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Sample records for 2c protein phosphatases

  1. Molecular cloning of rat type 2C (IA) protein phosphatase mRNA.

    Tamura, S; Lynch, K. R.; Larner, J.; Fox, J.; Yasui, A; Kikuchi, K.; Suzuki, Y.; Tsuiki, S

    1989-01-01

    A full-length cDNA encoding rat type 2C (IA) protein phosphatase was isolated from a kidney cDNA library. The cDNA was identified by screening the library with oligonucleotides based on a partial amino acid sequence determined from purified rat liver phosphatase. This clone is 2.35 kilobase pairs long and has a single extended translation reading frame that predicts a 382-amino acid protein of 42,416 daltons. The deduced amino acid sequence contains segments corresponding to three peptides fr...

  2. ABA Inducible Rice Protein Phosphatase 2C Confers ABA Insensitivity and Abiotic Stress Tolerance in Arabidopsis

    Singh, Amarjeet; Jha, Saroj K.; Bagri, Jayram; Pandey, Girdhar K.

    2015-01-01

    Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and t...

  3. ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis.

    Amarjeet Singh

    Full Text Available Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108, which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions.

  4. ABA inducible rice protein phosphatase 2C confers ABA insensitivity and abiotic stress tolerance in Arabidopsis.

    Singh, Amarjeet; Jha, Saroj K; Bagri, Jayram; Pandey, Girdhar K

    2015-01-01

    Arabidopsis PP2C belonging to group A have been extensively worked out and known to negatively regulate ABA signaling. However, rice (Oryza sativa) orthologs of Arabidopsis group A PP2C are scarcely characterized functionally. We have identified a group A PP2C from rice (OsPP108), which is highly inducible under ABA, salt and drought stresses and localized predominantly in the nucleus. Genetic analysis revealed that Arabidopsis plants overexpressing OsPP108 are highly insensitive to ABA and tolerant to high salt and mannitol stresses during seed germination, root growth and overall seedling growth. At adult stage, OsPP108 overexpression leads to high tolerance to salt, mannitol and drought stresses with far better physiological parameters such as water loss, fresh weight, chlorophyll content and photosynthetic potential (Fv/Fm) in transgenic Arabidopsis plants. Expression profile of various stress marker genes in OsPP108 overexpressing plants revealed interplay of ABA dependent and independent pathway for abiotic stress tolerance. Overall, this study has identified a potential rice group A PP2C, which regulates ABA signaling negatively and abiotic stress signaling positively. Transgenic rice plants overexpressing this gene might provide an answer to the problem of low crop yield and productivity during adverse environmental conditions. PMID:25886365

  5. Probing protein phosphatase substrate binding

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

    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...

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

    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.

  7. Pepper protein phosphatase type 2C, CaADIP1 and its interacting partner CaRLP1 antagonistically regulate ABA signalling and drought response.

    Lim, Chae Woo; Lee, Sung Chul

    2016-07-01

    Abscisic acid (ABA) is a key phytohormone that regulates plant growth and developmental processes, including seed germination and stomatal closing. Here, we report the identification and functional characterization of a novel type 2C protein phosphatase, CaADIP1 (Capsicum annuum ABA and Drought-Induced Protein phosphatase 1). The expression of CaADIP1 was induced in pepper leaves by ABA, drought and NaCl treatments. Arabidopsis plants overexpressing CaADIP1 (CaADIP1-OX) exhibited an ABA-hyposensitive and drought-susceptible phenotype. We used a yeast two-hybrid screening assay to identify CaRLP1 (Capsicum annuum RCAR-Like Protein 1), which interacts with CaADIP1 in the cytoplasm and nucleus. In contrast to CaADIP1-OX plants, CaRLP1-OX plants displayed an ABA-hypersensitive and drought-tolerant phenotype, which was characterized by low levels of transpirational water loss and increased expression of stress-responsive genes relative to those of wild-type plants. In CaADIP1-OX/CaRLP1-OX double transgenic plants, ectopic expression of the CaRLP1 gene led to strong suppression of CaADIP1-induced ABA hyposensitivity during the germinative and post-germinative stages, indicating that CaADIP1 and CaRLP1 act in the same signalling pathway and CaADIP1 functions downstream of CaRLP1. Our results indicate that CaADIP1 and its interacting partner CaRLP1 antagonistically regulate the ABA-dependent defense signalling response to drought stress. PMID:26825039

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

    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.

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

    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.

  10. Research progress of plant PP2C-type protein phosphatase in ABA signal transduction and adversity stress regulation mechanism%植物 PP2C 蛋白磷酸酶 ABA 信号转导及逆境胁迫调控机制研究进展

    张继红; 陶能国

    2015-01-01

    Protein phosphatase is the most important and pivotal enzymes in reversible protein phosphorylation regula-ting mechanisms.While the PP2C phosphatase is a kind of serine/threonine residues of protein phosphatase,is the largest protein phosphatase family in higher plants,there are 76 family members,widely exists in living organisms. So far,four kinds of PP2C protein phosphatases have been found in plants.Protein kinase and protein phosphatase catalyzed reversible protein phosphorylation,play an important role in plant signal transduction and physiological me-tabolism,protein phosphorylation exist in almost the signal transduction pathway.Numerous academic studies have shown that plant PP2Cs are involved in multiple signal transduction pathways including PP 2C involved in ABA sig-naling pathway,the response to drought,low temperature,salt stress,participated in the plant wound and seed dor-mancy or germination signal pathway,and exist the different regulation mechanism and the enzyme catalytic activity were dependent on the concentrations of Mg2+ or Mn2+ .In plant PP2Cs protein C-terminal,there are a highly con-served catalytic domains,as well as in their N-terminal,their function are different.The review would provide a brief overview of classification,structure of PP 2Cs ,the interaction between ABA receptor and PP2Cs protein,the recent progresses about their roles in ABA and other stress signal transduction pathway in higher plant.%蛋白磷酸酶(protein phosphatase,PP)是蛋白质可逆磷酸化调节机制中的关键酶,而 PP2C 磷酸酶是一类丝氨酸/苏氨酸残基蛋白磷酸酶,是高等植物中最大的蛋白磷酸酶家族,包含76个家族成员,广泛存在于生物体中。迄今为止,在植物体内已经发现了4种 PP2C 蛋白磷酸酶。蛋白激酶和蛋白磷酸酶协同催化蛋白质可逆磷酸化,在植物体内信号转导和生理代谢中起着重要的调节作用,蛋白质的磷酸化几乎存在于所有的信号转导途

  11. Structural Genomics of Protein Phosphatases

    Almo,S.; Bonanno, J.; Sauder, J.; Emtage, S.; Dilorenzo, T.; Malashkevich, V.; Wasserman, S.; Swaminathan, S.; Eswaramoorthy, S.; et al

    2007-01-01

    The New York SGX Research Center for Structural Genomics (NYSGXRC) of the NIGMS Protein Structure Initiative (PSI) has applied its high-throughput X-ray crystallographic structure determination platform to systematic studies of all human protein phosphatases and protein phosphatases from biomedically-relevant pathogens. To date, the NYSGXRC has determined structures of 21 distinct protein phosphatases: 14 from human, 2 from mouse, 2 from the pathogen Toxoplasma gondii, 1 from Trypanosoma brucei, the parasite responsible for African sleeping sickness, and 2 from the principal mosquito vector of malaria in Africa, Anopheles gambiae. These structures provide insights into both normal and pathophysiologic processes, including transcriptional regulation, regulation of major signaling pathways, neural development, and type 1 diabetes. In conjunction with the contributions of other international structural genomics consortia, these efforts promise to provide an unprecedented database and materials repository for structure-guided experimental and computational discovery of inhibitors for all classes of protein phosphatases.

  12. Searching for the role of protein phosphatases in eukaryotic microorganisms

    da-Silva A.M.

    1999-01-01

    Full Text Available Preference for specific protein substrates together with differential sensitivity to activators and inhibitors has allowed classification of serine/threonine protein phosphatases (PPs into four major types designated types 1, 2A, 2B and 2C (PP1, PP2A, PP2B and PP2C, respectively. Comparison of sequences within their catalytic domains has indicated that PP1, PP2A and PP2B are members of the same gene family named PPP. On the other hand, the type 2C enzyme does not share sequence homology with the PPP members and thus represents another gene family, known as PPM. In this report we briefly summarize some of our studies about the role of serine/threonine phosphatases in growth and differentiation of three different eukaryotic models: Blastocladiella emersonii, Neurospora crassa and Dictyostelium discoideum. Our observations suggest that PP2C is the major phosphatase responsible for dephosphorylation of amidotransferase, an enzyme that controls cell wall synthesis during Blastocladiella emersonii zoospore germination. We also report the existence of a novel acid- and thermo-stable protein purified from Neurospora crassa mycelia, which specifically inhibits the PP1 activity of this fungus and mammals. Finally, we comment on our recent results demonstrating that Dictyostelium discoideum expresses a gene that codes for PP1, although this activity has never been demonstrated biochemically in this organism.

  13. SERUM PROTEINS, TRANSAMINASES AND PHOSPHATASES IN MALNUTRITION

    H. Mohammadiha

    1976-07-01

    Full Text Available The levels of serum tota1 protein, albumin, transaminases and phosphatases were estimated in a group of children with severe Marasmus or mild malnutrition in order to identify some of the associated deficiencies in these syndromes. The biochemical pattern was similar in the normal and malnourished children.

  14. Structural and Biochemical Characterization of a Cyanobacterial PP2C Phosphatase Reveals Insights into Catalytic Mechanism and Substrate Recognition

    Yunlong Si

    2016-04-01

    Full Text Available PP2C-type phosphatases play roles in signal transduction pathways related to abiotic stress. The cyanobacterial PP2C-type phosphatase tPphA specifically dephosphorylates the PII protein, which is a key regulator in cyanobacteria adapting to nitrogen-deficient environments. Previous studies have shown that residue His39 of tPphA is critical for the enzyme’s recognition of the PII protein; however, the manner in which this residue determines tPphA substrate specificity is unknown. Here, we solved the crystal structure of H39A, a tPphA variant. The structure revealed that the mutation of residue His39 to alanine changes the conformation and the flexibility of the loop in which residue His39 is located, and these changes affect the substrate specificity of tPphA. Moreover, previous studies have assumed that the FLAP subdomain and the third metal (M3 of tPphA could mutually influence each other to regulate PP2C catalytic activity and substrate specificity. However, despite the variable conformations adopted by the FLAP subdomain, the position of M3 was consistent in the tPphA structure. These results indicate that the FLAP subdomain does not influence M3 and vice versa. In addition, a small screen of tPphA inhibitors was performed. Sanguinarine and Ni2+ were found to be the most effective inhibitors among the assayed chemicals. Finally, the dimeric form of tPphA was stabilized by cross-linkers and still exhibited catalytic activity towards p-nitrophenyl phosphate.

  15. Acid phosphatase purified from Mycoplasma fermentans has protein tyrosine phosphatase-like activity.

    Shibata, K; Noda, M.; Sawa, Y; Watanabe, T.

    1994-01-01

    Acid phosphatase purified from Mycoplasma fermentans dephosphorylated phosphotyrosine-containing lysozyme and Raytide, a peptide substrate for protein tyrosine phosphatases. The optimum pH for Raytide was about 5.5. Raytide phosphatase activity was inhibited by potassium fluoride, sodium molybdate, and sodium orthovanadate and was found to exist in some mycoplasmas.

  16. Specific dephosphorylation by phosphatases 1 and 2A of a nuclear protein structurally and immunologically related to nucleolin

    Schneider, H R; Mieskes, G; Issinger, O G

    1989-01-01

    A new nuclear substrate (N-60) for phosphatase 1 and 2Ac has been described. In contrast to nucleolin (C23), to which it is structurally and immunologically related, N-60 becomes dephosphorylated to 51% and 41% by phosphatases 1 and 2Ac, respectively, within 10 min. Incubation up to 20 min led to a...... complete dephosphorylation of N-60. The two other phosphatases tested (2B and 2C) did not dephosphorylate protein N-60 to the same extent as phosphatases 1 and 2Ac. In the case of nucleolin only 18% phosphate was released by all four phosphatases tested. The activity of both phosphatases, 1 and 2A, could...

  17. CLONING AND SEQUENCE ANALYSIS OF A PROTEIN PHOSPHATASE 2C CODING GENE FROM LEISHMANIA DONOVANI 1S%杜氏利什曼原虫蛋白磷酸酶-2C的基因克隆化

    成军; 夏小兵; 王刚; 刘妍; 钟彦伟; 王琳; 杨继珍

    2001-01-01

    目的克隆杜氏利什曼原虫(Leishmania donovani,Ld)1S株蛋白磷酸酶2C(PP2C)的编码基因,为应用这种编码T细胞抗原的基因进行基因疫苗研究奠定基础。方法体外培养杜氏利什曼原虫1S株无鞭毛体,常规方法从虫体提取制备基因组DNA。以夏科氏利什曼原虫(Leishmania chagasi,Lc)的PP2C基因序列为参照,设计并合成利什曼原虫PP2C基因序列特异性的引物。结果以杜氏利什曼原虫的基因组为模板,利用多聚酶链反应(PCR)技术,扩增获得了杜氏利什曼原虫PP2C的全长编码基因。基因序列测定结果表明,杜氏利什曼原虫1S株PP2C基因序列长度为1 317bp,开放读码框架由1 221bp组成,编码产物为406个氨基酸残基。获得的杜氏利什曼原虫1S株的PP2C基因与来源于夏科氏利什曼原虫的PP2C氨基酸残基序列的同源性为95%(387/406)。结论本研究克隆了杜氏利什曼原虫的PP2C基因,为应用诱导T细胞免疫应答抗原的编码基因进行杜氏利什曼原虫的基因疫苗研究奠定了基础。

  18. Partial purification and characterization of phosphotyrosyl-protein phosphatase(s) from human erythrocyte cytosol

    Phosphotyrosyl-protein phosphatase activity of human erythrocyte cytosol can be resolved into two fractions by DEAE-cellulose chromatography followed by P-cellulose chromatography. Both 32P-Tyr-phosphatases are able to dephosphorylate 32P-Tyr of poly (Glu-Tyr) 4:1 but no angiotensin II and synthetic peptide Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Gly, previously phosphorylated on tyrosine residues by rat spleen tyrosine-protein kinase. Both 32P-Tyr-phosphatase activities distinctly differ from either 32P-Ser-casein phosphatase activity or acid and alkaline p-nitrophenylphosphatase activities with regard to catalytic and physico-chemical properties such as substrate specificity, chromatographic behavior, response to various effectors

  19. Serum proteins, trace metals and phosphatases in psoriasis

    Bhatnagar M

    1994-01-01

    Full Text Available Serum proteins, zinc, copper, acid phosphatase (AcPase and alkaline phosphatase (AlPase were studied in both active and remission phases of psoriasis. Data were compared with healthy controls, ?1, ? and ? globulins were high in active phase while ?1 and ? globulins were at par in remission phase. Serum copper was low but zinc and alkaline phosphatase were significantly high in both active and remission phases of the disease. Acid phosphatase level was at par in all the experimental groups. Study suggests a positive correlation of globulin, zinc and Alpase in active and remission phase of psoriasis.

  20. Rhizobiales-like Phosphatase 2 from Arabidopsis thaliana Is a Novel Phospho-tyrosine-specific Phospho-protein Phosphatase (PPP) Family Protein Phosphatase.

    Uhrig, R Glen; Labandera, Anne-Marie; Muhammad, Jamshed; Samuel, Marcus; Moorhead, Greg B

    2016-03-11

    Cellular signaling through protein tyrosine phosphorylation is well established in mammalian cells. Although lacking the classic tyrosine kinases present in humans, plants have a tyrosine phospho-proteome that rivals human cells. Here we report a novel plant tyrosine phosphatase from Arabidopsis thaliana (AtRLPH2) that, surprisingly, has the sequence hallmarks of a phospho-serine/threonine phosphatase belonging to the PPP family. Rhizobiales/Rhodobacterales/Rhodospirillaceae-like phosphatases (RLPHs) are conserved in plants and several other eukaryotes, but not in animals. We demonstrate that AtRLPH2 is localized to the plant cell cytosol, is resistant to the classic serine/threonine phosphatase inhibitors okadaic acid and microcystin, but is inhibited by the tyrosine phosphatase inhibitor orthovanadate and is particularly sensitive to inhibition by the adenylates, ATP and ADP. AtRLPH2 displays remarkable selectivity toward tyrosine-phosphorylated peptides versus serine/threonine phospho-peptides and readily dephosphorylates a classic tyrosine phosphatase protein substrate, suggesting that in vivo it is a tyrosine phosphatase. To date, only one other tyrosine phosphatase is known in plants; thus AtRLPH2 represents one of the missing pieces in the plant tyrosine phosphatase repertoire and supports the concept of protein tyrosine phosphorylation as a key regulatory event in plants. PMID:26742850

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

    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 [32P]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

  2. Discovery of Small Molecule Inhibitors of the PH Domain Leucine-Rich Repeat Protein Phosphatase (PHLPP) by Chemical and Virtual Screening

    Sierecki, Emma; Sinko, William; McCammon, J. Andrew; Newton, Alexandra C.

    2010-01-01

    PH domain Leucine-rich repeat protein phosphatase (PHLPP) directly dephosphorylates and inactivates Akt and protein kinase C, poising it as a prime target for pharmacological intervention of two major survival pathways. Here we report on the discovery of small molecule inhibitors of the phosphatase activity of PHLPP, a member of the PP2C family of phosphatases for which there are no general pharmacological inhibitors. First, the Diversity Set of the NCI was screened for inhibition of the puri...

  3. Methods to distinguish various types of protein phosphatase activity

    To distinguish the action of protein Tyr(P) and protein Ser(P)/Thr(P) phosphatases on 32P-labeled phosphoproteins in subcellular fractions different inhibitors and activators are utilized. Comparison of the effects of added compounds provides a convenient, indirect method to characterize dephosphorylation reactions. Protein Tyr(P) phosphatases are specifically inhibited by micromolar Zn2+ or vanadate, and show maximal activity in the presence of EDTA. The other class of cellular phosphatases, specific for protein Ser(P) and Thr(P) residues, are inhibited by fluoride and EDTA. In this class of enzymes two major functional types can be distinguished: those sensitive to inhibition by the heat-stable protein inhibitor-2 and not stimulated by polycations, and those not sensitive to inhibition and stimulated by polycations. Preparation of 32P-labeled Tyr(P) and Ser(P) phosphoproteins also is presented for the direct measurement of phosphatase activities in preparations by the release of acid-soluble [32P]phosphate

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

    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.

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

    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.

  6. Purification and characterization of a protein phosphatase that dephosphorylates pyruvate kinase in an anoxia tolerant animal.

    Brooks, S P; Storey, K B

    1996-05-01

    A protein phosphatase that dephosphorylates pyruvate kinase (PK) in vitro was purified and characterized from the foot muscle of the anoxia tolerant gastropod mollusc Busycon canaliculatum. Purification involved three steps: negative chromatography through Blue Dextran and CM Sephadex, affinity chromatography on DEAE Sephadex and gel exclusion chromatography on Sephacryl S-400. Pyruvate kinase phosphatase (PK-Pase) activity was monitored by following changes in PK I50 values for L-alanine that had previously been linked to changes in the degree of PK phosphorylation. The purified PK-Pase gave a single band on SDS-polyacrylamide gel electrophoresis with a molecular weight of 41 +/- 1 kdaltons. Isoelectric focusing analysis showed that the PK-Pase had an isoelectric point of 4.2 +/- 0.1. Kinetic analysis showed that the enzyme was a Type 2C protein phosphatase with a pH optimum of 6.5. Maximal activity required the presence of magnesium ions (KM = 7.9 +/- 0.6 microM) although high concentrations of Mg2+ were inhibitory (I50 = 2.3 +/- 0.4 mM). The protein phosphatase activity was not affected by either spermine, cAMP, cGMP, potassium phosphate, tartrate, NaF, HgCl2, citrate or concentrations of CaCl2 less than 10 mM. The enzyme could also use ATP, ADP, and GTP as substrates. PMID:8739044

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

    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.

  8. Bioengineered protein phosphatase 2A: Update on need

    Rubiolo, Juan A.; López-Alonso, Henar; Alfonso, Amparo; Vega, Félix V.; Vieytes, Mercedes Rodríguez; Botana, Luis M

    2013-01-01

    Harmful algal blooms caused by phytoplankton can occur in all aquatic environments. Some of the algae present in these blooms are capable of producing extremely potent toxins. Due to climate change and eutrophication, harmful algal blooms are increasing on a global scale. One kind of toxin producing algae are those that produce okadaic acid, its derivatives (dinophysistoxin-1 and 2), and microcystins. These toxins are potent inhibitors of protein phosphatase 2A, so this protein is used to det...

  9. Inhibitors of the Ca2+/calmodulin-dependent protein kinase phosphatase family (CaMKP and CaMKP-N)

    Ca2+/calmodulin-dependent protein kinase phosphatase (CaMKP) and its nuclear isoform CaMKP-N are unique Ser/Thr protein phosphatases that negatively regulate the Ca2+/calmodulin-dependent protein kinase (CaMK) cascade by dephosphorylating multifunctional CaMKI, II, and IV. However, the lack of specific inhibitors of these phosphatases has hampered studies on these enzymes in vivo. In an attempt to obtain specific inhibitors, we searched inhibitory compounds and found that Evans Blue and Chicago Sky Blue 6B served as effective inhibitors for CaMKP. These compounds also inhibited CaMKP-N, but inhibited neither protein phosphatase 2C, another member of PPM family phosphatase, nor calcineurin, a typical PPP family phosphatase. The minimum structure required for the inhibition was 1-amino-8-naphthol-4-sulfonic acid. When Neuro2a cells cotransfected with CaMKIV and CaMKP-N were treated with these compounds, the dephosphorylation of CaMKIV was strongly suppressed, suggesting that these compounds could be used as potent inhibitors of CaMKP and CaMKP-N in vivo as well as in vitro

  10. Assembly and structure of protein phosphatase 2A

    2009-01-01

    Protein phosphatase 2A (PP2A) represents a conserved family of important protein serine/threonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, medi-ated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interac-tion with a large number of cellular cofactors. Recent biochemical and structural investigation reveals critical insights into the assembly and function of the PP2A core enzyme as well as two families of holoenzyme. This review focuses on the molecular mechanisms revealed by these latest advances.

  11. Assembly and structure of protein phosphatase 2A

    SHI YiGong

    2009-01-01

    Protein phosphatase 2A (PP2A) represents a conserved family of important protein serinetthreonine phosphatases in species ranging from yeast to human. The PP2A core enzyme comprises a scaffold subunit and a catalytic subunit. The heterotrimeric PP2A holoenzyme consists of the core enzyme and a variable regulatory subunit. The catalytic subunit of PP2A is subject to reversible methylation, mediated by two conserved enzymes. Both the PP2A core and holoenzymes are regulated through interaction with a large number of cellular cofactors. Recent biochemical and structural investigation reveals critical insights into the assembly and function of the PP2A core enzyme as well as two families of holoenzyme. This review focuses on the molecular mechanisms revealed by these latest advances.

  12. Protein phosphatase 2A, a key player in Alzheimer's disease

    Rong LIU; Qing TIAN

    2009-01-01

    Protein phosphatase 2A (PP2A) is the pre-dominant serine/threonine phosphatase in eukaryotic cells. In the brains of patients with Alzheimer's disease (AD), decreased PP2A activities were observed, which is suggested to be involved in neurofibrillary tangle (NFT) formation, disturbed amyloid precursor protein (APP) secretion and neurodegeneration in AD brain. Based on our research and other previous findings, decreased PP2Ac level, decreased PP2A holoenzyme composition, increased level of PP2A inhibitors, increased PP2Ac Leu309 demethylation and Tyr307 phosphorylation underlie PP2A inactivation in AD. β-amyloid (Aβ) over-production, estrogen deficiency and impaired homocys-teine metabolism are the possible up-stream factors that inactivate PP2A in AD neurons. Further studies are required to disclose the role of PP2A in Alzheimer's disease.

  13. Receptor-type protein tyrosine phosphatases in cancer

    Yu Du

    2015-02-01

    Full Text Available Protein tyrosine phosphatases (PTPs play an important role in regulating cell signaling events in coordination with tyrosine kinases to control cell proliferation, apoptosis, survival, migration, and invasion. Receptor-type protein tyrosine phosphatases (PTPRs are a subgroup of PTPs that share a transmembrane domain with resulting similarities in function and target specificity. In this review, we summarize genetic and epigenetic alterations including mutation, deletion, amplification, and promoter methylation of PTPRs in cancer and consider the consequences of PTPR alterations in different types of cancers. We also summarize recent developments using PTPRs as prognostic or predictive biomarkers and/or direct targets. Increased understanding of the role of PTPRs in cancer may provide opportunities to improve therapeutic approaches.

  14. Have We Overlooked the Importance of Serine/Threonine Protein Phosphatases in Pancreatic Beta-Cells? Role Played by Protein Phosphatase 2A in Insulin Secretion

    Esser V

    2005-07-01

    Full Text Available Genetic predisposition and environmental influences insidiously converge to cause glucose intolerance and hyperglycemia. Beta-cell compensates by secreting more insulin and when it fails, overt diabetes mellitus ensues. The need to understand the mechanisms involved in insulin secretion cannot be stressed enough. Phosphorylation of proteins plays an important role in regulating insulin secretion. In order to understand how a particular cellular process is regulated by protein phosphorylation the nature of the protein kinases and protein phosphatases involved and the mechanisms that determine when and where these enzymes are active should be investigated. While the actions of protein kinases have been intensely studied within the beta-cell, less emphasis has been placed on protein phosphatases even though they play an important regulatory role. This review focuses on the importance of protein phosphatase 2A in insulin secretion. Most of the present knowledge on protein phosphatase 2A originates from protein phosphatase inhibitor studies on islets and beta-cell lines. The ability of protein phosphatase 2A to change its activity in the presence of glucose and inhibitors provides clues to its role in regulating insulin secretion. An aggressive approach to elucidate the substrates and mechanisms of action of protein phosphatases is crucial to the understanding of phosphorylation events within the beta-cell. Characterizing protein phosphatase 2A within the beta-cell will certainly help us in understanding the mechanisms involved in insulin secretion and provide valuable information for drug development.

  15. Protein tyrosine phosphatases expression during development of mouse superior colliculus

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-01-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior col...

  16. Redox Regulation of Protein Tyrosine Phosphatase Activity by Hydroxyl Radical

    Meng, Fan-Guo; Zhang, Zhong-Yin

    2012-01-01

    Substantial evidence suggests that transient production of reactive oxygen species (ROS) such as hydrogen peroxide (H2O2) is an important signaling event triggered by the activation of various cell surface receptors. Major targets of H2O2 include protein tyrosine phosphatases (PTPs). Oxidation of the active site Cys by H2O2 abrogates PTP catalytic activity, thereby potentially furnishing a mechanism to ensure optimal tyrosine phosphorylation in response to a variety of physiological stimuli. ...

  17. Induction of Bone Matrix Protein Expression by Native Bone Matrix Proteins in C2C12 Culture

    ZHEN-MING HU; SEAN A. F. PEEL; STEPHEN K. C. HO; GEORGE K. B. SANDOR; CAMERON M. L. CLOKIE

    2009-01-01

    Objective To study the expression of bone matrix protein (BMP) induced by bovine bone morphogenetic proteins (BMPs) in vitro. Methods Type I collagen, osteopontin (OPN), osteonectin (ON), osteocalcin (OC), and bone sialoprotein (BSP) were detected by immunohistochemistry in C2C12 cultured from day 1 to day 28. Results The signaling of bone matrix protein expression became weaker except for type I collagen, OC and BSP after 5 days. Fourteen days after culture, the positive signaling of type I collagen, OPN, ON, OC, and BSP was gradually declined, and could be detected significantly as compared with that of the negative control on day 28. BMP assay showed that the Ikaline phosphatase (ALP) activity was higher in C2C12 culture than in the control during the 14-day culture. Also, total protein and DNA significantly increased during the 14-day culture. High levels of ALP were seen in preosteoblasts and osteoblsts in vivo and in differentiating ostcoblasts in vitro. ALP was well recognized as a marker reflecting osteoblastic activity. Conclusion Native bovine BMP induces conversion of myoblasts into osteoblasts, produces type 1 collagen, and plays significantly role in osteoinduction and bone matrix mineralization of C2C12 in vitro.

  18. Protein-tyrosine-phosphatase SHPTP2 couples platelet-derived growth factor receptor beta to Ras.

    Bennett, A.M.; Tang, T. L.; SUGIMOTO, S; Walsh, C T; Neel, B G

    1994-01-01

    Protein-tyrosine-phosphatase SHPTP2 (Syp/PTP-1D/PTP2C) is the homologue of the Drosophila corkscrew (csw) gene product, which transmits positive signals from receptor tyrosine kinases. Likewise, SHPTP2 has been implicated in positive signaling from platelet-derived growth factor receptor beta (PDGFR). Upon PDGF stimulation, SHPTP2 binds to the PDGFR and becomes tyrosine-phosphorylated. We have identified tyrosine-542 (pY542TNI) as the major in vivo site of SHPTP2 tyrosine phosphorylation. The...

