4, and 6 displayed significant inhibition with IC 50 values of 76.3, 43.2, and 6.6 µM, respectively. A detailed kinetic study was conducted by determining K m , V max , and the ratio of K ik and K iv , which revealed that all the compounds behaved as competitive inhibitors. Copyright © 2018 Elsevier Inc. All rights reserved.

  11. NCBI nr-aa BLAST: CBRC-XTRO-01-0674 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-XTRO-01-0674 ref|YP_001022362.1| putative heme O oxygenase (cytochrome aa3-controlling...) transmembrane protein [Methylibium petroleiphilum PM1] gb|ABM96127.1| putative heme O oxygenase (cytochrome aa3-controlling

  12. Dual-specificity phosphatase 3 deficiency or inhibition limits platelet activation and arterial thrombosis.

    Science.gov (United States)

    Musumeci, Lucia; Kuijpers, Marijke J; Gilio, Karen; Hego, Alexandre; Théâtre, Emilie; Maurissen, Lisbeth; Vandereyken, Maud; Diogo, Catia V; Lecut, Christelle; Guilmain, William; Bobkova, Ekaterina V; Eble, Johannes A; Dahl, Russell; Drion, Pierre; Rascon, Justin; Mostofi, Yalda; Yuan, Hongbin; Sergienko, Eduard; Chung, Thomas D Y; Thiry, Marc; Senis, Yotis; Moutschen, Michel; Mustelin, Tomas; Lancellotti, Patrizio; Heemskerk, Johan W M; Tautz, Lutz; Oury, Cécile; Rahmouni, Souad

    2015-02-17

    A limitation of current antiplatelet therapies is their inability to separate thrombotic events from bleeding occurrences. A better understanding of the molecular mechanisms leading to platelet activation is important for the development of improved therapies. Recently, protein tyrosine phosphatases have emerged as critical regulators of platelet function. This is the first report implicating the dual-specificity phosphatase 3 (DUSP3) in platelet signaling and thrombosis. This phosphatase is highly expressed in human and mouse platelets. Platelets from DUSP3-deficient mice displayed a selective impairment of aggregation and granule secretion mediated by the collagen receptor glycoprotein VI and the C-type lectin-like receptor 2. DUSP3-deficient mice were more resistant to collagen- and epinephrine-induced thromboembolism compared with wild-type mice and showed severely impaired thrombus formation on ferric chloride-induced carotid artery injury. Intriguingly, bleeding times were not altered in DUSP3-deficient mice. At the molecular level, DUSP3 deficiency impaired Syk tyrosine phosphorylation, subsequently reducing phosphorylation of phospholipase Cγ2 and calcium fluxes. To investigate DUSP3 function in human platelets, a novel small-molecule inhibitor of DUSP3 was developed. This compound specifically inhibited collagen- and C-type lectin-like receptor 2-induced human platelet aggregation, thereby phenocopying the effect of DUSP3 deficiency in murine cells. DUSP3 plays a selective and essential role in collagen- and C-type lectin-like receptor 2-mediated platelet activation and thrombus formation in vivo. Inhibition of DUSP3 may prove therapeutic for arterial thrombosis. This is the first time a protein tyrosine phosphatase, implicated in platelet signaling, has been targeted with a small-molecule drug. © 2014 American Heart Association, Inc.

  13. Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit.

    Science.gov (United States)

    Marsan, David W; Conrad, Stephen M; Stutts, Whitney L; Parker, Christine H; Deeds, Jonathan R

    2018-03-01

    The cyanobacterium Aphanizomenon flos-aquae (AFA), from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA) dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 μg/g microcystin (MC) for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP) regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 μg/g. The objectives of this study were (1) identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2) use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA), based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51) distinct AFA-BGA products had MC ≥0.25 μg/g (the detection limit of the kit), 10 products had MC concentrations between 0.5 and 1.0 μg/g, and 4 products exceeded the limit (1.1-2.8 μg/g). LC-MS/MS confirmed PPIA results ≥0.5 μg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 μg/g MC, most were at or below 1.0 μg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC

  14. Evaluation of microcystin contamination in blue-green algal dietary supplements using a protein phosphatase inhibition-based test kit

    Directory of Open Access Journals (Sweden)

