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Sample records for serine threonine kinase

  1. A retroviral oncogene, akt, encoding a serine-threonine kinase containing an SH2-like region.

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    Bellacosa, A; Testa, J R; Staal, S P; Tsichlis, P N

    1991-10-11

    The v-akt oncogene codes for a 105-kilodalton fusion phosphoprotein containing Gag sequences at its amino terminus. Sequence analysis of v-akt and biochemical characterization of its product revealed that it codes for a protein kinase C-related serine-threonine kinase whose cellular homolog is expressed in most tissues, with the highest amount found in thymus. Although Akt is a serine-threonine kinase, part of its regulatory region is similar to the Src homology-2 domain, a structural motif characteristic of cytoplasmic tyrosine kinases that functions in protein-protein interactions. This suggests that Akt may form a functional link between tyrosine and serine-threonine phosphorylation pathways.

  2. Cross-phosphorylation of bacterial serine/threonine and tyrosine protein kinases on key regulatory residues

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    Lei eShi

    2014-09-01

    Full Text Available Bacteria possess protein serine/threonine and tyrosine kinases which resemble eukaryal kinases in their capacity to phosphorylate multiple substrates. We hypothesized that the analogy might extend further, and bacterial kinases may also undergo mutual phosphorylation and activation, which is currently considered as a hallmark of eukaryal kinase networks. In order to test this hypothesis, we explored the capacity of all members of four different classes of serine/threonine and tyrosine kinases present in the firmicute model organism Bacillus subtilis to phosphorylate each other in vitro and interact with each other in vivo. The interactomics data suggested a high degree of connectivity among all types of kinases, while phosphorylation assays revealed equally wide-spread cross-phosphorylation events. Our findings suggest that the Hanks-type kinases PrkC, PrkD and YabT exhibit the highest capacity to phosphorylate other B. subtilis kinases, while the BY-kinase PtkA and the two-component-like kinases RsbW and SpoIIAB show the highest propensity to be phosphorylated by other kinases. Analysis of phosphorylated residues on several selected recipient kinases suggests that most cross-phosphorylation events concern key regulatory residues. Therefore, cross-phosphorylation events are very likely to influence the capacity of recipient kinases to phosphorylate substrates downstream in the signal transduction cascade. We therefore conclude that bacterial serine/threonine and tyrosine kinases probably engage in a network-type behavior previously described only in eukaryal cells.

  3. Distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases of Actinobacteria.

    Science.gov (United States)

    Ogawara, Hiroshi

    2016-09-01

    PASTA domains (penicillin-binding protein and serine/threonine kinase-associated domains) have been identified in penicillin-binding proteins and serine/threonine kinases of Gram-positive Firmicutes and Actinobacteria. They are believed to bind β-lactam antibiotics, and be involved in peptidoglycan metabolism, although their biological function is not definitively clarified. Actinobacteria, especially Streptomyces species, are distinct in that they undergo complex cellular differentiation and produce various antibiotics including β-lactams. This review focuses on the distribution of PASTA domains in penicillin-binding proteins and serine/threonine kinases in Actinobacteria. In Actinobacteria, PASTA domains are detectable exclusively in class A but not in class B penicillin-binding proteins, in sharp contrast to the cases in other bacteria. In penicillin-binding proteins, PASTA domains distribute independently from taxonomy with some distribution bias. Particularly interesting thing is that no Streptomyces species have penicillin-binding protein with PASTA domains. Protein kinases in Actinobacteria possess 0 to 5 PASTA domains in their molecules. Protein kinases in Streptomyces can be classified into three groups: no PASTA domain, 1 PASTA domain and 4 PASTA domain-containing groups. The 4 PASTA domain-containing groups can be further divided into two subgroups. The serine/threonine kinases in different groups may perform different functions. The pocket region in one of these subgroup is more dense and extended, thus it may be involved in binding of ligands like β-lactams more efficiently.

  4. Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

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    Pompeo, Frédérique; Foulquier, Elodie; Galinier, Anne

    2016-01-01

    Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

  5. Structural analysis of Staphylococcus aureus serine/threonine kinase PknB.

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    Sonja Rakette

    Full Text Available Effective treatment of infections caused by the bacterium Staphylococcus aureus remains a worldwide challenge, in part due to the constant emergence of new strains that are resistant to antibiotics. The serine/threonine kinase PknB is of particular relevance to the life cycle of S. aureus as it is involved in the regulation of purine biosynthesis, autolysis, and other central metabolic processes of the bacterium. We have determined the crystal structure of the kinase domain of PknB in complex with a non-hydrolyzable analog of the substrate ATP at 3.0 Å resolution. Although the purified PknB kinase is active in solution, it crystallized in an inactive, autoinhibited state. Comparison with other bacterial kinases provides insights into the determinants of catalysis, interactions of PknB with ligands, and the pathway of activation.

  6. Bacterial Serine/Threonine Protein Kinases in Host-Pathogen Interactions*

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    Canova, Marc J.; Molle, Virginie

    2014-01-01

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection. PMID:24554701

  7. Bacterial serine/threonine protein kinases in host-pathogen interactions.

    Science.gov (United States)

    Canova, Marc J; Molle, Virginie

    2014-04-04

    In bacterial pathogenesis, monitoring and adapting to the dynamically changing environment in the host and an ability to disrupt host immune responses are critical. The virulence determinants of pathogenic bacteria include the sensor/signaling proteins of the serine/threonine protein kinase (STPK) family that have a dual role of sensing the environment and subverting specific host defense processes. STPKs can sense a wide range of signals and coordinate multiple cellular processes to mount an appropriate response. Here, we review some of the well studied bacterial STPKs that are essential virulence factors and that modify global host responses during infection.

  8. Distribution of serine/threonine kinase SAD-B in mouse peripheral nerve synapse.

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    Hagiwara, Akari; Harada, Kenu; Hida, Yamato; Kitajima, Isao; Ohtsuka, Toshihisa

    2011-05-11

    The serine/threonine kinase SAD regulates neural functions such as axon/dendrite polarization and neurotransmitter release. In the vertebrate central nervous system, SAD-B, a homolog of Caenorhabditis elegans SAD-1, is associated with synaptic vesicles and the active zone cytomatrix in nerve terminals. However, the distribution of SAD-B in the peripheral nervous system remains elusive. Here, we show that SAD-B is specifically localized to neuromuscular junctions. Although the active zone protein bassoon showed a punctated signal indicating its localization to motor end plates, SAD-B shows relatively diffuse localization indicating its association with both the active zone and synaptic vesicles. Therefore, SAD kinase may regulate neurotransmitter release from motor end plates in a similar manner to its regulation of neurotransmitter release in the central nervous system.

  9. C. elegans serine-threonine kinase KIN-29 modulates TGFβ signaling and regulates body size formation

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    Cohen Stephen

    2005-04-01

    Full Text Available Background In C. elegans there are two well-defined TGFβ-like signaling pathways. The Sma/Mab pathway affects body size morphogenesis, male tail development and spicule formation while the Daf pathway regulates entry into and exit out of the dauer state. To identify additional factors that modulate TGFβ signaling in the Sma/Mab pathway, we have undertaken a genetic screen for small animals and have identified kin-29. Results kin-29 encodes a protein with a cytoplasmic serine-threonine kinase and a novel C-terminal domain. The kinase domain is a distantly related member of the EMK (ELKL motif kinase family, which interacts with microtubules. We show that the serine-threonine kinase domain has in vitro activity. kin-29 mutations result in small animals, but do not affect male tail morphology as do several of the Sma/Mab signal transducers. Adult worms are smaller than the wild-type, but also develop more slowly. Rescue by kin-29 is achieved by expression in neurons or in the hypodermis. Interaction with the dauer pathway is observed in double mutant combinations, which have been seen with Sma/Mab pathway mutants. We show that kin-29 is epistatic to the ligand dbl-1, and lies upstream of the Sma/Mab pathway target gene, lon-1. Conclusion kin-29 is a new modulator of the Sma/Mab pathway. It functions in neurons and in the hypodermis to regulate body size, but does not affect all TGFβ outputs, such as tail morphogenesis.

  10. HIV-1 incorporates and proteolytically processes human NDR1 and NDR2 serine-threonine kinases

    International Nuclear Information System (INIS)

    Devroe, Eric; Silver, Pamela A.; Engelman, Alan

    2005-01-01

    Mammalian genomes encode two related serine-threonine kinases, nuclear Dbf2 related (NDR)1 and NDR2, which are homologous to the Saccharomyces cerevisiae Dbf2 kinase. Recently, a yeast genetic screen implicated the Dbf2 kinase in Ty1 retrotransposition. Since several virion-incorporated kinases regulate the infectivity of human immunodeficiency virus type 1 (HIV-1), we speculated that the human NDR1 and NDR2 kinases might play a role in the HIV-1 life cycle. Here we show that the NDR1 and NDR2 kinases were incorporated into HIV-1 particles. Furthermore, NDR1 and NDR2 were cleaved by the HIV-1 protease (PR), both within virions and within producer cells. Truncation at the PR cleavage site altered NDR2 subcellular localization and inhibited NDR1 and NDR2 enzymatic activity. These studies identify two new virion-associated host cell enzymes and suggest a novel mechanism by which HIV-1 alters the intracellular environment of human cells

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

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

    2012-01-01

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

  12. Identification and characterization of a novel serine-threonine kinase gene from the Xp22 region.

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    Montini, E; Andolfi, G; Caruso, A; Buchner, G; Walpole, S M; Mariani, M; Consalez, G; Trump, D; Ballabio, A; Franco, B

    1998-08-01

    Eukaryotic protein kinases are part of a large and expanding family of proteins. Through our transcriptional mapping effort in the Xp22 region, we have isolated and sequenced the full-length transcript of STK9, a novel cDNA highly homologous to serine-threonine kinases. A number of human genetic disorders have been mapped to the region where STK9 has been localized including Nance-Horan (NH) syndrome, oral-facial-digital syndrome type 1 (OFD1), and a novel locus for nonsyndromic sensorineural deafness (DFN6). To evaluate the possible involvement of STK9 in any of the above-mentioned disorders, a 2416-bp full-length cDNA was assembled. The entire genomic structure of the gene, which is composed of 20 coding exons, was determined. Northern analysis revealed a transcript larger than 9.5 kb in several tissues including brain, lung, and kidney. The mouse homologue (Stk9) was identified and mapped in the mouse in the region syntenic to human Xp. This location is compatible with the location of the Xcat mutant, which shows congenital cataracts very similar to those observed in NH patients. Sequence homologies, expression pattern, and mapping information in both human and mouse make STK9 a candidate gene for the above-mentioned disorders. Copyright 1998 Academic Press.

  13. Nucleocytoplasmic shuttling of the HSV-2 serine/threonine kinase Us3

    International Nuclear Information System (INIS)

    Finnen, Renee L.; Johnston, Susan M.; Neron, Casey E.; Banfield, Bruce W.

    2011-01-01

    The alphaherpesvirus serine/threonine kinase Us3 plays diverse roles in virus multiplication and modifies both nuclear and cytoplasmic substrates. We recently reported that treatment of HSV-2 Us3-transfected and HSV-2-infected cells with leptomycin B, an inhibitor of nuclear export mediated by interaction of chromosomal regional maintenance protein (CRM1) with leucine rich nuclear export signals (NESs), resulted in nuclear trapping of Us3. Here, we utilized fluorescence loss in photobleaching to monitor nuclear export of HSV-2 Us3 and confirm that this process proceeds solely via a CRM1-mediated mechanism. Analysis of deletion derivatives of HSV-2 Us3 fused to a nuclear export reporter protein implicated the involvement of NES-like sequences in nuclear export. However, nuclear trapping of HSV-2 Us3 proteins carrying mutations in these potential NESs was not observed, indicating that these sequences are not functional in the context of full-length protein. Our analyses also revealed previously unidentified regions of HSV-2 Us3 that contribute to its kinase activity.

  14. Identification, sequence analysis, and characterization of serine/threonine protein kinase 17A from Clonorchis sinensis.

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    Huang, Lisi; Lv, Xiaoli; Huang, Yan; Hu, Yue; Yan, Haiyan; Zheng, Minghui; Zeng, Hua; Li, Xuerong; Liang, Chi; Wu, Zhongdao; Yu, Xinbing

    2014-05-01

    This is the first report of a novel protein from Clonorchis sinensis (C. sinensis), serine/threonine protein kinase 17A (CsSTK17A), which belongs to a member of the death-associated protein kinase (DAPK) family known to regulate diverse biological processes. The full-length sequence encoding CsSTK17A was isolated from C. sinensis adult cDNA plasmid library. Two transcribed isoforms of the gene were identified from the genome of C. sinensis. CsSTK17A contains a kinase domain at the N-terminus that shares a degree of conservation with the DAPK families. Besides, the catalytic domain contains 11 subdomains conserved among STKs and shares the highest identity with STK from Schistosoma mansoni (55.9%). Three-dimensional structure of CsSTK17A displays the canonical STK fold, including the helix C, P-loop, and the activation loop. We obtained recombinant CsSTK17A (rCsSTK17A) and anti-rCsSTK17A IgG. The rCsSTK17A could be probed by anti-rCsSTK17A rat serum, C. sinensis-infected rat serum and the sera from rats immunized with C. sinensis excretory-secretory products, indicating that it is a circulating antigen possessing a strong immunocompetence. Moreover, quantitative RT-PCR and western blotting analyses revealed that CsSTK17A exhibited the highest mRNA and protein expression level in eggs, followed by metacercariae and adult worms. Intriguingly, in the immunolocalization assay, CsSTK17A was intensively localized to the operculum region of eggs in uterus, as well as the vitelline gland of both adult worm and metacercaria, implying that the protein was associated with the reproduction and development of C. sinensis. Overall, these fundamental studies might contribute to further researches on signaling systems of the parasite.

  15. Regulation of hemolysin expression and virulence of Staphylococcus aureus by a serine/threonine kinase and phosphatase.

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    Kellie Burnside

    2010-06-01

    Full Text Available Exotoxins, including the hemolysins known as the alpha (alpha and beta (beta toxins, play an important role in the pathogenesis of Staphylococcus aureus infections. A random transposon library was screened for S. aureus mutants exhibiting altered hemolysin expression compared to wild type. Transposon insertions in 72 genes resulting in increased or decreased hemolysin expression were identified. Mutations inactivating a putative cyclic di-GMP synthetase and a serine/threonine phosphatase (Stp1 were found to reduce hemolysin expression, and mutations in genes encoding a two component regulator PhoR, LysR family transcriptional regulator, purine biosynthetic enzymes and a serine/threonine kinase (Stk1 increased expression. Transcription of the hla gene encoding alpha toxin was decreased in a Deltastp1 mutant strain and increased in a Deltastk1 strain. Microarray analysis of a Deltastk1 mutant revealed increased transcription of additional exotoxins. A Deltastp1 strain is severely attenuated for virulence in mice and elicits less inflammation and IL-6 production than the Deltastk1 strain. In vivo phosphopeptide enrichment and mass spectrometric analysis revealed that threonine phosphorylated peptides corresponding to Stk1, DNA binding histone like protein (HU, serine-aspartate rich fibrinogen/bone sialoprotein binding protein (SdrE and a hypothetical protein (NWMN_1123 were present in the wild type and not in the Deltastk1 mutant. Collectively, these studies suggest that Stk1 mediated phosphorylation of HU, SrdE and NWMN_1123 affects S. aureus gene expression and virulence.

  16. Serine/Threonine protein kinases from bacteria, archaea and eukarya share a common evolutionary origin deeply rooted in the tree of life

    DEFF Research Database (Denmark)

    Stancik, Ivan Andreas; Šestak, Martin Sebastijan; Ji, Boyang

    2018-01-01

    The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988. It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reaso...

  17. Bacillus subtilis single-stranded DNA-binding protein SsbA is phosphorylated at threonine 38 by the serine/threonine kinase YabT

    DEFF Research Database (Denmark)

    Derouiche, Abderahmane; Petranovic, Dina; Macek, Boris

    2016-01-01

    Background and purpose: Single-stranded DNA-binding proteins participate in all stages of DNA metabolism that involve single-stranded DNA, from replication, recombination, repair of DNA damage, to natural competence in species such as Bacillus subtilis. B. subtilis single-stranded DNA......-binding proteins have previously been found to be phosphorylated on tyrosine and arginine residues. While tyrosine phosphorylation was shown to enhance the DNA-binding properties of SsbA, arginine phosphorylation was not functionally characterized.Materials and methods: We used mass spectrometry analysis to detect...... phosphorylation of SsbA purified from B. subtilis cells. The detected phosphorylation site was assessed for its influence on DNA-binding in vitro, using electrophoretic mobility shift assays. The ability of B. subtilis serine/threonine kinases to phosphorylate SsbA was assessed using in vitro phosphorylation...

  18. Dual Regulation of a Chimeric Plant Serine/Threonine Kinase by Calcium and Calcium/Calmodulin

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    Takezawa, D.; Ramachandiran, S.; Paranjape, V.; Poovaiah, B. W.