  19. Protein phosphatase 1α is a Ras-activated Bad phosphatase that regulates interleukin-2 deprivation-induced apoptosis

    Ayllón, Verónica; Martínez-A, Carlos; García, Alphonse; Cayla, Xavier; Rebollo, Angelita

    2000-01-01

    Growth factor deprivation is a physiological mechanism to regulate cell death. We utilize an interleukin-2 (IL-2)-dependent murine T-cell line to identify proteins that interact with Bad upon IL-2 stimulation or deprivation. Using the yeast two-hybrid system, glutathione S-transferase (GST) fusion proteins and co-immunoprecipitation techniques, we found that Bad interacts with protein phosphatase 1α (PP1α). Serine phosphorylation of Bad is induced by IL-2 and its dephosphorylation correlates with appearance of apoptosis. IL-2 deprivation induces Bad dephosphorylation, suggesting the involvement of a serine phosphatase. A serine/threonine phosphatase activity, sensitive to the phosphatase inhibitor okadaic acid, was detected in Bad immunoprecipitates from IL-2-stimulated cells, increasing after IL-2 deprivation. This enzymatic activity also dephosphorylates in vivo 32P-labeled Bad. Treatment of cells with okadaic acid blocks Bad dephosphorylation and prevents cell death. Finally, Ras activation controls the catalytic activity of PP1α. These results strongly suggest that Bad is an in vitro and in vivo substrate for PP1α phosphatase and that IL-2 deprivation-induced apoptosis may operate by regulating Bad phosphorylation through PP1α phosphatase, whose enzymatic activity is regulated by Ras. PMID:10811615

  20. Functional Analysis of Protein Tyrosine Phosphatases in Thrombosis and Hemostasis.

    Rahmouni, Souad; Hego, Alexandre; Delierneux, Céline; Wéra, Odile; Musumeci, Lucia; Tautz, Lutz; Oury, Cécile

    2016-01-01

    Platelets are small blood cells derived from cytoplasmic fragments of megakaryocytes and play an essential role in thrombosis and hemostasis. Platelet activation depends on the rapid phosphorylation and dephosphorylation of key signaling molecules, and a number of kinases and phosphatases have been identified as major regulators of platelet function. However, the investigation of novel signaling proteins has suffered from technical limitations due to the anucleate nature of platelets and their very limited levels of mRNA and de novo protein synthesis. In the past, experimental methods were restricted to the generation of genetically modified mice and the development of specific antibodies. More recently, novel (phospho)proteomic technologies and pharmacological approaches using specific small-molecule inhibitors have added additional capabilities to investigate specific platelet proteins.In this chapter, we report methods for using genetic and pharmacological approaches to investigate the function of platelet signaling proteins. While the described experiments focus on the role of the dual-specificity phosphatase 3 (DUSP3) in platelet signaling, the presented methods are applicable to any signaling enzyme. Specifically, we describe a testing strategy that includes (1) aggregation and secretion experiments with mouse and human platelets, (2) immunoprecipitation and immunoblot assays to study platelet signaling events, (3) detailed protocols to use selected animal models in order to investigate thrombosis and hemostasis in vivo, and (4) strategies for utilizing pharmacological inhibitors on human platelets. PMID:27514813

  1. Protein kinase and phosphatase activities of thylakoid membranes

    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 Mg2+ ions. Dephosphorylation of the 8.3 kDa psb H gene product requires a Mg2+ 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

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

    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......-crystallize TC-PTP with the same set of inhibitors. This seems to be due to a multimerization process where residues 130-132, the DDQ loop, from one molecule is inserted into the active site of the neighboring molecule, resulting in a continuous string of interacting TC-PTP molecules. Importantly, despite the...

  3. Association of Protein Phosphatase 1γ1 with Spinophilin Suppresses Phosphatase Activity in a Parkinson Disease Model*

    Brown, Abigail M.; Baucum, Anthony J.; Bass, Martha A.; Roger J Colbran

    2008-01-01

    Sustained nigrostriatal dopamine depletion increases the serine/threonine phosphorylation of multiple striatal proteins that play a role in corticostriatal synaptic plasticity, including Thr286 phosphorylation of calcium/calmodulin-dependent protein kinase IIα (CaMKIIα). Mechanisms underlying these changes are unclear, but protein phosphatases play a critical role in the acute modulation of striatal protein phosphorylation. Here we show that dopamine depletion for periods ranging from 3 weeks...

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

    Reversible methylation of the protein phosphatase 2A catalytic subunit (PP2AC) 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-PP2AC antibodies revealed a good correlation with the methylation status of PP2AC, demethylated PP2AC being substantially nuclear. Throughout mitosis, demethylated PP2AC 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 PP2AC 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

  5. Significance of the photosystem II core phosphatase PBCP for plant viability and protein repair in thylakoid membranes.

    Puthiyaveetil, Sujith; Woodiwiss, Timothy; Knoerdel, Ryan; Zia, Ahmad; Wood, Magnus; Hoehner, Ricarda; Kirchhoff, Helmut

    2014-07-01

    PSII undergoes photodamage, which results in photoinhibition-the light-induced loss of photosynthetic activity. The main target of damage in PSII is the reaction center protein D1, which is buried in the massive 1.4 MDa PSII holocomplex. Plants have evolved a PSII repair cycle that degrades the damaged D1 subunit and replaces it with a newly synthesized copy. PSII core proteins, including D1, are phosphorylated in high light. This phosphorylation is important for the mobilization of photoinhibited PSII from stacked grana thylakoids to the repair machinery in distant unstacked stroma lamellae. It has been recognized that the degradation of the damaged D1 is more efficient after its dephosphorylation by a protein phosphatase. Recently a protein phosphatase 2C (PP2C)-type PSII core phosphatase (PBCP) has been discovered, which is involved in the dephosphorylation of PSII core proteins. Its role in PSII repair, however, is unknown. Using a range of spectroscopic and biochemical techniques, we report that the inactivation of the PBCP gene affects the growth characteristic of plants, with a decreased biomass and altered PSII functionality. PBCP mutants show increased phosphorylation of core subunits in dark and photoinhibitory conditions and a diminished degradation of the D1 subunit. Our results on D1 turnover in PBCP mutants suggest that dephosphorylation of PSII subunits is required for efficient D1 degradation. PMID:24793754

  6. Human neutrophil calmodulin-binding proteins: identification of the calmodulin-dependent protein phosphatase

    The molecular events in linking neutrophil activation and ligand binding to specific membrane receptors are mediated in part by an increase in intracellular Ca2+. One mechanism by which Ca2+ may trigger neutrophil activation is through Ca2+/calmodulin (CaM)-regulated proteins and enzymes. To determine which Ca2+/CaM-regulated enzymes may be present in the neutrophil, they have used Western blotting techniques and 125I-CaM to identify neutrophil CaM-binding proteins. Eleven proteins with molecular weights ranging from 230K to 13.5K bound 125I-CaM in a Ca2+-dependent manner. One predominant region of 125I-Cam binding was to a 59K protein; a protein with an identical mobility was labeled by an antisera against brain CaM-dependent phosphatase. Ca2+-dependent phosphatase activity, which was inhibited by the CaM antagonist trifluoperazine, was detected in a neutrophil extract; a radioimmunoassay for the phosphatase indicated that it was present in the extract at approximately 0.2 μg/mg protein. Most of the CaM-binding proteins, including the 59K protein, were rapidly degraded upon lysis of the neutrophil. There was a close correlation between the degradation of the 59K protein and the loss of Ca2+-dependent phosphatase activity in the neutrophil extract. Thus, human neutrophils contain numerous CaM-binding proteins which are presumably Ca2+/calmodulin-regulated enzymes and proteins; the 59K protein is a CaM-dependent phosphatase

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

    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 phosphotyrosin

  8. Redox and zinc signalling pathways converging on protein tyrosine phosphatases.

    Bellomo, Elisa; Hogstrand, Christer; Maret, Wolfgang

    2014-10-01

    Zinc ions, though redox-inert, have either pro-antioxidant or pro-oxidant functions at critical junctures in redox metabolism and redox signalling. They are released from cells and in cells, e.g. from metallothionein, a protein that transduces redox signals into zinc signals (1). The released zinc ions inhibit enzymes such as protein tyrosine phosphatases (PTPs), key regulatory enzymes of cellular phosphorylation signalling. The Ki(Zn) value for inhibition of receptor PTPB is 21pM (2). The binding is about as tight as the binding of zinc to zinc metalloenzymes and suggests tonic zinc inhibition. PTP1-B (PTPN1), an enzyme regulating the insulin and leptin receptors and involved in cancer and diabetes pathobiochemistry, has a Ki(Zn) value of about 5nM (3). Zinc ions bind to the enzyme in the closed conformation when additional metal-binding ligands are brought into the vicinity of the active site. In contrast, redox reactions target cysteines in the active sites of PTPs in the open conformation. This work provides a molecular basis how hydrogen peroxide and free zinc ions generated by growth factor signalling stimulate phosphorylation signalling differentially. (Supported by the Biotechnology and Biological Sciences Research Council UK, grant BB/K001442/1.). PMID:26461422

  9. Protein tyrosine phosphatases expression during development of mouse superior colliculus.

    Reinhard, Jacqueline; Horvat-Bröcker, Andrea; Illes, Sebastian; Zaremba, Angelika; Knyazev, Piotr; Ullrich, Axel; Faissner, Andreas

    2009-12-01

    Protein tyrosine phosphatases (PTPs) are key regulators of different processes during development of the central nervous system. However, expression patterns and potential roles of PTPs in the developing superior colliculus remain poorly investigated. In this study, a degenerate primer-based reverse transcription-polymerase chain reaction (RT-PCR) approach was used to isolate seven different intracellular PTPs and nine different receptor-type PTPs (RPTPs) from embryonic E15 mouse superior colliculus. Subsequently, the expression patterns of 11 PTPs (TC-PTP, PTP1C, PTP1D, PTP-MEG2, PTP-PEST, RPTPJ, RPTPε, RPTPRR, RPTPσ, RPTPκ and RPTPγ) were further analyzed in detail in superior colliculus from embryonic E13 to postnatal P20 stages by quantitative real-time RT-PCR, Western blotting and immunohistochemistry. Each of the 11 PTPs exhibits distinct spatiotemporal regulation of mRNAs and proteins in the developing superior colliculus suggesting their versatile roles in genesis of neuronal and glial cells and retinocollicular topographic mapping. At E13, additional double-immunohistochemical analysis revealed the expression of PTPs in collicular nestin-positive neural progenitor cells and RC-2-immunoreactive radial glia cells, indicating the potential functional importance of PTPs in neurogenesis and gliogenesis. PMID:19727691

  10. Protein Phosphatase-1 regulates Rift Valley fever virus replication.

    Baer, Alan; Shafagati, Nazly; Benedict, Ashwini; Ammosova, Tatiana; Ivanov, Andrey; Hakami, Ramin M; Terasaki, Kaori; Makino, Shinji; Nekhai, Sergei; Kehn-Hall, Kylene

    2016-03-01

    Rift Valley fever virus (RVFV), genus Phlebovirus family Bunyaviridae, is an arthropod-borne virus endemic throughout sub-Saharan Africa. Recent outbreaks have resulted in cyclic epidemics with an increasing geographic footprint, devastating both livestock and human populations. Despite being recognized as an emerging threat, relatively little is known about the virulence mechanisms and host interactions of RVFV. To date there are no FDA approved therapeutics or vaccines for RVF and there is an urgent need for their development. The Ser/Thr protein phosphatase 1 (PP1) has previously been shown to play a significant role in the replication of several viruses. Here we demonstrate for the first time that PP1 plays a prominent role in RVFV replication early on during the viral life cycle. Both siRNA knockdown of PP1α and a novel PP1-targeting small molecule compound 1E7-03, resulted in decreased viral titers across several cell lines. Deregulation of PP1 was found to inhibit viral RNA production, potentially through the disruption of viral RNA transcript/protein interactions, and indicates a potential link between PP1α and the viral L polymerase and nucleoprotein. These results indicate that PP1 activity is important for RVFV replication early on during the viral life cycle and may prove an attractive therapeutic target. PMID:26801627

  11. Protein phosphatase 2A in stretch-induced endothelial cell proliferation

    Murata, K.; Mills, I.; Sumpio, B. E.

    1996-01-01

    We previously proposed that activation of protein kinase C is a key mechanism for control of cell growth enhanced by cyclic strain [Rosales and Sumpio (1992): Surgery 112:459-466]. Here we examined protein phosphatase 1 and 2A activity in bovine aortic endothelial cells exposed to cyclic stain. Protein phosphatase 2A activity in the cytosol was decreased by 36.1% in response to cyclic strain for 60 min, whereas the activity in the membrane did not change. Treatment with low concentration (0.1 nM) of okadaic acid enhanced proliferation of both static and stretched endothelial cells in 10% fetal bovine serum. These data suggest that protein phosphatase 2A acts as a growth suppressor and cyclic strain may enhance cellular proliferation by inhibiting protein phosphatase 2A as well as stimulating protein kinase C.

  12. Protein tyrosine phosphatase 1B inhibitors isolated from Artemisia roxburghiana.

    Shah, Muhammad Raza; Ishtiaq; Hizbullah, Syed Muhammad; Habtemariam, Solomon; Zarrelli, Armando; Muhammad, Akhtar; Collina, Simona; Khan, Inamulllah

    2016-08-01

    Artemisia roxburghiana is used in traditional medicine for treating various diseases including diabetes. The present study was designed to evaluate the antidiabetic potential of active constituents by using protein tyrosine phosphatase 1B (PTP1B) as a validated target for management of diabetes. Various compounds were isolated as active principles from the crude methanolic extract of aerial parts of A. roxburghiana. All compounds were screened for PTP1B inhibitory activity. Molecular docking simulations were performed to investigate the mechanism behind PTP1B inhibition of the isolated compound and positive control, ursolic acid. Betulinic acid, betulin and taraxeryl acetate were the active PTP1B principles with IC50 values 3.49 ± 0.02, 4.17 ± 0.03 and 87.52 ± 0.03 µM, respectively. Molecular docking studies showed significant molecular interactions of the triterpene inhibitors with Gly220, Cys215, Gly218 and Asp48 inside the active site of PTP1B. The antidiabetic activity of A. roxburghiana could be attributed due to PTP1B inhibition by its triterpene constituents, betulin, betulinic acid and taraxeryl acetate. Computational insights of this study revealed that the C-3 and C-17 positions of the compounds needs extensive optimization for the development of new lead compounds. PMID:26118418

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

    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, PP1γ2, 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 PP1δ

  14. Inhibition of CDC25B Phosphatase Through Disruption of Protein-Protein Interaction

    Lund, George; Dudkin, Sergii; Borkin, Dmitry; Ni, Wendi; Grembecka, Jolanta; Cierpicki, Tomasz [Michigan

    2015-04-29

    CDC25 phosphatases are key cell cycle regulators and represent very attractive but challenging targets for anticancer drug discovery. Here, we explored whether fragment-based screening represents a valid approach to identify inhibitors of CDC25B. This resulted in identification of 2-fluoro-4-hydroxybenzonitrile, which directly binds to the catalytic domain of CDC25B. Interestingly, NMR data and the crystal structure demonstrate that this compound binds to the pocket distant from the active site and adjacent to the protein–protein interaction interface with CDK2/Cyclin A substrate. Furthermore, we developed a more potent analogue that disrupts CDC25B interaction with CDK2/Cyclin A and inhibits dephosphorylation of CDK2. Based on these studies, we provide a proof of concept that targeting CDC25 phosphatases by inhibiting their protein–protein interactions with CDK2/Cyclin A substrate represents a novel, viable opportunity to target this important class of enzymes.

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

    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.

  16. PAPP2C Interacts with the Atypical Disease Resistance Protein RPW8.2 and Negatively Regulates Salicylic Acid-Dependent Defense Responses in Arabidopsis

    Wen-Ming Wang; Xian-Feng Ma; Yi Zhang; Ming-Cheng Luo; Guo-Liang Wang; Maria Bellizzi; Xing-Yao Xiong; Shun-Yuan Xiao

    2012-01-01

    Many fungal and oomycete pathogens differentiate a feeding structure named the haustorium to extract nutrition from the plant epidermal cell.The atypical resistance (R) protein RPW8.2 activates salicylic acid (SA)-dependent,haustorium-targeted defenses against Golovinomyces spp.,the causal agents of powdery mildew diseases on multiple plant species.How RPW8.2 activates defense remains uncharacterized.Here,we report that RPW8.2 interacts with the phytochrome-associated protein phosphatase type 2C (PAPP2C) in yeast and in planta as evidenced by coimmunoprecipitation and bimolecular fluorescence complementation assays.Down-regulation of PAPP2C by RNA interference (RNAi) in Col-0 plants lacking RPW8.2 leads to leaf spontaneous cell death and enhanced disease resistance to powdery mildew via the SA-dependent signaling pathway.Moreover,down-regulation of PAPP2C by RNAi in the RPW8.2 background results in strong HR-like cell death,which correlates with elevated RPW8.2 expression.We further demonstrate that hemagglutinin (HA)-tagged PAPP2C prepared from tobacco leaf cells transiently transformed with HA-PAPP2C possesses phosphatase activity.In addition,silencing a rice gene (Os04g0452000) homologous to PAPP2C also results in spontaneous cell death in rice.Combined,our results suggest that RPW8.2 is functionally connected with PAPP2C and that PAPP2C negatively regulates SA-dependent basal defense against powdery mildew in Arabidopsis.

  17. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz; E. Wieczorek; B. Morawiecka

    2015-01-01

    Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.). The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance ...

  18. Deletion of conserved protein phosphatases reverses defects associated with mitochondrial DNA damage in Saccharomyces cerevisiae

    Garipler, Görkem; Mutlu, Nebibe; Lack, Nathan; Dunn, Cory David

    2014-01-01

    Mitochondrial biogenesis is regulated by signaling pathways sensitive to extracellular conditions and to the internal environment of the cell. Therefore, treatments for disease caused by mutation of mtDNA may emerge from studies of how signal transduction pathways command mitochondrial function. We have examined the role of phosphatases under the control of the conserved alpha 4/Tap42 protein in cells lacking a mitochondrial genome. We found that deletion of protein phosphatase 2A (PP2A) or o...

  19. STRIATAL-ENRICHED PROTEIN TYROSINE PHOSPHATASE (STEP) KNOCKOUT MICE HAVE ENHANCED HIPPOCAMPAL MEMORY

    Venkitaramani, Deepa V.; Moura, Paula J.; Picciotto, Marina R.; Lombroso, Paul J.

    2011-01-01

    STEP is a brain-specific phosphatase that opposes synaptic strengthening by the regulation of key synaptic signaling proteins. Previous studies suggest a possible role for STriatal-Enriched protein tyrosine Phosphatase (STEP) in learning and memory. To demonstrate the functional importance of STEP in learning and memory, we generated STEP knockout (KO) mice and examined the effect of deletion of STEP on behavioral performance, as well as the phosphorylation and expression of its substrates. H...

  20. Stimulation of protein phosphatase activity by insulin and growth factors in 3T3 cells

    Incubation of Swiss mouse 3T3-D1 cells with physiological concentrations of insulin resulted in a rapid and transient activation of protein phosphatase activity as measure by using [32P]phosphorylase α as substrate. Activation reached a maximum level (140% of control value) within 5 min of addition and returned to control levels within 20 min. The effect of insulin was dose-dependent with half-maximal activation occurring at ∼5 nM insulin. This activity could be completely inhibited by addition of the heat-stable protein inhibitor 2, which suggests the presence of an activated type-1 phosphatase. Similar effects on phosphatase activity were seen when epidermal growth factor and platelet-derived growth factor were tested. These results suggest that some of the intracellular effects caused by insulin and growth factors are mediated through the activation of a protein phosphatase

  1. The endogenous inhibitor of protein kinase-C in the rat ovary is a protein phosphatase.

    Eyster, K M; Waller, M S; Miller, T L; Miller, C J; Johnson, M J; Persing, J S

    1993-09-01

    Calcium- and lipid-dependent protein kinase (PKC) activity in the ovary of the pseudopregnant rat is masked by an endogenous inhibitor of PKC. These studies were undertaken to examine the mechanism of action of the endogenous inhibitor of PKC in the rat ovary. The addition of the phosphatase inhibitors calyculin-A (0.09 nM), microcystin-LR (6.4 nM), and okadaic acid (10 nM) resulted in the loss of PKC inhibitory activity and an increase in basal PKC activity in rat ovarian cytosol. In phosphatase assays, significant dephosphorylation of histone-III-S or myelin basic protein that had been phosphorylated by PKC occurred within 4 min after the addition of ovarian cytosol from the pseudopregnant rat. This dephosphorylation was prevented from the pseudopregnant rat. This dephosphorylation was prevented by the addition of calyculin-A (0.73 nM) and was removed by fractionation of ovarian cytosol on diethylaminoethyl cellulose. No inhibition of PKC activity was observed when the PKC-specific peptides AcMBP-(4-14) and [Ser25]PKC-(19-31) were used as the substrate for phosphorylation. In addition, rat ovarian cytosol did not exhibit phosphatase activity when the peptide AcMBP-(4-14) was used as the substrate. Addition of ovarian cytosol resulted in dephosphorylation of phosphorylase-alpha phosphorylated by phosphorylase kinase, but not dephosphorylation of histone-II-A or histone-VIII-S phosphorylated by PKA. The data suggest that the endogenous inhibitor of PKC in the rat ovary is a protein phosphatase. PMID:7689949

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

    MidoriShimada

    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.

  3. Protein Phosphatase 2Cβl Regulates Human Pregnane X Receptor-Mediated CYP3A4 Gene Expression in HepG2 Liver Carcinoma Cells

    Pondugula, Satyanarayana R.; Tong, Alexander A.; Wu, Jing; Cui, Jimmy; Chen, Taosheng

    2010-01-01

    The human pregnane X receptor (hPXR) regulates the expression of CYP3A4, which plays a vital role in hepatic drug metabolism and has considerably reduced expression levels in proliferating hepatocytes. We have recently shown that cyclin-dependent kinase 2 (CDK2) negatively regulates hPXR-mediated CYP3A4 gene expression. CDK2 can be dephosphorylated and inactivated by protein phosphatase type 2C beta isoform long (PP2Cβl), a unique phosphatase that was originally cloned from human liver. In th...

  4. One-Pot N2C/C2C/N2N Ligation To Trap Weak Protein-Protein Interactions.

    Zhao, Lei; Ehrt, Christiane; Koch, Oliver; Wu, Yao-Wen

    2016-07-01

    Weak transient protein-protein interactions (PPIs) play an essential role in cellular dynamics. However, it is challenging to obtain weak protein complexes owing to their short lifetime. Herein we present a general and facile method for trapping weak PPIs in an unbiased manner using proximity-induced ligations. To expand the chemical ligation spectrum, we developed novel N2N (N-terminus to N-terminus) and C2C (C-terminus to C-terminus) ligation approaches. By using N2C (N-terminus to C-terminus), N2N, and C2C ligations in one pot, the interacting proteins were linked. The weak Ypt1:GDI interaction drove C2C ligation with t1/2 of 4.8 min and near quantitative conversion. The Ypt1-GDI conjugate revealed that binding of Ypt1 G-domain causes opening of the lipid-binding site of GDI, which can accommodate one prenyl group, giving insights into Rab membrane recycling. Moreover, we used this strategy to trap the KRas homodimer, which plays an important role in Ras signaling. PMID:27213482

  5. HSP105 Recruits Protein Phosphatase 2A To Dephosphorylate β-Catenin

    Yu, Nancy; Kakunda, Michael; Pham, Victoria; Lill, Jennie R.; Du, Pan; Wongchenko, Matthew; Yan, Yibing; Firestein, Ron; Huang, Xiaodong

    2015-01-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are component...

  6. High-Throughput Screening of Substrate Specificity for Protein Tyrosine Phosphatases (PTPs) on Phosphopeptide Microarrays.

    Gao, Liqian; Lee, Su Seong; Chen, Jun; Sun, Hongyan; Zhao, Yuliang; Chai, Zhifang; Hu, Yi

    2016-01-01

    Phosphatases are a family of enzymes responsible for the dephosphorylation of biomolecules. Phosphatases play essential roles in cell cycle regulation, signal transduction, and cellular communication. In recent years, one type of phosphatases, protein tyrosine phosphatases (PTPs), emerges as important therapeutic targets for complex and devastating diseases. Nevertheless, the physiological roles, substrate specificity, and downstream targets for PTPs remain largely unknown. To demonstrate how microarrays can be applied to characterizing PTPs, we describe here a phosphopeptide microarray strategy for activity-based high-throughput screening of PTPs substrate specificity. This is followed by a kinetic microarray assay and microplate assay to determine the rate constants of dephosphorylation by PTPs. This microarray strategy has been successfully applied to identifying several potent and selective substrates against different PTPs. These substrates could be used to design potent and selective PTPs inhibitors in the future. PMID:26614076

  7. Latent phosphorylase phosphatases from rat liver: relationship with the heat-stable inhibitory protein.

    Jett, M F; Hers, H G

    1981-08-01

    A high-speed supernatant from rat liver contains at least two latent phosphorylase phosphatases the activities of which are revealed by treatment with ethanol, urea, mercaptoethanol or trypsin. This fraction also contains at least one protein which, after heating, inhibits to various degrees the activated form(s) of the two phosphatases. The two latent enzymes can be separated by cellulase-phosphate chromatography and can be differentiated by their preferential activation by ethanol or trypsin and by their different sensitivity to the inhibitory protein after ethanol activation. Activation of the latent phosphorylase phosphatases by ethanol, urea or mercaptoethanol is not accompanied by the destruction of the precursor of the inhibitory protein whereas activation by trypsin is. However, trypsin treatment of fractions previously activated by ethanol decreases their activity and also increases their sensitivity to the inhibitory protein in a way which is unrelated to the destruction of this inhibitor. Furthermore, some protein fractions, almost free of the precursor of the inhibitory protein can be readily activated by trypsin. In is concluded that the activation of the latent phosphorylase phosphorylase phosphatases is unrelated to the destruction of the inhibitory protein. PMID:6269850

  8. A novel role for protein tyrosine phosphatase 1B as a positive regulator of neuroinflammation

    Song, Gyun Jee; Jung, Myungsu; Kim, Jong-Heon; Park, Hana; Rahman, Md. Habibur; Zhang, Sheng; Zhang, Zhong-Yin; Park, Dong Ho; Kook, Hyun; Lee, In-Kyu; Suk, Kyoungho

    2016-01-01

    Background Protein tyrosine phosphatase 1B (PTP1B) is a member of the non-transmembrane phosphotyrosine phosphatase family. Recently, PTP1B has been proposed to be a novel target of anti-cancer and anti-diabetic drugs. However, the role of PTP1B in the central nervous system is not clearly understood. Therefore, in this study, we sought to define PTP1B’s role in brain inflammation. Methods PTP1B messenger RNA (mRNA) and protein expression levels were examined in mouse brain and microglial cel...

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

    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.

  10. Molecular Cloning and Functional Expression of a Protein-Serine/Threonine Phosphatase from the Hyperthermophilic Archaeon Pyrodictium abyssi TAG11

    Mai, Bianca; Frey, Gerhard; Swanson, Ronald V.; Mathur, Eric J.; Stetter, K. O.

    1998-01-01

    An open reading frame coding for a putative protein-serine/threonine phosphatase was identified in the hyperthermophilic archaeon Pyrodictium abyssi TAG11 and named Py-PP1. Py-PP1 was expressed in Escherichia coli, purified from inclusion bodies, and biochemically characterized. The phosphatase gene is part of an operon which may provide, for the first time, insight into a physiological role for archaeal protein phosphatases in vivo. PMID:9696747

  11. Kinetics and Mechanism of Protein Tyrosine Phosphatase 1B (PTP1B) Inactivation by Acrolein

    Seiner, Derrick R.; LaButti, Jason N.; Gates, Kent S.

    2007-01-01

    Human cells are exposed to the electrophilic α,β-unsaturated aldehyde acrolein from a variety of sources. Reaction of acrolein with functionally critical protein thiol residues can yield important biological consequences. Protein tyrosine phosphatases (PTPs) are an important class of cysteine-dependent enzymes whose reactivity with acrolein previously has not been well characterized. These enzymes catalyze the dephosphorylation of phosphotyrosine residues on proteins via a phosphocysteine int...

  12. Free Fatty Acids Inhibit Protein Tyrosine Phosphatase 1B and Activate Akt

    Eisuke Shibata

    2013-09-01

    Full Text Available Background/Aims: Accumulating evidence has suggested that free fatty acids (FFAs interact with protein kinases and protein phosphatases. The present study examined the effect of FFAs on protein phosphatases and Akt. Methods: Activities of protein phosphatase 1 (PP1, protein phosphatase 2A (PP2A, and protein tyrosine phosphatase 1B (PTP1B were assayed under the cell-free conditions. Phosphorylation of Akt was monitored in MSTO-211H human malignant pleural mesothelioma cells without and with knocking-down phosphatidylinositol 3 kinase (PI3K or 3-phosphoinositide-dependent protein kinase-1 (PDK1. Results: In the cell-free assay, unsaturated FFAs (uFFAs such as oleic, linoleic and linolenic acid and saturated FFAs (sFFAs such as stearic, palmitic, myristic, and behenic acid markedly reduced PTP1B activity, with the potential for uFFAs greater than that for sFFAs. All the investigated sFFAs inhibited PP2A activity, but otherwise no inhibition was obtained with uFFAs. Both uFFAs and sFFAs had no effect on PP1 activity. Oleic acid phosphorylated Akt both on Thr308 and Ser473, while stearic acid phosphorylated Akt on Thr308 alone. The effects of oleic and stearic acid on Akt phosphorylation were abrogated by the PI3K inhibitor wortmannin or the PDK1 inhibitor BX912 and also by knocking-down PI3K or PDK1. Conclusion: The results of the present study indicate that uFFAs and sFFAs could activate Akt through a pathway along a PI3K/PDK1/Akt axis in association with PTP1B inhibition.