    David W. Marsan

    2018-03-01

    Full Text Available The cyanobacterium Aphanizomenon flos-aquae (AFA, from Upper-Klamath Lake, Oregon, are used to produce blue-green algal (BGA dietary supplements. The periodic co-occurrence of hepatotoxin-producing contaminant species prompted the Oregon Health Division to establish a limit of 1 μg/g microcystin (MC for products sold in Oregon in 1997. At the federal level, the current good manufacturing practice (CGMP regulations for dietary supplements require manufacturers establish a specification, and test, for limits on contaminants that may adulterate finished products. Despite this, several previous international surveys reported MC in BGA supplements in excess of 1 μg/g. The objectives of this study were (1 identify a reliable, easy to use test kit for the detection of MC in dried BGA materials and (2 use this kit to assess the occurrence of MC contamination in AFA-BGA dietary supplements in the U.S. A commercial protein phosphatase inhibition assay (PPIA, based on the enzyme PP2A, was found to have acceptable relative enzyme inhibition and accuracy for the majority of MC variants tested, including those most commonly identified in commercial samples, making the kit fit for purpose. Using the PPIA kit, 51% (26 of 51 distinct AFA-BGA products had MC ≥0.25 μg/g (the detection limit of the kit, 10 products had MC concentrations between 0.5 and 1.0 μg/g, and 4 products exceeded the limit (1.1–2.8 μg/g. LC-MS/MS confirmed PPIA results ≥0.5 μg/g and determined that MC-LA and MC-LR were the main congeners present. PPIA is a reliable method for the detection of MC contamination in dried BGA dietary supplements produced in the U.S. While the majority of AFA-BGA products contained ≥0.25 μg/g MC, most were at or below 1.0 μg/g, suggesting that manufacturers have adopted this level as a specification in these products; however, variability in recommended serving sizes prevented further analysis of consumer exposure based on the concentrations of MC

  15. SH2 domain-containing phosphatase 1 regulates pyruvate kinase M2 in hepatocellular carcinoma.

    Science.gov (United States)

    Tai, Wei-Tien; Hung, Man-Hsin; Chu, Pei-Yi; Chen, Yao-Li; Chen, Li-Ju; Tsai, Ming-Hsien; Chen, Min-Husan; Shiau, Chung-Wai; Boo, Yin-Pin; Chen, Kuen-Feng

    2016-04-19

    Pyruvate kinase M2 (PKM2) is known to promote tumourigenesis through dimer formation of p-PKM2Y105. Here, we investigated whether SH2-containing protein tyrosine phosphatase 1 (SHP-1) decreases p-PKM2Y105 expression and, thus, determines the sensitivity of sorafenib through inhibiting the nuclear-related function of PKM2. Immunoprecipitation and immunoblot confirmed the effect of SHP-1 on PKM2Y105 dephosphorylation. Lactate production was assayed in cells and tumor samples to determine whether sorafenib reversed the Warburg effect. Clinical hepatocellular carcinoma (HCC) tumor samples were assessed for PKM2 expression. SHP-1 directly dephosphorylated PKM2 at Y105 and further decreased the proliferative activity of PKM2; similar effects were found in sorafenib-treated HCC cells. PKM2 was also found to determine the sensitivity of targeted drugs, such as sorafenib, brivanib, and sunitinib, by SHP-1 activation. Significant sphere-forming activity was found in HCC cells stably expressing PKM2. Clinical findings suggest that PKM2 acts as a predicting factor of early recurrence in patients with HCC, particularly those without known risk factors (63.6%). SHP-1 dephosphorylates PKM2 at Y105 to inhibit nuclear function of PKM2 and determines the efficacy of targeted drugs. Targeting PKM2 by SHP-1 might provide new therapeutic insights for patients with HCC.

  16. Evolution of Abscisic Acid Synthesis and Signaling Mechanisms

    Science.gov (United States)

    Hauser, Felix; Waadt, Rainer; Schroeder, Julian I.

    2011-01-01

    The plant hormone abscisic acid (ABA) mediates seed dormancy, controls seedling development and triggers tolerance to abiotic stresses, including drought. Core ABA signaling components consist of a recently identified group of ABA receptor proteins of the PYRABACTIN RESISTANCE (PYR)/REGULATORY COMPONENT OF ABA RECEPTOR (RCAR) family that act as negative regulators of members of the PROTEIN PHOSPHATASE 2C (PP2C) family. Inhibition of PP2C activity enables activation of SNF1-RELATED KINASE 2 (SnRK2) protein kinases, which target downstream components, including transcription factors, ion channels and NADPH oxidases. These and other components form a complex ABA signaling network. Here, an in depth analysis of the evolution of components in this ABA signaling network shows that (i) PYR/RCAR ABA receptor and ABF-type transcription factor families arose during land colonization of plants and are not found in algae and other species, (ii) ABA biosynthesis enzymes have evolved to plant- and fungal-specific forms, leading to different ABA synthesis pathways, (iii) existing stress signaling components, including PP2C phosphatases and SnRK kinases, were adapted for novel roles in this plant-specific network to respond to water limitation. In addition, evolutionarily conserved secondary structures in the PYR/RCAR ABA receptor family are visualized. PMID:21549957

  17. An acid phosphatase in the plasma membranes of human astrocytoma showing marked specificity toward phosphotyrosine protein.