    1996-01-01

    A chimeric Ca(2+)/calmodulin-dependent protein kinase (CCaMK) gene characterized by a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca(2+)-binding domain was recently cloned from plants. The Escherichia coli-expressed CCaMK phosphorylates various protein and peptide substrates in a Ca(2+)/calmodulin-dependent manner. The calmodulin-binding region of CCAMK has similarity to the calmodulin-binding region of the alpha-subunit of multifunctional Ca(2+)/calmodulin-dependent protein kinase (CaMKII). CCaMK exhibits basal autophosphorylation at the threonine residue(s) (0.098 mol of P-32/mol) that is stimulated 3.4-fold by Ca(2+) (0.339 mol of P-32/mol), while calmodulin inhibits Ca(2+)-stimulated autophosphorylation to the basal level. A deletion mutant lacking the visinin-like domain did not show Ca(2+)-simulated autophosphorylation activity but retained Ca(2+)/calmodulin-dependent protein kinase activity at a reduced level. Ca(2+)-dependent mobility shift assays using E.coli-expressed protein from residues 358-520 revealed that Ca(2+) binds to the visinin-like domain. Studies with site-directed mutants of the visinin-like domain indicated that EF-hands II and III are crucial for Ca(2+)-induced conformational changes in the visinin-like domain. Autophosphorylation of CCaMK increases Ca(2+)/calmodulin-dependent protein kinase activity by about 5-fold, whereas it did not affect its C(2+)-independent activity. This report provides evidence for the existence of a protein kinase in plants that is modulated by Ca(2+) and Ca(2+)/calmodulin. The presence of a visinin-like Ca(2+)-binding domain in CCaMK adds an additional Ca(2+)-sensing mechanism not previously known to exist in the Ca(2+)/calmodulin-mediated signaling cascade in plants.

  19. MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

    DEFF Research Database (Denmark)

    Pedersen, Anders Elm; Bregenholt, S; Johansen, B

    1999-01-01

    B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

  20. The external PASTA domain of the essential serine/threonine protein kinase PknB regulates mycobacterial growth.

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    Turapov, Obolbek; Loraine, Jessica; Jenkins, Christopher H; Barthe, Philippe; McFeely, Daniel; Forti, Francesca; Ghisotti, Daniela; Hesek, Dusan; Lee, Mijoon; Bottrill, Andrew R; Vollmer, Waldemar; Mobashery, Shahriar; Cohen-Gonsaud, Martin; Mukamolova, Galina V

    2015-07-01

    PknB is an essential serine/threonine protein kinase required for mycobacterial cell division and cell-wall biosynthesis. Here we demonstrate that overexpression of the external PknB_PASTA domain in mycobacteria results in delayed regrowth, accumulation of elongated bacteria and increased sensitivity to β-lactam antibiotics. These changes are accompanied by altered production of certain enzymes involved in cell-wall biosynthesis as revealed by proteomics studies. The growth inhibition caused by overexpression of the PknB_PASTA domain is completely abolished by enhanced concentration of magnesium ions, but not muropeptides. Finally, we show that the addition of recombinant PASTA domain could prevent regrowth of Mycobacterium tuberculosis, and therefore offers an alternative opportunity to control replication of this pathogen. These results suggest that the PknB_PASTA domain is involved in regulation of peptidoglycan biosynthesis and maintenance of cell-wall architecture.

  1. Prevention of pulmonary vascular and myocardial remodeling by the combined tyrosine and serine-/threonine kinase inhibitor, sorafenib, in pulmonary hypertension and right heart failure

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    M. Klein

    2008-06-01

    Full Text Available Inhibition of tyrosine kinases can reverse pulmonary hypertension but little is known about the role of serine-/threonine kinases in vascular and myocardial remodeling. We investigated the effects of sorafenib, an inhibitor of the tyrosine kinases VEGFR, PDGFR and c-kit as well as the serine-/threonine kinase Raf-1, in pulmonary hypertension and right ventricular (RV pressure overload. In monocrotaline treated rats, sorafenib (10 mg·kg–1·d–1 p.o. reduced pulmonary arterial pressure, pulmonary artery muscularization and RV hypertrophy, and improved systemic hemodynamics (table 1. Sorafenib prevented phosphorylation of Raf-1 and suppressed activation of downstream signaling pathways (Erk 1/2. After pulmonary banding, sorafenib, but not the PDGFR/c-KIT/ABL-inhibitor imatinib reduced RV mass and RV filling pressure significantly. Congruent with these results, sorafenib only prevented ERK phosphorylation and vasopressin induced hypertrophy of the cardiomyocyte cell line H9c2 dose dependently (IC50 = 300 nM. Combined inhibition of tyrosine and serine-/threonine kinases by sorafenib prevents vascular and cardiac remodeling in pulmonary hypertension, which is partly mediated via inhibition of the Raf kinase pathway.

  2. PASTA in Penicillin Binding Proteins and Serine/Threonine Kinases: A Recipe of Structural, Dynamic and Binding Properties.

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    Calvanese, Luisa; Falcigno, Lucia; Squeglia, Flavia; D'Auria, Gabriella; Berisio, Rita

    2017-11-24

    Penicillin binding proteins (PBPs) and Serine Threonine kinases (STPKs) are two classes of bacterial enzymes whose involvement in a series of vital processes in bacterial growth and division is well assessed. Many PBPs and STPKs show linked an ancillary domain named PASTA, whose functional role is not completely deciphered so far. It has been proposed that PASTAs are sensor modules that by binding opportune ligands (i.e. muropeptides) activate the cognate proteins to their functions. However, based on recent data, the sensor annotation sounds true for PASTA from STPKs, and false for PASTA from PBPs. Different PASTA domains, belonging or not to different protein classes, sharing or not appreciable sequence identities, always show identical folds. This survey of the structural, binding and dynamic properties of PASTA domains pursues the reasons why identical topologies may turn in different roles. Amino acid compositions, total charges and distribution of the hydrophobic/hydrophilic patches on the surface, significantly vary among PASTAs from STPKs and PBPs and appear to correlate with different functions. A possible criterion to discriminate between PASTA modules of STPKs or PBPs solely based on their sequences is proposed. Possibly reflecting different species as well as functional roles and evolutionary profile, our routine represents a fast even though approximate method to distinguish between PASTA belonging to different classes. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  3. Effect of Glucuronidation on the Potential of Kaempferol to Inhibit Serine/Threonine Protein Kinases

    NARCIS (Netherlands)

    Beekmann, Karsten; Haan, De Laura H.J.; Actis-Goretta, Lucas; Bladeren, Van Peter J.; Rietjens, Ivonne M.C.M.

    2016-01-01

    To study the effect of metabolic conjugation of flavonoids on the potential to inhibit protein kinase activity, the inhibitory effects of the dietary flavonol kaempferol and its major plasma conjugate kaempferol-3-O-glucuronide on protein kinases were studied. To this end, the inhibition of the

  4. The Serine/threonine kinase Stk33 exhibits autophosphorylation and phosphorylates the intermediate filament protein Vimentin

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    Herrmann Harald

    2008-09-01

    Full Text Available Abstract Background Colocalization of Stk33 with vimentin by double immunofluorescence in certain cells indicated that vimentin might be a target for phosphorylation by the novel kinase Stk33. We therefore tested in vitro the ability of Stk33 to phosphorylate recombinant full length vimentin and amino-terminal truncated versions thereof. In order to prove that Stk33 and vimentin are also in vivo associated proteins co-immunoprecipitation experiments were carried out. For testing the enzymatic activity of immunoprecipitated Stk33 we incubated precipitated Stk33 with recombinant vimentin proteins. To investigate whether Stk33 binds directly to vimentin, an in vitro co-sedimentation assay was performed. Results The results of the kinase assays demonstrate that Stk33 is able to specifically phosphorylate the non-α-helical amino-terminal domain of vimentin in vitro. Furthermore, co-immunoprecipitation experiments employing cultured cell extracts indicate that Stk33 and vimentin are associated in vivo. Immunoprecipitated Stk33 has enzymatic activity as shown by successful phosphorylation of recombinant vimentin proteins. The results of the co-sedimentation assay suggest that vimentin binds directly to Stk33 and that no additional protein mediates the association. Conclusion We hypothesize that Stk33 is involved in the in vivo dynamics of the intermediate filament cytoskeleton by phosphorylating vimentin.

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

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

    2017-03-08

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

  6. Serine/threonine kinase 15 gene polymorphism and risk of digestive system cancers: A meta-analysis.

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    Luo, Jianfei; Yan, Ruicheng; Zou, Li

    2015-01-01

    Previous studies have reported an association between the two coding polymorphisms (91T>A and 169G>A) of the serine/threonine kinase 15 (STK15) gene and the risk of digestive system cancers; however, the results are inconsistent. In the present study, a meta-analysis was carried out to assess the association between the two STK15 polymorphisms and the risk of digestive system cancers. Relevant studies were identified using PubMed, Web of Science, China National Knowledge Infrastructure, WanFang and VIP databases up to February 18, 2014. The pooled odds ratio (OR) with a 95% confidence interval (CI) was calculated using the fixed or random effects model. A total of 15 case-control studies from 14 publications were included. Of these, 15 studies concerned the 91T>A polymorphism and included 7,619 cases and 7,196 controls and four studies concerned the 161G>A polymorphism and included 826 cases and 713 controls. A significantly increased risk of digestive system cancers was observed for the 91T>A polymorphism (recessive model: OR, 1.19; 95% CI, 1.07-1.31). In subgroup analysis by ethnicity, a significant association was detected in Asian populations (recessive model: OR, 1.21; 95% CI, 1.08-1.36) but not in Caucasian and mixed populations. Stratification by tumor type indicated that the 91T>A polymorphism was associated with an increased risk of esophageal and colorectal cancers under the recessive model (OR, 1.19; 95% CI, 1.03-1.38; and OR, 1.24; 95% CI, 1.04-1.46; respectively); however, no significant association was observed between the 169G>A polymorphism and the risk of digestive system cancers in any of the genetic models. Furthermore, in subgroup analysis by ethnicity, similar results were observed in the Asian and Caucasian populations. The present meta-analysis demonstrated that the STK15 gene 91T>A polymorphism, but not the 169G>A polymorphism, may be a risk factor for digestive system cancers, particularly for esophageal and colorectal cancers.

  7. Serine/Threonine Kinase 35, a Target Gene of STAT3, Regulates the Proliferation and Apoptosis of Osteosarcoma Cells

    Directory of Open Access Journals (Sweden)

    Zhong Wu

    2018-01-01

    Full Text Available Background/Aims: Serine/threonine kinase 35 (STK35 may be associated with Parkinson disease and human colorectal cancer, but there have been no reports on the expression levels or roles of STK35 in osteosarcoma. Methods: STK35 mRNA expression was determined in osteosarcoma and bone cyst tissues by real-time PCR. Cell proliferation and apoptosis were assessed by Cell Counting Kit-8 (CCK-8 assay and flow cytometry analysis, respectively. Results: STK35 was up-regulated in osteosarcoma tissues as indicated by analyzing publicly available expression data (GEO dataset E-MEXP-3628 and real-time PCR analysis on our own cohort. We subsequently investigated the effects of STK35 knockdown on two osteosarcoma cell lines, MG63 and U2OS. STK35 knockdown inhibited the growth of osteosarcoma cells in vitro and in xenograft tumors. Meanwhile, STK35 knockdown enhanced apoptosis. Expression of the active forms and the activity of two major executioner caspases, caspase 3 and caspase 7, were also increased in osteosarcoma cells with STK35 silenced. Additionally, Gene Set Enrichment Analysis (GSEA identified that the JAK/STAT signaling pathway was positively correlated with STK35 expression. The mRNA expression of STK35 was repressed by STAT3 small interfering RNA (siRNA, but not by siRNA of STAT4, STAT5A or STAT6. A luciferase reporter assay further demonstrated that STAT3 transcriptionally regulated STK35 expression. A chromatin immunoprecipitation (ChIP assay confirmed the direct recruitment of STAT3 to the STK35 promoter. The promotion effects of STAT3 knockdown on cell apoptosis were partially abolished by STK35 overexpression. Furthermore, STK35 mRNA expression was positively correlated with STAT3 mRNA expression in osteosarcoma tissues by Pearson correlation analysis. Conclusions: These results collectively reveal that STAT3 regulates the transcription of STK35 in osteosarcoma. STK35 may exert an oncogenic role in osteosarcoma.

  8. A Single-Nucleotide Polymorphism in Serine-Threonine Kinase 11, the Gene Encoding Liver Kinase B1, Is a Risk Factor for Multiple Sclerosis

    Directory of Open Access Journals (Sweden)

    Anne I. Boullerne

    2015-02-01

    Full Text Available We identified a family in which five siblings were diagnosed with multiple sclerosis (MS or clinically isolated syndrome. Several women in the maternal lineage have comorbidities typically associated with Peutz Jeghers Syndrome, a rare autosomal-dominant disease caused by mutations in the serine-threonine-kinase 11 (STK11 gene, which encodes liver kinase B1. Sequence analysis of DNA from one sibling identified a single-nucleotide polymorphism (SNP within STK11 intron 5. This SNP (dbSNP ID: rs9282860 was identified by TaqMan polymerase chain reaction (PCR assays in DNA samples available from two other siblings. Further screening was carried out in samples from 654 relapsing-remitting MS patients, 100 primary progressive MS patients, and 661 controls. The STK11-SNP has increased frequency in all female patients versus controls (odds ratio = 1.66, 95% CI = 1.05, 2.64, p = .032. The STK11-SNP was not associated with disease duration or onset; however, it was significantly associated with reduced severity (assessed by MS severity scores, with the lowest scores in patients who also harbored the HLA-DRB1*1501 allele. In vitro studies showed that peripheral blood mononuclear cells from members of the family were more sensitive to the mitochondrial inhibitor metformin than cells from MS patients with the major STK11 allele. The increased association of SNP rs9282860 in women with MS defines this variant as a genetic risk factor. The lower disease severity observed in the context of HLA-DRB1*1501 combined with limited in vitro studies raises the provocative possibility that cells harboring the STK11-SNP could be targeted by drugs which increase metabolic stress.

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

    DEFF Research Database (Denmark)

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

    2000-01-01

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

  10. Nucleocytoplasmic shuttling of STK16 (PKL12), a Golgi-resident serine/threonine kinase involved in VEGF expression regulation

    International Nuclear Information System (INIS)

    Guinea, Barbara; Ligos, Jose Manuel; Lain de Lera, Teresa; Martin-Caballero, Juan; Flores, Juana; Gonzalez de la Pena, Manuel; Garcia-Castro, Javier; Bernad, Antonio

    2006-01-01

    PKL12/STK16 protein is the first identified mammalian member of a ser/thr kinase subfamily that is conserved across several kingdoms, with a broad expression pattern in murine tissues and cell types. Endogenous STK16 subcellular localization was evaluated by indirect immunofluorescence in NIH/3T3 and NRK cells, demonstrating a Golgi-associated pattern that appears to be independent of signals provided by integrin pathways. When cells were treated with brefeldin A (BFA) or nocodazole, drugs that promote Golgi disorganization, we observed STK16 translocation to the nuclear compartment. Constitutive overexpression of this protein by retroviral vectors also promotes accumulation of STK16 in the nuclear compartment, as shown by subfractionation studies. A kinase-dead STK16 mutant (E202A) was used to demonstrate that both the Golgi association and the nuclear translocation capabilities seem to be independent of the STK16 kinase activity. In addition, we show that STK16 overexpression in several cell lines enhances their capacity to produce and secrete VEGF. To confirm these data in vivo, we injected tumor cells overexpressing STK16 into immunodeficient BALBc/SCID mice. HT1080-derived tumors overexpressing STK16 showed increased volume and number of blood vessels compared to controls. Altogether, these data concur with previous reports suggesting a potential role for STK16 as a transcriptional co-activator

  11. Spatiotemporal proteomic analyses during pancreas cancer progression identifies serine/threonine stress kinase 4 (STK4) as a novel candidate biomarker for early stage disease.