  13. MALDI mass sequencing and biochemical characterization of Setaria cervi protein tyrosine phosphatase.

    Rai, Reeta; Singh, Neetu; Elesela, Srikanth; Tiwari, Savitri; Rathaur, Sushma

    2013-01-01

    A 30-kDa acid phosphatase with protein tyrosine phosphatase activity was identified in Setaria cervi (ScPTP). The enzyme was purified to homogeneity using three-step column chromatography. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis of purified ScPTP yielded a total of eight peptides matching most closely to phosphoprotein phosphatase of Ricinus communis (RcPP). A hydrophilicity plot of RcPP revealed the presence of these peptides in the hydrophilic region, suggesting their antigenic nature. The substrate specificity of ScPTP with ortho-phospho-L-tyrosine and inhibition with sodium orthovanadate and ammonium molybdate affirmed it as a protein tyrosine phosphatase. ScPTP was also found to be tartrate resistant. The Km and Vmax were 6.60 mM and 83.3 μM/ml/min, respectively, with pNPP and 8.0 mM and 111 μM/ml/min, respectively, with ortho-phospho-L-tyrosine as the substrate. The Ki value with sodium orthovanadate was calculated to be 16.10 mM. Active site modification with DEPC, EDAC and pHMB suggested the presence of histidine, cysteine and aspartate at its active site. Thus, on the basis of MALDI-TOF and biochemical studies, it was confirmed that purified acid phosphatase is a PTP. PMID:23052758

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

    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...

  15. Identification, purification, and characterization of phosphotyrosine-specific protein phosphatases from cultured chicken embryo fibroblasts

    Tyrosine phosphorylation catalyzed by a unique class of protein kinases is an important process in both normal cell proliferation and oncogenic transformation. In this study, phosphoprotein phosphatases specific for the dephosphorylation of phosphotyrosine residues were partially purified from secondary chicken embryo fibroblasts, using 32P-labeled immunoglobulin G. The soluble activity was purified by using DEAE-cellulose and carboxymethyl cellulose column chromatography and gel filtration, and at least three enzyme species of apparent Mr 55,000 (pTPI), 50,000 (pTPII), and 95,000 (pTPIII) were resolved. All three enzymes possessed somewhat similar properties. They had a pH optimum of about 7.4, they were inhibited by Zn2+, vanadate, ATP, and ADP, and they were unaffected by divalent metal cations, EDTA, and F- under standard assay conditions employing a physiological ionic strength. These properties suggest that they represent a class of enzymes distinct from well-known phosphoseryl-phosphothreonyl-protein phosphatases and that dephosphorylation of phosphotyrosine-containing proteins may be carried out by a unique family of phosphoprotein phosphatases. Transformation by Rous sarcoma virus resulted in a small increase in phosphotyrosyl-protein phosphatase activity

  16. Dexamethasone Causes Sustained Expression of Mitogen-Activated Protein Kinase (MAPK) Phosphatase 1 and Phosphatase-Mediated Inhibition of MAPK p38

    Lasa, Marina; Abraham, Sonya M.; Boucheron, Christine; Saklatvala, Jeremy; Clark, Andrew R.

    2002-01-01

    The stress-activated protein kinase p38 stabilizes a number of mRNAs encoding inflammatory mediators, such as cyclooxygenase 2 (Cox-2). In HeLa cells the anti-inflammatory glucocorticoid dexamethasone destabilizes Cox-2 mRNA by inhibiting p38 function. Here we demonstrate that this effect is phosphatase dependent. Furthermore, in HeLa cells dexamethasone induced the sustained expression of mitogen-activated protein kinase phosphatase 1 (MKP-1), a potent inhibitor of p38 function. The inhibiti...

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

    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 32P-ACC phosphorylated by the casein kinases was identified

  18. Protein-tyrosine phosphatase activity regulates osteoclast formation and function: inhibition by alendronate.

    Schmidt, A.; Rutledge, S J; Endo, N; Opas, E E; Tanaka, H; Wesolowski, G.; Leu, C T; Huang, Z; Ramachandaran, C; Rodan, S B; Rodan, G A

    1996-01-01

    Alendronate (ALN), an aminobisphosphonate used in the treatment of osteoporosis, is a potent inhibitor of bone resorption. Its molecular target is still unknown. This study examines the effects of ALN on the activity of osteoclast protein-tyrosine phosphatase (PTP; protein-tyrosine-phosphate phosphohydrolase, EC 3.1.3.48), called PTPepsilon. Using osteoclast-like cells generated by coculturing mouse bone marrow cells with mouse calvaria osteoblasts, we found by molecular cloning and RNA blot ...

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

    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. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase-5

    Swingle, M. R.; Honkanen, R.; Ciszak, E. M.

    2004-01-01

    Serinehhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth and cellular responses to stress. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 A. From this structure we resolved the mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a con served Aspn-271-M(sub 1):M(sub 2)-W(sup 1)-His-427-His-304-Asp-274 catalytic motif. The structure of PPSc provides a structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  1. Structural Basis for the Catalytic Activity of Human SER/THR Protein Phosphatase-5

    Swingle, M. R.; Honkanen, R.; Ciszak, E.

    2004-01-01

    Serinekhreonine protein phosphatase-5 (PP5) affects many signaling networks that regulate cell growth. Here we report the 1.6 Angstrom resolution crystal structure of PP5 catalytic domain with metal and phosphate ions in the active site. The structure reveals a mechanism for PPS-mediated catalysis that requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1),-M(sub 2)-His(sup 427)-W(sup 2)-His(sup 304)-Asp(sup 274) catalytic motif, and provides a structural basis for the exceptional catalytic proficiency of protein phosphatases placing them among the most powerful catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of PP5 should aid development of specific inhibitors.

  2. Acrylamide gel electrophoresis of proteins, acid phosphatases and RN-ases from three potato varieties

    A. Kubicz

    2015-05-01

    Full Text Available Studies on variety differences in the protein and acid phosphatase patterns as well as ribunuclease activity distribution were carried out by disc electrophoresis on saline extracts of three varieties of the potato Solanum tuberosum (L.. The protein bands varied in number, position and relative abundance. One main zone of the acid phosphatase activity was detected consisting of 2-3 electrophoretically different bands. Variety differences were concerned with the number and relative abundance of these bands. RNase activity was detected in 4 main zones, in some of them additional subbands were visible. Differences between the three examined varieties were reflected in the occurence of the particular activity zones or their subbands.

  3. Antioxidant effects of whey protein on muscle C2C12 cells.

    Kerasioti, Efthalia; Stagos, Dimitrios; Priftis, Alexandros; Aivazidis, Stefanos; Tsatsakis, Aristidis M; Hayes, A Wallace; Kouretas, Demetrios

    2014-07-15

    In the present study, the in vitro scavenging activity of sheep whey protein against free radicals, as well as its reducing power were determined and compared with that of beef protein, soy protein and cow whey protein. Moreover, the possible protective effects of sheep whey protein from tert-butyl hydroperoxide (tBHP)-induced oxidative stress in muscle C2C12 cells were determined by assessing oxidative stress markers by flow cytometry and spectrophotometry. The results showed that sheep whey protein scavenged DPPH, ABTS(+) and OH radicals with IC50 values of 3.1, 4.1 and 1.8 mg of protein/ml. Moreover, the reducing power activity assessed with potassium ferricyanide of sheep whey protein was 1.3mg/ml. As regards to the antioxidant effects in muscle cell line, sheep whey protein at 0.78, 1.56, 3.12 and 6.24 mg of protein/ml increased GSH levels up to 138%, lowered TBARS levels up to 25% and decreased ROS levels up to 41.4%. PMID:24594185

  4. The SAP, a new family of proteins, associate and function positively with the SIT4 phosphatase.

    Luke, M M; Della Seta, F; Di Como, C J; Sugimoto, H; Kobayashi, R.; Arndt, K T

    1996-01-01

    SIT4 is the catalytic subunit of a type 2A-related protein phosphatase in Saccharomyces cerevisiae that is required for G1 cyclin transcription and for bud formation. SIT4 associates with several high-molecular-mass proteins in a cell cycle-dependent fashion. We purified two SIT4-associated proteins, SAP155 and SAP190, and cloned the corresponding genes. By sequence homology, we isolated two additional SAP genes, SAP185 and SAP4. Through such an association is not yet proven for SAP4, each of...

  5. The role for protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2015-01-01

    Current hypothesis suggests that genetic, immunological and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease (IBD). Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of IBD. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signalling processes.Evidence emerges that expression levels of PTPN...

  6. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2016-01-01

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and...

  7. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation

    Spalinger, Marianne R.; MCCOLE, DECLAN F.; Rogler, Gerhard; Scharl, Michael

    2015-01-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that express...

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

    Anoushka Davé; Carol Cooley; Mansi Garg; Alessandro Bianchi

    2014-01-01

    Summary 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 re...

  9. Immunohistochemical Analysis of IA-2 Family of Protein Tyrosine Phosphatases in Rat Gastrointestinal Endocrine Cells

    Gomi, Hiroshi; Kubota-Murata, Chisato; Yasui, Tadashi; Tsukise, Azuma; Torii, Seiji

    2013-01-01

    Islet-associated protein–2 (IA-2) and IA-2β (also known as phogrin) are unique neuroendocrine-specific protein tyrosine phosphatases (PTPs). The IA-2 family of PTPs was originally identified from insulinoma cells and discovered to be major autoantigens in type 1 diabetes. Despite its expression in the neural and canonical endocrine tissues, data on expression of the IA-2 family of PTPs in gastrointestinal endocrine cells (GECs) are limited. Therefore, we immunohistochemically investigated the...

  10. Relevance of amyloid precursor-like protein 2 C-terminal fragments in pancreatic cancer cells

    PETERS, HALEY L.; Tuli, Amit; Wang, Xiaojian; Liu, Cuiling; Pan, Zenggang; Ouellette, Michel M.; Hollingsworth, Michael A.; MacDonald, Richard G.; Solheim, Joyce C.

    2012-01-01

    In some cellular systems, particularly neurons, amyloid precursor-like protein 2 (APLP2), and its highly homologous family member amyloid precursor protein (APP), have been linked to cellular growth. APLP2 and APP undergo regulated intramembrane proteolysis to produce C-terminal fragments. In this study, we found comprehensive expression of APLP2 C-terminal fragments in a panel of pancreatic cancer cell lines; however, APP C-terminal fragments were notably limited to the BxPC3 cell line. Exte...

  11. Glc7/Protein Phosphatase 1 Regulatory Subunits Can Oppose the Ipl1/Aurora Protein Kinase by Redistributing Glc7

    Pinsky, Benjamin A.; Kotwaliwale, Chitra V.; Tatsutani, Sean Y.; Breed, Christopher A.; Biggins, Sue

    2006-01-01

    Faithful chromosome segregation depends on the opposing activities of the budding yeast Glc7/PP1 protein phosphatase and Ipl1/Aurora protein kinase. We explored the relationship between Glc7 and Ipl1 and found that the phosphorylation of the Ipl1 substrate, Dam1, was altered by decreased Glc7 activity, whereas Ipl1 levels, localization, and kinase activity were not. These data strongly suggest that Glc7 ensures accurate chromosome segregation by dephosphorylating Ipl1 targets rather than regu...

  12. Activation of SPS from darkened spinach leaves by an endogenous protein phosphatase

    Sucrose-phosphate synthase from darkened spinach leaves has a low activation state but can undergo a time-dependent activation in desalted leaf extracts that is inhibited by Pi, molybdate, okadaic acid and vanadate, but stimulated by fluoride. SPS labeled in vivo with [32P]Pi in excised leaves in the dark loses incorporated 32P with time when extracts are incubated at 25 degree C. This loss is largely prevented by vanadate, suggesting that an endogenous protein phosphatase can use SPS as substrate. Changes in phosphorylation state are closely paralleled by changes in SPS activation state. The spontaneous activation achieved in the extracts can be reversed by addition of 2 mM MgATP. Feeding okadaic acid to darkened leaves prevents light activation of SPS suggesting that the endogenous protein phosphatase is similar to the type-1 enzyme of animal tissues. Overall, the results are consistent with the notion that light activation of SPS involves dephosphorylation of inhibitory phosphorylation site(s). Regulation of the protein phosphatase by Pi may be of physiological significance

  13. A novel role for Niemann-Pick disease type 2C protein in papillae formation.

    Masahiro Sugawara

    Full Text Available BACKGROUND: Despite the presence of papillary structures and papillary tumors in humans, the mechanism of papillae formation is unknown. We describe herein a novel role for Niemann-Pick disease type 2C (NPC2 protein, a cholesterol binding protein in the lysosome, in papillae formation. METHODOLOGY/PRINCIPAL FINDING: We examined NPC2 protein expression in surgical samples of papillary tissues by immunohistochemical stain, and all papillary tissues expressed NPC2 protein in the epithelium. To examine our hypothesis of NPC2 protein-mediated papillae formation, we carried out xenograft experiments using wild H460 cells (large cell lung carcinoma cell line that constitutively expressed abundant NPC2 protein and NPC2 protein-depleted H460 cells by NPC2 shRNA. The xenografts of wild H460 cells and empty shRNA vector cells showed distinct papillae formation, whereas NPC2 protein-depleted H460 cells displayed markedly reduced or no papillae. Since all papillary tissues have open spaces we examined whether NPC2 protein might also contribute to the creation of open spaces. The TUNEL assay in the xenografts of wild and empty shRNA vector H460 cells showed massive cell death, and NPC2 protein-depleted cells displayed minimal cell death. Measurement of caspase 3/7 activities in cultured H460 cells supported NPC2 protein-mediated apoptotic cell death. The presence of excess NPC2 protein, however, did not always produce papillae as seen in the xenografts of CHO cells that were stably transfected with NPC2. CONCLUSIONS/SIGNIFICANCE: The NPC2 protein of certain cells forms papillae coupled with apoptosis that creates open space. This protein may have future applications to modulate papillae formation and papillary growth in tumor tissues.

  14. INHIBITION OF PROTEIN TYROSINE PHOSPHATASE ACTIVITY MEDIATES EPIDERMAL GROWTH FACTOR RECEPTOR SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

    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...

  15. Pyruvate dehydrogenase/sub b/ phosphatase inhibition by NADH and dihydrolipoamide along with effects of and capacity for binding the phosphatase to the bovine kidney transacetylase-protein X subcomplex

    NADH inhibits PDH/sub b/ phosphatase activity when 32P-PDH is associated with the intact complex but not when 32P-PDH is prepared free of other components of the complex. Addition of the transacetylase-protein X (E2-X) subcomplex both activated the phosphatase and restored NADH inhibition. Low levels of dihydrolipoyl dehydrogenase associated with the subcomplex might be required for NADH inhibition. Dihydrolipoamide gave inhibition of the phosphatase equivalent to NADH and the combination did not give additional inhibition suggesting a common mechanism. Pretreatment of phosphorylated complex and phosphatase with 2.0 mM dithiothreitol nearly eliminated inhibition of the phosphatase by NADH or dihydrolipoamide. Strong arsenite inhibition of phosphatase activity occurred only in the presence of NADH suggesting modification of thiols reduced by NADH can alter phosphatase activity. Only about 6 molecules of purified phosphatase could be activated by 1 molecule of E2-X subcomplex (initial velocities measured in 15s period). Since that corresponded to the number of protein X rather than E2 subunits, protein X may contribute to the Ca2+-dependent binding of the phosphatase. Since protein X also contains a lipoyl moiety, it may also contribute to NADH inhibition of the phosphatase

  16. Dephosphorylation specificities of protein phosphatase for cardiac troponin I, troponin T, and sites within troponin T

    2006-03-01

    Full Text Available Protein dephosphorylation by protein phosphatase 1 (PP1, acting in concert with protein kinase C (PKC and protein kinase A (PKA, is a pivotal regulatory mechanism of protein phosphorylation. Isolated rat cardiac myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 were used in determining dephosphorylation specificities, Ca2+-stimulated Mg2+ATPase activities, and Ca2+ sensitivities. In reconstituted troponin (Tn complex, PP1 displayed distinct substrate specificity in dephosphorylation of TnT preferentially to TnI, in vitro. In situ phosphorylation of cardiomyocytes with calyculin A, a protein phosphatase inhibitor, resulted in an increase in the phosphorylation stiochiometry of TnT (0.3 to 0.5 (67%, TnI (2.6 to 3.6 (38%, and MLC2 (0.4 to 1.7 (325%. These results further confirmed that though MLC2 is the preferred target substrate for protein phosphatase in the thick filament, the Tn complex (TnI and TnT from thin filament and C-protein in the thick filament are also protein phosphatase substrates. Our in vitro dephosphorylation experiments revealed that while PP1 differentially dephosphorylated within TnT at multiple sites, TnI was uniformly dephosphorylated. Phosphopeptide maps from the in vitro experiments show that TnT phosphopeptides at spots 4A and 4B are much more resistant to PP1 dephosphorylation than other TnT phosphopeptides. Mg2+ATPase assays of myofibrils phosphorylated by PKC/PKA and dephosphorylated by PP1 delineated that while PKC and PKA phosphorylation decreased the Ca2+-stimulated Mg2+ATPase activities, dephosphorylation antagonistically restored it. PKC and PKA phosphorylation decreased Ca2+ sensitivity to 3.6 µM and 5.0 µM respectively. However, dephosphorylation restored the Mg2+ATPase activity of PKC (99% and PKA (95%, along with the Ca2+ sensitivities (3.3 µM and 3.0 µM, respectively.

  17. Identification of a human src homology 2-containing protein-tyrosine-phosphatase: a putative homolog of Drosophila corkscrew.

    Freeman, R. M.; Plutzky, J; Neel, B G

    1992-01-01

    src homology 2 (SH2) domains direct binding to specific phosphotyrosyl proteins. Recently, SH2-containing protein-tyrosine-phosphatases (PTPs) were identified. Using degenerate oligonucleotides and the PCR, we have cloned a cDNA for an additional PTP, SH-PTP2, which contains two SH2 domains and is expressed ubiquitously. When expressed in Escherichia coli, SH-PTP2 displays tyrosine-specific phosphatase activity. Strong sequence similarity between SH-PTP2 and the Drosophila gene corkscrew (csw...

  18. Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

    Verrastro, Ivan; Tveen-Jensen, Karina; Woscholski, Rudiger; Spickett, Corinne M; Pitt, Andrew R

    2016-01-01

    Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured. PMID:26561776

  19. Syndecan-2 is a novel ligand for the protein tyrosine phosphatase receptor CD148

    Whiteford, James R; Xian, Xiaojie; Chaussade, Claire; Vanhaesebroeck, Bart; Nourshargh, Sussan; Couchman, John R

    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...... phosphatase receptor CD148 is shown to be a key intermediary in cell adhesion to S2ED, with downstream ß1 integrin-mediated adhesion and cytoskeletal organization. We show that S2ED is a novel ligand for CD148 and identify the region proximal to the transmembrane domain of syndecan-2 as the site of...

  20. Epidermal growth factor stimulates substrate-selective protein-tyrosine-phosphatase activity.

    Hernández-Sotomayor, S M; Arteaga, C L; Soler, C. (Carlos); Carpenter, G

    1993-01-01

    This study investigates the regulation of protein-tyrosine-phosphatase (PTPase; EC 3.1.3.48) activity by epidermal growth factor (EGF). Cytosol from EGF-treated A-431 human epidermoid carcinoma cells was used as a source of PTPase activity, and tyrosine-phosphorylated ErbB2, EGF receptor, phospholipase C-gamma 1, and the Ras GTPase-activating protein were used as substrates to monitor PTPase activity. EGF stimulated PTPase activity that was selective toward these substrates, as it dephosphory...

  1. Expression of a Gibberellin-Induced Leucine-Rich Repeat Receptor-Like Protein Kinase in Deepwater Rice and Its Interaction with Kinase-Associated Protein Phosphatase1

    van der Knaap, Esther; Song, Wen-Yuan; Ruan, De-Ling; Sauter, Margret; Ronald, Pamela C.; Kende, Hans

    1999-01-01

    We identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active, autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively. PMID:10364408

  2. Expression of a gibberellin-induced leucine-rich repeat receptor-like protein kinase in deepwater rice and its interaction with kinase-associated protein phosphatase

    Knaap, E. van der; Sauter, M.; Kende, H. (Michigan State Univ., East Lansing, MI (United States). DOE Plant Research Lab.); Song, W.Y.; Ruan, D.L.; Ronald, P.C. (Univ. of California, Davis, CA (United States). Dept. of Plant Pathology)

    1999-06-01

    The authors identified in deepwater rice (Oryza sativa L.) a gene encoding a leucine-rich repeat receptor-like transmembrane protein kinase, OsTMK (O. sativa transmembrane kinase). The transcript levels of OsTMK increased in the rice internode in response to gibberellin. Expression of OsTMK was especially high in regions undergoing cell division and elongation. The kinase domain of OsTMK was enzymatically active autophosphorylating on serine and threonine residues. A cDNA encoding a rice ortholog of a kinase-associated type 2C protein phosphatase (OsKAPP) was cloned. KAPPs are putative downstream components in kinase-mediated signal transduction pathways. The kinase interaction domain of OsKAPP was phosphorylated in vitro by the kinase domain of OsTMK. RNA gel-blot analysis indicated that the expression of OsTMK and OsKAPP was similar in different tissues of the rice plant. In protein-binding assays, OsKAPP interacted with a receptor-like protein kinase, RLK5 of Arabidopsis, but not with the protein kinase domains of the rice and maize receptor-like protein kinases Xa21 and ZmPK1, respectively.

  3. Carboxyl-terminal receptor domains control the differential dephosphorylation of somatostatin receptors by protein phosphatase 1 isoforms.

    Andreas Lehmann

    Full Text Available We have recently identified protein phosphatase 1β (PP1β as G protein-coupled receptor (GPCR phosphatase for the sst2 somatostatin receptor using siRNA knockdown screening. By contrast, for the sst5 somatostatin receptor we identified protein phosphatase 1γ (PP1γ as GPCR phosphatase using the same approach. We have also shown that sst2 and sst5 receptors differ substantially in the temporal dynamics of their dephosphorylation and trafficking patterns. Whereas dephosphorylation and recycling of the sst2 receptor requires extended time periods of ∼30 min, dephosphorylation and recycling of the sst5 receptor is completed in less than 10 min. Here, we examined which receptor domains determine the selection of phosphatases for receptor dephosphorylation. We found that generation of tail-swap mutants between sst2 and sst5 was required and sufficient to reverse the patterns of dephosphorylation and trafficking of these two receptors. In fact, siRNA knockdown confirmed that the sst5 receptor carrying the sst2 tail is predominantly dephosphorylated by PP1β, whereas the sst2 receptor carrying the sst5 tail is predominantly dephosphorylated by PP1γ. Thus, the GPCR phosphatase responsible for dephosphorylation of individual somatostatin receptor subtypes is primarily determined by their different carboxyl-terminal receptor domains. This phosphatase specificity has in turn profound consequences for the dephosphorylation dynamics and trafficking patterns of GPCRs.

  4. Physical association of GPR54 C-terminal with protein phosphatase 2A

    KiSS1 was discovered as a metastasis suppressor gene and subsequently found to encode kisspeptins (KP), ligands for a G protein coupled receptor (GPCR), GPR54. This ligand-receptor pair was later shown to play a critical role in the neuro-endocrine regulation of puberty. The C-terminal cytoplasmic (C-ter) domain of GPR54 contains a segment rich in proline and arginine residues that corresponds to the primary structure of four overlapping SH3 binding motifs. Yeast two hybrid experiments identified the catalytic subunit of protein phosphatase 2A (PP2A-C) as an interacting protein. Pull-down experiments with GST fusion proteins containing the GPR54 C-ter confirmed binding to PP2A-C in cell lysates and these complexes contained phosphatase activity. The proline arginine rich segment is necessary for these interactions. The GPR54 C-ter bound directly to purified recombinant PP2A-C, indicating the GPR54 C-ter may form complexes involving the catalytic subunit of PP2A that regulate phosphorylation of critical signaling intermediates.

  5. Protein Phosphatase 2A Interacts with and Directly Dephosphorylates RelA*

    Yang, Jinming; Fan, Guo-Huang; Wadzinski, Brian E.; Sakurai, Hiroaki; Richmond, Ann

    2001-01-01

    Nuclear factor-κB (NF-κB)/Rel transcription factors are key regulators of a variety of genes involved in inflammatory responses, growth, differentiation, apoptosis, and development. There are increasing lines of evidence that NF-κB/Rel activity is controlled to a great extent by its phosphorylation state. In this study, we demonstrated that RelA physically associated with protein phosphatase 2A (PP2A) subunit A (PR65). Both the N- and C-terminal regions of RelA were responsible for the PP2A b...

  6. Mitogen-activated Protein Kinase Phosphatase (Mkp)-1 Protects Mice against Acetaminophen-induced Hepatic Injury

    Wancket, Lyn M.; Meng, Xiaomei; Rogers, Lynette K.; Liu, Yusen

    2012-01-01

    c-Jun N-terminal kinase (JNK) activation promotes hepatocyte death during acetaminophen overdose, a common cause of drug-induced liver failure. While mitogen-activated protein kinase (MAPK) phosphatase (Mkp)-1 is a critical negative regulator of JNK MAPK, little is known about the role of Mkp-1 during hepatotoxicity. In this study, we evaluated the role of Mkp-1 during acute acetaminophen toxicity. Mkp-1+/+ and Mkp-1−/− mice were dosed ip with vehicle or acetaminophen at 300 mg/kg (for mechan...

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

    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......, we characterize YwqE, compare the activities of the three B. subtilis PTPs (YwqE, YwlE, and YfkJ), and demonstrate that the two B. subtilis class II PTPs do not dephosphorylate the physiological substrates of YwqE....

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

    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...... the conformation and flexibility of active-site residues as well as the water-molecule network, is a key issue in understanding ligand binding and enzyme kinetics and in structure-based drug design. A 1.95 Angstrom apo PTP1B structure has been obtained, showing four highly coordinated water molecules...

  9. A novel protein tyrosine phosphatase 1B inhibitor from Tinospora sinensis

    Prasoon Gupta; Upasana Sharma; Gupta, Praveen K.; Rakesh Maurya

    2012-01-01

    Bioassay-directed fractionation led to the identification of a new compound, 4-hydroxy-heptadec-6-enoic acid ethyl ester (1) together with three known compounds (2-4) from Tinospora sinensis. The structure of 1 was determined by analysis of spectroscopic data. The isolated compounds were evaluated for their protein tyrosine phosphatase 1B (PTP1B) inhibitory activity. Compounds 1 and 2 displayed significant inhibitory activity with IC 50 values 61.1 and 74.2 μM, respectively

  10. Protein phosphatase 2A (PP2A) regulates interleukin-4-mediated STAT6 signaling

    Woetmann, Anders; Brockdorff, Johannes; Lovato, Paola;

    2002-01-01

    Interleukin-4 (IL-4) plays a pivotal role in the induction and maintenance of allergy by promoting Th2 differentiation and B cell isotype switching to IgE. Studies on STAT6-deficient mice have demonstrated the essential role of STAT6 in mediating the biological functions of IL-4. IL-4 induces...... of protein phosphatase 2A (PP2A) induces serine phosphorylation of STAT6 and severely inhibits DNA binding of STAT6. In contrast, IL-4-induced tyrosine phosphorylation of Janus kinase-1 and STAT6 is not affected, suggesting that PP2A acts downstream of Janus kinases in IL-4 signaling. In conclusion, we...

  11. A Mechanistic Study of Protein Phosphatase-1 (PP1), A Catalytically Promiscuous Enzyme

    McWhirter, Claire; Lund, Elizabeth A.; Eric A Tanifum; Feng, Guoqiang; Sheikh, Qaiser; Hengge, Alvan C.; Williams, Nicholas H

    2008-01-01

    The reaction catalyzed by the protein phosphatase-1 (PP1) has been examined by linear free energy relationships and kinetic isotope effects. With the substrate 4-nitrophenyl phosphate (4NPP), the reaction exhibits a bell-shaped pH-rate profile for kcat/KM indicative of catalysis by both acidic and basic residues, with kinetic pKas of 6.0 and 7.2. The enzymatic hydrolysis of a series of aryl monoester substrates yields a Brønsted βlg of -0.32, considerably less negative than that of the uncata...

  12. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    Janssens Veerle; Goris Jozef; Louis Justin V; Zwaenepoel Karen

    2008-01-01

    Abstract Background Protein phosphatase 2A (PP2A) is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specifici...