    OpenAIRE

    Leis, J F; Kaplan, N O

    1982-01-01

    The plasma membrane from the human tumor astrocytoma contains an active acid phosphatase activity based on hydrolysis of p-nitrophenyl phosphate. Other acid phosphatase substrates--beta-glycerophosphate, O-phosphorylcholine, and 5'-AMP--are not hydrolyzed significantly. The phosphatase activity is tartrate insensitive and is stimulated by Triton X-100 and EDTA. Of the three known phosphoamino acids, only free O-phosphotyrosine is hydrolyzed by the membrane phosphatase activity. Other acid pho...

  18. YbiV from E. coli K12 is a HAD phosphatase

    Energy Technology Data Exchange (ETDEWEB)

    Roberts, Anne; Lee, Seok-Yong; McCullagh, Emma; Silversmith, Ruth E.; Wemmer, David E.

    2004-03-16

    The protein YbiV from Escherichia coli K12 MG1655 is a hypothetical protein with sequence homology to the haloacid dehalogenase (HAD) superfamily of proteins. Although numerous members of this family have been identified, the functions of few are known. Using the crystal structure, sequence analysis, and biochemical assays, we have characterized ybiV as a HAD phosphatase. The crystal structure of YbiV reveals a two domain protein, one with the characteristic HAD hydrolase fold, the other an inserted a/b fold. In an effort to understand the mechanism we also solved and report the structures of YbiV in complex with beryllofluoride (BeF3-) and aluminum trifluoride (AlF3) which have been shown to mimic the phosphorylated intermediate and transition state for hydrolysis, respectively, in analogy to other HAD phosphatases. Analysis of the structures reveals the substrate binding cavity, which is hydrophilic in nature. Both structure and sequence homology indicate ybiV may be a sugar phosphatase, which is supported by biochemical assays which measured the release of free phosphate on a number of sugar-like substrates. We also investigated available genomic and functional data in an effort to determine the physiological substrate.

  19. Characterization of Human Bone Alkaline Phosphatase in Pichia Pastoris

    Science.gov (United States)

    Malone, Christine C.; Ciszak, Eva; Karr, Laurel J.

    1999-01-01

    A soluble form of human bone alkaline phosphatase has been expressed in a recombinant strain of the methylotrophic yeast Pichia pastoris. We constructed a plasmid containing cDNA encoding for human bone alkaline phosphatase, with the hydrophobic carboxyl terminal portion deleted. Alkaline phosphatase was secreted into the medium to a level of 32mg/L when cultured in shake flasks, and enzyme activity was 12U/mg, as measured by a spectrophotometric assay. By conversion to a fermentation system, a yield of 880mg/L has been achieved with an enzyme activity of 968U/mg. By gel electrophoresis analysis, it appears that greater than 50% of the total protein in the fermentation media is alkaline phosphatase. Although purification procedures are not yet completely optimized, they are expected to include filtration, ion exchange and affinity chromatography. Our presentation will focus on the purification and crystallization results up to the time of the conference. Structural data should provide additional information on the role of alkaline phosphatase in normal bone mineralization and in certain bone mineralization anomalies.

  20. Optimization of extraction parameters of PTP1β (protein tyrosine phosphatase 1β), inhibitory polyphenols, and anthocyanins from Zea mays L. using response surface methodology (RSM).

    Science.gov (United States)

    Hwang, Seung Hwan; Kwon, Shin Hwa; Wang, Zhiqiang; Kim, Tae Hyun; Kang, Young-Hee; Lee, Jae-Yong; Lim, Soon Sung

    2016-08-26

    Protein tyrosine phosphatase expressed in insulin-sensitive tissues (such as liver, muscle, and adipose tissue) has a key role in the regulation of insulin signaling and pathway activation, making protein tyrosine phosphatase a promising target for the treatment of type 2 diabetes mellitus and obesity and response surface methodology (RSM) is an effective statistical technique for optimizing complex processes using a multi-variant approach. In this study, Zea mays L. (Purple corn kernel, PCK) and its constituents were investigated for protein tyrosine phosphatase 1β (PTP1β) inhibitory activity including enzyme kinetic study and to improve total yields of anthocyanins and polyphenols, four extraction parameters, including temperature, time, solid-liquid ratio, and solvent volume, were optimized by RSM. Isolation of seven polyphenols and five anthocyanins was achieved by PTP1β assay. Among them, cyanidin-3-(6"malonylglucoside) and 3'-methoxyhirsutrin showed the highest PTP1β inhibition with IC50 values of 54.06 and 64.04 μM, respectively and 4.52 mg gallic acid equivalent/g (GAE/g) of total polyphenol content (TPC) and 43.02 mg cyanidin-3-glucoside equivalent/100 g (C3GE/100g) of total anthocyanin content (TAC) were extracted at 40 °C for 8 h with a 33 % solid-liquid ratio and a 1:15 solvent volume. Yields were similar to predictions of 4.58 mg GAE/g of TPC and 42.28 mg C3GE/100 g of TAC. These results indicated that PCK and 3'-methoxyhirsutrin and cyanidin-3-(6"malonylglucoside) might be active natural compounds and could be apply by optimizing of extraction process using response surface methodology.