    Science.gov (United States)

    Mirus, Justin E; Zhang, Yuzheng; Hollingsworth, Michael A; Solan, Joell L; Lampe, Paul D; Hingorani, Sunil R

    2014-12-01

    Pancreas cancer, or pancreatic ductal adenocarcinoma, is the deadliest of solid tumors, with a five-year survival rate of pancreas cancer. Mouse models that accurately recapitulate the human condition allow disease tracking from inception to invasion and can therefore be useful for studying early disease stages in which surgical resection is possible. Using a highly faithful mouse model of pancreas cancer in conjunction with a high-density antibody microarray containing ∼2500 antibodies, we interrogated the pancreatic tissue proteome at preinvasive and invasive stages of disease. The goal was to discover early stage tissue markers of pancreas cancer and follow them through histologically defined stages of disease using cohorts of mice lacking overt clinical signs and symptoms and those with end-stage metastatic disease, respectively. A panel of seven up-regulated proteins distinguishing pancreas cancer from normal pancreas was validated, and their levels were assessed in tissues collected at preinvasive, early invasive, and moribund stages of disease. Six of the seven markers also differentiated pancreas cancer from an experimental model of chronic pancreatitis. The levels of serine/threonine stress kinase 4 (STK4) increased between preinvasive and invasive stages, suggesting its potential as a tissue biomarker, and perhaps its involvement in progression from precursor pancreatic intraepithelial neoplasia to pancreatic ductal adenocarcinoma. Immunohistochemistry of STK4 at different stages of disease revealed a dynamic expression pattern further implicating it in early tumorigenic events. Immunohistochemistry of a panel of human pancreas cancers confirmed that STK4 levels were increased in tumor epithelia relative to normal tissue. Overall, this integrated approach yielded several tissue markers that could serve as signatures of disease stage, including early (resectable), and therefore clinically meaningful, stages. © 2014 by The American Society for

  12. MicroRNA-184 inhibits neuroblastoma cell survival through targeting the serine/threonine kinase AKT2

    Directory of Open Access Journals (Sweden)

    Murphy Derek M

    2010-04-01

    Full Text Available Abstract Background Neuroblastoma is a paediatric cancer of the sympathetic nervous system. The single most important genetic indicator of poor clinical outcome is amplification of the MYCN transcription factor. One of many down-stream MYCN targets is miR-184, which is either directly or indirectly repressed by this transcription factor, possibly due to its pro-apoptotic effects when ectopically over-expressed in neuroblastoma cells. The purpose of this study was to elucidate the molecular mechanism by which miR-184 conveys pro-apoptotic effects. Results We demonstrate that the knock-down of endogenous miR-184 has the opposite effect of ectopic up-regulation, leading to enhanced neuroblastoma cell numbers. As a mechanism of how miR-184 causes apoptosis when over-expressed, and increased cell numbers when inhibited, we demonstrate direct targeting and degradation of AKT2, a major downstream effector of the phosphatidylinositol 3-kinase (PI3K pathway, one of the most potent pro-survival pathways in cancer. The pro-apoptotic effects of miR-184 ectopic over-expression in neuroblastoma cell lines is reproduced by siRNA inhibition of AKT2, while a positive effect on cell numbers similar to that obtained by the knock-down of endogenous miR-184 can be achieved by ectopic up-regulation of AKT2. Moreover, co-transfection of miR-184 with an AKT2 expression vector lacking the miR-184 target site in the 3'UTR rescues cells from the pro-apoptotic effects of miR-184. Conclusions MYCN contributes to tumorigenesis, in part, by repressing miR-184, leading to increased levels of AKT2, a direct target of miR-184. Thus, two important genes with positive effects on cell growth and survival, MYCN and AKT2, can be linked into a common genetic pathway through the actions of miR-184. As an inhibitor of AKT2, miR-184 could be of potential benefit in miRNA mediated therapeutics of MYCN amplified neuroblastoma and other forms of cancer.

  13. Knockdown of human serine/threonine kinase 33 suppresses human small cell lung carcinoma by blocking RPS6/BAD signaling transduction.

    Science.gov (United States)

    Sun, E L; Liu, C X; Ma, Z X; Mou, X Y; Mu, X A; Ni, Y H; Li, X L; Zhang, D; Ju, Y R

    2017-01-01

    Small cell lung cancer (SCLC) is characterized by rapid growth rate and a tendency to metastasize to distinct sites of patients' bodies. The human serine/threonine kinase 33 (STK33) gene has shown its potency as a therapeutic target for prevention of lung carcinomas including non-small cell lung cancer (NSCLC), but its function in the oncogenesis and development of SCLC remains unrevealed. In the current study, it was hypothesized that STK33 played a key role in the proliferation, survival, and invasion of SCLC cells. The expression of STK33 in human SCLC cell lines NCI-H466 and DMS153 was inhibited by specific shRNA. The cell proliferation, cell apoptosis, and cell invasion of the cells were assessed with a series of in vitro assays. To explore the mechanism through which STK33 gene exerted its function in the carcinogenesis of SCLC cells, the effect of STK33 knockdown on the activity of S6K1/RPS6/BAD signaling was detected. Then the results were further confirmed with STK33 inhibitor ML281 and in vivo assays. The results demonstrated that inhibition of STK33 in SCLC cells suppressed the cell proliferation and invasion while induced cell apoptosis. Associated with the change in the phenotypic features, knockdown of STK33 also decreased the phosphorylation of RPS6 and BAD while increased the expression of cleaved caspase 9, indicating that apoptosis induced by STK33 suppression was mediated via mitochondrial pathway. Similar to the results of STK33 knockdown, incubating NCI-H466 cells with STK33 inhibitor also reduced the cell viability by suppressing RPS6/BAD pathways. Additionally, STK33 knockdown also inhibited tumor growth and RPS6/BAD activity in mice models. Findings outlined in our study were different from that in NSCLC to some extent: knockdown of STK33 in SCLC cells induced the apoptosis through mitochondrial pathway but independent of S6K1 function, inferring that the function of STK33 might be cancer type specific.

  14. Distinct kinetics of serine and threonine dephosphorylation are essential for mitosis

    DEFF Research Database (Denmark)

    Hein, Jamin B; Hertz, Emil P T; Garvanska, Dimitriya H

    2017-01-01

    Protein phosphatase 2A (PP2A) in complex with B55 regulatory subunits reverses cyclin-dependent kinase 1 (Cdk1) phosphorylations at mitotic exit. Interestingly, threonine and serine residues phosphorylated by Cdk1 display distinct phosphorylation dynamics, but the biological significance remains ...

  15. The MurC ligase essential for peptidoglycan biosynthesis is regulated by the serine/threonine protein kinase PknA in Corynebacterium glutamicum.

    Science.gov (United States)

    Fiuza, Maria; Canova, Marc J; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A

    2008-12-26

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (L-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis.

  16. The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

    Science.gov (United States)

    Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

    2008-01-01

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recently emerged as a major physiological mechanism of regulation in prokaryotes. Herein, the hypothesis of a phosphorylation-dependent mechanism of regulation of the MurC activity was investigated in Corynebacterium glutamicum. We showed that MurC was phosphorylated in vitro by the PknA protein kinase. An analysis of the phosphoamino acid content indicated that phosphorylation exclusively occurred on threonine residues. Six phosphoacceptor residues were identified by mass spectrometry analysis, and we confirmed that mutagenesis to alanine residues totally abolished PknA-dependent phosphorylation of MurC. In vitro and in vivo ligase activity assays showed that the catalytic activity of MurC was impaired following mutation of these threonine residues. Further in vitro assays revealed that the activity of the MurC-phosphorylated isoform was severely decreased compared with the non-phosphorylated protein. To our knowledge, this is the first demonstration of a MurC ligase phosphorylation in vitro. The finding that phosphorylation is correlated with a decrease in MurC enzymatic activity could have significant consequences in the regulation of peptidoglycan biosynthesis. PMID:18974047

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

    Science.gov (United States)

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

    2014-02-14

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

  18. The MurC Ligase Essential for Peptidoglycan Biosynthesis Is Regulated by the Serine/Threonine Protein Kinase PknA in Corynebacterium glutamicum*

    OpenAIRE

    Fiuza, Maria; Canova, Marc J.; Patin, Delphine; Letek, Michal; Zanella-Cléon, Isabelle; Becchi, Michel; Mateos, Luís M.; Mengin-Lecreulx, Dominique; Molle, Virginie; Gil, José A.

    2008-01-01

    The Mur ligases play an essential role in the biosynthesis of bacterial cell-wall peptidoglycan and thus represent attractive targets for the design of novel antibacterials. These enzymes catalyze the stepwise formation of the peptide moiety of the peptidoglycan disaccharide peptide monomer unit. MurC is responsible of the addition of the first residue (l-alanine) onto the nucleotide precursor UDP-MurNAc. Phosphorylation of proteins by Ser/Thr protein kinases has recen...

  19. The Mycobacterium tuberculosis transcriptional repressor EthR is negatively regulated by Serine/Threonine phosphorylation.

    Science.gov (United States)

    Leiba, Jade; Carrère-Kremer, Séverine; Blondiaux, Nicolas; Dimala, Martin Moune; Wohlkönig, Alexandre; Baulard, Alain; Kremer, Laurent; Molle, Virginie

    2014-04-18

    Recent efforts have underlined the role of Serine/Threonine Protein Kinases (STPKs) in growth, pathogenesis and cell wall metabolism in mycobacteria. Herein, we demonstrated that the Mycobacterium tuberculosis EthR, a transcriptional repressor that regulates the activation process of the antitubercular drug ethionamide (ETH) is a specific substrate of the mycobacterial kinase PknF. ETH is a prodrug that must undergo bioactivation by the monooxygenease EthA to exert its antimycobacterial activity and previous studies reported that EthR represses transcription of ethA by binding to the ethA-ethR intergenic region. Mass spectrometry analyses and site-directed mutagenesis identified a set of four phosphoacceptors, namely Thr2, Thr3, Ser4 and Ser7. This was further supported by the complete loss of PknF-dependent phosphorylation of a phosphoablative EthR mutant protein. Importantly, a phosphomimetic version of EthR, in which all phosphosites were replaced by Asp residues, exhibited markedly decreased DNA-binding activity compared with the wild-type protein. Together, these findings are the first demonstration of EthR phosphorylation and indicate that phosphorylation negatively affects its DNA-binding activity, which may impact ETH resistance levels in M. tb. Copyright © 2014 Elsevier Inc. All rights reserved.

  20. Generation of serine/threonine check points in HN(C)N spectra

    Indian Academy of Sciences (India)

    Administrator

    ing to generate alanine. 6 and serine/threonine specific peak patterns. 7 have enhanced the speed of assign- ment quite substantially. These developments involved a simple modification to the pulse sequence. Continuing such efforts for rapid resonance as- signments, we have implemented here the tuning ideas.

  1. Oxytocin analogues with O-glycosylated serine and threonine in position 4

    Czech Academy of Sciences Publication Activity Database

    Marcinkowska, A.; Borovičková, Lenka; Slaninová, Jiřina; Grzonka, Z.

    2007-01-01

    Roč. 81, č. 7 (2007), s. 1335-1344 ISSN 0137- 5083 Institutional research plan: CEZ:AV0Z40550506; CEZ:AV0Z90210515 Keywords : oxytocin * glycosylated serin * glycosylated threonin * position 4 Subject RIV: CE - Biochemistry Impact factor: 0.483, year: 2007

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

    Directory of Open Access Journals (Sweden)

    Sven I Walaas

    2011-08-01

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

  3. Phosphopeptide derivatization signatures to identify serine and threonine phosphorylated peptides by mass spectrometry.

    Science.gov (United States)

    Molloy, M P; Andrews, P C

    2001-11-15

    The development of rapid, global methods for monitoring states of protein phosphorylation would provide greater insight for understanding many fundamental biological processes. Current best practices use mass spectrometry (MS) to profile digests of purified proteins for evidence of phosphorylation. However, this approach is beset by inherent difficulties in both identifying phosphopeptides from within a complex mixture containing many other unmodified peptides and ionizing phosphopeptides in positive-ion MS. We have modified an approach that uses barium hydroxide to rapidly eliminate the phosphoryl group of serine and threonine modified amino acids, creating dehydroamino acids that are susceptible to nucleophilic derivatization. By derivatizing a protein digest with a mixture of two different alkanethiols, phosphopeptide-specific derivatives were readily distinguished by MS due to their characteristic ion-pair signature. The resulting tagged ion pairs accommodate simple and rapid screening for phosphopeptides in a protein digest, obviating the use of isotopically labeled samples for qualitative phosphopeptide detection. MALDI-MS is used in a first pass manner to detect derivatized phosphopeptides, while the remaining sample is available for tandem MS to reveal the site of derivatization and, thus, phosphorylation. We demonstrated the technique by identifying phosphopeptides from beta-casein and ovalbumin. The approach was further used to examine in vitro phosphorylation of recombinant human HSP22 by protein kinase C, revealing phosphorylation of Thr-63.

  4. Membrane skeletal proteins and their integral membrane protein anchors are targets for tyrosine and threonine kinases in Euglena.

    Science.gov (United States)

    Fazio, M J; Da Silva, A C; Rosiere, T K; Bouck, G B

    1995-01-01

    Proteins of the membrane skeleton of Euglena gracilis were extensively phosphorylated in vivo and in vitro after incubation with [32P]-orthophosphate or gamma-[32P] ATP. Endogenous protein threonine/serine activity phosphorylated the major membrane skeletal proteins (articulins) and the putative integral membrane protein (IP39) anchor for articulins. The latter was also the major target for endogenous protein tyrosine kinase activity. A cytoplasmic domain of IP39 was specifically phosphorylated, and removal of this domain with papain eliminated the radiolabeled phosphoamino acids and eliminated or radically shifted the PI of the multiple isoforms of IP39. In gel kinase assays IP39 autophosphorylated and a 25 kDa protein which does not autophosphorylate was identified as a threonine/serine (casein) kinase. Plasma membranes from the membrane skeletal protein complex contained threonine/serine (casein) kinase activity, and cross-linking experiments suggested that IP39 was the likely source for this membrane activity. pH optima, cation requirements and heparin sensitivity of the detergent solubilized membrane activity were determined. Together these results suggest that protein kinases may be important modulators of protein assembly and function of the membrane skeleton of these protistan cells.

  5. Realizing Serine/Threonine Ligation: Scope and Limitations and Mechanistic Implication Thereof

    Directory of Open Access Journals (Sweden)

    Clarence T. T. Wong

    2014-05-01

    Full Text Available Serine/Threonine ligation (STL has emerged as an alternative tool for protein chemical synthesis, bioconjugations as well as macrocyclization of peptides of various sizes. Owning to the high abundance of Ser/Thr residues in natural peptides and proteins, STL is expected to find a wide range of applications in chemical biology research. Herein, we have fully investigated the compatibility of the serine/threonine ligation strategy for X-Ser/Thr ligation sites, where X is any of the 20 naturally occurring amino acids. Our studies have shown that 17 amino acids are suitable for ligation, while Asp, Glu, and Lys are not compatible. Among the working 17 C-terminal amino acids, the retarded reaction resulted from the bulky β-branched amino acid (Thr, Val and Ile is not seen under the current ligation condition. We have also investigated the chemoselectivity involving the amino group of the internal lysine which may compete with the N-terminal Ser/Thr for reaction with the C-terminal salicylaldehyde (SAL ester aldehyde group. The result suggested that the free internal amino group does not adversely slow down the ligation rate.

  6. Two novel pyrrolooxazole pigments formed by the Maillard reaction between glucose and threonine or serine.

    Science.gov (United States)

    Noda, Kyoko; Murata, Masatsune

    2017-02-01

    Pyrrolothiazolate formed by the Maillard reaction between l-cysteine and d-glucose has a pyrrolothiazole skeleton as a chromophore. We searched for a Maillard pigment having a pyrrolooxazole skeleton formed from l-threonine or l-serine instead of l-cysteine in the presence of d-glucose. As a result, two novel yellow pigments, named pyrrolooxazolates A and B, were isolated from model solutions of the Maillard reaction containing l-threonine and d-glucose, and l-serine and d-glucose, respectively, and identified as (2R,3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-2,5,7a-trimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid and (3S,7aS)-2,3,7,7a-tetrahydro-6-hydroxy-5,7a-dimethyl-7-oxo-pyrrolo[2,1-b]oxazole-3-calboxylic acid by instrumental analyses. These compounds were pyrrolooxazole derivatives carrying a carboxy group, and showed the absorption maxima at 300-360 nm under acidic and neutral conditions and at 320-390 nm under alkaline conditions.

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

    International Nuclear Information System (INIS)

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

    2006-01-01

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

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

    Energy Technology Data Exchange (ETDEWEB)

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

    2006-09-01

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

  9. LFA-1 and Mac-1 integrins bind to the serine/threonine-rich domain of thrombomodulin

    Energy Technology Data Exchange (ETDEWEB)

    Kawamoto, Eiji [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Okamoto, Takayuki, E-mail: okamotot@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Takagi, Yoshimi [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan); Honda, Goichi [Medical Affairs Department, Asahi Kasei Pharma Corporation, 1-105 Kanda Jinbo-cho, Chiyoda-ku, Tokyo 101-8101 (Japan); Suzuki, Koji [Faculty of Pharmaceutical Science, Suzuka University of Medical Science, 3500-3, Minamitamagaki-cho, Suzuka, Mie 513-8679 (Japan); Imai, Hiroshi [Emergency and Critical Care Center, Mie University Hospital, 2-174 Edobashi, Tsu 514-8507 (Japan); Shimaoka, Motomu, E-mail: shimaoka@doc.medic.mie-u.ac.jp [Department of Molecular Pathobiology and Cell Adhesion Biology, Mie University Graduate School of Medicine, 2-174 Edobashi, Tsu, Mie 514-8507 (Japan)

    2016-05-13

    LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins regulate leukocyte trafficking in health and disease by binding primarily to IgSF ligand ICAM-1 and ICAM-2 on endothelial cells. Here we have shown that the anti-coagulant molecule thrombomodulin (TM), found on the surface of endothelial cells, functions as a potentially new ligand for leukocyte integrins. We generated a recombinant extracellular domain of human TM and Fc fusion protein (TM-domains 123-Fc), and showed that pheripheral blood mononuclear cells (PBMCs) bind to TM-domains 123-Fc dependent upon integrin activation. We then demonstrated that αL integrin-blocking mAb, αM integrin-blocking mAb, and β2 integrin-blocking mAb inhibited the binding of PBMCs to TM-domains 123-Fc. Furthermore, we show that the serine/threonine-rich domain (domain 3) of TM is required for the interaction with the LFA-1 (αLβ2) and Mac-1 (αMβ2) integrins to occur on PBMCs. These results demonstrate that the LFA-1 and Mac-1 integrins on leukocytes bind to TM, thereby establishing the molecular and structural basis underlying LFA-1 and Mac-1 integrin interaction with TM on endothelial cells. In fact, integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells. - Highlights: • LFA-1 and Mac-1 integrins bind to the anti-coagulant molecule thrombomodulin. • The serine/threonine-rich domain of thrombomodulin is essential to interact with the LFA-1 and Mac-1 integrins on PBMCs. • Integrin-TM interactions might be involved in the dynamic regulation of leukocyte adhesion with endothelial cells.