  13. Effect of gamma-irradiation on nucleic acids, proteins, respiration and phosphatase activity of carrot callus cultures

    Callus tissue cultures were subjected to 60Co qamma irradiation at 0.5 Krad and analysed for nucleic acids, proteins, respiration rate and phosphatase activity on 0, 10, 20 and 30 days. The RNA contents and respiratory rates were enhanced as a result of irradiation. The RNA contents were reduced than their non-irradiated counterparts. The acid phosphatase activity was enhanced immediately after irradiation, declined on 10th and 20th day and more thereafter. (author)

  14. HSP105 recruits protein phosphatase 2A to dephosphorylate β-catenin.

    Yu, Nancy; Kakunda, Michael; Pham, Victoria; Lill, Jennie R; Du, Pan; Wongchenko, Matthew; Yan, Yibing; Firestein, Ron; Huang, XiaoDong

    2015-04-01

    The Wnt/β-catenin pathway causes accumulation of β-catenin in the cytoplasm and its subsequent translocation into the nucleus to initiate the transcription of the target genes. Without Wnt stimulation, β-catenin forms a complex with axin (axis inhibitor), adenomatous polyposis coli (APC), casein kinase 1α (CK1α), and glycogen synthase kinase 3β (GSK3β) and undergoes phosphorylation-dependent ubiquitination. Phosphatases, such as protein phosphatase 2A (PP2A), interestingly, also are components of this degradation complex; therefore, a balance must be reached between phosphorylation and dephosphorylation. How this balance is regulated is largely unknown. Here we show that a heat shock protein, HSP105, is a previously unidentified component of the β-catenin degradation complex. HSP105 is required for Wnt signaling, since depletion of HSP105 compromises β-catenin accumulation and target gene transcription upon Wnt stimulation. Mechanistically, HSP105 depletion disrupts the integration of PP2A into the β-catenin degradation complex, favoring the hyperphosphorylation and degradation of β-catenin. HSP105 is overexpressed in many types of tumors, correlating with increased nuclear β-catenin protein levels and Wnt target gene upregulation. Furthermore, overexpression of HSP105 is a prognostic biomarker that correlates with poor overall survival in breast cancer patients as well as melanoma patients participating in the BRIM2 clinical study. PMID:25645927

  15. Low molecular weight protein tyrosine phosphatase: Multifaceted functions of an evolutionarily conserved enzyme.

    Caselli, Anna; Paoli, Paolo; Santi, Alice; Mugnaioni, Camilla; Toti, Alessandra; Camici, Guido; Cirri, Paolo

    2016-10-01

    Originally identified as a low molecular weight acid phosphatase, LMW-PTP is actually a protein tyrosine phosphatase that acts on many phosphotyrosine-containing cellular proteins that are primarily involved in signal transduction. Differences in sequence, structure, and substrate recognition as well as in subcellular localization in different organisms enable LMW-PTP to exert many different functions. In fact, during evolution, the LMW-PTP structure adapted to perform different catalytic actions depending on the organism type. In bacteria, this enzyme is involved in the biosynthesis of group 1 and 4 capsules, but it is also a virulence factor in pathogenic strains. In yeast, LMW-PTPs dephosphorylate immunophilin Fpr3, a peptidyl-prolyl-cis-trans isomerase member of the protein chaperone family. In humans, LMW-PTP is encoded by the ACP1 gene, which is composed of three different alleles, each encoding two active enzymes produced by alternative RNA splicing. In animals, LMW-PTP dephosphorylates a number of growth factor receptors and modulates their signalling processes. The involvement of LMW-PTP in cancer progression and in insulin receptor regulation as well as its actions as a virulence factor in a number of pathogenic bacterial strains may promote the search for potent, selective and bioavailable LMW-PTP inhibitors. PMID:27421795

  16. Mutations in a new Arabidopsis cyclophilin disrupt its interaction with protein phosphatase 2A

    Jackson, K.; Soll, D.; Evans, M. L. (Principal Investigator)

    1999-01-01

    The heterotrimeric protein phosphatase 2A (PP2A) is a component of multiple signaling pathways in eukaryotes. Disruption of PP2A activity in Arabidopsis is known to alter auxin transport and growth response pathways. We demonstrated that the regulatory subunit A of an Arabidopsis PP2A interacts with a novel cyclophilin, ROC7. The gene for this cyclophilin encodes a protein that contains a unique 30-amino acid extension at the N-terminus, which distinguishes the gene product from all previously identified Arabidopsis cyclophilins. Altered forms of ROC7 cyclophilin with mutations in the conserved DENFKL domain did not bind to PP2A. Unlike protein phosphatase 2B, PP2A activity in Arabidopsis extracts was not affected by the presence of the cyclophilin-binding molecule cyclosporin. The ROC7 transcript was expressed to high levels in all tissues tested. Expression of an ROC7 antisense transcript gave rise to increased root growth. These results indicate that cyclophilin may have a role in regulating PP2A activity, by a mechanism that differs from that employed for cyclophilin regulation of PP2B.

  17. Identification and Biochemical Characterization of Protein Phosphatase 5 from the Cantharidin-Producing Blister Beetle, Epicauta chinensis

    Xi'en Chen

    2013-12-01

    Full Text Available Protein phosphatase 5 (PP5 is a unique member of serine/threonine phosphatases which has been recognized in regulation of diverse cellular processes. A cDNA fragment encoding PP5 (EcPP5 was cloned and characterized from the cantharidin-producing blister beetle, E. chinensis. EcPP5 contains an open reading frame of 1500 bp that encodes a protein of 56.89 kDa. The deduced amino acid sequence shares 88% and 68% identities to the PP5 of Tribolium castaneum and humans, respectively. Analysis of the primary sequence shows that EcPP5 has three TPR (tetratricopeptide repeat motifs at its N-terminal region and contains a highly conserved C-terminal catalytic domain. RT-PCR reveals that EcPP5 is expressed in all developmental stages and in different tissues. The recombinant EcPP5 (rEcPP5 was produced in Escherichia coli and purified to homogeneity. The purified protein exhibited phosphatase activity towards pNPP (p-nitrophenyl phosphate and phosphopeptides, and its activity can be enhanced by arachidonic acid. In vitro inhibition study revealed that protein phosphatase inhibitors, okadaic acid, cantharidin, norcantharidin and endothall, inhibited its activity. Further, protein phosphatase activity of total soluble protein extract from E. chinensis adults could be impeded by these inhibitors suggesting there might be some mechanism to protect this beetle from being damaged by its self-produced cantharidin.

  18. Sodium selenate retards epileptogenesis in acquired epilepsy models reversing changes in protein phosphatase 2A and hyperphosphorylated tau.

    Liu, Shi-Jie; Zheng, Ping; Wright, David K; Dezsi, Gabi; Braine, Emma; Nguyen, Thanh; Corcoran, Niall M; Johnston, Leigh A; Hovens, Christopher M; Mayo, Jamie N; Hudson, Matthew; Shultz, Sandy R; Jones, Nigel C; O'Brien, Terence J

    2016-07-01

    There are no treatments in clinical practice known to mitigate the neurobiological processes that convert a healthy brain into an epileptic one, a phenomenon known as epileptogenesis. Downregulation of protein phosphatase 2A, a protein that causes the hyperphosphorylation of tau, is implicated in neurodegenerative diseases commonly associated with epilepsy, such as Alzheimer's disease and traumatic brain injury. Here we used the protein phosphatase 2A activator sodium selenate to investigate the role of protein phosphatase 2A in three different rat models of epileptogenesis: amygdala kindling, post-kainic acid status epilepticus, and post-traumatic epilepsy. Protein phosphatase 2A activity was decreased, and tau phosphorylation increased, in epileptogenic brain regions in all three models. Continuous sodium selenate treatment mitigated epileptogenesis and prevented the biochemical abnormalities, effects which persisted after drug withdrawal. Our studies indicate that limbic epileptogenesis is associated with downregulation of protein phosphatase 2A and the hyperphosphorylation of tau, and that targeting this mechanism with sodium selenate is a potential anti-epileptogenic therapy. PMID:27289302

  19. PTEN Protein Phosphatase Activity Correlates with Control of Gene Expression and Invasion, a Tumor-Suppressing Phenotype, But Not with AKT Activity

    Tibarewal, P; Zilidis, G; Spinelli, L.; et al.

    2012-01-01

    The tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) has a well-characterized lipid phosphatase activity and a poorly characterized protein phosphatase activity. We show that both activities are required for PTEN to inhibit cellular invasion and to mediate most of its largest effects on gene expression. PTEN appears to dephosphorylate itself at threonine 366, and mutation of this site makes lipid phosphatase activity sufficient for PTEN to inhibit invasion. We p...

  20. Structural basis for recognition of synaptic vesicle protein 2C by botulinum neurotoxin A

    Benoit, Roger M.; Frey, Daniel; Hilbert, Manuel; Kevenaar, Josta T.; Wieser, Mara M.; Stirnimann, Christian U.; McMillan, David; Ceska, Tom; Lebon, Florence; Jaussi, Rolf; Steinmetz, Michel O.; Schertler, Gebhard F. X.; Hoogenraad, Casper C.; Capitani, Guido; Kammerer, Richard A.

    2014-01-01

    Botulinum neurotoxin A (BoNT/A) belongs to the most dangerous class of bioweapons. Despite this, BoNT/A is used to treat a wide range of common medical conditions such as migraines and a variety of ocular motility and movement disorders. BoNT/A is probably best known for its use as an antiwrinkle agent in cosmetic applications (including Botox and Dysport). BoNT/A application causes long-lasting flaccid paralysis of muscles through inhibiting the release of the neurotransmitter acetylcholine by cleaving synaptosomal-associated protein 25 (SNAP-25) within presynaptic nerve terminals. Two types of BoNT/A receptor have been identified, both of which are required for BoNT/A toxicity and are therefore likely to cooperate with each other: gangliosides and members of the synaptic vesicle glycoprotein 2 (SV2) family, which are putative transporter proteins that are predicted to have 12 transmembrane domains, associate with the receptor-binding domain of the toxin. Recently, fibroblast growth factor receptor 3 (FGFR3) has also been reported to be a potential BoNT/A receptor. In SV2 proteins, the BoNT/A-binding site has been mapped to the luminal domain, but the molecular details of the interaction between BoNT/A and SV2 are unknown. Here we determined the high-resolution crystal structure of the BoNT/A receptor-binding domain (BoNT/A-RBD) in complex with the SV2C luminal domain (SV2C-LD). SV2C-LD consists of a right-handed, quadrilateral β-helix that associates with BoNT/A-RBD mainly through backbone-to-backbone interactions at open β-strand edges, in a manner that resembles the inter-strand interactions in amyloid structures. Competition experiments identified a peptide that inhibits the formation of the complex. Our findings provide a strong platform for the development of novel antitoxin agents and for the rational design of BoNT/A variants with improved therapeutic properties.

  1. Identification of a Low Molecular Weight Protein Tyrosine Phosphatase and Its Potential Physiological Substrates in Synechocystis sp. PCC 6803

    Mukhopadhyay, Archana

    2006-01-01

    The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the gross functional properties of this hypothetical protein, open reading frame slr0328 was cloned, and its predicted protein product was expressed in E. coli. The recombinant protein, SynPTP, was purified by metal ion column chromatography. The catalytic act...

  2. Residue 182 influences the second step of protein-tyrosine phosphatase-mediated catalysis

    Pedersen, A.K.; Guo, X.; Møller, K.B.;

    2004-01-01

    Previous enzyme kinetic and structural studies have revealed a critical role for Asp(181) (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp(181) functions as a general acid, while in the E-P hydrolysis step it acts as a gene......Previous enzyme kinetic and structural studies have revealed a critical role for Asp(181) (PTP1B numbering) in PTP (protein-tyrosine phosphatase)-mediated catalysis. In the E-P (phosphoenzyme) formation step, Asp(181) functions as a general acid, while in the E-P hydrolysis step it acts...... as a general base. Most of our understanding of the role of Asp(181). is derived from studies with the Yersinia PTP and the mammalian PTP1B, and to some extent also TC (T-cell)-PTP and, the related PTPalpha and PTPepsilon. The neighbouring residue 182 is a phenylalanine in these four mammalian enzymes......, in comparison with Phe(182)-PTPs, have significantly decreased k(cat) values, and to a lesser degree, decreased k(cat)/K-m values. Combined enzyme kinetic, X-ray crystallographic and molecular dynamics studies indicate that the effect of His(182) is due to interactions with Asp(181) and with Gln(262). We...

  3. Selective binding modes and allosteric inhibitory effects of lupane triterpenes on protein tyrosine phosphatase 1B.

    Jin, Tiantian; Yu, Haibo; Huang, Xu-Feng

    2016-01-01

    Protein Tyrosine Phosphatase 1B (PTP1B) has been recognized as a promising therapeutic target for treating obesity, diabetes, and certain cancers for over a decade. Previous drug design has focused on inhibitors targeting the active site of PTP1B. However, this has not been successful because the active site is positively charged and conserved among the protein tyrosine phosphatases. Therefore, it is important to develop PTP1B inhibitors with alternative inhibitory strategies. Using computational studies including molecular docking, molecular dynamics simulations, and binding free energy calculations, we found that lupane triterpenes selectively inhibited PTP1B by targeting its more hydrophobic and less conserved allosteric site. These findings were verified using two enzymatic assays. Furthermore, the cell culture studies showed that lupeol and betulinic acid inhibited the PTP1B activity stimulated by TNFα in neurons. Our study indicates that lupane triterpenes are selective PTP1B allosteric inhibitors with significant potential for treating those diseases with elevated PTP1B activity. PMID:26865097

  4. A mutation in protein phosphatase 2A regulatory subunit A affects auxin transport in Arabidopsis

    Garbers, C.; DeLong, A.; Deruere, J.; Bernasconi, P.; Soll, D.; Evans, M. L. (Principal Investigator)

    1996-01-01

    The phytohormone auxin controls processes such as cell elongation, root hair development and root branching. Tropisms, growth curvatures triggered by gravity, light and touch, are also auxin-mediated responses. Auxin is synthesized in the shoot apex and transported through the stem, but the molecular mechanism of auxin transport is not well understood. Naphthylphthalamic acid (NPA) and other inhibitors of auxin transport block tropic curvature responses and inhibit root and shoot elongation. We have isolated a novel Arabidopsis thaliana mutant designated roots curl in NPA (rcn1). Mutant seedlings exhibit altered responses to NPA in root curling and hypocotyl elongation. Auxin efflux in mutant seedlings displays increased sensitivity to NPA. The rcn1 mutation was transferred-DNA (T-DNA) tagged and sequences flanking the T-DNA insert were cloned. Analysis of the RCN1 cDNA reveals that the T-DNA insertion disrupts a gene for the regulatory A subunit of protein phosphatase 2A (PP2A-A). The RCN1 gene rescues the rcn1 mutant phenotype and also complements the temperature-sensitive phenotype of the Saccharomyces cerevisiae PP2A-A mutation, tpd3-1. These data implicate protein phosphatase 2A in the regulation of auxin transport in Arabidopsis.

  5. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the activation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modification but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  6. Negative regulation of caspase 3-cleaved PAK2 activity by protein phosphatase 1

    2008-01-01

    The p21-activated kinase 2 (PAK2) is activated by binding of small G proteins, Cdc42 and Rac, or through proteolytic cleavage by caspases or caspase-like proteases. Activation by both small G protein and caspase requires autophosphorylation at Thr-402 of PAK2. Although activation of PAK2 has been investigated for nearly a decade, the mechanism of PAK2 downregulation is unclear. In this study, we have applied the kinetic theory of substrate reaction during modification of enzyme activity to study the regulation mechanism of PAK2 activity by the catalytic subunit of protein phosphatase 1 (PP1α). On the basis of the kinetic equation of the substrate reaction during the reversible phosphorylation of PAK2, all microscopic kinetic constants for the free enzyme and enzyme-substrate(s) complexes have been determined. The results indicate that (1) PP1α can act directly on phosphorylated Thr-402 in the acti-vation loop of PAK2 and down-regulate its kinase activity; (2) binding of the exogenous protein/peptide substrates at the active site of PAK2 decreases both the rates of PAK2 autoactivation and inactivation. The present method provides a novel approach for studying reversible phosphorylation reactions. The advantage of this method is not only its usefulness in study of substrate effects on enzyme modifica-tion but also its convenience in study of modification reaction directly involved in regulation of enzyme activity. This initial study should provide a foundation for future structural and mechanistic work of protein kinases and phosphatases.

  7. Recent progress on the structure of Ser/Thr protein phosphatases

    WANG BaiJing; ZHANG Peng; WEI Quni

    2008-01-01

    PP1, PP2A and PP2B, belonging to the PPP family of Ser/Thr protein phosphatases, participate in regulating many important physiological processes, such as cell cycle control, regulation of cell growth and division regulation, etc. The sequence homology between them is relatively high, and ter-tiary structure is conserved. Because of the complexity of the structure of PP2A and the diversity of its regulatory subunits, its structure is less well known than those of PP1 and PP2B. The PP2A holoen-zyme consists of a heterodimeric core enzyme, comprising a scaffolding subunit and a catalytic sub-unit, as well as a variable regulatory subunit. In this study, the subunit compositions, similarities and differences between the Ser/Thr protein phsphatases structures are summarized.

  8. Disulfide bonds in the catalytic subunit of skeletal muscle protein phosphatase

    The protein phosphatase in skeletal muscle that inactivates phosphorylase and phosphorylase kinase and activates glycogen synthase is both inhibited and reversibly activated/inactivated by a heat-stable protein called inhibitor-2 (I2). This enzyme, called type-1 or MgATP-dependent protein phosphatase, has a catalytic subunit (C) of M/sub r/ = 38,000. Conversion of the inactive to the active conformer of C occurs by reaction with Mn2+ or Co2+, or by dephosphorylation of I2 bound to the C. The C active conformer is cleaved by trypsin to give a stable catalytic fragment of M/sub r/ = 33,000 which resists denaturation by 80% ethanol. This fragment of C was purified by polylysine and Sephadex chromatography to over 20,000 U/mg. After boiling the catalytic fragment of C in 0.1% dodecyl sulfate containing dithiothreitol (DTT), reaction with [14C]-iodoacetate labeled about 1 residue/molecule. However, the authors found the same extent of labeling without DTT in 6M guanidine at pH 9 and in parallel reactions that contained DTT, labeling was increased over 10-fold. The fully labeled protein migrated at M/sub r/ = 33,000 during gel electrophoresis. Chymotryptic digestion in dodecyl sulfate yielded eight major 14C peptides that were resolved by reversed-phase HPLC. The results are interpreted as evidence that C contains one free Cys residue and as many as 5 disulfide bonds. Multiple disulfides would account for the unusual stability of C and intramolecular disulfide interchange is proposed as the mechanism for conversion of the conformers

  9. Striatal-enriched protein tyrosine phosphatase modulates nociception: evidence from genetic deletion and pharmacological inhibition.

    Azkona, Garikoitz; Saavedra, Ana; Aira, Zigor; Aluja, David; Xifró, Xavier; Baguley, Tyler; Alberch, Jordi; Ellman, Jonathan A; Lombroso, Paul J; Azkue, Jon J; Pérez-Navarro, Esther

    2016-02-01

    The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception. PMID:26270590

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

    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.

  11. Energy-requiring translocation of the OmpA protein and alkaline phosphatase of Escherichia coli into inner membrane vesicles.

    Rhoads, D B; Tai, P C; Davis, B D

    1984-01-01

    In developing a reliable in vitro system for translocating bacterial proteins, we found that the least dense subfraction of the membrane of Escherichia coli was superior to the total inner membrane, both for a secreted protein (alkaline phosphatase) and for an outer membrane protein (OmpA). Compounds that eliminated the proton motive force inhibited translocation, as already observed in cells; since protein synthesis continued, the energy for translocation appears to be derived from the energ...

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

    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.

  13. Lentivirus-Mediated Short-Hairpin RNA Targeting Protein Phosphatase 4 Regulatory Subunit 1 Inhibits Growth in Breast Cancer

    Qi, Yuying; Hu, Tinghui; Li, Kai; Ye, Renqing; Ye, Zuodong

    2015-01-01

    Purpose Protein phosphatase 4 regulatory subunit 1 (PP4R1), as an interaction partner of the catalytic serine/threonine-protein phosphatase 4 catalytic subunit has been shown to involve in cellular processes and nuclear factor κB signaling. However, the functions of PP4R1 in human breast cancers remain unclear. This study is designed to explore the effect of PP4R1 knockdown on the biological characteristics of breast cancer cells. Methods A lentivirus-mediated short hairpin RNA (shRNA) was de...

  14. Protein-protein interactions in crystals of the human receptor-type protein tyrosine phosphatase ICA512 ectodomain.

    María E Primo

    Full Text Available ICA512 (or IA-2 is a transmembrane protein-tyrosine phosphatase located in secretory granules of neuroendocrine cells. Initially, it was identified as one of the main antigens of autoimmune diabetes. Later, it was found that during insulin secretion, the cytoplasmic domain of ICA512 is cleaved and relocated to the nucleus, where it stimulates the transcription of the insulin gene. The role of the other parts of the receptor in insulin secretion is yet to be unveiled. The structures of the intracellular pseudocatalytic and mature extracellular domains are known, but the transmembrane domain and several intracellular and extracellular parts of the receptor are poorly characterized. Moreover the overall structure of the receptor remains to be established. We started to address this issue studying by X-ray crystallography the structure of the mature ectodomain of ICA512 (ME ICA512 and variants thereof. The variants and crystallization conditions were chosen with the purpose of exploring putative association interfaces, metal binding sites and all other structural details that might help, in subsequent works, to build a model of the entire receptor. Several structural features were clarified and three main different association modes of ME ICA512 were identified. The results provide essential pieces of information for the design of new experiments aimed to assess the structure in vivo.

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

    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.

  16. Mechanisms underlying the inhibitory effects of arsenic compounds on protein tyrosine phosphatase (PTP)

    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 (iAsIII) and its intermediate metabolites such as monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII) 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., MMAIII and DMAIII) but not by iAsIII. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS) has clearly confirmed that the organic intermediate, DMAIII 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 DMAIII strongly inhibited activity of PTP1B. ► DMAIII directly bound to PTP1B, resulting in inhibition of enzyme

  17. Characterization of Two Putative Protein Phosphatase Genes and Their Involvement in Phosphorus Efficiency in Phaseolus vulgari

    Cui-Yue Liang; Zhi-Jian Chen; Zhu-Fang Yao; Jiang Tian; Hong Liao

    2012-01-01

    Protein dephosphorylation mediated by protein phosphatases plays a major role in signal transduction of plant responses to environmental stresses.In this study,two putative protein phosphatases,PvPS2:1 and PvPS2:2 were identified and characterized in bean (Phaseolus vulgaris).The two PvPS2 members were found to be localized to the plasma membrane and the nucleus by transient expression of PvPS2:GFP in onion epidermal cells.Transcripts of the two PvPS2 genes were significantly increased by phosphate (Pi) starvation in the two bean genotypes,G19833 (a P-efficient genotype) and DOR364 (a P-inefficient genotype).However,G19833 exhibited higher PvPS2:1 expression levels than DOR364 in both leaves and roots during P1 starvation.Increased transcription of PvPS2:1 in response to Pi starvation was further verified through histochemical analysis of PvPS2:1 promoter fusion β-glucuronidase (GUS) in transgenic Arabidopsis plants.Analysis of PvPS2∶1 overexpression lines in bean hairy roots and Arabidopsis showed that PvS2:1 was involved in root growth and P accumulation.Furthermore,expression levels of two P(1) starvation responsive genes were upregulated and the APase activities were enhanced in the overexpressing PvPS2∶1 Arabidopsis lines.Taken together,our results strongly suggested that PvPS2∶1positively regulated plant responses to P1 starvation,and could be further targeted as a candidate gene to improve crop P efficiency.

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

    Kang, Yoonsung [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Cheong, Hyang-Min [Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Lee, Jung-Hee [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Song, Peter I. [Department of Dermatology, University of Arkansas for Medical Science, 4301 West Markham, Slot 576, Little Rock, AR 72205 (Korea, Republic of); Lee, Kwang-Ho [Department of Life Science, College of Natural Science, Chung-Ang University, 221 Heuksuk-Dong, Dongjak-Ku, Seoul 156-756 (Korea, Republic of); Kim, Sang-Yong [Division of Endocrinology, Department of Internal Medicine, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); Jun, Jae Yeoul [Department of Physiology, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of); You, Ho Jin, E-mail: hjyou@chosun.ac.kr [Department of Pharmacology, DNA Repair Research Center, Chosun University School of Medicine, 375 Seosuk-Dong, Gwangju 501-759 (Korea, Republic of)

    2011-01-07

    Research highlights: {yields} 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. {yields} However, it is not clear exactly how PP5 participates in this process. {yields} 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.

  19. Regulation of human CYP2C9 expression by electrophilic stress involves activator protein 1 activation and DNA looping.

    Makia, Ngome L; Surapureddi, Sailesh; Monostory, Katalin; Prough, Russell A; Goldstein, Joyce A

    2014-08-01

    Cytochrome P450 (CYP)2C9 and CYP2C19 are important human enzymes that metabolize therapeutic drugs, environmental chemicals, and physiologically important endogenous compounds. Initial studies using primary human hepatocytes showed induction of both the CYP2C9 and CYP2C19 genes by tert-butylhydroquinone (tBHQ). As a pro-oxidant, tBHQ regulates the expression of cytoprotective genes by activation of redox-sensing transcription factors, such as the nuclear factor E2-related factor 2 (Nrf2) and members of the activator protein 1 (AP-1) family of proteins. The promoter region of CYP2C9 contains two putative AP-1 sites (TGAGTCA) at positions -2201 and -1930, which are also highly conserved in CYP2C19. The CYP2C9 promoter is activated by ectopic expression of cFos and JunD, whereas Nrf2 had no effect. Using specific kinase inhibitors for mitogen-activated protein kinase, we showed that extracellular signal-regulated kinase and Jun N-terminal kinase are essential for tBHQ-induced expression of CYP2C9. Electrophoretic mobility shift assays demonstrate that cFos distinctly interacts with the distal AP-1 site and JunD with the proximal site. Because cFos regulates target genes as heterodimers with Jun proteins, we hypothesized that DNA looping might be required to bring the distal and proximal AP-1 sites together to activate the CYP2C9 promoter. Chromosome conformation capture analyses confirmed the formation of a DNA loop in the CYP2C9 promoter, possibly allowing interaction between cFos at the distal site and JunD at the proximal site to activate CYP2C9 transcription in response to electrophiles. These results indicate that oxidative stress generated by exposure to electrophilic xenobiotics and metabolites induces the expression of CYP2C9 and CYP2C19 in human hepatocytes. PMID:24830941

  20. Foot-and-Mouth Disease Virus 2C Is a Hexameric AAA+ Protein with a Coordinated ATP Hydrolysis Mechanism

    Sweeney, Trevor; Cisnetto, Valentina; Bose, Daniel;

    2010-01-01

    Foot-and-mouth disease virus (FMDV), a positive sense, single-stranded RNA virus, causes a highly contagious disease in cloven-hoofed livestock. Like other picornaviruses, FMDV has a conserved 2C protein assigned to the superfamily 3 helicases a group of AAA+ ATPases that has a predicted N....... Unliganded 2C(34–318) exhibits concentration-dependent self-association to yield oligomeric forms, the largest of which is tetrameric. Strikingly, in the presence of ATP and RNA, FMDV 2C(34–318) containing the N207A mutation, which binds but does not hydrolyze ATP, was found to oligomerize specifically...... into hexamers. Visualization of FMDV 2C-ATP-RNA complexes by negative stain electron microscopy revealed hexameric ring structures with 6-fold symmetry that are characteristic of AAA+ ATPases. ATPase assays performed by mixing purified active and inactive 2C(34–318) subunits revealed a coordinated mechanism...

  1. Family of receptor-linked protein tyrosine phosphatases in humans and Drosophila

    To understand the regulation of cell proliferation by tyrosine phosphorylation, characterization of protein tyrosine phosphatases is essential. The human genes LCA (leukocyte common antigen) and LAR encode putative receptor-linked PTPases. By using consensus sequence probes, two additional receptor-linked PTPase genes, DLAR and DPTP, were isolated from Drosophila melanogaster. The extra-cellular segments of both DLAR and DPTP are composed of multiple immunoglobulin-like domains and fibronectin type III-like domains. The cytoplasmic region of DLAR and DPTP, as well as human LCA and LAR, are composed of two tandemly repeated PTPase domains. PTPase activities of immunoprecipitated LCA and LAR were demonstrated by measuring the release of phosphate from a 32P-labeled [Tyr(P)]peptide. Furthermore, the cytoplasmic domains of LCA, LAR, DLAR, and DPTP, expressed in Escherichia coli, have PTPase activity. Site-directed mutagenesis showed that a conserved cysteine residue is essential for PTPase activity

  2. Protein phosphatase 2A isotypes regulate cell surface expression of the T cell receptor

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

    2001-01-01

    The mechanisms underlying T cell receptor (TCR) down-regulation have been extensively studied during the last decade. Whereas the importance of phosphorylation in this process has been established, it is less certain whether dephosphorylation plays a role in TCR down-regulation. In this study, we...... show that inhibition of the serine/threonine protein phosphatase PP2A family had a biphasic effect on TCR expression. Thus, low concentrations of PP2A inhibitors induced TCR down-regulation, whereas higher concentrations of PP2A inhibitors induced TCR up-regulation. The effect of PP2A inhibition was...... independent of phosphorylation of the CD3gamma endocytosis motif. Whereas TCR down-regulation was caused by a partial inhibition of exocytosis, TCR up-regulation was caused by an inhibition of endocytosis. The effects on exocytosis and endocytosis were not restricted to the TCR, indicating a more general...

  3. Cell Death Inducing Microbial Protein Phosphatase Inhibitors—Mechanisms of Action

    Rune Kleppe

    2015-10-01

    Full Text Available Okadaic acid (OA and microcystin (MC as well as several other microbial toxins like nodularin and calyculinA are known as tumor promoters as well as inducers of apoptotic cell death. Their intracellular targets are the major serine/threonine protein phosphatases. This review summarizes mechanisms believed to be responsible for the death induction and tumor promotion with focus on the interdependent production of reactive oxygen species (ROS and activation of Ca2+/calmodulin kinase II (CaM-KII. New data are presented using inhibitors of specific ROS producing enzymes to curb nodularin/MC-induced liver cell (hepatocyte death. They indicate that enzymes of the arachidonic acid pathway, notably phospholipase A2, 5-lipoxygenase, and cyclooxygenases, may be required for nodularin/MC-induced (and presumably OA-induced cell death, suggesting new ways to overcome at least some aspects of OA and MC toxicity.

  4. Expression, purification and characterization of recombinant protein tyrosine phosphatase from Thermus thermophilus HB27

    Yejing Wang; Fanguo Meng; Yingmei Zhang

    2009-01-01

    The low-molecular-weight protein tyrosine phospha-tases (PTPase) exist ubiquitously in prokaryotes and eukaryotes and play important roles in the regulation of physiological activities. We report here the expression, purification and characterization of an active and soluble PTPase from Thermus thermophilus HB27 in Escherichia coli. This PTPase has an optimum pH range of 2.8-4.8 when using p-nitrophenyl phos-phate as the substrate. The thermal inactivation results indicate a high thermal stability of this enzyme, with the optimum temperature of 75℃ for activity. It can be activated by Mn2+, Mg2+, Ca2+, Ba2+, and Ni2+, but inhibited by Zn2+, Cu2+, Cl-, and SO2-4. These results suggest that this heat-resistant PTPase may play impor-tant roles in vivo in the adaptation of the microorgan-ism to extreme temperatures and specific nutritional conditions.

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

    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. PMID:26462736

  6. Structural Basis for the Catalytic Activity of Human Serine/Threonine Protein Phosphatase type 5 (PP5)

    Swingle, Mark R.; Ciszak, Ewa M.; Honkanen, Richard E.

    2004-01-01

    Serine/threonine protein phosphatase-5 (PP5) is a member of the PPP-gene family of protein phosphatases that is widely expressed in mammalian tissues and is highly conserved among eukaryotes. PP5 associates with several proteins that affect signal transduction networks, including the glucocorticoid receptor (GR)-heat shock protein-90 (Hsp90)-heterocomplex, the CDC16 and CDC27 subunits of the anaphase-promoting complex, elF2alpha kinase, the A subunit of PP2A, the G12-alpha / G13-alpha subunits of heterotrimeric G proteins and DNA-PK. The catalytic domain of PP5 (PP5c) shares 35-45% sequence identity with the catalytic domains of other PPP-phosphatases, including protein phosphatase-1 (PP1), -2A (PP2A), -2B / calcineurin (PP2B), -4 (PP4), -6 (PP6), and -7 (PP7). Like PP1, PP2A and PP4, PP5 is also sensitive to inhibition by okadaic acid, microcystin, cantharidin, tautomycin, and calyculin A. Here we report the crystal structure of the PP5 catalytic domain (PP5c) at a resolution of 1.6 angstroms. From this structure we propose a mechanism for PP5-mediated hydrolysis of phosphoprotein substrates, which requires the precise positioning of two metal ions within a conserved Asp(sup 271)-M(sub 1):M(sub 2)-W(sup 1)-His(sup 304)-Asp(sup 274) catalytic motif. The structure of PP5c provides a possible structural basis for explaining the exceptional catalytic proficiency of protein phosphatases, which are among the most powerful known catalysts. Resolution of the entire C-terminus revealed a novel subdomain, and the structure of the PP5c should also aid development of type-specific inhibitors.

  7. Phosphatases in plants.

    Schweighofer, Alois; Meskiene, Irute

    2015-01-01

    Reversible protein phosphorylation is an essential posttranslational modification mechanism executed by opposing actions of protein phosphatases and protein kinases. About 1,000 predicted kinases in Arabidopsis thaliana kinome predominate the number of protein phosphatases, of which there are only ~150 members in Arabidopsis. Protein phosphatases were often referred to as "housekeeping" enzymes, which act to keep eukaryotic systems in balance by counteracting the activity of protein kinases. However, recent investigations reveal the crucial and specific regulatory functions of phosphatases in cell signaling. Phosphatases operate in a coordinated manner with the protein kinases, to execute their important function in determining the cellular response to a physiological stimulus. Closer examination has established high specificity of phosphatases in substrate recognition and important roles in plant signaling pathways, such as pathogen defense and stress regulation, light and hormonal signaling, cell cycle and differentiation, metabolism, and plant growth. In this minireview we provide a compact overview about Arabidopsis protein phosphatase families, as well as members of phosphoglucan and lipid phosphatases, and highlight the recent discoveries in phosphatase research. PMID:25930691

  8. The myeloperoxidase-derived oxidant hypothiocyanous acid inhibits protein tyrosine phosphatases via oxidation of key cysteine residues

    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 (S...

  9. Interaction of NCOR/SMRT Repressor Complexes with Papillomavirus E8^E2C Proteins Inhibits Viral Replication.

    Dreer, Marcel; Fertey, Jasmin; van de Poel, Saskia; Straub, Elke; Madlung, Johannes; Macek, Boris; Iftner, Thomas; Stubenrauch, Frank

    2016-04-01

    Infections with high-risk human papillomaviruses (HR-HPV) such as HPV16 and 31 can lead to ano-genital and oropharyngeal cancers and HPV types from the beta genus have been implicated in the development of non-melanoma skin cancer. HPV replicate as nuclear extrachromosomal plasmids at low copy numbers in undifferentiated cells. HPV16 and 31 mutants have indicated that these viruses express an E8^E2C protein which negatively regulates genome replication. E8^E2C shares the DNA-binding and dimerization domain (E2C) with the essential viral replication activator E2 and the E8 domain replaces the replication/transcription activation domain of E2. The HR-HPV E8 domain is required for inhibiting viral transcription and the replication of the viral origin mediated by viral E1 and E2 proteins. We show now that E8^E2C also limits replication of HPV1, a mu-PV and HPV8, a beta-PV, in normal human keratinocytes. Proteomic analyses identified all NCoR/SMRT corepressor complex components (HDAC3, GPS2, NCoR, SMRT, TBL1 and TBLR1) as co-precipitating host cell proteins for HPV16 and 31 E8^E2C proteins. Co-immunoprecipitation and co-localization experiments revealed that NCoR/SMRT components interact with HPV1, 8, 16 and 31 E8^E2C proteins in an E8-dependent manner. SiRNA knock-down experiments confirm that NCoR/SMRT components are critical for both the inhibition of transcription and HPV origin replication by E8^E2C proteins. Furthermore, a dominant-negative NCoR fragment activates transcription and replication only from HPV16 and 31 wt but not from mutant genomes encoding NCoR/SMRT-binding deficient E8^E2C proteins. In summary, our data suggest that the repressive function of E8^E2C is highly conserved among HPV and that it is mediated by an E8-dependent interaction with NCoR/SMRT complexes. Our data also indicate for the first time that NCoR/SMRT complexes not only are involved in inhibiting cellular and viral transcription but also in controlling the replication of HPV origins

  10. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Stabel Silvia

    2002-04-01

    Full Text Available Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro. Results We generated recombinant 124-v-Mos using the baculovirus expression system in Spodoptera frugiperda cells and demonstrated constitutive kinase activity by the ability of 124-v-Mos to auto-phosphorylate and to phosphorylate vimentin, a known substrate of c-Mos. Using this approach we analyzed a panel of acidic and basic substrates in immunocomplex protein kinase assays and identified novel in vitro substrates for 124-v-Mos, the protein tyrosine phosphatase 1B (PTP1B, alpha-casein and beta-casein. We controlled mos-specific phosphorylation of PTP1B and casein in comparative assays using a synthetic kinase-inactive 124-v-Mos mutant and further, tryptic digests of mos-phosphorylated beta-casein identified a phosphopeptide specifically targeted by wild-type 124-v-Mos. Two-dimensional phosphoamino acid analyses showed that 124-v-mos targets serine and threonine residues for phosphorylation in casein at a 1:1 ratio but auto-phosphorylation occurs predominantly on serine residues. Conclusion The mos substrates identified in this study represent a basis to approach the identification of the mos-consensus phosphorylation motif, important for the development of specific inhibitors of the Mos protein kinase.

  11. Inhibition of Setaria cervi protein tyrosine phosphatases by Phenylarsine oxide: A proteomic and biochemical study.

    Singh, Neetu; Wadhawan, Mohit; Tiwari, Savitri; Kumar, Ranjeet; Rathaur, Sushma

    2016-07-01

    Phenylarsine oxide (PAO), a specific protein tyrosine phosphatase (PTP) inhibitor significantly decreased the motility and viability of Setaria cervi ultimately leading to its death. The PTP activity present in the cytosolic and detergent soluble fractions as well as on surface of these parasites was significantly inhibited by PAO. A marked alteration in protein spots abundance after proteomic analysis showed 14 down-regulated and 9 upregulated spots in the treated parasites as compared to the control. The PTP inhibition led to increase in the cytosolic and mitochondrial calpain activity in these parasites. PAO also blocked the ATP generation in the parasite depicted by reduced activity of phosphoglycerate kinase and expression of enolase. An increased ROS level, induced lipid peroxidation/protein carbonyl formation and decreased activity of different antioxidant enzymes like thioredoxin reductase, glutathione reductase and glutathione transferases was also observed in the PAO treated parasites. PAO, thus disturbs the overall homeostasis of the filarial parasite by inhibiting PTPs. Thereby suggesting that these molecules could be used as a good chemotherapeutic target for lymphatic filariasis. PMID:26965172

  12. BRCA1 affects protein phosphatase 6 signalling through its interaction with ANKRD28.

    Vincent, Anne; Berthel, Elise; Dacheux, Estelle; Magnard, Clémence; Venezia, Nicole L Dalla

    2016-04-01

    The tumour suppressor BRCA1 (breast and ovarian cancer-susceptibility gene 1) is implicated in several nuclear processes including DNA repair, transcription regulation and chromatin remodelling. BRCA1 also has some cytoplasmic functions including a pro-apoptotic activity. We identified ANKRD28 (ankyrin repeat domain 28) as a novel BRCA1-interacting protein in a yeast two-hybrid screen and confirmed this interaction by reciprocal immunoprecipitations of the two overexpressed proteins. Endogenous interaction between BRCA1 and ANKRD28 was also observed by co-immunoprecipitation and located in the cytoplasm by proximity ligation assay. The main site of interaction of ANKRD28 on BRCA1 is located in its intrinsically disordered scaffold central region. Whereas ANKRD28 silencing results in a destabilization of IκBε (inhibitor of nuclear factor κBε) through its activation of PP6 (protein phosphatase 6) co-regulator upon TNFα (tumour necrosis factor α) stimulation, BRCA1 overexpression stabilizes IκBε. A truncated form of BRCA1 that does not interact with ANKRD28 has no such effect. Our findings suggest that BRCA1 is a novel modulator of PP6 signalling via its interaction with ANKRD28. This new cytoplasmic process might participate in BRCA1 tumour-suppressor function. PMID:27026398

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

    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.

  14. Unbiased identification of substrates of protein tyrosine phosphatase ptp-3 in C. elegans.

    Mitchell, Christopher J; Kim, Min-Sik; Zhong, Jun; Nirujogi, Raja Sekhar; Bose, Anjun K; Pandey, Akhilesh

    2016-06-01

    The leukocyte antigen related (LAR) family of receptor-like protein tyrosine phosphatases has three members in humans - PTPRF, PTPRD and PTPRS - that have been implicated in diverse processes including embryonic development, inhibition of cell growth and axonal guidance. Mutations in the LAR family are associated with developmental defects such as cleft palate as well as various cancers including breast, neck, lung, colon and brain. Although this family of tyrosine phosphatases is important for many developmental processes, little is known of their substrates. This is partially due to functional redundancy within the LAR family, as deletion of a single gene in the LAR family does not have an appreciable phenotype, but a dual knockout is embryonically lethal in mouse models. To circumvent the inability to knockout multiple members of the LAR family in mouse models, we used a knockout of ptp-3, which is the only known ortholog of the LAR family in Caenorhabditis elegans and allows for the study of the LAR family at the organismal level. Using SILAC-based quantitative phosphoproteomics, we identified 255 putative substrates of ptp-3, which included four of the nine known annotated substrates of the LAR family. A motif analysis of the identified phosphopeptides allowed for the determination of sequences that appear to be preferentially dephosphorylated. Finally, we discovered that kinases were overrepresented in the list of identified putative substrates and tyrosine residues whose phosphorylation is known to increase kinase activity were dephosphorylated by ptp-3. These data are suggestive of ptp-3 as a potential negative regulator of several kinase families, such as the mitogen activated kinases (MAPKs), and multiple tyrosine kinases including FER, MET, and NTRK2. PMID:27067626

  15. Protein-tyrosine phosphatase activity of Coxiella burnetii that inhibits human neutrophils

    Supernatants prepared from disrupted Coxiella burnetii posses acid phosphatase (ACP) activity that apparently accounts for the inhibition of the metabolic burst of formyl-Met-Leu-Phe(fMLP)-stimulated human neutrophils. Results are presented regarding purification and biochemical-biological characterization of the ACP. The highly purified enzyme, which exhibited an apparent M of 91 K and optimal activity at pH 5.0, also inhibited neutrophils. The enzyme retained full activity at pH 4.5, 5.5, and 7.4, when incubated overnight at 0 grad C and room temperature; at pH 5.5, it retained full activity after overnight incubation at 37 grad C. Apparently, the enzyme contains asparagine-linked but not serine- or threonine-liked glycan residues since its treatment with N-glycosidase F decreased its Mr to 87 K and no changes were detected with O-glycosidase. The enzyme's capacity to hydrolyze phosphate from a number of phosphate-containing compounds was examined; five phospho-compounds were significantly hydrolyzed: 5'-CMP > fructose 1,6-diphosphate > tyrosine phosphate > 3'-AMP >5'-AMP. The ACP also dephosphorylated 32P-Raytide, a phosphotyrosine-containing peptide. Dephosphorylation of Raytide was inhibited by the following phosphatase inhibitors: sodium molybdate, potassium fluoride, sodium ortho-vanadate and D2, a heteropolymolybdate compound. These results indicate that C.burnetii ACP may play a role in disrupting tyrosine phosphorylation/dephosphorylation reactions associated with the signal transduction pathway culminating in the metabolic burst. Interestingly, Western blot analysis of ACP-inhibited neutrophils showed a marked increase in tyrosine phosphorylation of a 44 K protein as compared to uninhibited cells. (author)

  16. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro

  17. Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

    Nguyen, Ngoc Uyen Nhi; Liang, Vincent Roderick; Wang, Hao-Ven, E-mail: hvwang@mail.ncku.edu.tw

    2014-09-26

    Highlights: • Palladin is involved in myogenesis in vitro. • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation. • Palladin knockdown decreases C2C12 myoblast migration ability. - Abstract: The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

  18. Elongation factor-1A1 is a novel substrate of the protein phosphatase 1-TIMAP complex.

    Boratkó, Anita; Péter, Margit; Thalwieser, Zsófia; Kovács, Előd; Csortos, Csilla

    2015-12-01

    TIMAP (TGF-β inhibited membrane associated protein) is a protein phosphatase 1 (PP1) regulatory subunit highly abundant in endothelial cells and it is involved in the maintenance of pulmonary endothelial barrier function. It localizes mainly in the plasma membrane, but it is also present in the nuclei and cytoplasm. Direct interaction of TIMAP with the eukaryotic elongation factor 1 A1 (eEF1A1) is shown by pull-down, LC-MS/MS, Far-Western and immunoprecipitations. In connection with the so called moonlighting functions of the elongation factor, eEF1A is thought to establish protein-protein interactions through a transcription-dependent nuclear export motif, TD-NEM, and to aid nuclear export of TD-NEM containing proteins. We found that a TD-NEM-like motif of TIMAP has a critical role in its specific binding to eEF1A1. However, eEF1A1 is not or not exclusively responsible for the nuclear export of TIMAP. On the contrary, TIMAP seems to regulate membrane localization of eEF1A1 as the elongation factor co-localized with TIMAP in the plasma membrane fraction of control endothelial cells, but it has disappeared from the membrane in TIMAP depleted cells. It is demonstrated that membrane localization of eEF1A1 depends on the phosphorylation state of its Thr residue(s); and ROCK phosphorylated eEF1A1 is a novel substrate for TIMAP-PP1 underlining the complex regulatory role of TIMAP in the endothelium. The elongation factor seems to be involved in the regulation of endothelial cell attachment and spreading as silencing of eEF1A1 positively affected these processes which were monitored by transendothelial resistance measurements. PMID:26497934

  19. Alkaline phosphatase and OmpA protein can be translocated posttranslationally into membrane vesicles of Escherichia coli.

    Chen, L.; Rhoads, D; Tai, P C

    1985-01-01

    We previously described a system for translocating the periplasmic enzyme alkaline phosphatase and the outer membrane protein OmpA into inverted membrane vesicles of Escherichia coli. We have now optimized and substantially improved the translocation system by including polyamines and by reducing the amount of membrane used. Under these conditions, efficient translocation was seen even posttranslationally, i.e., when vesicles were not added until after protein synthesis was stopped. This was ...

  20. A low molecular weight protein tyrosine phosphatase from Synechocystis sp. strain PCC 6803: enzymatic characterization and identification of its potential substrates

    Mukhopadhyay, Archana; Kennelly, Peter J.

    2011-01-01

    The predicted protein product of open reading frame slr0328 from Synechocystis sp. PCC 6803, SynPTP, possesses significant amino acid sequence similarity with known low molecular weight protein tyrosine phosphatases (PTPs). To determine the functional properties of this hypothetical protein, open reading frame slr0328 was expressed in Escherichia coli. The purified recombinant protein, SynPTP, displayed its catalytic phosphatase activity towards several tyrosine, but not serine, phosphorylate...

  1. Protein Tyrosine Phosphatase-1B Negatively Impacts Host Defense against Pseudomonas aeruginosa Infection.

    Yue, Lei; Xie, Zhongping; Li, Hua; Pang, Zheng; Junkins, Robert D; Tremblay, Michel L; Chen, Xiaochun; Lin, Tong-Jun

    2016-05-01

    Pseudomonas aeruginosa is a major opportunistic pathogen in immune-compromised individuals. Mechanisms governing immune responses to P. aeruginosa infection remain incompletely defined. Herein, we demonstrate that protein tyrosine phosphatase-1B (PTP1B) is a critical negative regulator in P. aeruginosa infection. PTP1B-deficient mice display greatly enhanced bacterial clearance and reduced disease scores, which are accompanied by increased neutrophil infiltration and cytokine production. Interestingly, PTP1B deficiency mainly up-regulates the production of interferon-stimulated response elements-regulated cytokines and chemokines, including chemokine ligand 5 (regulated on activation normal T cell expressed and secreted), CXCL10 (interferon γ-inducible protein 10), and interferon-β production. Further studies reveal that PTP1B deficiency leads to increased interferon regulatory factor 7 (IRF7) expression and activation. These findings demonstrate a novel regulatory mechanism of the immune response to P. aeruginosa infection through PTP1B-IRF7 interaction. This novel PTP1B-IRF7-interferon-stimulated response elements pathway may have broader implications in Toll-like receptor-mediated innate immunity. PMID:27105736

  2. Regulation of Ikaros function by casein kinase 2 and protein phosphatase 1

    Amy; K; Erbe; Aleksandar; Savic; Sinisa; Dovat

    2011-01-01

    The Ikaros gene encodes a zinc finger,DNA-binding protein that regulates gene transcription and chromatin remodeling.Ikaros is a master regulator of hematopoiesis and an established tumor suppressor.Moderate alteration of Ikaros activity (e.g.haploinsufficiency) appears to be sufficient to promote malignant transformation in human hematopoietic cells.This raises questions about the mechanisms that normally regulate Ikaros function and the potential of these mechanisms to contribute to the development of leukemia.The focus of this review is the regulation of Ikaros function by phosphorylation/dephosphorylation.Site-specific phosphorylation of Ikaros by casein kinase 2 (CK2) controls Ikaros DNA-binding ability and subcellular localization.As a consequence,the ability of Ikaros to regulate cell cycle progression,chromatin remodeling,target gene expression,and thymocyte differentiation are controlled by CK2.In addition,hyperphosphorylation of Ikaros by CK2 leads to decreased Ikaros levels due to ubiquitinmediated degradation.Dephosphorylation of Ikaros by protein phosphatase 1 (PP1) acts in opposition to CK2 to increase Ikaros stability and restore Ikaros DNA binding ability and pericentromeric localization.Thus,the CK2 and PP1 pathways act in concert to regulate Ikaros activity in hematopoiesis and as a tumor suppressor.This highlights the importance of these signal transduction pathways as potential mediators of leukemogenesis via their role in regulating the activities of Ikaros.

  3. Inactivation of Protein Tyrosine Phosphatases Enhances Interferon Signaling in Pancreatic Islets.

    Stanley, William J; Litwak, Sara A; Quah, Hong Sheng; Tan, Sih Min; Kay, Thomas W H; Tiganis, Tony; de Haan, Judy B; Thomas, Helen E; Gurzov, Esteban N

    2015-07-01

    Type 1 diabetes (T1D) is the result of an autoimmune assault against the insulin-producing pancreatic β-cells, where chronic local inflammation (insulitis) leads to β-cell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T-cell invasion and activation. Cytokine-signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from NOD mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling compared with NOD mice that do not have insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and β-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation-induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis. PMID:25732191

  4. B′-protein phosphatase 2A is a functional binding partner of delta-retroviral integrase

    Maertens, Goedele N.

    2016-01-01

    To establish infection, a retrovirus must insert a DNA copy of its RNA genome into host chromatin. This reaction is catalysed by the virally encoded enzyme integrase (IN) and is facilitated by viral genus-specific host factors. Herein, cellular serine/threonine protein phosphatase 2A (PP2A) is identified as a functional IN binding partner exclusive to δ-retroviruses, including human T cell lymphotropic virus type 1 and 2 (HTLV-1 and HTLV-2) and bovine leukaemia virus (BLV). PP2A is a heterotrimer composed of a scaffold, catalytic and one of any of four families of regulatory subunits, and the interaction is specific to the B′ family of the regulatory subunits. B′-PP2A and HTLV-1 IN display nuclear co-localization, and the B′ subunit stimulates concerted strand transfer activity of δ-retroviral INs in vitro. The protein–protein interaction interface maps to a patch of highly conserved residues on B′, which when mutated render B′ incapable of binding to and stimulating HTLV-1 and -2 IN strand transfer activity. PMID:26657642

  5. IA-2, a transmembrane protein of the protein tyrosine phosphatase family, is a major autoantigen in insulin-dependent diabetes mellitus.

    Lan, M S; Wasserfall, C; Maclaren, N K; Notkins, A L

    1996-01-01

    IA-2 is a 105,847 Da transmembrane protein that belongs to the protein tyrosine phosphatase family. Immunoperoxidase staining with antibody raised against IA-2 showed that this protein is expressed in human pancreatic islet cells. In this study, we expressed the full-length cDNA clone of IA-2 in a rabbit reticulocyte transcription/translation system and used the recombinant radiolabeled IA-2 protein to detect autoantibodies by immunoprecipitation. Coded sera (100) were tested: 50 from patient...

  6. Protein phosphatases and chromatin modifying complexes in the inflammatory cascade in acute pancreatitis

    Javier Escobar

    2010-06-01

    Full Text Available Acute pancreatitis is an inflammation of the pancreas that may lead to systemic inflammatory response syndrome and death due to multiple organ failure. Acinar cells, together with leukocytes, trigger the inflammatory cascade in response to local damage of the pancreas. Amplification of the inflammatory cascade requires up-regulation of pro-inflammatory cytokines and this process is mediated not only by nuclear factor κB but also by chromatin modifying complexes and chromatin remodeling. Among the different families of histone acetyltransferases, the p300/CBP family seems to be particularly associated with the inflammatory process. cAMP activates gene expression via the cAMP-responsive element (CRE and the transcription factor CRE-binding protein (CREB. CREB can be phosphorylated and activated by different kinases, such as protein kinase A and MAPK, and then it recruits the histone acetyltransferase co-activator CREB-binding protein (CBP and its homologue p300. The recruitment of CBP/p300 and changes in the level of histone acetylation are required for transcription activation. Transcriptional repression is also a dynamic and essential mechanism of down-regulation of genes for resolution of inflammation, which seems to be mediated mainly by protein phosphatases (PP1, PP2A and MKP1 and histone deacetylases (HDACs. Class II HDACs are key transcriptional regulators whose activities are controlled via phosphorylation-dependent nucleo/cytoplasmic shuttling. PP2A is responsible for dephosphorylation of class II HDACs, triggering nuclear localization and repression of target genes, whereas phosphorylation triggers cytoplasmic localization leading to activation of target genes. The potential benefit from treatment with phosphodiesterase inhibitors and histone deacetylase inhibitors is discussed.

  7. Protein phosphatases and chromatin modifying complexes in the inflammatory cascade in acute pancreatitis

    Javier; Escobar; Javier; Pereda; Alessandro; Arduini; Juan; Sastre; Juan; Sandoval; Luis; Aparisi; Gerardo; López-Rodas; Luis; Sabater

    2010-01-01

    Acute pancreatitis is an inflammation of the pancreas that may lead to systemic inflammatory response syndrome and death due to multiple organ failure. Acinar cells, together with leukocytes, trigger the inflammatory cascade in response to local damage of the pancreas. Amplification of the inflammatory cascade requires up-regulation of proinflammatory cytokines and this process is mediated not only by nuclear factor κB but also by chromatinmodifying complexes and chromatin remodeling. Among the different families of histone acetyltransferases, the p300/CBP family seems to be particularly associated with the inflammatory process. cAMP activates gene expression via the cAMP-responsive element (CRE) and the transcription factor CRE-binding protein (CREB). CREB can be phosphorylated and activated by different kinases, such as protein kinase A and MAPK, and then it recruits the histone acetyltransferase co-activator CREB-binding protein (CBP) and its homologue p300. The recruitment of CBP/p300 and changes in the level of histone acetylation are required for transcription activation. Transcriptional repression is also a dynamic and essential mechanism of down-regulation of genes for resolution of inflammation, which seems to be mediated mainly by protein phosphatases (PP1, PP2A and MKP1) and histone deacetylases(HDACs) .Class HDACs are key transcriptional regulators whose activities are controlled via phosphorylationdependent nucleo/cytoplasmic shuttling. PP2A is responsible for dephosphorylation of class HDACs, triggeringnuclear localization and repression of target genes, whereas phosphorylation triggers cytoplasmic localization leading to activation of target genes. The potential benefit from treatment with phosphodiesterase inhibitors and histone deacetylase inhibitors is discussed.

  8. Phosphorylation of SET protein at Ser171 by protein kinase D2 diminishes its inhibitory effect on protein phosphatase 2A.

    Atsushi Irie

    Full Text Available We previously reported that protein kinase D2 (PKD2 in T cells is promptly activated after T-cell receptor (TCR stimulation and involved in the activation of interleukin-2 promoter and T cell death, and that one of its candidate substrate is SET protein, a natural inhibitor for protein phosphatase 2A (PP2A. In this study, we investigated the target amino acid residues of SET phosphorylated by PKD2 and the effects of phosphorylation of SET on PP2A phosphatase activity. In vitro kinase assay using various recombinant SET mutants having Ser/Thr to Ala substitutions revealed that Ser171 of SET is one of the sites phosphorylated by PKD2. Recombinant SET with phosphorylation-mimic Ser171 to Glu substitution reduced its inhibitory effects on PP2A phosphatase activity compared with Ser171 to Ala substituted or wild-type SET. In addition, knockdown of PKD2 in Jurkat cells by RNAi or treatment of human CD4(+ T cell clone with the PKD2 inhibitor Gö6976 resulted in reduced PP2A activity after TCR-stimulation judged from phosphorylation status of Tyr307 of the catalytic subunit of PP2A. These results suggest that PKD2 is involved in the regulation of PP2A activity in activated T cells through phosphorylation of Ser171 of SET.

  9. Expression of a truncated receptor protein tyrosine phosphatase kappa in the brain of an adult transgenic mouse

    Shen, P; Canoll, P D; Sap, J;

    1999-01-01

    Receptor protein tyrosine phosphatases (RPTPs) comprise a family of proteins that feature intracellular phosphatase domains and an ectodomain with putative ligand-binding motifs. Several RPTPs are expressed in the brain, including RPTP-kappa which participates in homophilic cell-cell interactions...... developmental processes such as axonal growth and target recognition, as has been demonstrated for certain Drosophila RPTPs. The brain distribution of RPTP-kappa-expressing cells has not been determined, however. In a gene-trap mouse model with a beta-gal+neo (beta-geo) insertion in the endogenous RPTP......-kappa gene, the consequent loss of RPTP-kappa's enzymatic activity does not produce any obvious phenotypic defects [W.C. Skarnes, J.E. Moss, S.M. Hurtley, R.S.P. Beddington, Capturing genes encoding membrane and secreted proteins important for mouse development, Proc. Natl. Acad. Sci. U.S.A. 92 (1995) 6592...

  10. Pancreatic Protein Tyrosine Phosphatase 1B Deficiency Exacerbates Acute Pancreatitis in Mice.

    Bettaieb, Ahmed; Koike, Shinichiro; Chahed, Samah; Bachaalany, Santana; Griffey, Stephen; Sastre, Juan; Haj, Fawaz G

    2016-08-01

    Acute pancreatitis (AP) is a common and devastating gastrointestinal disorder that causes significant morbidity. The disease starts as local inflammation in the pancreas that may progress to systemic inflammation and complications. Protein tyrosine phosphatase 1B (PTP1B) is implicated in inflammatory signaling, but its significance in AP remains unclear. To investigate whether PTP1B may have a role in AP, we used pancreas PTP1B knockout (panc-PTP1B KO) mice and determined the effects of pancreatic PTP1B deficiency on cerulein- and arginine-induced acute pancreatitis. We report that PTP1B protein expression was increased in the early phase of AP in mice and rats. In addition, histological analyses of pancreas samples revealed enhanced features of AP in cerulein-treated panc-PTP1B KO mice compared with controls. Moreover, cerulein- and arginine-induced serum amylase and lipase were significantly higher in panc-PTP1B KO mice compared with controls. Similarly, pancreatic mRNA and serum concentrations of the inflammatory cytokines IL-1B, IL-6, and tumor necrosis factor-α were increased in panc-PTP1B KO mice compared with controls. Furthermore, panc-PTP1B KO mice exhibited enhanced cerulein- and arginine-induced NF-κB inflammatory response accompanied with increased mitogen-activated protein kinases activation and elevated endoplasmic reticulum stress. Notably, these effects were recapitulated in acinar cells treated with a pharmacological inhibitor of PTP1B. These findings reveal a novel role for pancreatic PTP1B in cerulein- and arginine-induced acute pancreatitis. PMID:27461362

  11. Screening of a Library of FDA-Approved Drugs Identifies Several Enterovirus Replication Inhibitors That Target Viral Protein 2C.