  10. Basal Levels of AMPA Receptor GluA1 Subunit Phosphorylation at Threonine 840 and Serine 845 in Hippocampal Neurons

    Science.gov (United States)

    Babiec, Walter E.; Guglietta, Ryan; O'Dell, Thomas J.

    2016-01-01

    Dephosphorylation of AMPA receptor (AMPAR) GluA1 subunits at two sites, serine 845 (S845) and threonine 840 (T840), is thought to be involved in NMDA receptor-dependent forms of long-term depression (LTD). Importantly, the notion that dephosphorylation of these sites contributes to LTD assumes that a significant fraction of GluA1 subunits are…

  11. Molecular characterization and functional analysis of serine/threonine protein phosphatase of Toxocara canis.

    Science.gov (United States)

    Ma, Guang Xu; Zhou, Rong Qiong; Hu, Shi Jun; Huang, Han Cheng; Zhu, Tao; Xia, Qing You

    2014-06-01

    Toxocara canis (T. canis) is a widely prevalent zoonotic parasite that infects a wide range of mammalian hosts, including humans. We generated the full-length complementary DNA (cDNA) of the serine/threonine phosphatase gene of T. canis (Tc stp) using 5' rapid amplification of the cDNA ends. The 1192-bp sequence contained a continuous 942-nucleotide open reading frame, encoding a 313-amino-acid polypeptide. The Tc STP polypeptide shares a high level of amino-acid sequence identity with the predicted STPs of Loa loa (89%), Brugia malayi (86%), Oesophagostomum columbianum (76%), and Oesophagostomumdentatum (76%). The Tc STP contains GDXHG, GDXVDRG, GNHE motifs, which are characteristic of members of the phosphoprotein phosphatase family. Our quantitative real-time polymerase chain reaction analysis showed that the Tc STP was expressed in six different tissues in the adult male, with high-level expression in the spermary, vas deferens, and musculature, but was not expressed in the adult female, suggesting that Tc STP might be involved in spermatogenesis and mating behavior. Thus, STP might represent a potential molecular target for controlling T. canis reproduction. Copyright © 2014 Elsevier Inc. All rights reserved.

  12. Serine/Threonine Protein Phosphatase PstP of Mycobacterium tuberculosis Is Necessary for Accurate Cell Division and Survival of Pathogen*

    Science.gov (United States)

    Sharma, Aditya K.; Arora, Divya; Singh, Lalit K.; Gangwal, Aakriti; Sajid, Andaleeb; Molle, Virginie; Singh, Yogendra; Nandicoori, Vinay Kumar

    2016-01-01

    Protein phosphatases play vital roles in phosphorylation-mediated cellular signaling. Although there are 11 serine/threonine protein kinases in Mycobacterium tuberculosis, only one serine/threonine phosphatase, PstP, has been identified. Although PstP has been biochemically characterized and multiple in vitro substrates have been identified, its physiological role has not yet been elucidated. In this study, we have investigated the impact of PstP on cell growth and survival of the pathogen in the host. Overexpression of PstP led to elongated cells and partially compromised survival. We find that depletion of PstP is detrimental to cell survival, eventually leading to cell death. PstP depletion results in elongated multiseptate cells, suggesting a role for PstP in regulating cell division events. Complementation experiments performed with PstP deletion mutants revealed marginally compromised survival, suggesting that all of the domains, including the extracellular domain, are necessary for complete rescue. On the other hand, the catalytic activity of PstP is absolutely essential for the in vitro growth. Mice infection experiments establish a definitive role for PstP in pathogen survival within the host. Depletion of PstP from established infections causes pathogen clearance, indicating that the continued presence of PstP is necessary for pathogen survival. Taken together, our data suggest an important role for PstP in establishing and maintaining infection, possibly via the modulation of cell division events. PMID:27758870

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

    Directory of Open Access Journals (Sweden)

    Adams Brian

    2001-11-01

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

  14. Regulation of tumor cell migration by protein tyrosine phosphatase (PTP)-proline-, glutamate-, serine-, and threonine-rich sequence (PEST)

    Science.gov (United States)

    Zheng, Yanhua; Lu, Zhimin

    2013-01-01

    Protein tyrosine phosphatase (PTP)–proline-, glutamate-, serine-, and threonine-rich sequence (PEST) is ubiquitously expressed and is a critical regulator of cell adhesion and migration. PTP-PEST activity can be regulated transcriptionally via gene deletion or mutation in several types of human cancers or via post-translational modifications, including phosphorylation, oxidation, and caspase-dependent cleavage. PTP-PEST interacts with and dephosphorylates cytoskeletal and focal adhesion-associated proteins. Dephosphorylation of PTP-PEST substrates regulates their enzymatic activities and/or their interaction with other proteins and plays an essential role in the tumor cell migration process. PMID:23237212

  15. Human ribosomal protein L37 has motifs predicting serine/threonine phosphorylation and a zinc-finger domain.

    Science.gov (United States)

    Barnard, G F; Staniunas, R J; Puder, M; Steele, G D; Chen, L B

    1994-08-02

    Ribosomal protein L37 mRNA is overexpressed in colon cancer. The nucleotide sequences of human L37 from several tumor and normal, colon and liver cDNA sources were determined to be identical. L37 mRNA was approximately 375 nucleotides long encoding 97 amino acids with M(r) = 11,070, pI = 12.6, multiple potential serine/threonine phosphorylation sites and a zinc-finger domain. The human sequence is compared to other species.

  16. The interaction between tropomyosin-related kinase B receptors and serine kinases modulates acetylcholine release in adult neuromuscular junctions.

    Science.gov (United States)

    Santafé, Manel M; Garcia, Neus; Tomàs, Marta; Obis, Teresa; Lanuza, Maria A; Besalduch, Nuria; Tomàs, Josep

    2014-02-21

    We conducted an electrophysiological study of the functional link between the tropomyosin-related kinase B (trkB) receptor signaling mechanism and serine-threonine kinases, both protein kinase C (PKC) and protein kinase A (PKA). We describe their coordinated role in transmitter release at the neuromuscular junction (NMJ) of the Levator auris longus muscle of the adult mouse. The trkB receptor normally seems to be coupled to stimulate ACh release because inhibiting the trkB receptor with K-252a results in a significant reduction in the size of EPPs. We found that the intracellular PKC pathway can operate as in basal conditions (to potentiate ACh release) without the involvement of the trkB receptor function, although the trkB pathway needs an operative PKC pathway if it is to couple to the release mechanism and potentiate it. To actively stimulate PKA (which also results in ACh release potentiation), the operativity of trkB is a necessary condition, and one effect of trkB may be PKA stimulation. Copyright © 2014 Elsevier Ireland Ltd. All rights reserved.

  17. Tracking protons from respiratory chain complexes to ATP synthase c-subunit: The critical role of serine and threonine residues.

    Science.gov (United States)

    Panfoli, Isabella; Ponassi, Marco; Ravera, Silvia; Calzia, Daniela; Beitia, Maider; Morelli, Alessandro; Rosano, Camillo

    2017-01-22

    F 1 F o -ATP synthase is a multisubunit enzyme responsible for the synthesis of ATP. Among its multiple subunits (8 in E. coli, 17 in yeast S. cerevisiae, 16 in vertebrates), two subunits a and c are known to play a central role controlling the H + flow through the inner mitochondrial membrane which allows the subsequent synthesis of ATP, but the pathway followed by H + within the two proteins is still a matter of debate. In fact, even though the structure of ATP synthase is now well defined, the molecular mechanisms determining the function of both F 1 and F O domains are still largely unknown. In this study, we propose a pathway for proton migration along the ATP synthase by hydrogen-bonded chain mechanism, with a key role of serine and threonine residues, by X-ray diffraction data on the subunit a of E. coli Fo. Copyright © 2016 Elsevier Inc. All rights reserved.

  18. A CROSS-SECTIONAL RESEARCH ON THE WEAK ASSOCIATION OF SERENE THREONINE KINASE – 39 GENE TO ESSENTIAL HYPERTENSION (EHTN) WITH RESPECT TO INCREASING AGE FACTOR, BMI AND DIABETES IN THE ABSENCE OF EXERCISE AND PHYSICAL ACTIVITY

    OpenAIRE

    Dr Muhammad Waqar Anjum, Dr. Sehar Fatima , Dr. Mehak Ali

    2018-01-01

    Objective: This research was aimed to study the (Serine Threonine Kinase-39) A-G prevalence polymorphism in the population that is settled in the rural areas. Methods: Nature of the research was cross-sectional, random which was completed in the time span of 2015 – 2017 on 528 participants. We excluded secondary hypertension cases from the record of this research. Controls and cases were respectively Normotensives and Hypertensives. We performed genotyping by tetra-primer amplification re...

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

    Science.gov (United States)

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

    2011-06-01

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

  20. Influence of the O-phosphorylation of serine, threonine and tyrosine in proteins on the amidic N-15 chemical shielding anisotropy tensors

    Czech Academy of Sciences Publication Activity Database

    Emmer, J.; Vavrinská, A.; Sychrovský, Vladimír; Benda, Ladislav; Kříž, Z.; Koča, J.; Boelens, R.; Sklenář, V.; Trantírek, L.

    2013-01-01

    Roč. 55, č. 1 (2013), s. 59-70 ISSN 0925-2738 R&D Projects: GA ČR GAP205/10/0228 Grant - others:CEITEC(XE) CZ.1.05/1.1.00/02.0068 Institutional support: RVO:61388963 Keywords : CSA * phosphorylation * amidic nitrogen * serine * threonine * tyrosine * protein * NMR Subject RIV: CE - Biochemistry Impact factor: 3.305, year: 2013

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

    International Nuclear Information System (INIS)

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

    2015-01-01

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

  2. Stromal serine protein kinase activity in spinach chloroplasts

    International Nuclear Information System (INIS)

    Cortez, N.; Lucero, H.A.; Vallejos, R.H.

    1987-01-01

    At least twelve 32 P-labeled stromal proteins were detected by electrophoresis under denaturing conditions when intact chloroplasts were incubated with 32 Pi, in the light but only three were detected in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) or in the dark. Incubation of isolated stroma with [gamma- 32 P]ATP resulted in the preferential phosphorylation of one of them, a 70-kDa polypeptide, in serine residues. Thylakoid membranes in the dark promoted the phosphorylation of two additional stromal polypeptides of 55 and 40 kDa. Illumination during the phosphorylation of stroma in the presence of thylakoids stimulated severalfold the labeling of the 40-kDa polypeptide but not when DCMU was added. The protein kinase activity present in isolated stroma phosphorylated exogenous substrates like histone III, phosvitin, histone II, and casein with specific activities of 3, 1.8, 0.7, and 0.2 pmol X mg-1 X min-1. Histone III polypeptides were phosphorylated differently by stroma and by thylakoids in the dark. Moreover, histone III phosphorylated by thylakoids in the dark yielded a pattern of phosphopeptides after V8 protease treatment that was different from the pattern obtained when histone III was phosphorylated by stroma

  3. Mutations that cause threonine sensitivity identify catalytic and regulatory regions of the aspartate kinase of Saccharomyces cerevisiae

    DEFF Research Database (Denmark)

    Arévalo-Rodríguez, M; Calderón, I L; Holmberg, S

    1999-01-01

    The HOM3 gene of Saccharomyces cerevisiae encodes aspartate kinase, which catalyses the first step in the branched pathway leading to the synthesis of threonine and methionine from aspartate. Regulation of the carbon flow into this pathway takes place mainly by feedback inhibition of this enzyme ...

  4. DMPD: The serine/threonine kinase Pim-1. [Dynamic Macrophage Pathway CSML Database

    Lifescience Database Archive (English)

    Full Text Available le (.html) CSML File (.csml) Open .csml file with CIOPlayer Open .csml file with CIOPlayer - ※CIO Playerのご利用上の注意 Open .csml file with CIO Open .csml file with CIO - ※CIOのご利用上の注意 ...

  5. Preclinical Evaluation of Serine/Threonine Kinase Inhibitors Against Prostate Cancer Metastases

    Science.gov (United States)

    2008-11-01

    Invest, 103:197- 206, 1999. Yin JJ, Mohammad KS, Kakonen SM, Harris S, Wu-Wong JR, Wessale JL, Padley RJ, Garrett IR , Chirgwin JM, Guise TA. A...coefficient of organic, mm is attenuation coefficient of mineral, and C represents the density of hydroxyapatite . Nanoindentation Dissected male mouse tibiae

  6. Regulation of Serine-Threonine Kinase Akt Activation by NAD+-Dependent Deacetylase SIRT7

    Directory of Open Access Journals (Sweden)

    Jia Yu

    2017-01-01

    Full Text Available The Akt pathway is a central regulator that promotes cell survival in response to extracellular signals. Depletion of SIRT7, an NAD+-dependent deacetylase that is the least-studied sirtuin, is known to significantly increase Akt activity in mice through unknown mechanisms. In this study, we demonstrate that SIRT7 depletion in breast cancer cells results in Akt hyper-phosphorylation and increases cell survival following genotoxic stress. Mechanistically, SIRT7 specifically interacts with and deacetylates FKBP51 at residue lysines 28 and 155 (K28 and K155, resulting in enhanced interactions among FKBP51, Akt, and PHLPP, as well as Akt dephosphorylation. Mutating both lysines to arginines abolishes the effect of SIRT7 on Akt activity through FKBP51 deacetylation. Finally, energy stress strengthens SIRT7-mediated effects on Akt dephosphorylation through FKBP51 and thus sensitizes cancer cells to cytotoxic agents. These results reveal a direct role of SIRT7 in Akt regulation and raise the possibility of using the glucose analog 2-deoxy-D-glucose (2DG as a chemo-sensitizing agent.

  7. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine

  8. Threonine 89 Is an Important Residue of Profilin-1 That Is Phosphorylatable by Protein Kinase A.

    Directory of Open Access Journals (Sweden)

    David Gau

    Full Text Available Dynamic regulation of actin cytoskeleton is at the heart of all actin-based cellular events. In this study, we sought to identify novel post-translational modifications of Profilin-1 (Pfn1, an important regulator of actin polymerization in cells.We performed in vitro protein kinase assay followed by mass-spectrometry to identify Protein Kinase A (PKA phosphorylation sites of Pfn1. By two-dimensional gel electrophoresis (2D-GE analysis, we further examined the changes in the isoelectric profile of ectopically expressed Pfn1 in HEK-293 cells in response to forskolin (FSK, an activator of cAMP/PKA pathway. Finally, we combined molecular dynamics simulations (MDS, GST pull-down assay and F-actin analyses of mammalian cells expressing site-specific phosphomimetic variants of Pfn1 to predict the potential consequences of phosphorylation of Pfn1.We identified several PKA phosphorylation sites of Pfn1 including Threonine 89 (T89, a novel site. Consistent with PKA's ability to phosphorylate Pfn1 in vitro, FSK stimulation increased the pool of the most negatively charged form of Pfn1 in HEK-293 cells which can be attenuated by PKA inhibitor H89. MDS predicted that T89 phosphorylation destabilizes an intramolecular interaction of Pfn1, potentially increasing its affinity for actin. The T89D phosphomimetic mutation of Pfn1 elicits several changes that are hallmarks of proteins folded into alternative three-dimensional conformations including detergent insolubility, protein aggregation and accelerated proteolysis, suggesting that T89 is a structurally important residue of Pfn1. Expression of T89D-Pfn1 induces actin:T89D-Pfn1 co-clusters and dramatically reduces overall actin polymerization in cells, indicating an actin-sequestering action of T89D-Pfn1. Finally, rendering T89 non-phosphorylatable causes a positive charge shift in the isoelectric profile of Pfn1 in a 2D gel electrophoresis analysis of cell extracts, a finding that is consistent with

  9. Selective inhibition reveals cyclin-dependent kinase 2 as another kinase that phosphorylates the androgen receptor at serine 81

    Czech Academy of Sciences Publication Activity Database

    Jorda, Radek; Bučková, Zuzana; Řezníčková, Eva; Bouchal, J.; Kryštof, Vladimír

    2018-01-01

    Roč. 1865, č. 2 (2018), s. 354-363 ISSN 0167-4889 R&D Projects: GA MŠk(CZ) LO1204; GA MŠk(CZ) LO1304 Institutional support: RVO:61389030 Keywords : Androgen receptor * Cyclin-dependent kinase * Inhibitor * Phosphorylation * Serine 81 Subject RIV: EB - Genetics ; Molecular Biology OBOR OECD: Biochemistry and molecular biology Impact factor: 4.521, year: 2016

  10. The small serine-threonine protein SIP2 interacts with STE12 and is involved in ascospore germination in Sordaria macrospora.