    Ulferts, Rachel; de Boer, S Matthijn; van der Linden, Lonneke; Bauer, Lisa; Lyoo, Hey Rhyoung; Maté, Maria J; Lichière, Julie; Canard, Bruno; Lelieveld, Daphne; Omta, Wienand; Egan, David; Coutard, Bruno; van Kuppeveld, Frank J M

    2016-05-01

    Enteroviruses (EVs) represent many important pathogens of humans. Unfortunately, no antiviral compounds currently exist to treat infections with these viruses. We screened the Prestwick Chemical Library, a library of approved drugs, for inhibitors of coxsackievirus B3, identified pirlindole as a potent novel inhibitor, and confirmed the inhibitory action of dibucaine, zuclopenthixol, fluoxetine, and formoterol. Upon testing of viruses of several EV species, we found that dibucaine and pirlindole inhibited EV-B and EV-D and that dibucaine also inhibited EV-A, but none of them inhibited EV-C or rhinoviruses (RVs). In contrast, formoterol inhibited all enteroviruses and rhinoviruses tested. All compounds acted through the inhibition of genome replication. Mutations in the coding sequence of the coxsackievirus B3 (CV-B3) 2C protein conferred resistance to dibucaine, pirlindole, and zuclopenthixol but not formoterol, suggesting that 2C is the target for this set of compounds. Importantly, dibucaine bound to CV-B3 protein 2C in vitro, whereas binding to a 2C protein carrying the resistance mutations was reduced, providing an explanation for how resistance is acquired. PMID:26856848

  12. Molecular dynamics simulations of interaction between protein-tyrosine phosphatase 1B and a bidentate inhibitor

    Gui-xia LIU; Jin-zhi TAN; Chun-ying NIU; Jian-hua SHEN; Xiao-min LUO; Xu SHEN; Kai-xian CHEN; Hua-liang JIANG

    2006-01-01

    Aim: To investigate the dynamic properties of protein-tyrosine phosphatase (PTP)1B and reveal the structural factors responsible for the high inhibitory potency and selectivity of the inhibitor SNA for PTP1B. Methods: We performed molecular dynamics (MD) simulations using a long time-scale for both PTP1B and PTP1B complexed with the inhibitor SNA, the most potent and selective PTP1B inhibitor reported to date. The trajectories were analyzed by using principal component analysis. Results: Trajectory analyses showed that upon binding the ligand, the flexibility of the entire PTP1B molecule decreases. The most notable change is the movement of the WPD-loop. Our simulation results also indicated that electrostatic interactions contribute more to PTP1B-SNA complex conformation than the van der Waals interactions, and that Lys41, Arg47, and Asp48 play important roles in determining the conformation of the inhibitor SNA and in the potency and selectivity of the inhibitor. Of these, Arg47 contributed most. These results were in agreement with previous experimental results. Conclusion: The information presented here suggests that potent and selective PTP1B inhibitors can be designed by targeting the surface residues, for example the region containing Lys41,Arg47, and Asp48, instead of the second phosphate binding site (besides the active phosphate binding site).

  13. NLRP3 tyrosine phosphorylation is controlled by protein tyrosine phosphatase PTPN22.

    Spalinger, Marianne R; Kasper, Stephanie; Gottier, Claudia; Lang, Silvia; Atrott, Kirstin; Vavricka, Stephan R; Scharl, Sylvie; Gutte, Petrus M; Grütter, Markus G; Beer, Hans-Dietmar; Contassot, Emmanuel; Chan, Andrew C; Dai, Xuezhi; Rawlings, David J; Mair, Florian; Becher, Burkhard; Falk, Werner; Fried, Michael; Rogler, Gerhard; Scharl, Michael

    2016-05-01

    Inflammasomes form as the result of the intracellular presence of danger-associated molecular patterns and mediate the release of active IL-1β, which influences a variety of inflammatory responses. Excessive inflammasome activation results in severe inflammatory conditions, but physiological IL-1β secretion is necessary for intestinal homeostasis. Here, we have described a mechanism of NLRP3 inflammasome regulation by tyrosine phosphorylation of NLRP3 at Tyr861. We demonstrated that protein tyrosine phosphatase non-receptor 22 (PTPN22), variants in which are associated with chronic inflammatory disorders, dephosphorylates NLRP3 upon inflammasome induction, allowing efficient NLRP3 activation and subsequent IL-1β release. In murine models, PTPN22 deficiency resulted in pronounced colitis, increased NLRP3 phosphorylation, but reduced levels of mature IL-1β. Conversely, patients with inflammatory bowel disease (IBD) that carried an autoimmunity-associated PTPN22 variant had increased IL-1β levels. Together, our results identify tyrosine phosphorylation as an important regulatory mechanism for NLRP3 that prevents aberrant inflammasome activation. PMID:27043286

  14. Protein tyrosine phosphatase non-receptor type 2 and inflammatory bowel disease.

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2016-01-21

    Genome wide association studies have associated single nucleotide polymorphisms within the gene locus encoding protein tyrosine phosphatase non-receptor type 2 (PTPN2) with the onset of inflammatory bowel disease (IBD) and other inflammatory disorders. Expression of PTPN2 is enhanced in actively inflamed intestinal tissue featuring a marked up-regulation in intestinal epithelial cells. PTPN2 deficient mice suffer from severe intestinal and systemic inflammation and display aberrant innate and adaptive immune responses. In particular, PTPN2 is involved in the regulation of inflammatory signalling cascades, and critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses, and finally for maintaining intestinal homeostasis. On one hand, dysfunction of PTPN2 has drastic effects on innate host defence mechanisms, including increased secretion of pro-inflammatory cytokines, limited autophagosome formation in response to invading pathogens, and disruption of the intestinal epithelial barrier. On the other hand, PTPN2 function is crucial for controlling adaptive immune functions, by regulating T cell proliferation and differentiation as well as maintaining T cell tolerance. In this way, dysfunction of PTPN2 contributes to the manifestation of IBD. The aim of this review is to present an overview of recent findings on the role of PTPN2 in intestinal homeostasis and the impact of dysfunctional PTPN2 on intestinal inflammation. PMID:26811645

  15. Role of Receptor Protein Tyrosine Phosphatase γ in Sensing Extracellular CO2 and HCO3.

    Zhou, Yuehan; Skelton, Lara A; Xu, Lumei; Chandler, Margaret P; Berthiaume, Jessica M; Boron, Walter F

    2016-09-01

    Regulation of blood pH-critical for virtually every facet of life-requires that the renal proximal tubule (PT) adjust its rate of H(+) secretion (nearly the same as the rate of HCO3 (-) reabsorption, JHCO3 ) in response to changes in blood [CO2] and [HCO3 (-)]. Yet CO2/HCO3 (-) sensing mechanisms remain poorly characterized. Because receptor tyrosine kinase inhibitors render JHCO3 in the PT insensitive to changes in CO2 concentration, we hypothesized that the structural features of receptor protein tyrosine phosphatase-γ (RPTPγ) that are consistent with binding of extracellular CO2 or HCO3 (-) facilitate monitoring of blood CO2/HCO3 (-) concentrations. We now report that PTs express RPTPγ on blood-facing membranes. Moreover, RPTPγ deletion in mice eliminated the CO2 and HCO3 (-) sensitivities of JHCO3 as well as the normal defense of blood pH during whole-body acidosis. Thus, RPTPγ appears to be a novel extracellular CO2/HCO3 (-) sensor critical for pH homeostasis. PMID:26839367

  16. A Novel Molecular Diagnostic of Glioblastomas: Detection of an Extracellular Fragment of Protein Tyrosine Phosphatase μ

    Susan M. Burden-Gulley

    2010-04-01

    Full Text Available We recently found that normal human brain and low-grade astrocytomas express the receptor protein tyrosine phosphatase mu (PTPμ and that the more invasive astrocytomas, glioblastoma multiforme (GBM, downregulate full-length PTPμ expression. Loss of PTPμ expression in GBMs is due to proteolytic cleavage that generates an intracellular and potentially a cleaved and released extracellular fragment of PTPμ. Here, we identify that a cleaved extracellular fragment containing the domains required for PTPμ-mediated adhesion remains associated with GBM tumor tissue. We hypothesized that detection of this fragment would make an excellent diagnostic tool for the localization of tumor tissue within the brain. To this end, we generated a series of fluorescently tagged peptide probes that bind the PTPμ fragment. The peptide probes specifically recognize GBM cells in tissue sections of surgically resected human tumors. To test whether the peptide probes are able to detect GBM tumors in vivo, the PTPμ peptide probes were tested in both mouse flank and intracranial xenograft human glioblastoma tumor model systems. The glial tumors were molecularly labeled with the PTPμ peptide probes within minutes of tail vein injection using the Maestro FLEX In Vivo Imaging System. The label was stable for at least 3 hours. Together, these results indicate that peptide recognition of the PTPμ extracellular fragment provides a novel molecular diagnostic tool for detection of human glioblastomas. Such a tool has clear translational applications and may lead to improved surgical resections and prognosis for patients with this devastating disease.

  17. Oleanane triterpenes as protein tyrosine phosphatase 1B (PTP1B) inhibitors from Camellia japonica.

    Uddin, Mohammad Nasir; Sharma, Govinda; Yang, Jun-Li; Choi, Hong Seok; Lim, Seong-Il; Kang, Keon Wook; Oh, Won Keun

    2014-07-01

    Protein tyrosine phosphatase 1B (PTP1B) plays a key role in metabolic signaling, thereby making it an exciting drug target for type 2 diabetes and obesity. Besides, there is substantial evidence that shows its overexpression is involved in breast cancer, which suggests that selective PTP1B inhibition might be effective in breast cancer treatment. As part of our continuous research on PTP1B inhibitors from medicinal plants, four oleanane-type triterpenes were isolated from an EtOAc-soluble extract of fruit peels of Camellia japonica (Theaceae), together with 6 previously known compounds of this class. Their structures were determined on the basis of spectroscopic data analysis (UV, IR, (1)H and (13)CNMR, HMBC, HSQC, NOESY, and MS). All isolates were evaluated for their inhibitory effects on PTP1B, as well as their cytotoxic effects against human breast cancer cell lines MCF7, MCF7/ADR, and MDA-MB-231. Several compounds with OH-3 or/and COOH-28 functionalities showed strong PTP1B inhibitory activity (IC50 values ranging from 3.77±0.11 to 6.40±0.81 μM) as well as significant cytotoxicity (IC50 values ranging from 0.51±0.05 to 13.55±1.44 μM). PMID:24815008

  18. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress

    Shiori Sekine

    2016-03-01

    Full Text Available Phosphoglycerate mutase family member 5 (PGAM5 is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT, a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21 that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria.

  19. The Ablation of Mitochondrial Protein Phosphatase Pgam5 Confers Resistance Against Metabolic Stress.

    Sekine, Shiori; Yao, Akari; Hattori, Kazuki; Sugawara, Sho; Naguro, Isao; Koike, Masato; Uchiyama, Yasuo; Takeda, Kohsuke; Ichijo, Hidenori

    2016-03-01

    Phosphoglycerate mutase family member 5 (PGAM5) is a mitochondrial protein phosphatase that has been reported to be involved in various stress responses from mitochondrial quality control to cell death. However, its roles in vivo are largely unknown. Here, we show that Pgam5-deficient mice are resistant to several metabolic insults. Under cold stress combined with fasting, Pgam5-deficient mice better maintained body temperature than wild-type mice and showed an extended survival rate. Serum triglycerides and lipid content in brown adipose tissue (BAT), a center of adaptive thermogenesis, were severely reduced in Pgam5-deficient mice. Moreover, although Pgam5 deficiency failed to maintain proper mitochondrial integrity in BAT, it reciprocally resulted in the dramatic induction of fibroblast growth factor 21 (FGF21) that activates various functions of BAT including thermogenesis. Thus, the enhancement of lipid metabolism and FGF21 may contribute to the cold resistance of Pgam5-deficient mice under fasting condition. Finally, we also found that Pgam5-deficient mice are resistant to high-fat-diet-induced obesity. Our study uncovered that PGAM5 is involved in the whole-body metabolism in response to stresses that impose metabolic challenges on mitochondria. PMID:27077115

  20. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    Kamolrat, Torkamol [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom); Gray, Stuart R., E-mail: s.r.gray@abdn.ac.uk [Musculoskeletal Research Programme, Institute of Medical Sciences, University of Aberdeen, AB25 2ZD (United Kingdom)

    2013-03-22

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using {sup 3}H-labelled phenylalanine. Protein breakdown was measured using {sup 3}H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion.

  1. The effect of eicosapentaenoic and docosahexaenoic acid on protein synthesis and breakdown in murine C2C12 myotubes

    Highlights: ► EPA can enhance protein synthesis and retard protein breakdown in muscle cells. ► These effects were concurrent with increases in p70s6k and FOXO3a phosphorylation. ► EPA may be a useful tool in the treatment of muscle wasting conditions. -- Abstract: Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been found to stimulate protein synthesis with little information regarding their effects on protein breakdown. Furthermore whether there are distinct effects of EPA and DHA remains to be established. The aim of the current study was to determine the distinct effects of EPA and DHA on protein synthesis, protein breakdown and signalling pathways in C2C12 myotubes. Fully differentiated C2C12 cells were incubated for 24 h with 0.1% ethanol (control), 50 μM EPA or 50 μM DHA prior to experimentation. After serum (4 h) and amino acid (1 h) starvation cells were stimulated with 2 mM L-leucine and protein synthesis measured using 3H-labelled phenylalanine. Protein breakdown was measured using 3H-labelled phenylalanine and signalling pathways (Akt, mTOR, p70S6k, 4EBP1, rps6 and FOXO3a) via Western blots. Data revealed that after incubation with EPA protein synthesis was 25% greater (P < 0.05) compared to control cells, with no effect of DHA. Protein breakdown was 22% (P < 0.05) lower, compared to control cells, after incubation with EPA, with no effect of DHA. Analysis of signalling pathways revealed that both EPA and DHA incubation increased (P < 0.05) p70s6k phosphorylation, EPA increased (P < 0.05) FOXO3a phosphorylation, with no alteration in other signalling proteins. The current study has demonstrated distinct effects of EPA and DHA on protein metabolism with EPA showing a greater ability to result in skeletal muscle protein accretion

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

    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

  3. Heart failure-inducible gene therapy targeting protein phosphatase 1 prevents progressive left ventricular remodeling.

    Yosuke Miyazaki

    Full Text Available BACKGROUND: The targeting of Ca(2+ cycling has emerged as a potential therapy for the treatment of severe heart failure. These approaches include gene therapy directed at overexpressing sarcoplasmic reticulum (SR Ca(2+ ATPase, or ablation of phospholamban (PLN and associated protein phosphatase 1 (PP1 protein complexes. We previously reported that PP1β, one of the PP1 catalytic subunits, predominantly suppresses Ca(2+ uptake in the SR among the three PP1 isoforms, thereby contributing to Ca(2+ downregulation in failing hearts. In the present study, we investigated whether heart-failure-inducible PP1β-inhibition by adeno-associated viral-9 (AAV9 vector mediated gene therapy is beneficial for preventing disease progression in genetic cardiomyopathic mice. METHODS: We created an adeno-associated virus 9 (AAV9 vector encoding PP1β short-hairpin RNA (shRNA or negative control (NC shRNA. A heart failure inducible gene expression system was employed using the B-type natriuretic protein (BNP promoter conjugated to emerald-green fluorescence protein (EmGFP and the shRNA sequence. AAV9 vectors (AAV9-BNP-EmGFP-PP1βshRNA and AAV9-BNP-EmGFP-NCshRNA were injected into the tail vein (2×10(11 GC/mouse of muscle LIM protein deficient mice (MLPKO, followed by serial analysis of echocardiography, hemodynamic measurement, biochemical and histological analysis at 3 months. RESULTS: In the MLPKO mice, BNP promoter activity was shown to be increased by detecting both EmGFP expression and the induced reduction of PP1β by 25% in the myocardium. Inducible PP1βshRNA delivery preferentially ameliorated left ventricular diastolic function and mitigated adverse ventricular remodeling. PLN phosphorylation was significantly augmented in the AAV9-BNP-EmGFP-PP1βshRNA injected hearts compared with the AAV9-BNP-EmGFP-NCshRNA group. Furthermore, BNP production was reduced, and cardiac interstitial fibrosis was abrogated at 3 months. CONCLUSION: Heart failure

  4. Tau pathology involves protein phosphatase 2A in parkinsonism-dementia of Guam.

    Arif, Mohammad; Kazim, Syed Faraz; Grundke-Iqbal, Inge; Garruto, Ralph M; Iqbal, Khalid

    2014-01-21

    Parkinsonism-dementia (PD) of Guam is a neurodegenerative disease with parkinsonism and early-onset Alzheimer-like dementia associated with neurofibrillary tangles composed of hyperphosphorylated microtubule-associated protein, tau. β-N-methylamino-l-alanine (BMAA) has been suspected of being involved in the etiology of PD, but the mechanism by which BMAA leads to tau hyperphosphorylation is not known. We found a decrease in protein phosphatase 2A (PP2A) activity associated with an increase in inhibitory phosphorylation of its catalytic subunit PP2Ac at Tyr(307) and abnormal hyperphosphorylation of tau in brains of patients who had Guam PD. To test the possible involvement of BMAA in the etiopathogenesis of PD, we studied the effect of this environmental neurotoxin on PP2A activity and tau hyperphosphorylation in mouse primary neuronal cultures and metabolically active rat brain slices. BMAA treatment significantly decreased PP2A activity, with a concomitant increase in tau kinase activity resulting in elevated tau hyperphosphorylation at PP2A favorable sites. Moreover, we found an increase in the phosphorylation of PP2Ac at Tyr(307) in BMAA-treated rat brains. Pretreatment with metabotropic glutamate receptor 5 (mGluR5) and Src antagonists blocked the BMAA-induced inhibition of PP2A and the abnormal hyperphosphorylation of tau, indicating the involvement of an Src-dependent PP2A pathway. Coimmunoprecipitation experiments showed that BMAA treatment dissociated PP2Ac from mGluR5, making it available for phosphorylation at Tyr(307). These findings suggest a scenario in which BMAA can lead to tau pathology by inhibiting PP2A through the activation of mGluR5, the consequent release of PP2Ac from the mGluR5-PP2A complex, and its phosphorylation at Tyr(307) by Src. PMID:24395787

  5. Coumarins from Angelica decursiva inhibit α-glucosidase activity and protein tyrosine phosphatase 1B.

    Ali, Md Yousof; Jannat, Susoma; Jung, Hyun Ah; Jeong, Hyong Oh; Chung, Hae Young; Choi, Jae Sue

    2016-05-25

    In the present study, we investigated the anti-diabetic potential of six natural coumarins, 4-hydroxy Pd-C-III (1), 4'-methoxy Pd-C-I (2), decursinol (3), decursidin (4), umbelliferone 6-carboxylic acid (5), and 2'-isopropyl psoralene (6) isolated from Angelica decursiva and evaluated their inhibitory activities against protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, and ONOO(-)-mediated protein tyrosine nitration. Coumarins 1-6 showed potent PTP1B and α-glucosidase inhibitory activities with ranges of IC50 values of 5.39-58.90 μM and 65.29-172.10 μM, respectively. In the kinetic study for PTP1B enzyme inhibition, compounds 1, 5, and 6 were competitive, whereas 2 and 4 showed mixed type, and 3 displayed noncompetitive type inhibition. For α-glucosidase enzyme inhibition, compounds 1 and 3 exhibited good mixed-type, while 2, 5, and 6 showed noncompetitive and 4 displayed competitive type inhibition. Furthermore, these coumarins also effectively suppressed ONOO(-)-mediated tyrosine nitration in a dose-dependent manner. To further investigate PTP1B inhibition, we generated a 3D structure of PTP1B using Autodock 4.2 and simulated the binding of compounds 1-6. Docking simulations showed that different residues of PTP1B interacted with different functional groups of compounds 1-6 through hydrogen and hydrophobic interactions. In addition, the binding energies of compounds 1-6 were negative, suggesting that hydrogen bonding may stabilize the open form of the enzyme and potentiate tight binding of the active site of PTP1B, thereby resulting in more effective PTP1B inhibition. These results demonstrate that the whole plant of A. decursiva and its coumarins are useful as potential functional food ingredients for the prevention and treatment of type 2 diabetes. PMID:27085377

  6. Role of protein tyrosine phosphatases in regulating the immune system: implications for chronic intestinal inflammation.

    Spalinger, Marianne R; McCole, Declan F; Rogler, Gerhard; Scharl, Michael

    2015-03-01

    Current hypothesis suggests that genetic, immunological, and bacterial factors contribute essentially to the pathogenesis of inflammatory bowel disease. Variations within the gene loci encoding protein tyrosine phosphatases (PTPs) have been associated with the onset of inflammatory bowel disease. PTPs modulate the activity of their substrates by dephosphorylation of tyrosine residues and are critical for the regulation of fundamental cellular signaling processes. Evidence emerges that expression levels of PTPN2, PTPN11, and PTPN22 are altered in actively inflamed intestinal tissue. PTPN2 seems to be critical for protecting intestinal epithelial barrier function, regulating innate and adaptive immune responses and finally for maintaining intestinal homeostasis. These observations have been confirmed in PTPN2 knockout mice in vivo. Those animals are clearly more susceptible to intestinal and systemic inflammation and feature alterations in innate and adaptive immune responses. PTPN22 controls inflammatory signaling in lymphocytes and mononuclear cells resulting in aberrant cytokine secretion pattern and autophagosome formation. PTPN22 deficiency in vivo results in more severe colitis demonstrating the relevance of PTPN22 for intestinal homeostasis in vivo. Of note, loss of PTPN22 promotes mitogen-activated protein kinase-induced cytokine secretion but limits secretion of nuclear factor κB-associated cytokines and autophagy in mononuclear cells. Loss of PTPN11 is also associated with increased colitis severity in vivo. In summary, dysfunction of those PTPs results in aberrant and uncontrolled immune responses that result in chronic inflammatory conditions. This way, it becomes more and more evident that dysfunction of PTPs displays an important factor in the pathogenesis of chronic intestinal inflammation, in particular inflammatory bowel disease. PMID:25581833

  7. Identification of genomic regions that interact with a viable allele of the Drosophila protein tyrosine phosphatase corkscrew.

    Firth, L; Manchester, J; Lorenzen, J A; Baron, M.; Perkins, L A

    2000-01-01

    Signaling by receptor tyrosine kinases (RTKs) is critical for a multitude of developmental decisions and processes. Among the molecules known to transduce the RTK-generated signal is the nonreceptor protein tyrosine phosphatase Corkscrew (Csw). Previously, Csw has been demonstrated to function throughout the Drosophila life cycle and, among the RTKs tested, Csw is essential in the Torso, Sevenless, EGF, and Breathless/FGF RTK pathways. While the biochemical function of Csw remains to be unamb...

  8. The protein phosphatase PhzA of A. fumigatus is involved in oxidative stress tolerance and fungal virulence

    Muszkieta, Laetitia; de Jesus Carrion, Steven; Robinet, Pauline; Beau, Rémi; Elbim, Carole; Pearlman, Eric; Latgé, Jean-Paul

    2014-01-01

    Protein phosphatases Z that are unique to the fungal kingdom have been associated to resistance to high salt concentration, cell wall integrity, cell cycle regulation, and oxidative stress in fungi. In Aspergillus fumigatus it was shown that PHZA is under the control of the transcription factor Skn7 and is only involved in the control of the oxidative stress. Accordingly, the ΔphzA mutant showed a defect in virulence in an experimental model of corneal infection in immuno...

  9. Homo sapiens Dullard Protein Phosphatase Shows Preference Toward Insulin-dependent Phosphorylation Site of Lipin1†

    Wu, Rui; Garland, Megan; Dunaway-Mariano, Debra; Allen, Karen N.

    2011-01-01

    Human lipin1 catalyzes the highly regulated conversion of phosphatidic acids to diacylglycerides. Lipin’s cellular location, protein partners, and biological function are directed by phosphorylation/dephosphorylation events catalyzed by the phosphoserine phosphatase dullard. To define the determinants of dullard substrate recognition and catalysis, and hence, lipin regulation, steady-state kinetic analysis was performed on phosphoserine-bearing nonapeptides based on the phosphorylation sites ...

  10. Functional dissection of T. brucei Protein Tyrosine Phosphatase 1 and investigation of its development as a therapeutic target

    Ruberto, Irene

    2011-01-01

    Trypanosoma brucei undergoes developmentally regulated morphological and biochemical changes during its life cycle, being transmitted between the mammalian host and the tsetse fly. It is generally recognized that cellular responses to environmental changes are mediated through signalling pathways, but our understanding of trypanosome signal transduction during differentiation is limited. Protein Tyrosine Phosphatase 1 (TbPTP1) is the one of the few factors identified to b...

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

    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.

    2014-01-01

    Recent evidence points to a role for Rif1 (Rap1-interacting factor) in DNA replication and genomic stability; however, the mechanism by which Rif1 functions has remained elusive. Hiraga el al. now report that budding yeast Rif1 controls DNA replication genome-wide and describe how Rif1 opposes Dbf4-dependent kinase (DDK) function by directing Protein Phosphatase 1 (PP1)-mediated dephosphorylation of the MCM complex. PP1 interaction sites are evolutionarily conserved within the Rif1 sequence; ...

  12. Force-inhibiting effect of Ser/Thr protein phosphatase 2A inhibitors on bovine ciliary muscle.

    石田, 美織

    2015-01-01

    Ciliary muscle is a smooth muscle characterized by a rapid response to muscarinic receptor stimulation and sustained contraction. Although it is evident that these contractions are Ca(2+)-dependent, detailed molecular mechanisms are still unknown. In order to elucidate the role of Ser/Thr protein phosphatase 2A (PP2A) in ciliary muscle contraction, we examined the effects of okadaic acid and other PP2A inhibitors on contractions induced by carbachol (CCh) and ionomycin in bovine ciliary muscl...

  13. SRK2C, a SNF1-related protein kinase 2, improves drought tolerance by controlling stress-responsive gene expression in Arabidopsis thaliana

    Umezawa, Taishi; Yoshida, Riichiro; Maruyama, Kyonoshin; Yamaguchi-Shinozaki, Kazuko; Shinozaki, Kazuo

    2004-01-01

    Protein phosphorylation/dephosphorylation are major signaling events induced by osmotic stress in higher plants. Here, we showed that a SNF1-related protein kinase 2 (SnRK2), SRK2C, is an osmotic-stress-activated protein kinase in Arabidopsis thaliana that can significantly impact drought tolerance of Arabidopsis plants. Knockout mutants of SRK2C exhibited drought hypersensitivity in their roots, suggesting that SRK2C is a positive regulator of drought tolerance in Arabidopsis roots. Addition...

  14. Protein phosphatases PP2A, PP4 and PP6: mediators and regulators in development and responses to environmental cues.

    Lillo, Cathrine; Kataya, Amr R A; Heidari, Behzad; Creighton, Maria T; Nemie-Feyissa, Dugassa; Ginbot, Zekarias; Jonassen, Else M

    2014-12-01

    The three closely related groups of serine/threonine protein phosphatases PP2A, PP4 and PP6 are conserved throughout eukaryotes. The catalytic subunits are present in trimeric and dimeric complexes with scaffolding and regulatory subunits that control activity and confer substrate specificity to the protein phosphatases. In Arabidopsis, three scaffolding (A subunits) and 17 regulatory (B subunits) proteins form complexes with five PP2A catalytic subunits giving up to 255 possible combinations. Three SAP-domain proteins act as regulatory subunits of PP6. Based on sequence similarities with proteins in yeast and mammals, two putative PP4 regulatory subunits are recognized in Arabidopsis. Recent breakthroughs have been made concerning the functions of some of the PP2A and PP6 regulatory subunits, for example the FASS/TON2 in regulation of the cellular skeleton, B' subunits in brassinosteroid signalling and SAL proteins in regulation of auxin transport. Reverse genetics is starting to reveal also many more physiological functions of other subunits. A system with key regulatory proteins (TAP46, TIP41, PTPA, LCMT1, PME-1) is present in all eukaryotes to stabilize, activate and inactivate the catalytic subunits. In this review, we present the status of knowledge concerning physiological functions of PP2A, PP4 and PP6 in Arabidopsis, and relate these to yeast and mammals. PMID:24810976

  15. Protein phosphatase 2A regulatory subunits perform distinct functional roles in the maize pathogen Fusarium verticillioides.