    Science.gov (United States)

    Elleuche, Skander; Bernhards, Yasmine; Schäfers, Christian; Varghese, Jans Manjali; Nolting, Nicole; Pöggeler, Stefanie

    2010-12-01

    In fungi, the homoeodomain protein STE12 controls diverse developmental processes, and derives its regulatory specificity from different protein interactions. We recently showed that in the homothallic ascomycete Sordaria macrospora, STE12 is essential for ascospore development, and is able to interact with the alpha-domain mating-type protein SMTA-1 and the MADS box protein MCM1. To further evaluate the functional roles of STE12, we used the yeast two-hybrid approach to identify new STE12-interacting partners. Using STE12 as bait, a small, serine-threonine-rich protein (designated STE12-interacting protein 2, SIP2) was identified. SIP2 is conserved among members of the fungal class Sordariomycetes. In vivo localization studies revealed that SIP2 was targeted to the nucleus and cytoplasm. The STE12/SIP2 interaction was further confirmed in vivo by bimolecular fluorescence complementation. Nuclear localization of SIP2 was apparently mediated by STE12. Unlike deletion of ste12, deletion of sip2 in S. macrospora led to only a slight decrease in ascospore germination, and no other obvious morphological phenotype. In comparison to the Δste12 single knockout strain, ascospore germination was significantly increased in a Δsip2/ste12 double knockout strain. Our data provide evidence for a regulatory role of the novel fungal protein SIP2 in ascospore germination. Copyright © 2010 Elsevier GmbH. All rights reserved.

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

    Directory of Open Access Journals (Sweden)

    Ari M. P. Koskinen

    2010-01-01

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

  12. Phorbol ester-induced serine phosphorylation of the insulin receptor decreases its tyrosine kinase activity.

    Science.gov (United States)

    Takayama, S; White, M F; Kahn, C R

    1988-03-05

    The effect of 12-O-tetradecanoylphorbol-13-acetate (TPA) on the function of the insulin receptor was examined in intact hepatoma cells (Fao) and in solubilized extracts purified by wheat germ agglutinin chromatography. Incubation of ortho[32P]phosphate-labeled Fao cells with TPA increased the phosphorylation of the insulin receptor 2-fold after 30 min. Analysis of tryptic phosphopeptides from the beta-subunit of the receptor by reverse-phase high performance liquid chromatography and determination of their phosphoamino acid composition suggested that TPA predominantly stimulated phosphorylation of serine residues in a single tryptic peptide. Incubation of the Fao cells with insulin (100 nM) for 1 min stimulated 4-fold the phosphorylation of the beta-subunit of the insulin receptor. Prior treatment of the cells with TPA inhibited the insulin-stimulated tyrosine phosphorylation by 50%. The receptors extracted with Triton X-100 from TPA-treated Fao cells and purified on immobilized wheat germ agglutinin retained the alteration in kinase activity and exhibited a 50% decrease in insulin-stimulated tyrosine autophosphorylation and phosphotransferase activity toward exogenous substrates. This was due primarily to a decrease in the Vmax for these reactions. TPA treatment also decreased the Km of the insulin receptor for ATP. Incubation of the insulin receptor purified from TPA-treated cells with alkaline phosphatase decreased the phosphate content of the beta-subunit to the control level and reversed the inhibition, suggesting that the serine phosphorylation of the beta-subunit was responsible for the decreased tyrosine kinase activity. Our results support the notion that the insulin receptor is a substrate for protein kinase C in the Fao cell and that the increase in serine phosphorylation of the beta-subunit of the receptor produced by TPA treatment inhibited tyrosine kinase activity in vivo and in vitro. These data suggest that protein kinase C may regulate the function

  13. Protein kinase A phosphorylates serine 267 in the homeodomain of engrailed-2 leading to decreased DNA binding

    DEFF Research Database (Denmark)

    Hjerrild, Majbrit; Stensballe, Allan; Jensen, Ole N

    2004-01-01

    Engrailed-2 (En-2) belongs to an evolutionarily conserved family of DNA binding homeodomain-containing proteins that are expressed in mammalian brain during development. Here, we demonstrate that serine 267 in the homeodomain of En-2 is phosphorylated by protein kinase A (PKA) in forskolin......-treated COS-7 cells. Furthermore, we analyze the physiological function of En-2 phosphorylation by PKA. The nuclear localization of En-2 is not influenced by the phosphorylation of serine 267. However, substitution of serine 267 with alanine resulted in increased binding of En-2 to DNA, while replacing serine...

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

    Science.gov (United States)

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

    2015-09-01

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

  15. Receptor-interacting protein (RIP) kinase family

    OpenAIRE

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, incl...

  16. Un-catalyzed peptide bond formation between two monomers of glycine, alanine, serine, threonine, and aspartic acid in gas phase: a density functional theory study

    Science.gov (United States)

    Bhunia, Snehasis; Singh, Ajeet; Ojha, Animesh K.

    2016-05-01

    In the present report, un-catalyzed peptide bond formation between two monomers of glycine (Gly), alanine (Ala), serine (Ser), threonine (Thr), and aspartic acid (Asp) has been investigated in gas phase via two steps reaction mechanism and concerted mechanism at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. The peptide bond is formed through a nucleophilic reaction via transition states, TS1 and TS2 in stepwise mechanism. The TS1 reveals formation of a new C-N bond while TS2 illustrate the formation of C=O bond. In case of concerted mechanism, C-N bond is formed by a single four-centre transition state (TS3). The energy barrier is used to explain the involvement of energy at each step of the reaction. The energy barrier (20-48 kcal/mol) is required for the transformation of reactant state R1 to TS1 state and intermediate state I1 to TS2 state. The large value of energy barrier is explained in terms of distortion and interaction energies for stepwise mechanism. The energy barrier of TS3 in concerted mechanism is very close to the energy barrier of the first transition state (TS1) of the stepwise mechanism for the formation of Gly-Gly and Ala-Ala di- peptide. However, in case of Ser-Ser, Thr-Thr and Asp-Asp di-peptide, the energy barrier of TS3 is relatively high than that of the energy barrier of TS1 calculated at B3LYP/6-31G(d,p) and M062X/6-31G(d,p) level of theories. In both the mechanisms, the value of energy barrier calculated at B3LYP/6-31G(d,p) level of theory is greater than that of the value calculated at M062X/6-31G(d,p) level of theory.

  17. A cytoplasmic serine protein kinase binds and may regulate the Fanconi anemia protein FANCA.

    Science.gov (United States)

    Yagasaki, H; Adachi, D; Oda, T; Garcia-Higuera, I; Tetteh, N; D'Andrea, A D; Futaki, M; Asano, S; Yamashita, T

    2001-12-15

    Fanconi anemia (FA) is an autosomal recessive disease with congenital anomalies, bone marrow failure, and susceptibility to leukemia. Patient cells show chromosome instability and hypersensitivity to DNA cross-linking agents. At least 8 complementation groups (A-G) have been identified and 6 FA genes (for subtypes A, C, D2, E, F, and G) have been cloned. Increasing evidence indicates that a protein complex assembly of multiple FA proteins, including FANCA and FANCG, plays a crucial role in the FA pathway. Previously, it was reported that FANCA was phosphorylated in lymphoblasts from normal controls, whereas the phosphorylation was defective in those derived from patients with FA of multiple complementation groups. The present study examined phosphorylation of FANCA ectopically expressed in FANCA(-) cells. Several patient-derived mutations abrogated in vivo phosphorylation of FANCA in this system, suggesting that FANCA phosphorylation is associated with its function. In vitro phosphorylation studies indicated that a physiologic protein kinase for FANCA (FANCA-PK) forms a complex with the substrate. Furthermore, at least a part of FANCA-PK as well as phosphorylated FANCA were included in the FANCA/FANCG complex. Thus, FANCA-PK appears to be another component of the FA protein complex and may regulate function of FANCA. FANCA-PK was characterized as a cytoplasmic serine kinase sensitive to wortmannin. Identification of the protein kinase is expected to elucidate regulatory mechanisms that control the FA pathway.

  18. Phosphorylation of SAF-A/hnRNP-U Serine 59 by Polo-Like Kinase 1 Is Required for Mitosis.

    Science.gov (United States)

    Douglas, Pauline; Ye, Ruiqiong; Morrice, Nicholas; Britton, Sébastien; Trinkle-Mulcahy, Laura; Lees-Miller, Susan P

    2015-08-01

    Scaffold attachment factor A (SAF-A), also called heterogenous nuclear ribonuclear protein U (hnRNP-U), is phosphorylated on serine 59 by the DNA-dependent protein kinase (DNA-PK) in response to DNA damage. Since SAF-A, DNA-PK catalytic subunit (DNA-PKcs), and protein phosphatase 6 (PP6), which interacts with DNA-PKcs, have all been shown to have roles in mitosis, we asked whether DNA-PKcs phosphorylates SAF-A in mitosis. We show that SAF-A is phosphorylated on serine 59 in mitosis, that phosphorylation requires polo-like kinase 1 (PLK1) rather than DNA-PKcs, that SAF-A interacts with PLK1 in nocodazole-treated cells, and that serine 59 is dephosphorylated by protein phosphatase 2A (PP2A) in mitosis. Moreover, cells expressing SAF-A in which serine 59 is mutated to alanine have multiple characteristics of aberrant mitoses, including misaligned chromosomes, lagging chromosomes, polylobed nuclei, and delayed passage through mitosis. Our findings identify serine 59 of SAF-A as a new target of both PLK1 and PP2A in mitosis and reveal that both phosphorylation and dephosphorylation of SAF-A serine 59 by PLK1 and PP2A, respectively, are required for accurate and timely exit from mitosis. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  19. Regulation of Autophagy by Kinases

    International Nuclear Information System (INIS)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets

  20. Regulation of Autophagy by Kinases

    Energy Technology Data Exchange (ETDEWEB)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda, E-mail: alakananda.basu@unthsc.edu [Department of Molecular Biology and Immunology, Institute for Cancer Research, University of North Texas Health Science Center, Fort Worth, TX 76107 (United States)

    2011-06-09

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  1. Regulation of Autophagy by Kinases

    Science.gov (United States)

    Sridharan, Savitha; Jain, Kirti; Basu, Alakananda

    2011-01-01

    Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated protein kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK) and protein kinase C that are often deregulated in cancer and are important therapeutic targets. PMID:24212825

  2. Regulation of Autophagy by Kinases

    Directory of Open Access Journals (Sweden)

    Savitha Sridharan

    2011-06-01

    Full Text Available Autophagy is a process of self-degradation that maintains cellular viability during periods of metabolic stress. Although autophagy is considered a survival mechanism when faced with cellular stress, extensive autophagy can also lead to cell death. Aberrations in autophagy are associated with several diseases, including cancer. Therapeutic exploitation of this process requires a clear understanding of its regulation. Although the core molecular components involved in the execution of autophagy are well studied there is limited information on how cellular signaling pathways, particularly kinases, regulate this complex process. Protein kinases are integral to the autophagy process. Atg1, the first autophagy-related protein identified, is a serine/threonine kinase and it is regulated by another serine/threonine kinase mTOR. Emerging studies suggest the participation of many different kinases in regulating various components/steps of this catabolic process. This review focuses on the regulation of autophagy by several kinases with particular emphasis on serine/threonine protein kinases such as mTOR, AMP-activated kinase, Akt, mitogen-activated protein kinase (ERK, p38 and JNK and protein kinase C that are often deregulated in cancer and are important therapeutic targets.

  3. A mass spectrometry-based proteomic approach for identification of serine/threonine-phosphorylated proteins by enrichment with phospho-specific antibodies

    DEFF Research Database (Denmark)

    Grønborg, Mads; Kristiansen, Troels Zakarias; Stensballe, Allan

    2002-01-01

    /threonine-phosphorylated proteins including drebrin 1, alpha-actinin 4, and filamin-1. We also identified a protein, poly(A)-binding protein 2, which was previously not known to be phosphorylated, in addition to a novel protein without any obvious domains that we designate as Frigg. Frigg is widely expressed and was demonstrated...

  4. Staphylococcal PknB as the First Prokaryotic Representative of the Proline-Directed Kinases

    NARCIS (Netherlands)

    Miller, Malgorzata; Donat, Stefanie; Rakette, Sonja; Stehle, Thilo; Kouwen, Thijs R. H. M.; Diks, Sander H.; Dreisbach, Annette; Reilman, Ewoud; Gronau, Katrin; Becher, Doerte; Peppelenbosch, Maikel P.; van Dijl, Jan Maarten; Ohlsen, Knut

    2010-01-01

    In eukaryotic cell types, virtually all cellular processes are under control of proline-directed kinases and especially MAP kinases. Serine/threonine kinases in general were originally considered as a eukaryote-specific enzyme family. However, recent studies have revealed that orthologues of

  5. Regulation of AMP-activated protein kinase by LKB1 and CaMKK in adipocytes

    DEFF Research Database (Denmark)

    Gormand, Amélie; Henriksson, Emma; Ström, Kristoffer

    2011-01-01

    AMP-activated protein kinase (AMPK) is a serine/threonine kinase that regulates cellular and whole body energy homeostasis. In adipose tissue, activation of AMPK has been demonstrated in response to a variety of extracellular stimuli. However, the upstream kinase that activates AMPK in adipocytes...

  6. KSR1 is a functional protein kinase capable of serine autophosphorylation and direct phosphorylation of MEK1

    International Nuclear Information System (INIS)

    Goettel, Jeremy A.; Liang, Dongchun; Hilliard, Valda C.; Edelblum, Karen L.; Broadus, Matthew R.; Gould, Kathleen L.; Hanks, Steven K.; Polk, D. Brent

    2011-01-01

    The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1 -/- colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.

  7. The kinase domain residue serine 173 of Schizosaccharomyces pombe Chk1 kinase is critical for the response to DNA replication stress

    Directory of Open Access Journals (Sweden)

    Naomi Coulton

    2017-12-01

    Full Text Available While mammalian Chk1 kinase regulates replication origins, safeguards fork integrity and promotes fork progression, yeast Chk1 acts only in G1 and G2. We report here that the mutation of serine 173 (S173A in the kinase domain of fission yeast Chk1 abolishes the G1-M and S-M checkpoints with little impact on the G2-M arrest. This separation-of-function mutation strongly reduces the Rad3-dependent phosphorylation of Chk1 at serine 345 during logarithmic growth, but not when cells experience exogenous DNA damage. Loss of S173 lowers the restrictive temperature of a catalytic DNA polymerase epsilon mutant (cdc20.M10 and is epistatic with a mutation in DNA polymerase delta (cdc6.23 when DNA is alkylated by methyl-methanesulfate (MMS. The chk1-S173A allele is uniquely sensitive to high MMS concentrations where it displays a partial checkpoint defect. A complete checkpoint defect occurs only when DNA replication forks break in cells without the intra-S phase checkpoint kinase Cds1. Chk1-S173A is also unable to block mitosis when the G1 transcription factor Cdc10 (cdc10.V50 is impaired. We conclude that serine 173, which is equivalent to lysine 166 in the activation loop of human Chk1, is only critical in DNA polymerase mutants or when forks collapse in the absence of Cds1.