    Shin, Joon-Hee; Kim, Jung-Eun; Malapi-Wight, Martha; Choi, Yoon-E; Shaw, Brian D; Shim, Won-Bo

    2013-06-01

    Fusarium verticillioides is a pathogen of maize causing ear rot and stalk rot. The fungus also produces fumonisins, a group of mycotoxins linked to disorders in animals and humans. A cluster of genes, designated FUM genes, plays a key role in the synthesis of fumonisins. However, our understanding of the regulatory mechanism of fumonisin biosynthesis is still incomplete. We have demonstrated previously that Cpp1, a protein phosphatase type 2A (PP2A) catalytic subunit, negatively regulates fumonisin production and is involved in cell shape maintenance. In general, three PP2A subunits, structural A, regulatory B and catalytic C, make up a heterotrimer complex to perform regulatory functions. Significantly, we identified two PP2A regulatory subunits in the F. verticillioides genome, Ppr1 and Ppr2, which are homologous to Saccharomyces cerevisiae Cdc55 and Rts1, respectively. In this study, we hypothesized that Ppr1 and Ppr2 are involved in the regulation of fumonisin biosynthesis and/or cell development in F. verticillioides, and generated a series of mutants to determine the functional role of Ppr1 and Ppr2. The PPR1 deletion strain (Δppr1) resulted in drastic growth defects, but increased microconidia production. The PPR2 deletion mutant strain (Δppr2) showed elevated fumonisin production, similar to the Δcpp1 strain. Germinating Δppr1 conidia formed abnormally swollen cells with a central septation site, whereas Δppr2 showed early hyphal branching during conidia germination. A kernel rot assay showed that the mutants were slow to colonize kernels, but this is probably a result of growth defects rather than a virulence defect. Results from this study suggest that two PP2A regulatory subunits in F. verticillioides carry out distinct roles in the regulation of fumonisin biosynthesis and fungal development. PMID:23452277

  16. Nuclear Export and Centrosome Targeting of the Protein Phosphatase 2A Subunit B56α

    Flegg, Cameron P.; Sharma, Manisha; Medina-Palazon, Cahora; Jamieson, Cara; Galea, Melanie; Brocardo, Mariana G.; Mills, Kate; Henderson, Beric R.

    2010-01-01

    Protein phosphatase (PP) 2A is a heterotrimeric enzyme regulated by specific subunits. The B56 (or B′/PR61/PPP2R5) class of B-subunits direct PP2A or its substrates to different cellular locations, and the B56α, -β, and -ϵ isoforms are known to localize primarily in the cytoplasm. Here we studied the pathways that regulate B56α subcellular localization. We detected B56α in the cytoplasm and nucleus, and at the nuclear envelope and centrosomes, and show that cytoplasmic localization is dependent on CRM1-mediated nuclear export. The inactivation of CRM1 by leptomycin B or by siRNA knockdown caused nuclear accumulation of ectopic and endogenous B56α. Conversely, CRM1 overexpression shifted B56α to the cytoplasm. We identified a functional nuclear export signal at the C terminus (NES; amino acids 451–469), and site-directed mutagenesis of the NES (L461A) caused nuclear retention of full-length B56α. Active NESs were identified at similar positions in the cytoplasmic B56-β and ϵ isoforms, but not in the nuclear-localized B56-δ or γ isoforms. The transient expression of B56α induced nuclear export of the PP2A catalytic (C) subunit, and this was blocked by the L461A NES mutation. In addition, B56α co-located with the PP2A active (A) subunit at centrosomes, and its centrosome targeting involved sequences that bind to the A-subunit. Fluorescence Recovery after Photobleaching (FRAP) assays revealed dynamic and immobile pools of B56α-GFP, which was rapidly exported from the nucleus and subject to retention at centrosomes. We propose that B56α can act as a PP2A C-subunit chaperone and regulates PP2A activity at diverse subcellular locations. PMID:20378546

  17. Regulation of brown fat adipogenesis by protein tyrosine phosphatase 1B.

    Kosuke Matsuo

    Full Text Available Protein-tyrosine phosphatase 1B (PTP1B is a physiological regulator of insulin signaling and energy balance, but its role in brown fat adipogenesis requires additional investigation.To precisely determine the role of PTP1B in adipogenesis, we established preadipocyte cell lines from wild type and PTP1B knockout (KO mice. In addition, we reconstituted KO cells with wild type, substrate-trapping (D/A and sumoylation-resistant (K/R PTP1B mutants, then characterized differentiation and signaling in these cells. KO, D/A- and WT-reconstituted cells fully differentiated into mature adipocytes with KO and D/A cells exhibiting a trend for enhanced differentiation. In contrast, K/R cells exhibited marked attenuation in differentiation and lipid accumulation compared with WT cells. Expression of adipogenic markers PPARγ, C/EBPα, C/EBPδ, and PGC1α mirrored the differentiation pattern. In addition, the differentiation deficit in K/R cells could be reversed completely by the PPARγ activator troglitazone. PTP1B deficiency enhanced insulin receptor (IR and insulin receptor substrate 1 (IRS1 tyrosyl phosphorylation, while K/R cells exhibited attenuated insulin-induced IR and IRS1 phosphorylation and glucose uptake compared with WT cells. In addition, substrate-trapping studies revealed that IRS1 is a substrate for PTP1B in brown adipocytes. Moreover, KO, D/A and K/R cells exhibited elevated AMPK and ACC phosphorylation compared with WT cells.These data indicate that PTP1B is a modulator of brown fat adipogenesis and suggest that adipocyte differentiation requires regulated expression of PTP1B.

  18. Protein tyrosine phosphatase receptor delta acts as a neuroblastoma tumor suppressor by destabilizing the aurora kinase a oncogene

    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.

  19. Molecular dynamics simulations of protein-tyrosine phosphatase 1B: II. Substrate-enzyme interactions and dynamics

    Peters, Günther H.j.; Frimurer, T. M.; Andersen, J. N.; Olsen, O. H.

    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 and...... 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...

  20. Interaction of the Papillomavirus E8∧E2C Protein with the Cellular CHD6 Protein Contributes to Transcriptional Repression▿ †

    Fertey, Jasmin; Ammermann, Ingo; Winkler, Michael; Stöger, Reinhard; Iftner, Thomas; Stubenrauch, Frank

    2010-01-01

    Expression of the E6 and E7 oncogenes of high-risk human papillomaviruses (HPV) is controlled by cellular transcription factors and by viral E2 and E8∧E2C proteins, which are both derived from the HPV E2 gene. Both proteins bind to and repress the HPV E6/E7 promoter. Promoter inhibition has been suggested to be due to binding site competition with cellular transcription factors and to interactions of different cellular transcription modulators with the different amino termini of E2 and E8∧E2C...

  1. Analysis of Two Putative Candida albicans Phosphopantothenoylcysteine Decarboxylase / Protein Phosphatase Z Regulatory Subunits Reveals an Unexpected Distribution of Functional Roles

    Petrényi, Katalin; Molero, Cristina; Kónya, Zoltán; Erdődi, Ferenc; Ariño, Joaquin; Dombrádi, Viktor

    2016-01-01

    Protein phosphatase Z (Ppz) is a fungus specific enzyme that regulates cell wall integrity, cation homeostasis and oxidative stress response. Work on Saccharomyces cerevisiae has shown that the enzyme is inhibited by Hal3/Vhs3 moonlighting proteins that together with Cab3 constitute the essential phosphopantothenoylcysteine decarboxylase (PPCDC) enzyme. In Candida albicans CaPpz1 is also involved in the morphological changes and infectiveness of this opportunistic human pathogen. To reveal the CaPpz1 regulatory context we searched the C. albicans database and identified two genes that, based on the structure of their S. cerevisiae counterparts, were termed CaHal3 and CaCab3. By pull down analysis and phosphatase assays we demonstrated that both of the bacterially expressed recombinant proteins were able to bind and inhibit CaPpz1 as well as its C-terminal catalytic domain (CaPpz1-Cter) with comparable efficiency. The binding and inhibition were always more pronounced with CaPpz1-Cter, indicating a protective effect against inhibition by the N-terminal domain in the full length protein. The functions of the C. albicans proteins were tested by their overexpression in S. cerevisiae. Contrary to expectations we found that only CaCab3 and not CaHal3 rescued the phenotypic traits that are related to phosphatase inhibition by ScHal3, such as tolerance to LiCl or hygromycin B, requirement for external K+ concentrations, or growth in a MAP kinase deficient slt2 background. On the other hand, both of the Candida proteins turned out to be essential PPCDC components and behaved as their S. cerevisiae counterparts: expression of CaCab3 and CaHal3 rescued the cab3 and hal3 vhs3 S. cerevisiae mutations, respectively. Thus, both CaHal3 and CaCab3 retained the PPCDC related functions and have the potential for CaPpz1 inhibition in vitro. The fact that only CaCab3 exhibits its phosphatase regulatory potential in vivo suggests that in C. albicans CaCab3, but not CaHal3, acts as a

  2. Archaeal signal transduction: impact of protein phosphatase deletions on cell size, motility, and energy metabolism in Sulfolobus acidocaldarius.

    Reimann, Julia; Esser, Dominik; Orell, Alvaro; Amman, Fabian; Pham, Trong Khoa; Noirel, Josselin; Lindås, Ann-Christin; Bernander, Rolf; Wright, Phillip C; Siebers, Bettina; Albers, Sonja-Verena

    2013-12-01

    In this study, the in vitro and in vivo functions of the only two identified protein phosphatases, Saci-PTP and Saci-PP2A, in the crenarchaeal model organism Sulfolobus acidocaldarius were investigated. Biochemical characterization revealed that Saci-PTP is a dual-specific phosphatase (against pSer/pThr and pTyr), whereas Saci-PP2A exhibited specific pSer/pThr activity and inhibition by okadaic acid. Deletion of saci_pp2a resulted in pronounced alterations in growth, cell shape and cell size, which could be partially complemented. Transcriptome analysis of the three strains (Δsaci_ptp, Δsaci_pp2a and the MW001 parental strain) revealed 155 genes that were differentially expressed in the deletion mutants, and showed significant changes in expression of genes encoding the archaella (archaeal motility structure), components of the respiratory chain and transcriptional regulators. Phosphoproteome studies revealed 801 unique phosphoproteins in total, with an increase in identified phosphopeptides in the deletion mutants. Proteins from most functional categories were affected by phosphorylation, including components of the motility system, the respiratory chain, and regulatory proteins. In the saci_pp2a deletion mutant the up-regulation at the transcript level, as well as the observed phosphorylation pattern, resembled starvation stress responses. Hypermotility was also observed in the saci_pp2a deletion mutant. The results highlight the importance of protein phosphorylation in regulating essential cellular processes in the crenarchaeon S. acidocaldarius. PMID:24078887

  3. Characterization of Saccharomyces cerevisiae protein Ser/Thr phosphatase T1 and comparison to its mammalian homolog PP5

    Park Jung-Min

    2003-03-01

    Full Text Available Abstract Background Protein Ser/Thr phosphatase 5 (PP5 and its Saccharomyces cerevisiae homolog protein phosphatase T1 (Ppt1p each contain an N-terminal domain consisting of several tetratricopeptide repeats (TPRs and a C-terminal catalytic domain that is related to the catalytic subunits of protein phosphatases 1 and 2A, and calcineurin. Analysis of yeast Ppt1p could provide important clues to the function of PP5 and its homologs, however it has not yet been characterized at the biochemical or cellular level. Results The specific activity of recombinant Ppt1p toward the artificial substrates 32P-myelin basic protein (MBP and 32P-casein was similar to that of PP5. Dephosphorylation of 32P-MBP, but not 32P-casein, was stimulated by unsaturated fatty acids and by arachidoyl coenzyme A. Limited proteolysis of Ppt1p removed the TPR domain and abrogated lipid stimulation. The remaining catalytic fragment exhibited a two-fold increase in activity toward 32P-MBP, but not 32P-casein. Removal of the C terminus increased Ppt1p activity toward both substrates two fold, but did not prevent further stimulation of activity toward 32P-MBP by lipid treatment. Ppt1p was localized throughout the cell including the nucleus. Levels of PPT1 mRNA and protein peaked in early log phase growth. Conclusions Many characteristics of Ppt1p are similar to those of PP5, including stimulation of phosphatase activity with some substrates by lipids, and peak expression during periods of rapid cell growth. Unlike PP5, however, proteolytic removal of the TPR domain or C-terminal truncation only modestly increased its activity. In addition, C-terminal truncation did not prevent further activation by lipid. This suggests that these regions play only a minor role in controlling its activity compared to PP5. Ppt1p is present in both the nucleus and cytoplasm, indicating that it may function in multiple compartments. The observation that Ppt1p is most highly expressed during early log

  4. Cardiac sodium channel Na(v)1.5 interacts with and is regulated by the protein tyrosine phosphatase PTPH1

    Jespersen, Thomas; Gavillet, Bruno; van Bemmelen, Miguel X; Cordonier, Sophie; Thomas, Marc A; Staub, Olivier; Abriel, Hugues

    2006-01-01

    In order to identify proteins interacting with the cardiac voltage-gated sodium channel Na(v)1.5, we used the last 66 amino acids of the C-terminus of the channel as bait to screen a human cardiac cDNA library. We identified the protein tyrosine phosphatase PTPH1 as an interacting protein. Pull-d...

  5. Regulation of Platelet Derived Growth Factor Signaling by Leukocyte Common Antigen-related (LAR) Protein Tyrosine Phosphatase: A Quantitative Phosphoproteomics Study.

    Sarhan, Adil R; Patel, Trushar R; Creese, Andrew J; Tomlinson, Michael G; Hellberg, Carina; Heath, John K; Hotchin, Neil A; Cunningham, Debbie L

    2016-06-01

    Intracellular signaling pathways are reliant on protein phosphorylation events that are controlled by a balance of kinase and phosphatase activity. Although kinases have been extensively studied, the role of phosphatases in controlling specific cell signaling pathways has been less so. Leukocyte common antigen-related protein (LAR) is a member of the LAR subfamily of receptor-like protein tyrosine phosphatases (RPTPs). LAR is known to regulate the activity of a number of receptor tyrosine kinases, including platelet-derived growth factor receptor (PDGFR). To gain insight into the signaling pathways regulated by LAR, including those that are PDGF-dependent, we have carried out the first systematic analysis of LAR-regulated signal transduction using SILAC-based quantitative proteomic and phosphoproteomic techniques. We haveanalyzed differential phosphorylation between wild-type mouse embryo fibroblasts (MEFs) and MEFs in which the LAR cytoplasmic phosphatase domains had been deleted (LARΔP), and found a significant change in abundance of phosphorylation on 270 phosphosites from 205 proteins because of the absence of the phosphatase domains of LAR. Further investigation of specific LAR-dependent phosphorylation sites and enriched biological processes reveal that LAR phosphatase activity impacts on a variety of cellular processes, most notably regulation of the actin cytoskeleton. Analysis of putative upstream kinases that may play an intermediary role between LAR and the identified LAR-dependent phosphorylation events has revealed a role for LAR in regulating mTOR and JNK signaling. PMID:27074791

  6. Selective Serotonin Reuptake Inhibitor Fluoxetine Inhibits Replication of Human Enteroviruses B and D by Targeting Viral Protein 2C

    Ulferts, R.; van der Linden, L.; Thibaut, H.J.; Lanke, K. H. W.; Leyssen, P.; Coutard, B.; De Palma, A.M.; Canard, B; Neyts, J.; Van Kuppeveld, F. J. M.

    2013-01-01

    Although the genus Enterovirus contains many important human pathogens, there is no licensed drug for either the treatment or the prophylaxis of enterovirus infections. We report that fluoxetine (Prozac)-a selective serotonin reuptake inhibitor-inhibits the replication of human enterovirus B (HEV-B) and HEV-D but does not affect the replication of HEV-A and HEV-C or human rhinovirus A or B. We show that fluoxetine interferes with viral RNA replication, and we identified viral protein 2C as th...

  7. Organization and alternate splice products of the gene encoding nuclear inhibitor of protein phosphatase-1 (NIPP-1).

    Van Eynde, A; Pérez-Callejón, E; Schoenmakers, E; Jacquemin, M; Stalmans, W; Bollen, M

    1999-04-01

    Nuclear inhibitor of protein phosphatase-1 (NIPP-1) is one of two major regulatory subunits of protein phosphatase-1 in mammalian nuclei. We report here the cloning and structural characterization of the human NIPP-1 genes, designated PPP1R8P and PPP1R8 in human gene nomenclature. PPP1R8P (1.2 kb) is a processed pseudogene and was localized by in situ hybridization to chromosome 1p33-32. PPP1R8 is an authentic NIPP-1 gene and was localized to chromosome 1p35. PPP1R8 (25.2 kb) is composed of seven exons and encodes four different transcripts, as determined from cDNA library screening, reverse transcriptase-PCR (RT-PCR) and/or EST (expressed sequence tag) database search analysis. NIPP-1alpha mRNA represents the major transcript in human tissues and various cell lines, and encodes a polypeptide of 351 residues that only differs from the previously cloned calf thymus NIPP-1 by a single residue. The other transcripts, termed NIPP-1beta, gamma and delta, are generated by alternative 5'-splice site usage, by exon skipping and/or by alternative polyadenylation. The NIPP-1beta/delta and NIPP-1gamma mRNAs are expected to encode fragments of NIPP-1alpha that differ from the latter by the absence of the first 142 and 224 residues, respectively. NIPP-1gamma corresponds to 'activator of RNA decay-1' (Ard-1) which, unlike NIPP-1alpha, displays in vitro and endoribonuclease activity and lacks an RVXF consensus motif for interaction with protein phosphatase-1. While the NIPP-1alpha/beta/delta-transcripts were found to be present in various human tissues, the NIPP-1gamma transcript could only be detected in human transformed B-lymphocytes. PMID:10103062

  8. Applying a Targeted Label-free Approach using LC-MS AMT Tags to Evaluate Changes in Protein Phosphorylation Following Phosphatase Inhibition

    Yang, Feng; Jaitly, Navdeep; Jayachandran, Hemalatha; Lou, Quanzhou; Monroe, Matthew E.; Du, Xiuxia; Gritsenko, Marina A.; Zhang, Rui; Anderson, David J.; Purvine, Samuel O.; Adkins, Joshua N.; Moore, Ronald J.; Mottaz, Heather M.; Ding, Shi-Jian; Lipton, Mary S.; Camp, David G.; Udseth, Harold R.; Smith, Richard D.; Rossie, Sandra S.

    2007-10-12

    To identify phosphoproteins regulated by the phosphoprotein phosphatase (PPP) family of S/T phosphatases, we performed a large-scale characterization of changes in protein phosphorylation on extracts from HeLa cells treated with or without calyculin A, a potent PPP enzyme inhibitor. A label-free comparative Phosphoproteomics approach using immobilized metal ion affinity chromatography and targeted tandem mass spectrometry was employed to discover and identify signatures based upon distinctive changes in abundance. Overall, 232 proteins were identified as either direct or indirect targets for PPP enzyme regulation. Most of the present identifications represent novel PPP enzyme targets at the level of both phosphorylation site and protein. These include phosphorylation sites within signaling proteins such as p120 Catenin, A Kinase Anchoring Protein 8, JunB, and Type II Phosphatidyl Inositol 4 Kinase. These data can be used to define underlying signaling pathways and events regulated by the PPP family of S/T phosphatases.

  9. Improved Glucose Homeostasis in Mice with Muscle-Specific Deletion of Protein-Tyrosine Phosphatase 1B▿

    Delibegovic, Mirela; Bence, Kendra K.; Mody, Nimesh; Hong, Eun-Gyoung; Ko, Hwi Jin; Jason K Kim; Kahn, Barbara B.; Neel, Benjamin G

    2007-01-01

    Obesity and type 2 diabetes are characterized by insulin resistance. Mice lacking the protein-tyrosine phosphatase PTP1B in all tissues are hypersensitive to insulin but also have diminished fat stores. Because adiposity affects insulin sensitivity, the extent to which PTP1B directly regulates glucose homeostasis has been unclear. We report that mice lacking PTP1B only in muscle have body weight and adiposity comparable to those of controls on either chow or a high-fat diet (HFD). Muscle trig...

  10. Time dependent physiological characterization of yeast oxidative stress response and growth modulation of protein kinase/phosphatase mutants

    Altintas, Ali; Workman, Christopher

    mutants were selected for their known activities in various stress response pathways, including oxidative stress, and were investigated for their response to oxidative stress. Hydrogen peroxide was us ed as the oxidizing agent at a number of different concentrations ranging from mild to moderate stress (0......The objective of the project was to investigate the time-dependent batch growth effects of oxidative environmental conditions on protein ki nase (PK) and phosphatase (PP) deletion mutants and relevant wild type strains of Saccharomyces cerevisiae . To achieve this goal, 44 different PK and PP...

  11. Inhibitor-3 ensures bipolar mitotic spindle attachment by limiting association of SDS22 with kinetochore-bound protein phosphatase-1

    Eiteneuer, Annika; Seiler, Jonas; Weith, Matthias; Beullens, Monique; Lesage, Bart; Krenn, Veronica; Musacchio, Andrea; Bollen, Mathieu; Meyer, Hemmo

    2014-01-01

    Faithful chromosome segregation during mitosis is tightly regulated by opposing activities of Aurora B kinase and protein phosphatase-1 (PP1). PP1 function at kinetochores has been linked to SDS22, but the exact localization of SDS22 and how it affects PP1 are controversial. Here, we confirm that SDS22 is required for PP1 activity, but show that SDS22 does not normally localize to kinetochores. Instead, SDS22 is kept in solution by formation of a ternary complex with PP1 and inhibitor-3 (I3)....

  12. A Rapid Lateral Flow Immunoassay for the Detection of Tyrosine Phosphatase-Like Protein IA-2 Autoantibodies in Human Serum

    Ingrid Kikkas; Roberto Mallone; Etienne Larger; Hervé Volland; Nathalie Morel

    2014-01-01

    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...

  13. Electronic structural flexibility of heterobimetallic Mn/Fe cofactors: R2lox and R2c proteins.

    Shafaat, Hannah S; Griese, Julia J; Pantazis, Dimitrios A; Roos, Katarina; Andersson, Charlotta S; Popović-Bijelić, Ana; Gräslund, Astrid; Siegbahn, Per E M; Neese, Frank; Lubitz, Wolfgang; Högbom, Martin; Cox, Nicholas

    2014-09-24

    The electronic structure of the Mn/Fe cofactor identified in a new class of oxidases (R2lox) described by Andersson and Högbom [Proc. Natl. Acad. Sci. U.S.A. 2009, 106, 5633] is reported. The R2lox protein is homologous to the small subunit of class Ic ribonucleotide reductase (R2c) but has a completely different in vivo function. Using multifrequency EPR and related pulse techniques, it is shown that the cofactor of R2lox represents an antiferromagnetically coupled Mn(III)/Fe(III) dimer linked by a μ-hydroxo/bis-μ-carboxylato bridging network. The Mn(III) ion is coordinated by a single water ligand. The R2lox cofactor is photoactive, converting into a second form (R2loxPhoto) upon visible illumination at cryogenic temperatures (77 K) that completely decays upon warming. This second, unstable form of the cofactor more closely resembles the Mn(III)/Fe(III) cofactor seen in R2c. It is shown that the two forms of the R2lox cofactor differ primarily in terms of the local site geometry and electronic state of the Mn(III) ion, as best evidenced by a reorientation of its unique (55)Mn hyperfine axis. Analysis of the metal hyperfine tensors in combination with density functional theory (DFT) calculations suggests that this change is triggered by deprotonation of the μ-hydroxo bridge. These results have important consequences for the mixed-metal R2c cofactor and the divergent chemistry R2lox and R2c perform. PMID:25153930

  14. Characterization of an adapter subunit to a phosphatidylinositol (3)P 3-phosphatase: Identification of a myotubularin-related protein lacking catalytic activity

    Nandurkar, H. H.; Caldwell, K K; Whisstock, J C; Layton, M. J.; Gaudet, E. A.; Norris, F. A.; Majerus, P W; Mitchell, C. A.

    2001-01-01

    The D3-phosphoinositides act as second messengers by recruiting, and thereby activating, diverse signaling proteins. We have previously described the purification of a rat phosphatidylinositol 3-phosphate [PtdIns(3)P] 3-phosphatase, comprising a heterodimer of a 78-kDa adapter subunit in complex with a 65-kDa catalytic subunit. Here, we have cloned and characterized the cDNA encoding the human 3-phosphatase adapter subunit (3-PAP). Sequence alignment showed that 3-PAP ...

  15. Identification and characterization of novel membrane-bound PRL protein tyrosine phosphatases from Setaria cervi, a bovine filarial parasite.

    Singh, Neetu; Yadav, Smita; Rathaur, Sushma

    2015-11-01

    A significant amount of protein tyrosine phosphatase (PTP) activity was detected in the detergent-soluble membrane-bound fraction of Setaria cervi, a bovine filarial parasite. The membrane-bound PTP activity was significantly inhibited when the adult parasites were exposed to compounds having antifilarial activity like aspirin and SK7 as well as phenylarsine oxide, a specific PTP inhibitor suggesting that this activity is stress regulated. Further, this enzyme was purified as a single protein of apparently 21 kDa using two different chromatographic techniques. The MALDI-MS/MS analysis of its peptides showed closest match with protein tyrosine phosphatase PRL (Aedes aegypti). This purified enzyme (named as PRL) showed maximum activity at pH 5.5/37 °C and hydrolysed para nitro phenyl phosphate (pNPP) at the highest rate followed by O-P-L-tyrosine and O-P-L-threonine. It showed significant inhibition by specific inhibitors of PTP such as sodium orthovanadate, phenylarsine oxide and ammonium molybdate and was activated by dithiothreitol (DTT). The active site modification studies suggested involvement of cysteine, arginine, histidine and aspartic acid in the catalytic activity of PRL. The activity of S. cervi PRL was also found to be resistant towards the external oxidative stress. Thus, S. cervi PRL could be taken as a potential target for the management of human lymphatic filariasis. PMID:26341797

  16. Characterization of a eukaryotic type serine/threonine protein kinase and protein phosphatase of Streptococcus pneumoniae and identification of kinase substrates

    Nováková, Linda; Přenosilová, Lenka; Pallová, Petra; Janeček, Jiří; Novotná, Jana; Ulrych, Aleš; Echenique, J.; Trombe, M.-C.; Branny, Pavel

    2005-01-01

    Roč. 272, - (2005), s. 1243-1254. E-ISSN 1742-4658 R&D Projects: GA ČR GA204/99/1534; GA ČR GA204/02/1423 Grant ostatní: UK 188/2004/B-BIO/PrF Institutional research plan: CEZ:AV0Z50200510 Keywords : phosphoglucosamine mutase * phosphoproteome * protein phosphatase Subject RIV: EE - Microbiology, Virology

  17. Gene order for rubella virus structural proteins is NH/sub 2/-C-E2-E1-COOH

    Oker-Blom, C.

    1984-08-01

    The order of translation in vivo of the genes coding for rubella virus structural proteins was studied in infected B-Vero cells. The proteins were sequentially pulse-chase labeled with (/sup 35/S)methionine after synchronization of translation initiation with hypertonic salt treatment. A sequential labeling procedure (window-labeling) to specifically label defined segments of the structural proteins was also used. The labeled proteins were identified by sodium dodecyl sulfate-gel electrophoresis after immunoprecipitation with specific antisera directed against the two virion glycoproteins (E1 and E2a/E2b) and the nucleocapsid (C) protein. The order of translation was found to be NH/sub 2/-C-E2-E1-COOH. We have previously shown that the structural proteins are synthesized in vitro from a cytoplasmic 24S subgenomic mRNA as a 110,000-dalton (p110) precursor. Here, it is shown that p110 is precipitated with anti-C, anti-E2, and anti-E1 sera, indicating that p110 is the precursor of all three structural proteins. Two major in vitro translation products (M/sub r/s, 66,000 and 62,000) that could represent preterminated polypeptide chains or proteolytic cleavage products were precipitated with anti-C and anti-Es sera, but not with anti-E1 serum, indicating, in conformity with the in vivo results, that the genes for the C and E2 proteins are adjacent to each other. Using these specific antisera, we have also confirmed the identity of the unglycosylated forms of E1 (M/sub r/, 53,000) and E2 (M/sub r/ 30,000) immunoprecipitated from tunicamycin-treated infected cells. 18 references, 6 figures.

  18. Cathepsin D-mediated yolk protein degradation is blocked by acid phosphatase inhibitors.

    Fialho, Eliane; Nakamura, Angelica; Juliano, Luiz; Masuda, Hatisaburo; Silva-Neto, Mário A C

    2005-04-15

    Vitellin (VT) is a lipoglycophosphoprotein stored inside the eggs of every oviparous organism during oogenesis. In the blood-sucking bug Rhodnius prolixus, VT is deposited inside growing oocytes together with two acid hydrolases: acid phosphatase (AP) and cathepsin D (CD). Egg fertilization triggers AP activity and VT proteolysis in vivo [Insect Biochem. Mol. Biol. 2002 (32) 847]. Here, we show that CD is the main protease targeting VT proteolysis during egg development. CD activity in total egg homogenates is blocked by the classical aspartyl protease inhibitor, pepstatin A. Surprisingly, AP inhibitors such as NaF, Na+/K+ tartrate, and inorganic phosphate also block VT proteolysis, whereas this effect is not observed when tyrosine phosphatase inhibitors such as vanadate and phenylarsine oxide or an inhibitor of alkaline phosphatases such as levamisole are used in a VT proteolysis assay. NaF concentrations that block isolated AP activity do not affect the activity of partially purified CD. Therefore, a specific repressor of VT proteolysis must be dephosphorylated by AP in vivo. In conclusion, these results demonstrate for the first time that acid hydrolases act cooperatively to promote yolk degradation during egg development in arthropods. PMID:15797237

  19. Implication of protein tyrosine phosphatase 1B in MCF-7 cell proliferation and resistance to 4-OH tamoxifen

    Blanquart, Christophe; Karouri, Salah-Eddine [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris (France); Inserm, U567, Paris (France); Issad, Tarik, E-mail: tarik.issad@inserm.fr [Institut Cochin, Universite Paris Descartes, CNRS (UMR 8104), Paris (France); Inserm, U567, Paris (France)

    2009-10-02

    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.

  20. FIN13, a novel growth factor-inducible serine-threonine phosphatase which can inhibit cell cycle progression.

    Guthridge, M A; Bellosta, P; Tavoloni, N; Basilico, C.

    1997-01-01

    We have identified a novel type 2C serine-threonine phosphatase, FIN13, whose expression is induced by fibroblast growth factor 4 and serum in late G1 phase. The protein encoded by FIN13 cDNA includes N- and C-terminal domains with significant homologies to type 2C phosphatases, a domain homologous to collagen, and an acidic domain. FIN13 expression predominates in proliferating tissues. Bacterially expressed FIN13 and FIN13 expressed in mammalian cells exhibit serine-threonine phosphatase ac...