  8. Partial molar volumes of L-alanine, DL-serine, DL-threonine, L-histidine, glycine, and glycylglycine in water, NaCl, and DMSO aqueous solutions at T 298.15 K

    International Nuclear Information System (INIS)

    Yuan Quan; Li Zhifen; Wang Baohuai

    2006-01-01

    The apparent molar volumes of L-alanine, DL-serine, DL-threonine, L-histidine, glycine, and glycylglycine in water and in the aqueous solutions of NaCl and DMSO with various concentrations at T = 298.15 K have been measured by the precise vibrating-tube digital densimeter. The calculated partial molar volumes at infinite dilution have been used to obtain corresponding transfer volumes from water to various solutions. The experimental results show that the standard partial molar volumes of the above amino acids and peptide at the dilute DMSO aqueous solutions are very close to those in water. However, the volumes show several types of variations with the increase of the concentrations of DMSO due to different types of side chain of amino acids, which should be discussed specifically. The NaCl changes considerably the infinite dilution standard partial molar volumes of the above amino acids and peptide in the aqueous solutions. The infinite dilution standard partial molar volumes of the each amino acids and peptide increase with the concentrations of NaCl. The experimental results have been rationalized by a cosphere overlap model

  9. The role of DNA dependent protein kinase in synapsis of DNA ends

    NARCIS (Netherlands)

    E.P.W.C. Weterings (Eric); N.S. Verkaik (Nicole); H.T. Brüggenwirth (Hennie); D.C. van Gent (Dik); J.H.J. Hoeijmakers (Jan)

    2003-01-01

    textabstractDNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks

  10. Guanylate kinase domains of the MAGUK family scaffold proteins as specific phospho-protein-binding modules

    OpenAIRE

    Zhu, Jinwei; Shang, Yuan; Xia, Caihao; Wang, Wenning; Wen, Wenyu; Zhang, Mingjie

    2011-01-01

    Membrane-associated guanylate kinases (MAGUK) family proteins contain an inactive guanylate kinase (GK) domain, whose function has been elusive. Here, this domain is revealed as a new type of phospho-peptide-binding module, in which the GMP-binding site has evolved to accommodate phospho-serines or -threonines.

  11. Proteínas quinases: características estruturais e inibidores químicos Kinase protein: structural features and chemical inhibitors

    Directory of Open Access Journals (Sweden)

    Bárbara V. Silva

    2009-01-01

    Full Text Available Protein kinases are one of the largest protein families and they are responsible for regulation of a great number of signal transduction pathways in cells, through the phosphorylation of serine, threonine, or tyrosine residues. Deregulation of these enzymes is associated with several diseases including cancer, diabetes and inflammation. For this reason, specific inhibition of tyrosine or serine/threonine kinases may represent an interesting therapeutic approach. The most important types of protein kinases, their structural features and chemical inhibitors are discussed in this paper. Emphasis is given to the small-molecule drugs that target the ATP-binding sites of these enzymes.

  12. Polo-like kinase 2-dependent phosphorylation of NPM/B23 on serine 4 triggers centriole duplication.

    Directory of Open Access Journals (Sweden)

    Annekatrin Krause

    Full Text Available Duplication of the centrosome is well controlled during faithful cell division while deregulation of this process leads to supernumary centrosomes, chromosome missegregation and aneuploidy, a hallmark of many cancer cells. We previously reported that Polo-like kinase 2 (Plk2 is activated near the G1/S phase transition, and regulates the reproduction of centrosomes. In search for Plk2 interacting proteins we have identified NPM/B23 (Nucleophosmin as a novel Plk2 binding partner. We find that Plk2 and NPM/B23 interact in vitro in a Polo-box dependent manner. An association between both proteins was also observed in vivo. Moreover, we show that Plk2 phosphorylates NPM/B23 on serine 4 in vivo in S-phase. Notably, expression of a non-phosphorylatable NPM/B23 S4A mutant interferes with centriole reduplication in S-phase arrested cells and leads to a dilution of centriole numbers in unperturbed U2OS cells. The corresponding phospho-mimicking mutants have the opposite effect and their expression leads to the accumulation of centrioles. These findings suggest that NPM/B23 is a direct target of Plk2 in the regulation of centriole duplication and that phosphorylation on serine 4 can trigger this process.

  13. Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

    Directory of Open Access Journals (Sweden)

    Jessica W Luzwick

    Full Text Available Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

  14. Structure-function similarities between a plant receptor-like kinase and the human interleukin-1 receptor-associated kinase-4.

    Science.gov (United States)

    Klaus-Heisen, Dörte; Nurisso, Alessandra; Pietraszewska-Bogiel, Anna; Mbengue, Malick; Camut, Sylvie; Timmers, Ton; Pichereaux, Carole; Rossignol, Michel; Gadella, Theodorus W J; Imberty, Anne; Lefebvre, Benoit; Cullimore, Julie V

    2011-04-01

    Phylogenetic analysis has previously shown that plant receptor-like kinases (RLKs) are monophyletic with respect to the kinase domain and share an evolutionary origin with the animal interleukin-1 receptor-associated kinase/Pelle-soluble kinases. The lysin motif domain-containing receptor-like kinase-3 (LYK3) of the legume Medicago truncatula shows 33% amino acid sequence identity with human IRAK-4 over the kinase domain. Using the structure of this animal kinase as a template, homology modeling revealed that the plant RLK contains structural features particular to this group of kinases, including the tyrosine gatekeeper and the N-terminal extension α-helix B. Functional analysis revealed the importance of these conserved features for kinase activity and suggests that kinase activity is essential for the biological role of LYK3 in the establishment of the root nodule nitrogen-fixing symbiosis with rhizobia bacteria. The kinase domain of LYK3 has dual serine/threonine and tyrosine specificity, and mass spectrometry analysis identified seven serine, eight threonine, and one tyrosine residue as autophosphorylation sites in vitro. Three activation loop serine/threonine residues are required for biological activity, and molecular dynamics simulations suggest that Thr-475 is the prototypical phosphorylated residue that interacts with the conserved arginine in the catalytic loop, whereas Ser-471 and Thr-472 may be secondary sites. A threonine in the juxtamembrane region and two threonines in the C-terminal lobe of the kinase domain are important for biological but not kinase activity. We present evidence that the structure-function similarities that we have identified between LYK3 and IRAK-4 may be more widely applicable to plant RLKs in general.

  15. Oncoprotein protein kinase antibody kit

    Science.gov (United States)

    Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

    2008-12-23

    An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

  16. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    International Nuclear Information System (INIS)

    Josephson, Matthew P.; Sikkink, Laura A.; Penheiter, Alan R.; Burghardt, Thomas P.; Ajtai, Katalin

    2011-01-01

    Highlights: ► Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. ► Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. ► It is a widely believed that MYL2 is a poor substrate for smMLCK. ► In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. ► Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca 2+ sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis–Menten V max and K M for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant kinase in cardiac tissue on the basis of its specificity, kinetics, and tissue expression.

  17. Smooth muscle myosin light chain kinase efficiently phosphorylates serine 15 of cardiac myosin regulatory light chain

    Energy Technology Data Exchange (ETDEWEB)

    Josephson, Matthew P.; Sikkink, Laura A. [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Penheiter, Alan R. [Molecular Medicine Program, Mayo Clinic, Rochester, MN 55905 (United States); Burghardt, Thomas P., E-mail: burghardt@mayo.edu [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States); Department of Physiology and Biomedical Engineering, Mayo Clinic, Rochester, MN 55905 (United States); Ajtai, Katalin [Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, MN 55905 (United States)

    2011-12-16

    Highlights: Black-Right-Pointing-Pointer Cardiac myosin regulatory light chain (MYL2) is phosphorylated at S15. Black-Right-Pointing-Pointer Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase. Black-Right-Pointing-Pointer It is a widely believed that MYL2 is a poor substrate for smMLCK. Black-Right-Pointing-Pointer In fact, smMLCK efficiently and rapidly phosphorylates S15 in MYL2. Black-Right-Pointing-Pointer Phosphorylation kinetics measured by novel fluorescence method without radioactivity. -- Abstract: Specific phosphorylation of the human ventricular cardiac myosin regulatory light chain (MYL2) modifies the protein at S15. This modification affects MYL2 secondary structure and modulates the Ca{sup 2+} sensitivity of contraction in cardiac tissue. Smooth muscle myosin light chain kinase (smMLCK) is a ubiquitous kinase prevalent in uterus and present in other contracting tissues including cardiac muscle. The recombinant 130 kDa (short) smMLCK phosphorylated S15 in MYL2 in vitro. Specific modification of S15 was verified using the direct detection of the phospho group on S15 with mass spectrometry. SmMLCK also specifically phosphorylated myosin regulatory light chain S15 in porcine ventricular myosin and chicken gizzard smooth muscle myosin (S20 in smooth muscle) but failed to phosphorylate the myosin regulatory light chain in rabbit skeletal myosin. Phosphorylation kinetics, measured using a novel fluorescence method eliminating the use of radioactive isotopes, indicates similar Michaelis-Menten V{sub max} and K{sub M} for regulatory light chain S15 phosphorylation rates in MYL2, porcine ventricular myosin, and chicken gizzard myosin. These data demonstrate that smMLCK is a specific and efficient kinase for the in vitro phosphorylation of MYL2, cardiac, and smooth muscle myosin. Whether smMLCK plays a role in cardiac muscle regulation or response to a disease causing stimulus is unclear but it should be considered a potentially significant

  18. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE.

    Science.gov (United States)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A; Moraes, Carlos T; Sanderson, Thomas H; Stemmler, Timothy L; Grossman, Lawrence I; Kagan, Valerian E; Brunzelle, Joseph S; Salomon, Arthur R; Edwards, Brian F P; Hüttemann, Maik

    2017-01-06

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr 28 , leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨ m ), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr 28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr 28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr 28 in vivo We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via "controlled respiration," preventing ΔΨ m hyperpolarization, a known cause of ROS and trigger of apoptosis. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  19. Phosphorylation of Cytochrome c Threonine 28 Regulates Electron Transport Chain Activity in Kidney: IMPLICATIONS FOR AMP KINASE

    Energy Technology Data Exchange (ETDEWEB)

    Mahapatra, Gargi; Varughese, Ashwathy; Ji, Qinqin; Lee, Icksoo; Liu, Jenney; Vaishnav, Asmita; Sinkler, Christopher; Kapralov, Alexandr A.; Moraes, Carlos T.; Sanderson, Thomas H.; Stemmler, Timothy L.; Grossman, Lawrence I.; Kagan, Valerian E.; Brunzelle, Joseph S.; Salomon, Arthur R.; Edwards, Brian F. P.; Hüttemann, Maik

    2016-10-07

    Mammalian cytochrome c (Cytc) plays a key role in cellular life and death decisions, functioning as an electron carrier in the electron transport chain and as a trigger of apoptosis when released from the mitochondria. However, its regulation is not well understood. We show that the major fraction of Cytc isolated from kidneys is phosphorylated on Thr28, leading to a partial inhibition of respiration in the reaction with cytochrome c oxidase. To further study the effect of Cytc phosphorylation in vitro, we generated T28E phosphomimetic Cytc, revealing superior behavior regarding protein stability and its ability to degrade reactive oxygen species compared with wild-type unphosphorylated Cytc. Introduction of T28E phosphomimetic Cytc into Cytc knock-out cells shows that intact cell respiration, mitochondrial membrane potential (ΔΨm), and ROS levels are reduced compared with wild type. As we show by high resolution crystallography of wild-type and T28E Cytc in combination with molecular dynamics simulations, Thr28 is located at a central position near the heme crevice, the most flexible epitope of the protein apart from the N and C termini. Finally, in silico prediction and our experimental data suggest that AMP kinase, which phosphorylates Cytc on Thr28 in vitro and colocalizes with Cytc to the mitochondrial intermembrane space in the kidney, is the most likely candidate to phosphorylate Thr28 in vivo. We conclude that Cytc phosphorylation is mediated in a tissue-specific manner and leads to regulation of electron transport chain flux via “controlled respiration,” preventing ΔΨm hyperpolarization, a known cause of ROS and trigger of apoptosis.

  20. Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

    International Nuclear Information System (INIS)

    Grose, C.; Jackson, W.; Traugh, J.A.

    1989-01-01

    Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an in vitro assay containing [γ- 32 P]ATP. The same glycoprotein was phosphorylated when [ 32 P]GTP was substituted for [ 32 P]ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein

  1. Protein kinase C signaling and cell cycle regulation

    OpenAIRE

    Black, Adrian R.; Black, Jennifer D.

    2013-01-01

    A link between T cell proliferation and the protein kinase C (PKC) family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. Th...

  2. Autoregulation of kinase dephosphorylation by ATP binding in AGC protein kinases.

    Science.gov (United States)

    Chan, Tung O; Pascal, John M; Armen, Roger S; Rodeck, Ulrich

    2012-02-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non- ATP-competitive kinase inhibitors that discriminate within and between protein kinase families.

  3. Autoregulation of kinase dephosphorylation by ATP binding to AGC protein kinases

    Science.gov (United States)

    Pascal, John M; Armen, Roger S

    2012-01-01

    AGC kinases, including the three Akt (protein kinase B) isoforms, protein kinase A (PKA) and all protein kinase C (PKC) isoforms, require activation loop phosphorylation (threonine 308 in Akt1) as well as phosphorylation of a C-terminal residue (serine 473 in Akt1) for catalytic activity and phosphorylation of downstream targets. Conversely, phosphatases reverse these phosphorylations. Virtually all cellular processes are affected by AGC kinases, a circumstance that has led to intense scrutiny of the molecular mechanisms that regulate phosphorylation of these kinases. Here, we review a new layer of control of phosphorylation in Akt, PKA and PKC pointing to ATP binding pocket occupancy as a means to decelerate dephosphorylation of these and, potentially, other kinases. This additional level of kinase regulation opens the door to search for new functional motifs for the rational design of non-ATP-competitive kinase inhibitors that discriminate within and between protein kinase families. PMID:22262182

  4. Maintained activity of glycogen synthase kinase-3β despite of its phosphorylation at serine-9 in okadaic acid-induced neurodegenerative model

    International Nuclear Information System (INIS)

    Lim, Yong-Whan; Yoon, Seung-Yong; Choi, Jung-Eun; Kim, Sang-Min; Lee, Hui-Sun; Choe, Han; Lee, Seung-Chul; Kim, Dong-Hou

    2010-01-01

    Glycogen synthase kinase-3β (GSK3β) is recognized as one of major kinases to phosphorylate tau in Alzheimer's disease (AD), thus lots of AD drug discoveries target GSK3β. However, the inactive form of GSK3β which is phosphorylated at serine-9 is increased in AD brains. This is also inconsistent with phosphorylation status of other GSK3β substrates, such as β-catenin and collapsin response mediator protein-2 (CRMP2) since their phosphorylation is all increased in AD brains. Thus, we addressed this paradoxical condition of AD in rat neurons treated with okadaic acid (OA) which inhibits protein phosphatase-2A (PP2A) and induces tau hyperphosphorylation and cell death. Interestingly, OA also induces phosphorylation of GSK3β at serine-9 and other substrates including tau, β-catenin and CRMP2 like in AD brains. In this context, we observed that GSK3β inhibitors such as lithium chloride and 6-bromoindirubin-3'-monoxime (6-BIO) reversed those phosphorylation events and protected neurons. These data suggest that GSK3β may still have its kinase activity despite increase of its phosphorylation at serine-9 in AD brains at least in PP2A-compromised conditions and that GSK3β inhibitors could be a valuable drug candidate in AD.

  5. A novel, non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3)

    OpenAIRE

    Fuller, Stephen J; McGuffin, Liam J; Marshall, Andrew K; Giraldo, Alejandro; Pikkarainen, Sampsa; Clerk, Angela; Sugden, Peter

    2012-01-01

    The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr178), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative ‘non-canonical’ pathway of MST3 activation (regulated primarily thro...

  6. Bacterial Protein-Tyrosine Kinases

    DEFF Research Database (Denmark)

    Shi, Lei; Kobir, Ahasanul; Jers, Carsten

    2010-01-01

    in exopolysaccharide production, virulence, DNA metabolism, stress response and other key functions of the bacterial cell. BY-kinases act through autophosphorylation (mainly in exopolysaccharide production) and phosphorylation of other proteins, which have in most cases been shown to be activated by tyrosine......Bacteria and Eukarya share essentially the same family of protein-serine/threonine kinases, also known as the Hanks-type kinases. However, when it comes to protein-tyrosine phosphorylation, bacteria seem to have gone their own way. Bacterial protein-tyrosine kinases (BY-kinases) are bacterial...... and highlighted their importance in bacterial physiology. Having no orthologues in Eukarya, BY-kinases are receiving a growing attention from the biomedical field, since they represent a particularly promising target for anti-bacterial drug design....

  7. In vitro phosphorylation of the movement protein of tomato mosaic tobamovirus by a cellular kinase.

    Science.gov (United States)

    Matsushita, Y; Hanazawa, K; Yoshioka, K; Oguchi, T; Kawakami, S; Watanabe, Y; Nishiguchi, M; Nyunoya, H

    2000-08-01

    The movement protein (MP) of tomato mosaic virus (ToMV) was produced in E. coli as a soluble fusion protein with glutathione S-transferase. When immobilized on glutathione affinity beads, the recombinant protein was phosphorylated in vitro by incubating with cell extracts of Nicotiana tabacum and tobacco suspension culture cells (BY-2) in the presence of [gamma-(32)P]ATP. Phosphorylation occurred even after washing the beads with a detergent-containing buffer, indicating that the recombinant MP formed a stable complex with some protein kinase(s) during incubation with the cell extract. Phosphoamino acid analysis revealed that the MP was phosphorylated on serine and threonine residues. Phosphorylation of the MP was decreased by addition of kinase inhibitors such as heparin, suramin and quercetin, which are known to be effective for casein kinase II (CK II). The phosphorylation level was not changed by other types of inhibitor. In addition, as shown for animal and plant CK II, [gamma-(32)P]GTP was efficiently used as a phosphoryl donor. Phosphorylation was not affected by amino acid replacements at serine-37 and serine-238, but was completely inhibited by deletion of the carboxy-terminal 9 amino acids, including threonine-256, serine-257, serine-261 and serine-263. These results suggest that the MP of ToMV could be phosphorylated in plant cells by a host protein kinase that is closely related to CK II.

  8. Threonine phosphorylation of rat liver glycogen synthase

    International Nuclear Information System (INIS)

    Arino, J.; Arro, M.; Guinovart, J.J.

    1985-01-01

    32 P-labeled glycogen synthase specifically immunoprecipitated from 32 P-phosphate incubated rat hepatocytes contains, in addition to [ 32 P] phosphoserine, significant levels of [ 32 P] phosphothreonine. When the 32 P-immunoprecipitate was cleaved with CNBr, the [ 32 P] phosphothreonine was recovered in the large CNBr fragment (CB-2, Mapp 28 Kd). Homogeneous rat liver glycogen synthase was phosphorylated by all the protein kinases able to phosphorylate CB-2 in vitro. After analysis of the immunoprecipitated enzyme for phosphoaminoacids, it was observed that only casein kinase II was able to phosphorylate on threonine and 32 P-phosphate was only found in CB-2. These results demonstrate that rat liver glycogen synthase is phosphorylated at threonine site(s) contained in CB-2 and strongly indicate that casein kinase II may play a role in the ''in vivo'' phosphorylation of liver glycogen synthase. This is the first protein kinase reported to phosphorylate threonine residues in liver glycogen synthase

  9. Quantitative and Dynamic Imaging of ATM Kinase Activity.

    Science.gov (United States)

    Nyati, Shyam; Young, Grant; Ross, Brian Dale; Rehemtulla, Alnawaz

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including DNA double-strand breaks (DSBs). ATM activation results in the initiation of a complex cascade of events facilitating DNA damage repair, cell cycle checkpoint control, and survival. Traditionally, protein kinases have been analyzed in vitro using biochemical methods (kinase assays using purified proteins or immunological assays) requiring a large number of cells and cell lysis. Genetically encoded biosensors based on optical molecular imaging such as fluorescence or bioluminescence have been developed to enable interrogation of kinase activities in live cells with a high signal to background. We have genetically engineered a hybrid protein whose bioluminescent activity is dependent on the ATM-mediated phosphorylation of a substrate. The engineered protein consists of the split luciferase-based protein complementation pair with a CHK2 (a substrate for ATM kinase activity) target sequence and a phospho-serine/threonine-binding domain, FHA2, derived from yeast Rad53. Phosphorylation of the serine residue within the target sequence by ATM would lead to its interaction with the phospho-serine-binding domain, thereby preventing complementation of the split luciferase pair and loss of reporter activity. Bioluminescence imaging of reporter expressing cells in cultured plates or as mouse xenografts provides a quantitative surrogate for ATM kinase activity and therefore the cellular DNA damage response in a noninvasive, dynamic fashion.

  10. SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

    Science.gov (United States)

    Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

    1997-10-31

    Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

  11. Overexpression of protein kinase STK25 in mice exacerbates ectopic lipid accumulation, mitochondrial dysfunction and insulin resistance in skeletal muscle

    DEFF Research Database (Denmark)

    Chursa, Urszula; Nuñez-Durán, Esther; Cansby, Emmelie

    2017-01-01

    AIMS/HYPOTHESIS: Understanding the molecular networks controlling ectopic lipid deposition and insulin responsiveness in skeletal muscle is essential for developing new strategies to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a critical regulator...... in skeletal muscle, highlighting the potential of STK25 antagonists for type 2 diabetes treatment....

  12. Control of cell division in Streptococcus pneumoniae by the conserved Ser/Thr protein kinase StkP

    Czech Academy of Sciences Publication Activity Database

    Beilharz, K.; Nováková, Linda; Fadda, D.; Branny, Pavel; Massida, O.; Veening, J.-W.

    2012-01-01

    Roč. 109, č. 15 (2012), s. 905-913 ISSN 0027-8424 R&D Projects: GA AV ČR IAA600200801; GA MŠk LH12055 Institutional research plan: CEZ:AV0Z50200510 Keywords : PENICILLIN-BINDING PROTEINS * SERINE/THREONINE KINASE * MYCOBACTERIUM- TUBERCULOSIS Subject RIV: EE - Microbiology, Virology Impact factor: 9.737, year: 2012

  13. A Eukaryotic-Type Serine/Threonine Protein Kinase Is Required for Biofilm Formation, Genetic Competence, and Acid Resistance in Streptococcus mutans

    Czech Academy of Sciences Publication Activity Database

    Hussain, H.; Branny, Pavel; Allan, E.

    2006-01-01

    Roč. 188, č. 4 (2006), s. 1628-1632 ISSN 0021-9193 Institutional research plan: CEZ:AV0Z50200510 Keywords : streptococcus mutans * stpk * pathogenesis Subject RIV: EE - Microbiology, Virology Impact factor: 3.993, year: 2006

  14. Phosphorylation of the norepinephrine transporter at threonine 258 and serine 259 is linked to protein kinase C-mediated transporter internalization

    DEFF Research Database (Denmark)

    Jayanthi, Lankupalle D; Annamalai, Balasubramaniam; Samuvel, Devadoss J

    2006-01-01

    . Most interestingly, the plasma membrane insertion of the WT-hNET and hNET double mutant were not affected by beta-PMA. Although the WT-hNET showed increased endocytosis and redistribution from caveolin-rich plasma membrane domains following beta-PMA treatment, the hNET double mutant was completely......-regulation. These results suggest that Thr-258 and Ser-259 serve as a PKC-specific phospho-acceptor site and that phosphorylation of this motif is linked to PKC-induced NET internalization....

  15. Src kinase regulation by phosphorylation and dephosphorylation

    International Nuclear Information System (INIS)

    Roskoski, Robert

    2005-01-01

    Src and Src-family protein-tyrosine kinases are regulatory proteins that play key roles in cell differentiation, motility, proliferation, and survival. The initially described phosphorylation sites of Src include an activating phosphotyrosine 416 that results from autophosphorylation, and an inhibiting phosphotyrosine 527 that results from phosphorylation by C-terminal Src kinase (Csk) and Csk homologous kinase. Dephosphorylation of phosphotyrosine 527 increases Src kinase activity. Candidate phosphotyrosine 527 phosphatases include cytoplasmic PTP1B, Shp1 and Shp2, and transmembrane enzymes include CD45, PTPα, PTPε, and PTPλ. Dephosphorylation of phosphotyrosine 416 decreases Src kinase activity. Thus far PTP-BL, the mouse homologue of human PTP-BAS, has been shown to dephosphorylate phosphotyrosine 416 in a regulatory fashion. The platelet-derived growth factor receptor protein-tyrosine kinase mediates the phosphorylation of Src Tyr138; this phosphorylation has no direct effect on Src kinase activity. The platelet-derived growth factor receptor and the ErbB2/HER2 growth factor receptor protein-tyrosine kinases mediate the phosphorylation of Src Tyr213 and activation of Src kinase activity. Src kinase is also a substrate for protein-serine/threonine kinases including protein kinase C (Ser12), protein kinase A (Ser17), and CDK1/cdc2 (Thr34, Thr46, and Ser72). Of the three protein-serine/threonine kinases, only phosphorylation by CDK1/cdc2 has been demonstrated to increase Src kinase activity. Although considerable information on the phosphoprotein phosphatases that catalyze the hydrolysis of Src phosphotyrosine 527 is at hand, the nature of the phosphatases that mediate the hydrolysis of phosphotyrosine 138 and 213, and phosphoserine and phosphothreonine residues has not been determined

  16. Receptor-interacting protein (RIP) kinase family

    Science.gov (United States)

    Zhang, Duanwu; Lin, Juan; Han, Jiahuai

    2010-01-01

    Receptor-interacting protein (RIP) kinases are a group of threonine/serine protein kinases with a relatively conserved kinase domain but distinct non-kinase regions. A number of different domain structures, such as death and caspase activation and recruitment domain (CARD) domains, were found in different RIP family members, and these domains should be keys in determining the specific function of each RIP kinase. It is known that RIP kinases participate in different biological processes, including those in innate immunity, but their downstream substrates are largely unknown. This review will give an overview of the structures and functions of RIP family members, and an update of recent progress in RIP kinase research. PMID:20383176

  17. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    OpenAIRE

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic bindin...

  18. Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis

    OpenAIRE

    Ackah, Eric; Yu, Jun; Zoellner, Stefan; Iwakiri, Yasuko; Skurk, Carsten; Shibata, Rei; Ouchi, Noriyuki; Easton, Rachael M.; Galasso, Gennaro; Birnbaum, Morris J.; Walsh, Kenneth; Sessa, William C.

    2005-01-01

    Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Ak...

  19. Identification of a novel phosphorylation site on TBC1D4 regulated by AMP-activated protein kinase in skeletal muscle

    DEFF Research Database (Denmark)

    Treebak, Jonas Thue; Taylor, Eric B.; Witczak, Carol A.

    2010-01-01

    TBC1D4 (also known as AS160) regulates GLUT4 translocation and glucose uptake in adipocytes and skeletal muscle. Its mode of action involves phosphorylation of Serine (S)/Threonine (T) residues by upstream kinases resulting in inactivation of Rab-GAP activity leading to GLUT4 mobilization...

  20. Protein kinase C, focal adhesions and the regulation of cell migration

    DEFF Research Database (Denmark)

    Fogh, Betina S; Multhaupt, Hinke A B; Couchman, John Robert

    2014-01-01

    in their intracellular compartment. Among these are tyrosine kinases, which have received a great deal of attention, whereas the serine/threonine kinase protein kinase C has received much less. Here the status of protein kinase C in focal adhesions and cell migration is reviewed, together with discussion of its roles...... and adhesion turnover. Focal adhesions, or focal contacts, are widespread organelles at the cell-matrix interface. They arise as a result of receptor interactions with matrix ligands, together with clustering. Recent analysis shows that focal adhesions contain a very large number of protein components...

  1. ACTIVATION OF A CRYPTIC D-SERINE DEAMINASE (DSD) GENE FROM PSEUDOMONAS CEPACIA 17616

    Science.gov (United States)

    D-serine inhibits growth of P. cepacia 17616; however, resistant mutants able to express an ordinarily cryptic D-serine deaminase (dsd) gene were isolated readily. The resistant strains formed high levels of a D-serine deaminase active on D-threonine as well as D-serine. IS eleme...

  2. Bacillus subtilis Two-Component System Sensory Kinase DegS Is Regulated by Serine Phosphorylation in Its Input Domain

    DEFF Research Database (Denmark)

    Jers, Carsten; Kobir, Ahasanul; Søndergaard, Elsebeth Oline

    2011-01-01

    Bacillus subtilis two-component system DegS/U is well known for the complexity of its regulation. The cytosolic sensory kinase DegS does not receive a single predominant input signal like most two-component kinases, instead it integrates a wide array of metabolic inputs that modulate its activity......S phosphorylation can be carried out by at least two B. subtilis Hanks-type kinases in vitro, and this stimulates the phosphate transfer towards DegU. The consequences of this process were studied in vivo, using phosphomimetic (Ser76Asp) and non-phosphorylatable (Ser76Ala) mutants of DegS. In a number...

  3. A framework for classification of prokaryotic protein kinases.

    Directory of Open Access Journals (Sweden)

    Nidhi Tyagi

    Full Text Available BACKGROUND: Overwhelming majority of the Serine/Threonine protein kinases identified by gleaning archaeal and eubacterial genomes could not be classified into any of the well known Hanks and Hunter subfamilies of protein kinases. This is owing to the development of Hanks and Hunter classification scheme based on eukaryotic protein kinases which are highly divergent from their prokaryotic homologues. A large dataset of prokaryotic Serine/Threonine protein kinases recognized from genomes of prokaryotes have been used to develop a classification framework for prokaryotic Ser/Thr protein kinases. METHODOLOGY/PRINCIPAL FINDINGS: We have used traditional sequence alignment and phylogenetic approaches and clustered the prokaryotic kinases which represent 72 subfamilies with at least 4 members in each. Such a clustering enables classification of prokaryotic Ser/Thr kinases and it can be used as a framework to classify newly identified prokaryotic Ser/Thr kinases. After series of searches in a comprehensive sequence database we recognized that 38 subfamilies of prokaryotic protein kinases are associated to a specific taxonomic level. For example 4, 6 and 3 subfamilies have been identified that are currently specific to phylum proteobacteria, cyanobacteria and actinobacteria respectively. Similarly subfamilies which are specific to an order, sub-order, class, family and genus have also been identified. In addition to these, we also identify organism-diverse subfamilies. Members of these clusters are from organisms of different taxonomic levels, such as archaea, bacteria, eukaryotes and viruses. CONCLUSION/SIGNIFICANCE: Interestingly, occurrence of several taxonomic level specific subfamilies of prokaryotic kinases contrasts with classification of eukaryotic protein kinases in which most of the popular subfamilies of eukaryotic protein kinases occur diversely in several eukaryotes. Many prokaryotic Ser/Thr kinases exhibit a wide variety of modular

  4. Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-1 kinase

    International Nuclear Information System (INIS)

    Qian, Kevin C.; Studts, Joey; Wang, Lian; Barringer, Kevin; Kronkaitis, Anthony; Peng, Charline; Baptiste, Alistair; LaFrance, Roger; Mische, Sheenah; Farmer, Bennett

    2004-01-01

    Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P6 5 , with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit

  5. Crystallization and initial X-ray diffraction study of the three PASTA domains of the Ser/Thr kinase Stk1 from the human pathogen Staphylococcus aureus

    International Nuclear Information System (INIS)

    Paracuellos, Patricia; Ballandras, Allison; Robert, Xavier; Cozzone, Alain J.; Duclos, Bertrand; Gouet, Patrice

    2009-01-01

    Crystallization conditions have been determined for an extracellular portion of the Ser/Thr kinase Stk1 from the human pathogen S. aureus that contains three PASTA subunits. Synchrotron data have been collected to a resolution of 2.9 Å. Phasing is in progress. PASTA subunits (∼70 amino acids) are specific to bacterial serine/threonine kinases and to penicillin-binding proteins (PBPs) and are involved in the synthesis of peptidoglycan. The human pathogen Staphylococcus aureus contains a serine/threonine kinase, Stk1, which plays a major role in virulence. A recombinant His-tagged portion of the extracellular domain of Stk1 containing three PASTA subunits has been crystallized using zinc sulfate as a crystallizing agent. The crystals belonged to the tetragonal space group P4 1 22, with unit-cell parameters a = 68.0, b = 68.0, c = 158.1 Å. Structure determination by the MAD method is now in progress

  6. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    OpenAIRE

    Jette, Nicholas; Lees-Miller, Susan P.

    2014-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemi...

  7. Recent Progress on Liver Kinase B1 (LKB1: Expression, Regulation, Downstream Signaling and Cancer Suppressive Function

    Directory of Open Access Journals (Sweden)

    Ren-You Gan

    2014-09-01

    Full Text Available Liver kinase B1 (LKB1, known as a serine/threonine kinase, has been identified as a critical cancer suppressor in many cancer cells. It is a master upstream kinase of 13 AMP-activated protein kinase (AMPK-related protein kinases, and possesses versatile biological functions. LKB1 gene is mutated in many cancers, and its protein can form different protein complexes with different cellular localizations in various cell types. The expression of LKB1 can be regulated through epigenetic modification, transcriptional regulation and post-translational modification. LKB1 dowcnstream pathways mainly include AMPK, microtubule affinity regulating kinase (MARK, salt-inducible kinase (SIK, sucrose non-fermenting protein-related kinase (SNRK and brain selective kinase (BRSK signalings, etc. This review, therefore, mainly discusses recent studies about the expression, regulation, downstream signaling and cancer suppressive function of LKB1, which can be helpful for better understanding of this molecular and its significance in cancers.

  8. Preparation of kinase-biased compounds in the search for lead inhibitors of kinase targets.

    Science.gov (United States)

    Lai, Justine Y Q; Langston, Steven; Adams, Ruth; Beevers, Rebekah E; Boyce, Richard; Burckhardt, Svenja; Cobb, James; Ferguson, Yvonne; Figueroa, Eva; Grimster, Neil; Henry, Andrew H; Khan, Nawaz; Jenkins, Kerry; Jones, Mark W; Judkins, Robert; Major, Jeremy; Masood, Abid; Nally, James; Payne, Helen; Payne, Lloyd; Raphy, Gilles; Raynham, Tony; Reader, John; Reader, Valérie; Reid, Alison; Ruprah, Parminder; Shaw, Michael; Sore, Hannah; Stirling, Matthew; Talbot, Adam; Taylor, Jess; Thompson, Stephen; Wada, Hiroki; Walker, David

    2005-05-01

    This work describes the preparation of approximately 13,000 compounds for rapid identification of hits in high-throughput screening (HTS). These compounds were designed as potential serine/threonine or tyrosine kinase inhibitors. The library consists of various scaffolds, e.g., purines, oxindoles, and imidazoles, whereby each core scaffold generally includes the hydrogen bond acceptor/donor properties known to be important for kinase binding. Several of these are based upon literature kinase templates, or adaptations of them to provide novelty. The routes to their preparation are outlined. A variety of automation techniques were used to prepare >500 compounds per scaffold. Where applicable, scavenger resins were employed to remove excess reagents and when necessary, preparative high performance liquid chromatography (HPLC) was used for purification. These compounds were screened against an 'in-house' kinase panel. The success rate in HTS was significantly higher than the corporate compound collection. Copyright (c) 2004 Wiley Periodicals, Inc.