  1. Cervix carcinoma is associated with an up-regulation and nuclear localization of the dual-specificity protein phosphatase VHR

    The 21-kDa Vaccinia virus VH1-related (VHR) dual-specific protein phosphatase (encoded by the DUSP3 gene) plays a critical role in cell cycle progression and is itself regulated during the cell cycle. We have previously demonstrated using RNA interference that cells lacking VHR arrest in the G1 and G2 phases of the cell cycle and show signs of beginning of cell senescence. In this report, we evaluated successfully the expression levels of VHR protein in 62 hysterectomy or conization specimens showing the various (pre) neoplastic cervical epithelial lesions and 35 additional cases of hysterectomy performed for non-cervical pathologies, from patients under 50 years of age. We used a tissue microarray and IHC technique to evaluate the expression of the VHR phosphatase. Immunofluorescence staining under confocal microscopy, Western blotting and RT-PCR methods were used to investigate the localization and expression levels of VHR. We report that VHR is upregulated in (pre) neoplastic lesions (squamous intraepithelial lesions; SILs) of the uterine cervix mainly in high grade SIL (H-SIL) compared to normal exocervix. In the invasive cancer, VHR is also highly expressed with nuclear localization in the majority of cells compared to normal tissue where VHR is always in the cytoplasm. We also report that this phosphatase is highly expressed in several cervix cancer cell lines such as HeLa, SiHa, CaSki, C33 and HT3 compared to primary keratinocytes. The immunofluorescence technique under confocal microscopy shows that VHR has a cytoplasmic localization in primary keratinocytes, while it localizes in both cytoplasm and nucleus of the cancer cell lines investigated. We report that the up-regulation of this phosphatase is mainly due to its post-translational stabilization in the cancer cell lines compared to primary keratinocytes rather than increases in the transcription of DUSP3 locus. These results together suggest that VHR can be considered as a new marker for cancer

  2. Sensitive and direct electrochemical detection of double-stranded DNA utilizing alkaline phosphatase-labelled zinc finger proteins.

    Noh, Soodong; Ha, Dat Thinh; Yang, Haesik; Kim, Moon-Soo

    2015-06-21

    Direct detection of double-stranded DNA (dsDNA) using zinc finger proteins (ZFPs) is of great importance in biomedical applications such as identifying pathogens and circulating DNAs. However, its sensitivity is still not sufficiently high because limited signalling labels can be conjugated or fused. Herein, we report sensitive and direct detection of dsDNA using (i) alkaline phosphatase (ALP) as a fast catalytic label conjugated to ZFPs along with (ii) electrochemical measurement of an ALP product (l-ascorbic acid) at the indium-tin oxide electrode with a high signal-to-background ratio. ALP is simply conjugated to a ZFP through lysine residues in a ZFP purification tag, a maltose binding protein (MBP). Sandwich-type electrochemical detection of dsDNA allows a detection limit of ca. 100 fM without using DNA amplification. PMID:25969923

  3. 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

    Jiang, Y P; Wang, H; D'Eustachio, P; Musacchio, J M; Schlessinger, J; Sap, J

    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, ...

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

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca2+ 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 32P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated 32P-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

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

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

    1986-05-01

    Rat skeletal muscle contains a glycogen synthase kinase (GSK-M) which is not stimulated by Ca/sup 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 /sup 32/P-labeled I-2 with trypsin and separating the peptides by reversed phase HPLC. Two partially separated /sup 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.

  6. Potential role of lncRNA cyp2c91-protein interactions on diseases of the immune system.

    Suravajhala, Prashanth; Kogelman, Lisette J A; Mazzoni, Gianluca; Kadarmideen, Haja N

    2015-01-01

    With unprecedented increase in next generation sequencing technologies, there has been a persistent interest on transcript profiles of long non-coding RNAs (lncRNAs) and protein-coding genes forming an interaction network. Apart from protein-protein interaction (PPI), gene network models such as Weighted Gene Co-expression Network Analysis (WGCNA) are used to functionally annotate lncRNAs in identifying their potential disease associations. To address this, studies have led to characterizing transcript structures and understanding expression profiles mediating regulatory roles. In the current exploratory analysis, we show how a lncRNA - cyp2c91 contributes to the transcriptional regulation localized to cytoplasm thereby making refractory environment for transcription. By applying network methods and pathway analyses on genes related to a disease such as obesity and systemic lupus erythematosus, we show that we can gain deeper insight in biological processes such as the perturbances in immune system, and get a better understanding of the systems biology of diseases. PMID:26284111

  7. Potential role of lncRNA cyp2c91-protein interactions on diseases of the immune system

    Prashanth eSuravajhala

    2015-07-01

    Full Text Available With unprecedented increase in next generation sequencing (NGS technologies, there has been a persistent interest on transcript profiles of long noncoding RNAs (lncRNAs and protein-coding genes forming an interaction network. Apart from protein-protein interaction (PPI, gene network models such as Weighted Gene Co-expression Network Analysis are used to functionally annotate lncRNAs in identifying their potential disease associations. To address this, studies have led to characterizing transcript structures and understanding expression profiles mediating regulatory roles. In the current exploratory analysis, we show how a lncRNA - cyp2c91 contributes to the transcriptional regulation localized to cytoplasm thereby making refractory environment for transcription. By applying network methods and pathway analyses on genes related to a disease such as obesity and systemic lupus erythematosus, we show that we can gain deeper insight in biological processes such as the perturbances in immune system, and get a better understanding of the systems biology of diseases.

  8. Localization in rat heart mitochondria of outer and inner pools of camp dependent protein kinase, a kinase anchor proteins and PP2C serine phosphatase

    Sardanalli, A. M.; Dobrová, Zuzana; Signorile, A.; Nuzzi, R.; Drahota, Z.; Papa, S.

    Milano, 2003, s. 250. [Join Symposia with the British Biochemical Society /SIP 2003./. Milano (IT), 15.09.2003-18.09.2003] Institutional research plan: CEZ:AV0Z5020903 Keywords : pcamp * akap * aqdq Subject RIV: EE - Microbiology, Virology

  9. Crystal Structure of the Catalytic Domain of a Serine Threonine Protein Phosphatase

    Swinglel, Mark; Honkanel, Richard; Ciszak, Ewa

    2003-01-01

    Reversible phosphorylation of serine and threonine residues is a well-recognized mechanism in eukaryotic cells for the regulation of cell-cycle progression, cell growth and metabolism. Human serine/threonine phosphatases can be placed into two major families, PPP and PPM. To date the structure on one PPP family member (PPl) has been determined. Here we present the structure of a 323-residue catalytic domain of a second phosphatase belonging to the PPP family of enzyme. catalytic domain of the enzyme has been determined to 1.60Angstrom resolution and refined to R=17.5 and Rfree = 20.8%. The catalytic domain possesses a unique fold consisting of a largely monolithic structure, divisible into closely-associated helical and sheet regions. The catalytic site contains two manganese ions that are involved in substrate binding and catalysis. The enzyme crystallizes as a dimer that completely buries catalytic surfaces of both monomers, Also, the structure shows evidence of some flexibility around the active site cleft that may be related to substrate specificity of this enzyme.

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

    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

  11. Electroacupuncture Improves Insulin Resistance by Reducing Neuroprotein Y/Agouti-Related Protein Levels and Inhibiting Expression of Protein Tyrosine Phosphatase 1B in Diet-induced Obese Rats.

    Liu, Xia; He, Jun-Feng; Qu, Ya-Ting; Liu, Zhi-Jun; Pu, Qing-Yang; Guo, Sheng-Tong; Du, Jia; Jiang, Peng-Fei

    2016-04-01

    Electroacupuncture (EA) has been shown to exert beneficial effects on obesity, but the mechanism is unclear. This study investigated the effects of EA on diet-induced obese (DIO) rats. Fifty male Sprague-Dawley rats were randomly divided into low-fat diet (LFD, 10 rats) and high-fat diet (HFD, 40 rats) groups. After the DIO models had been established, successful model rats were randomly divided into HFD, EA, and orlistat (OLST) groups. The EA group received EA at Zusanli (ST36) and Quchi (LI11) for 20 minutes once per day for 28 days. The OLST group was treated with orlistat by gavage. The body weight, homeostasis model assessment-insulin resistance index, adipocyte diameters, and neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B levels were significantly lower in the EA group than in the HFD group. The rats of the OLST group showed watery stools and yellow hairs whereas those of the EA group had regular stools and sleek coats. The effect of EA on weight loss may be related to improved insulin resistance caused by changes in the adipocyte size and by reductions in the expressions of neuroprotein Y/agouti-related protein and protein tyrosine phosphatase 1B. This study indicates that EA may be a better method of alternative therapy for treating obesity and other metabolic diseases. PMID:27079226

  12. Cloning and sequence analysis of a full-length cDNA of SmPP1cb encoding turbot protein phosphatase 1 beta catalytic subunit

    Qi, Fei; Guo, Huarong; Wang, Jian

    2008-02-01

    Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is an important and versatile mechanism by which eukaryotic cells regulate almost all the signaling processes. Protein phosphatase 1 (PP1) is the first and well-characterized member of the protein serine/threonine phosphatase family. In the present study, a full-length cDNA encoding the beta isoform of the catalytic subunit of protein phosphatase 1(PP1cb), was for the first time isolated and sequenced from the skin tissue of flatfish turbot Scophthalmus maximus, designated SmPP1cb, by the rapid amplification of cDNA ends (RACE) technique. The cDNA sequence of SmPP1cb we obtained contains a 984 bp open reading frame (ORF), flanked by a complete 39 bp 5' untranslated region and 462 bp 3' untranslated region. The ORF encodes a putative 327 amino acid protein, and the N-terminal section of this protein is highly acidic, Met-Ala-Glu-Gly-Glu-Leu-Asp-Val-Asp, a common feature for PP1 catalytic subunit but absent in protein phosphatase 2B (PP2B). And its calculated molecular mass is 37 193 Da and pI 5.8. Sequence analysis indicated that, SmPP1cb is extremely conserved in both amino acid and nucleotide acid levels compared with the PP1cb of other vertebrates and invertebrates, and its Kozak motif contained in the 5'UTR around ATG start codon is GXXAXXGXX ATGG, which is different from mammalian in two positions A-6 and G-3, indicating the possibility of different initiation of translation in turbot, and also the 3'UTR of SmPP1cb is highly diverse in the sequence similarity and length compared with other animals, especially zebrafish. The cloning and sequencing of SmPP1cb gene lays a good foundation for the future work on the biological functions of PP1 in the flatfish turbot.

  13. Taperin (c9orf75, a mutated gene in nonsyndromic deafness, encodes a vertebrate specific, nuclear localized protein phosphatase one alpha (PP1α docking protein

    Tony Ferrar

    2012-02-01

    The promiscuous activity of protein phosphatase one (PP1 is controlled in the cell by associated proteins termed regulatory or targeting subunits. Using biochemical and proteomic approaches we demonstrate that the autosomal recessive nonsyndromic hearing loss gene, taperin (C9orf75, encodes a protein that preferentially docks the alpha isoform of PP1. Taperin associates with PP1 through a classic ‘RVxF’ motif and suppresses the general phosphatase activity of the enzyme. The steady-state localization of taperin is predominantly nuclear, however we demonstrate here that the protein can shuttle between the nucleus and cytoplasm and that it is found complexed to PP1 in both of these cellular compartments. Although originally identified as a hearing loss gene, Western blot analyses with taperin-specific antibodies revealed that the protein is widely expressed across mammalian tissues as multiple splice variants. Taperin is a recent proteome addition appearing during the vertebrate lineage with the PP1 binding site embedded within the most conserved region of the protein. Taperin also shares an ancestral relationship with the cytosolic actin binding protein phostensin, another PP1 interacting partner. Quantitative Stable Isotope Labeling by Amino acids in Culture (SILAC-based mass spectrometry was employed to uncover additional taperin binding partners, and revealed an interaction with the DNA damage response proteins Ku70, Ku80, PARP and topoisomerases I and IIα. Consistent with this, we demonstrate the active recruitment of taperin to sites of DNA damage. This makes taperin a new addition to the family of PP1 targeting subunits involved in the DNA damage repair pathway.

  14. Protein tyrosine phosphatase 1B (PTP1B)-inhibiting constituents from the leaves of Syzygium polyanthum.

    Saifudin, Azis; Tanaka, Ken; Kadota, Shigetoshi; Tezuka, Yasuhiro

    2012-08-01

    A methanol extract of the leaves of Syzygium polyanthum (Wight) Walp. afforded four new acylbenzene derivatives (1-4) together with seven known compounds (5-11). The structures of 1-11 were elucidated by extensive spectroscopic methods and comparison with the literature data. The new compounds 1-3 and a known compound, campest-4-en-3-one (10), exhibited a significant protein tyrosine phosphatase 1B inhibitory activity with IC₅₀ values of 13.1 ± 0.1, 5.77 ± 0.15, 4.01 ± 0.26, and 10.4 ± 0.5 µM, respectively. The inhibitory potency of the new compounds 2 and 3 was comparable to that of a positive control RK-682 (IC₅₀, 5.51 ± 0.04 µM). PMID:22763740

  15. Use of an Anaerobic Chamber Environment for the Assay of Endogenous Cellular Protein-Tyrosine Phosphatase Activities

    Zhu Li

    2002-01-01

    Full Text Available Protein-tyrosine phosphatases (PTPases have a catalytic cysteine residue whose reduced state is integral to the reaction mechanism. Since exposure to air can artifactually oxidize this highly reactive thiol, PTPase assays have typically used potent reducing agents to reactivate the enzymes present; however, this approach does not allow for the measurement of the endogenous PTPase activity directly isolated from the in vivo cellular environment. Here we provide a method for using an anaerobic chamber to preserve the activity of the total PTPase complement in a tissue lysate or of an immunoprecipitated PTPase homolog to characterize their endogenous activation state. Comparison with a sample treated with biochemical reducing agents allows the determination of the activatable (reducible fraction of the endogenous PTPase pool.

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

    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.

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

    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

  18. Inhibition of receptor tyrosine kinase signalling by small molecule agonist of T-cell protein tyrosine phosphatase

    T-cell protein tyrosine phosphatase (TCPTP/TC45) is a ubiquitously expressed intra-cellular non-receptor protein tyrosine phosphatase involved in the negative regulation of several cancer relevant cellular signalling pathways. We have previously shown that interaction between the α-cytoplasmic tail of α1β1 integrin and TCPTP activates TCPTP by disrupting an inhibitory intra-molecular bond in TCPTP. Thus, inhibition of the regulatory interaction in TCPTP is a desirable strategy for TCPTP activation and attenuation of oncogenic RTK signalling. However, this is challenging with low molecular weight compounds. We developed a high-throughput compatible assay to analyse activity of recombinant TCPTP in vitro. Using this assay we have screened 64280 small molecules to identify novel agonists for TCPTP. Dose-dependent response to TCPTP agonist was performed using the in vitro assay. Inhibition effects and specificity of TCPTP agonists were evaluated using TCPTP expressing and null mouse embryonic fibroblasts. Western blot analysis was used to evaluate attenuation of PDGFRβ and EGFR phosphorylation. Inhibition of VEGF signalling was analysed with VEGF-induced endothelial cell sprouting assays. From the screen we identified six TCPTP agonists. Two compounds competed with α1-cytoplasmic domain for binding to TCPTP, suggesting that they activate TCPTP similar to α1-cyt by disrupting the intra-molecular bond in TCPTP. Importantly, one of the compounds (spermidine) displayed specificity towards TCPTP in cells, since TCPTP -/- cells were 43-fold more resistant to the compound than TCPTP expressing cells. This compound attenuates PDGFRβ and VEGFR2 signalling in cells in a TCPTP-dependent manner and functions as a negative regulator of EGFR phosphorylation in cancer cells. In this study we showed that small molecules mimicking TCPTP-α1 interaction can be used as TCPTP agonists. These data provide the first proof-of-concept description of the use of high-throughput screening

  19. Integrin α1β1 Promotes Caveolin-1 Dephosphorylation by Activating T Cell Protein-tyrosine Phosphatase*

    Borza, Corina M.; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R.; Zent, Roy; Pozzi, Ambra

    2010-01-01

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  20. Integrin {alpha}1{beta}1 promotes caveolin-1 dephosphorylation by activating T cell protein-tyrosine phosphatase.

    Borza, Corina M; Chen, Xiwu; Mathew, Sijo; Mont, Stacey; Sanders, Charles R; Zent, Roy; Pozzi, Ambra

    2010-12-17

    Integrin α1β1 is a collagen receptor that down-regulates collagen and reactive oxygen species (ROS) production, and mice lacking this receptor show increased ROS levels and exacerbated glomerular sclerosis following injury. Caveolin-1 (Cav-1) is a multifunctional protein that is tyrosine-phosphorylated in response to injury and has been implicated in ROS-mediated injury. Cav-1 interacts with integrins, and integrin α1β1 binds/activates T cell protein-tyrosine phosphatase (TCPTP), which is homologous to the tyrosine phosphatase PTP1B known to dephosphorylate Cav-1. In this study, we analyzed whether phosphorylated Cav-1 (pCav-1) is a substrate of TCPTP and if integrin α1β1 is essential for promoting TCPTP-mediated Cav-1 dephosphorylation. We found that Cav-1 phosphorylation is significantly higher in cells lacking integrin α1β1 at base line and following oxidative stress. Overexpression of TCPTP leads to reduced pCav-1 levels only in cells expressing integrin α1β1. Using solid phase binding assays, we demonstrated that 1) purified Cav-1 directly interacts with TCPTP and the integrin α1 subunit, 2) pCav-1 is a substrate of TCPTP, and 3) TCPTP-mediated Cav-1 dephosphorylation is highly increased by the addition of purified integrin α1β1 or an integrin α1 cytoplasmic peptide to which TCPTP has been shown to bind. Thus, our results demonstrate that pCav-1 is a new substrate of TCPTP and that integrin α1β1 acts as a negative regulator of Cav-1 phosphorylation by activating TCPTP. This could explain the protective function of integrin α1β1 in oxidative stress-mediated damage and why integrin α1-null mice are more susceptible to fibrosis following injury. PMID:20940300

  1. Diversity in genomic organisation, developmental regulation and distribution of the murine PR72/B" subunits of protein phosphatase 2A

    Janssens Veerle

    2008-08-01

    Full Text Available Abstract Background Protein phosphatase 2A (PP2A is a serine/threonine-specific phosphatase displaying vital functions in growth and development through its role in various signalling pathways. PP2A holoenzymes comprise a core dimer composed of a catalytic C and a structural A subunit, which can associate with a variable B-type subunit. The importance of the B-type subunits for PP2A regulation cannot be overestimated as they determine holoenzyme localisation, activity and substrate specificity. Three B-type subunit families have been identified: PR55/B, PR61/B' and PR72/B", of which the latter is currently the least characterised. Results We deduced the sequences and genomic organisation of the different murine PR72/B" isoforms: three genes encode nine isoforms, five of which are abundantly expressed and give rise to genuine PP2A subunits. Thereby, one novel subunit was identified. Using Northern blotting, we examined the tissue-specific and developmental expression of these subunits. All subunits are highly expressed in heart, suggesting an important cardiac function. Immunohistochemical analysis revealed a striated expression pattern of PR72 and PR130 in heart and skeletal muscle, but not in bladder smooth muscle. The subcellular localisation and cell cycle regulatory ability of several PR72/B" isoforms were determined, demonstrating differences as well as similarities. Conclusion In contrast to PR55/B and PR61/B', the PR72/B" family seems evolutionary more divergent, as only two of the murine genes have a human orthologue. We have integrated these results in a more consistent nomenclature of both human and murine PR72/B" genes and their transcripts/proteins. Our results provide a platform for the future generation of PR72/B" knockout mice.

  2. Overexpression of protein tyrosine phosphatase-alpha (PTP-alpha) but not PTP-kappa inhibits translocation of GLUT4 in rat adipose cells

    Cong, L N; Chen, H; Li, Y;

    1999-01-01

    Protein tyrosine phosphatases (PTPases) are likely to play important roles in insulin action. We recently demonstrated that the nontransmembrane PTPase PTP1B can act as a negative modulator of insulin-stimulated translocation of GLUT4. We now examine the role of PTP-alpha and PTP-kappa (two trans......-stimulated glucose transport....

  3. Protein tyrosine phosphatase PTPN14 is a regulator of lymphatic function and choanal development in humans.

    Au, Audrey C; Hernandez, Paolo A; Lieber, Ernest; Nadroo, Ali M; Shen, Yu-Ming; Kelley, Kevin A; Gelb, Bruce D; Diaz, George A

    2010-09-10

    The lymphatic vasculature is essential for the recirculation of extracellular fluid, fat absorption, and immune function and as a route of tumor metastasis. The dissection of molecular mechanisms underlying lymphangiogenesis has been accelerated by the identification of tissue-specific lymphatic endothelial markers and the study of congenital lymphedema syndromes. We report the results of genetic analyses of a kindred inheriting a unique autosomal-recessive lymphedema-choanal atresia syndrome. These studies establish linkage of the trait to chromosome 1q32-q41 and identify a loss-of-function mutation in PTPN14, which encodes a nonreceptor tyrosine phosphatase. The causal role of PTPN14 deficiency was confirmed by the generation of a murine Ptpn14 gene trap model that manifested lymphatic hyperplasia with lymphedema. Biochemical studies revealed a potential interaction between PTPN14 and the vascular endothelial growth factor receptor 3 (VEGFR3), a receptor tyrosine kinase essential for lymphangiogenesis. These results suggest a unique and conserved role for PTPN14 in the regulation of lymphatic development in mammals and a nonconserved role in choanal development in humans. PMID:20826270

  4. Receptor-type Protein Tyrosine Phosphatase β Regulates Met Phosphorylation and Function in Head and Neck Squamous Cell Carcinoma

    Yiru Xu

    2012-11-01

    Full Text Available Head and neck squamous cell carcinoma (HNSCC is the sixth most common cancer and has a high rate of mortality. Emerging evidence indicates that hepatocyte growth factor receptor (or Met pathway plays a pivotal role in HNSCC metastasis and resistance to chemotherapy. Met function is dependent on tyrosine phosphorylation that is under direct control by receptor-type protein tyrosine phosphatase β (RPTP-β. We report here that RPTP-β expression is significantly downregulated in HNSCC cells derived from metastatic tumors compared to subject-matched cells from primary tumors. Knockdown of endogenous RPTP-β in HNSCC cells from primary tumor potentiated Met tyrosine phosphorylation, downstream mitogen-activated protein (MAP kinase pathway activation, cell migration, and invasion. Conversely, restoration of RPTP-β expression in cells from matched metastatic tumor decreased Met tyrosine phosphorylation and downstream functions. Furthermore, we observed that six of eight HNSCC tumors had reduced levels of RPTP-β protein in comparison with normal oral tissues. Collectively, the results demonstrate the importance of RPTP-β in tumor biology of HNSCC through direct dephosphorylation of Met and regulation of downstream signal transduction pathways. Reduced RPTP-β levels, with or without Met overexpression, could promote Met activation in HNSCC tumors.

  5. Light-switched inhibitors of protein tyrosine phosphatase PTP1B based on phosphonocarbonyl phenylalanine as photoactive phosphotyrosine mimetic.

    Wagner, Stefan; Schütz, Anja; Rademann, Jörg

    2015-06-15

    Phosphopeptide mimetics containing the 4-phosphonocarbonyl phenylalanine (pcF) as a photo-active phosphotyrosine isoster are developed as potent, light-switchable inhibitors of the protein tyrosine phosphatase PTP1B. The photo-active inhibitors 6-10 are derived from phosphopeptide substrates and are prepared from the suitably protected pcF building block 12 by Fmoc-based solid phase peptide synthesis. All pcF-containing peptides are moderate inhibitors of PTP1B with KI values between 10 and 50μM. Irradiation of the inhibitors at 365nm in the presence of the protein PTP1B amplify the inhibitory activity of pcF-peptides up to 120-fold, switching the KI values of the best inhibitors to the sub-micromolar range. Photo-activation of the inhibitors results in the formation of triplet intermediates of the benzoylphosphonate moiety, which deactivate PTP1B following an oxidative radical mechanism. Deactivation of PTP1B proceeds without covalent crosslinking of the protein target with the photo-switched inhibitors and can be reverted by subsequent addition of reducing agent dithiothreitol (DTT). PMID:25907367

  6. Identification of protein tyrosine phosphatase 1B and casein as substrates for 124-v-Mos

    Stabel Silvia; Proikas-Cezanne Tassula; Riethmacher Dieter

    2002-01-01

    Abstract Background The mos proto-oncogene encodes a cytoplasmic serine/threonine-specific protein kinase with crucial function during meiotic cell division in vertebrates. Based on oncogenic amino acid substitutions the viral derivative, 124-v-Mos, displays constitutive protein kinase activity and functions independent of unknown upstream effectors of mos protein kinase. We have utilized this property of 124-v-Mos and screened for novel mos substrates in immunocomplex kinase assays in vitro....

  7. Effect of microcystin-LR on protein phosphatase 2A and its function in human amniotic epithelial cells

    Jing LIANG; Tan LI; Ya-li ZHANG; Zong-lou GUO; Li-hong XU

    2011-01-01

    Due to their toxicity,the increased distribution of microcystins (MCs) has become an important worldwide problem.MCs have been recognized as inhibitors of protein phosphatase 2A (PP2A) through their binding to the PP2A catalytic subunit.However,the exact mechanism of MC toxicity has not been elucidated,especially concerning the cellular response and its autoregulation.To further dissect the role of PP2A in MC-induced toxicity,the present study was undertaken to determine the response of PP2A in human amniotic epithelial (FL) cells treated with microcystin-LR (MCLR),one of the MC congeners.The results show that a low-dose treatment of MCLR in FL cells for 6 h induced an increase in PP2A activity,and a high-dose treatment of MCLR for 24 h decreased the activity of PP2A,as expected.The increased mRNA and protein levels of the PP2A C subunit may explain the increased activity of PP2A.Furthermore,MCLR altered microtubule post-translational modifications through PP2A.These results further clarify the underlying mechanism how MCLR affects PP2A and may be helpful for elucidating the complex toxicity of MCLR.

  8. Hypothermic Preconditioning Reverses Tau Ontogenesis in Human Cortical Neurons and is Mimicked by Protein Phosphatase 2A Inhibition.

    Rzechorzek, Nina M; Connick, Peter; Livesey, Matthew R; Borooah, Shyamanga; Patani, Rickie; Burr, Karen; Story, David; Wyllie, David J A; Hardingham, Giles E; Chandran, Siddharthan

    2016-01-01

    Hypothermia is potently neuroprotective, but the molecular basis of this effect remains obscure. Changes in neuronal tau protein are of interest, since tau becomes hyperphosphorylated in injury-resistant, hypothermic brains. Noting inter-species differences in tau isoforms, we have used functional cortical neurons differentiated from human pluripotent stem cells (hCNs) to interrogate tau modulation during hypothermic preconditioning at clinically-relevant temperatures. Key tau developmental transitions (phosphorylation status and splicing shift) are recapitulated during hCN differentiation and subsequently reversed by mild (32 °C) to moderate (28 °C) cooling--conditions which reduce oxidative and excitotoxic stress-mediated injury in hCNs. Blocking a major tau kinase decreases hCN tau phosphorylation and abrogates hypothermic neuroprotection, whilst inhibition of protein phosphatase 2A mimics cooling-induced tau hyperphosphorylation and protects normothermic hCNs from oxidative stress. These findings indicate a possible role for phospho-tau in hypothermic preconditioning, and suggest that cooling drives human tau towards an earlier ontogenic phenotype whilst increasing neuronal resilience to common neurotoxic insults. This work provides a critical step forward in understanding how we might exploit the neuroprotective benefits of cooling without cooling patients. PMID:26870825

  9. Knockdown of microRNA-195 contributes to protein phosphatase-2A inactivation in rats with chronic brain hypoperfusion.

    Liu, Cheng-Di; Wang, Qin; Zong, De-Kang; Pei, Shuang-Chao; Yan, Yan; Yan, Mei-Ling; Sun, Lin-Lin; Hao, Yang-Yang; Mao, Meng; Xing, Wen-Jing; Ren, Huan; Ai, Jing

    2016-09-01

    Reduction of protein phosphatase-2A (PP2A) activity is a common clinical feature of Alzheimer's disease and vascular dementia. In this study, we observed that chronic brain hypoperfusion induced by bilateral common carotid artery occlusion of rats led to PP2A inactivation based on the increase in tyrosine-307 phosphorylation and leucine-309 demethylation of PP2AC and the depression in PP2ABα. Knockdown of miR-195 using overexpression of its antisense molecule oligonucleotide (pre-AMO-miR-195) delivered by a lentivirus (lenti-pre-AMO-miR-195) increased tyrosine-307 phosphorylation and decreased both PP2ABα expression and leucine-309 methylation; these effects were prevented by the overexpression of miR-195 using lenti-pre-miR-195 and controlled by an increase in methylesterase (PME-1) and a decrease in leucine carboxyl methyltransferase-1. In vitro studies demonstrated that miR-195 regulated PME-1 expression by binding to the Ppme1 gene 3'-untranslated region (3'UTR) domain. Masking the miR-195 binding sites in the amyloid precursor protein (APP) and β-site APP cleaving enzyme 1 genes prevented miR-195-induced leucine carboxyl methyltransferase-1 elevation. We concluded that the miR-195 downregulation in chronic brain hypoperfusion involved PP2A inactivity, which was mediated by the post-transcriptional regulation PME-1, APP, and β-site APP cleaving enzyme 1 expression. PMID:27459928

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

    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.