  9. Genetic Disruption of Protein Kinase STK25 Ameliorates Metabolic Defects in a Diet-Induced Type 2 Diabetes Model

    OpenAIRE

    Amrutkar, Manoj; Cansby, Emmelie; Chursa, Urszula; Nu?ez-Dur?n, Esther; Chancl?n, Bel?n; St?hlman, Marcus; Frid?n, Vincent; Manner?s-Holm, Louise; Wickman, Anna; Smith, Ulf; B?ckhed, Fredrik; Bor?n, Jan; Howell, Brian W.; Mahlapuu, Margit

    2015-01-01

    Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type litte...

  10. Effects of inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate induced insulin resistance in L6 myotubes.

    Directory of Open Access Journals (Sweden)

    Agnieszka Mikłosz

    Full Text Available BACKGROUND: The objective of this study was to examine the effects of short (2 h and prolonged (18 h inhibition of serine palmitoyltransferase (SPT and sphingosine kinase 1 (SphK1 on palmitate (PA induced insulin resistance in L6 myotubes. METHODS: L6 myotubes were treated simultaneously with either PA and myriocin (SPT inhibitor or PA and Ski II (SphK1inhibitor for different time periods (2 h and 18 h. Insulin stimulated glucose uptake was measured using radioactive isotope. Expression of insulin signaling proteins was determined using Western blot analyses. Intracellular sphingolipids content [sphinganine (SFA, ceramide (CER, sphingosine (SFO, sphingosine-1-phosphate (S1P] were estimated by HPLC. RESULTS: Our results revealed that both short and prolonged time of inhibition of SPT by myriocin was sufficient to prevent ceramide accumulation and simultaneously reverse palmitate induced inhibition of insulin-stimulated glucose transport. In contrast, prolonged inhibition of SphK1 intensified the effect of PA on insulin-stimulated glucose uptake and attenuated further the activity of insulin signaling proteins (pGSK3β/GSK3β ratio in L6 myotubes. These effects were related to the accumulation of sphingosine in palmitate treated myotubes. CONCLUSION: Myriocin is more effective in restoration of palmitate induced insulin resistance in L6 myocytes, despite of the time of SPT inhibition, comparing to SKII (a specific SphK1 inhibitor. Observed changes in insulin signaling proteins were related to the content of specific sphingolipids, namely to the reduction of ceramide. Interestingly, inactivation of SphK1 augmented the effect of PA induced insulin resistance in L6 myotubes, which was associated with further inhibition of insulin stimulated PKB and GSK3β phosphorylation, glucose uptake and the accumulation of sphingosine.

  11. Autocrine motility factor (neuroleukin, phosphohexose isomerase) induces cell movement through 12-lipoxygenase-dependent tyrosine phosphorylation and serine dephosphorylation events.

    Science.gov (United States)

    Timár, J; Tóth, S; Tóvári, J; Paku, S; Raz, A

    1999-01-01

    Autocrine motility factor (AMF) is one of the motility cytokines regulating tumor cell migration, therefore identification of the signaling pathway coupled with it has critical importance. Previous studies revealed several elements of this pathway predominated by lipoxygenase-PKC activations but the role for tyrosine kinases remained questionable. Motility cytokines frequently have mitogenic effect as well, producing activation of overlapping signaling pathways therefore we have used B16a melanoma cells as models where AMF has exclusive motility effect. Our studies revealed that in B16a cells AMF initiated rapid (1-5 min) activation of the protein tyrosine kinase (PTK) cascade inducing phosphorylation of 179, 125, 95 and 40/37 kD proteins which was mediated by upstream cyclo- and lipoxygenases. The phosphorylated proteins were localized to the cortical actin-stress fiber attachment zones in situ by confocal microscopy. On the other hand, AMF receptor activation induced significant decrease in overall serine-phosphorylation level of cellular proteins accompanied by serine phosphorylation of 200, 90, 78 and 65 kd proteins. The decrease in serine phosphorylation was independent of PTKs, PKC as well as cyclo- and lipoxygenases. However, AMF induced robust translocation of PKCalpha to the stress fibers and cortical actin suggesting a critical role for this kinase in the generation of the motility signal. Based on the significant decrease in serine phosphorylation after AMF stimulus in B16a cells we postulated the involvement of putative serine/threonine phosphatase(s) upstream lipoxygenase and activation of the protein tyrosine kinase cascade downstream cyclo- and lipoxygenase(s) in the previously identified autocrine motility signal.

  12. Pim kinases are upregulated during Epstein-Barr virus infection and enhance EBNA2 activity

    International Nuclear Information System (INIS)

    Rainio, Eeva-Marja; Ahlfors, Helena; Carter, Kara L.; Ruuska, Marja; Matikainen, Sampsa; Kieff, Elliott; Koskinen, Paeivi J.

    2005-01-01

    Latent Epstein-Barr virus (EBV) infection is strongly associated with B-cell proliferative diseases such as Burkitt's lymphoma. Here we show that the oncogenic serine/threonine kinases Pim-1 and Pim-2 enhance the activity of the viral transcriptional activator EBNA2. During EBV infection of primary B-lymphocytes, the mRNA expression levels of pim genes, especially of pim-2, are upregulated and remain elevated in latently infected B-cell lines. Thus, EBV-induced upregulation of Pim kinases and Pim-stimulated EBNA2 transcriptional activity may contribute to the ability of EBV to immortalize B-cells and predispose them to malignant growth

  13. Death-associated protein kinase (DAPK) and signal transduction: regulation in cancer.

    Science.gov (United States)

    Michie, Alison M; McCaig, Alison M; Nakagawa, Rinako; Vukovic, Milica

    2010-01-01

    Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine protein kinase that is dysregulated in a wide variety of cancers. The mechanism by which this occurs has largely been attributed to promoter hypermethylation, which results in gene silencing. However, recent studies indicate that DAPK expression can be detected in some cancers, but its function is still repressed, suggesting that DAPK activity can be subverted at a post-translational level in cancer cells. This review will focus on recent data describing potential mechanisms that may alter the expression, regulation or function of DAPK.

  14. Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

    Science.gov (United States)

    Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

    2016-07-01

    The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

  15. SOcK, MiSTs, MASK and STicKs: the GCKIII (germinal centre kinase III) kinases and their heterologous protein-protein interactions.

    Science.gov (United States)

    Sugden, Peter H; McGuffin, Liam J; Clerk, Angela

    2013-08-15

    The GCKIII (germinal centre kinase III) subfamily of the mammalian Ste20 (sterile 20)-like group of serine/threonine protein kinases comprises SOK1 (Ste20-like/oxidant-stress-response kinase 1), MST3 (mammalian Ste20-like kinase 3) and MST4. Initially, GCKIIIs were considered in the contexts of the regulation of mitogen-activated protein kinase cascades and apoptosis. More recently, their participation in multiprotein heterocomplexes has become apparent. In the present review, we discuss the structure and phosphorylation of GCKIIIs and then focus on their interactions with other proteins. GCKIIIs possess a highly-conserved, structured catalytic domain at the N-terminus and a less-well conserved C-terminal regulatory domain. GCKIIIs are activated by tonic autophosphorylation of a T-loop threonine residue and their phosphorylation is regulated primarily through protein serine/threonine phosphatases [especially PP2A (protein phosphatase 2A)]. The GCKIII regulatory domains are highly disorganized, but can interact with more structured proteins, particularly the CCM3 (cerebral cavernous malformation 3)/PDCD10 (programmed cell death 10) protein. We explore the role(s) of GCKIIIs (and CCM3/PDCD10) in STRIPAK (striatin-interacting phosphatase and kinase) complexes and their association with the cis-Golgi protein GOLGA2 (golgin A2; GM130). Recently, an interaction of GCKIIIs with MO25 has been identified. This exhibits similarities to the STRADα (STE20-related kinase adaptor α)-MO25 interaction (as in the LKB1-STRADα-MO25 heterotrimer) and, at least for MST3, the interaction may be enhanced by cis-autophosphorylation of its regulatory domain. In these various heterocomplexes, GCKIIIs associate with the Golgi apparatus, the centrosome and the nucleus, as well as with focal adhesions and cell junctions, and are probably involved in cell migration, polarity and proliferation. Finally, we consider the association of GCKIIIs with a number of human diseases, particularly

  16. Plasma membrane Ca2+-ATPase 4: interaction with constitutive nitric oxide synthases in human sperm and prostasomes which carry Ca2+/CaM-dependent serine kinase.

    Science.gov (United States)

    Andrews, Rachel E; Galileo, Deni S; Martin-DeLeon, Patricia A

    2015-11-01

    Deletion of the gene encoding the widely conserved plasma membrane calcium ATPase 4 (PMCA4), a major Ca(2+) efflux pump, leads to loss of sperm motility and male infertility in mice. PMCA4's partners in sperm and how its absence exerts its effect on fertility are unknown. We hypothesize that in sperm PMCA4 interacts with endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) which are rapidly activated by Ca(2+), and that these fertility-modulating proteins are present in prostasomes, which deliver them to sperm. We show that in human sperm PMCA4 is present on the acrosome, inner acrosomal membrane, posterior head, neck, midpiece and the proximal principal piece. PMCA4 localization showed inter- and intra-individual variation and was most abundant at the posterior head/neck junction, co-localizing with NOSs. Co-immunoprecipitations (Co-IP) revealed a close association of PMCA4 and the NOSs in Ca(2+) ionophore-treated sperm but much less so in uncapacitated untreated sperm. Fluorescence resonance energy transfer (FRET) showed a similar Ca(2+)-related association: PMCA4 and the NOSs are within 10 nm apart, and preferentially so in capacitated, compared with uncapacitated, sperm. FRET efficiencies varied, being significantly (P < 0.001) higher at high cytosolic Ca(2+) concentration ([Ca(2+)]c) in capacitated sperm than at low [Ca(2+)]c in uncapacitated sperm for the PMCA4-eNOS complex. These dynamic interactions were not seen for PMCA4-nNOS complexes, which had the highest FRET efficiencies. Further, along with Ca(2+)/CaM-dependent serine kinase (CASK), PMCA4 and the NOSs are present in the seminal plasma, specifically in prostasomes where Co-IP showed complexes similar to those in sperm. Finally, flow cytometry demonstrated that following co-incubation of sperm and seminal plasma, PMCA4 and the NOSs can be delivered in vitro to sperm via prostasomes. Our findings indicate that PMCA4 interacts simultaneously with the NOSs preferentially at

  17. KFC, a Ste20-like kinase with mitogenic potential and capability to activate the SAPK/JNK pathway.

    Science.gov (United States)

    Yustein, J T; Li, D; Robinson, D; Kung, H J

    2000-02-03

    The Sterile-20 (Ste20) family of serine-threonine kinases has been implicated in the activation of the stress-activated protein kinase pathways. However, the physiological role has remained ambiguous for most of the investigated mammalian Ste20's. Here we report the cloning of a novel Ste20-like kinase, from chicken embryo fibroblast (CEF) cells, which we have named KFC, for Kinase From Chicken. The 898 amino acid full-length KFC protein contains an amino-terminal kinase domain, an adjacent downstream serine-rich region, and a C-terminal tail containing a coiled-coil domain. Here we show that the coiled-coil domain of KFC negatively regulates the intrinsic kinase activity. We have also identified a splice variant of KFC in which there is a 207 nucleotide in-frame deletion. This deletion of 69 amino acids encompasses the serine-rich region. These two isoforms, called KFCL, for full-length, and KFCS for spliced (or short) form, not only differ in structure, but also in biological properties. Stable CEF cells overexpressing KFCL, but not KFCS, have a significant increase in growth rate when compared to parental cells. This mitogenic effect is the first such reported for this family of kinases. Finally, we found that KFC, when activated by truncation of the regulatory C-terminus, has a specific activation of the stress-activated protein kinase (SAPK/JNK) pathway.

  18. Minor Threonine Dehydratase Encoded Within the Threonine Synthetic Region of Bacillus subtilis1

    Science.gov (United States)

    Vapnek, Daniel; Greer, Sheldon

    1971-01-01

    Challenging auxotrophs on metabolites that are precursors of a biosynthetic step involving a mutated enzyme has revealed a new class of suppressor mutations which act by derepressing a minor enzyme activity not normally detected in the wild-type strain. These indirect, partial suppressor mutations which allow isoleucine auxotrophs to grow on homoserine or threonine have been analyzed to determine their effect on enzymes involved in the biosynthesis of these amino acids. It has been found that one class of these suppressor mutations (sprA) leads to the derepression of homoserine kinase, homoserine dehydrogenase, and a minor threonine dehydratase that is not sufficiently active to be detected in the wild-type strain. The gene encoding this second threonine dehydratase activity has been found to be located between the structural genes for homoserine kinase and homoserine dehydrogenase. The results of these experiments indicate that plating of auxotrophs on precursors of a biosynthetic step involving mutated enzymes could prove to be a valuable method for the detection of regulatory mutants as well as a possible tool in studying the evolution of biochemical pathways. PMID:4997544

  19. Physical and functional interactions between ZIP kinase and UbcH5

    International Nuclear Information System (INIS)

    Ohbayashi, Norihiko; Okada, Katsuya; Kawakami, Shiho; Togi, Sumihito; Sato, Noriko; Ikeda, Osamu; Kamitani, Shinya; Muromoto, Ryuta; Sekine, Yuichi; Kawai, Taro; Akira, Shizuo; Matsuda, Tadashi

    2008-01-01

    Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination

  20. Phosphorylations of Serines 21/9 in Glycogen Synthase Kinase 3α/β Are Not Required for Cell Lineage Commitment or WNT Signaling in the Normal Mouse Intestine.

    Directory of Open Access Journals (Sweden)

    Fiona Hey

    Full Text Available The WNT signalling pathway controls many developmental processes and plays a key role in maintenance of intestine renewal and homeostasis. Glycogen Synthase Kinase 3 (GSK3 is an important component of the WNT pathway and is involved in regulating β-catenin stability and expression of WNT target genes. The mechanisms underpinning GSK3 regulation in this context are not completely understood, with some evidence suggesting this occurs through inhibitory N-terminal serine phosphorylation in a similar way to GSK3 inactivation in insulin signaling. To investigate this in a physiologically relevant context, we have analysed the intestinal phenotype of GSK3 knockin mice in which N-terminal serines 21/9 of GSK3α/β have been mutated to non-phosphorylatable alanine residues. We show that these knockin mutations have very little effect on overall intestinal integrity, cell lineage commitment, β-catenin localization or WNT target gene expression although a small increase in apoptosis at villi tips is observed. Our results provide in vivo evidence that GSK3 is regulated through mechanisms independent of N-terminal serine phosphorylation in order for β-catenin to be stabilised.

  1. The Arabidopsis SOS2 protein kinase physically interacts with and is activated by the calcium-binding protein SOS3

    OpenAIRE

    Halfter, Ursula; Ishitani, Manabu; Zhu, Jian-Kang

    2000-01-01

    The Arabidopsis thaliana SOS2 and SOS3 genes are required for intracellular Na+ and K+ homeostasis and plant tolerance to high Na+ and low K+ environments. SOS3 is an EF hand type calcium-binding protein having sequence similarities with animal neuronal calcium sensors and the yeast calcineurin B. SOS2 is a serine/threonine protein kinase in the SNF1/AMPK family. We report here that SOS3 physically interacts with and activates SOS2 protein kinase. Genetically, sos2sos3 double mutant analysis ...

  2. The Pim kinases: new targets for drug development.

    Science.gov (United States)

    Swords, Ronan; Kelly, Kevin; Carew, Jennifer; Nawrocki, Stefan; Mahalingam, Devalingam; Sarantopoulos, John; Bearss, David; Giles, Francis

    2011-12-01

    The three Pim kinases are a small family of serine/threonine kinases regulating several signaling pathways that are fundamental to cancer development and progression. They were first recognized as pro-viral integration sites for the Moloney Murine Leukemia virus. Unlike other kinases, they possess a hinge region which creates a unique binding pocket for ATP. Absence of a regulatory domain means that these proteins are constitutively active once transcribed. Pim kinases are critical downstream effectors of the ABL (ableson), JAK2 (janus kinase 2), and Flt-3 (FMS related tyrosine kinase 1) oncogenes and are required by them to drive tumorigenesis. Recent investigations have established that the Pim kinases function as effective inhibitors of apoptosis and when overexpressed, produce resistance to the mTOR (mammalian target of rapamycin) inhibitor, rapamycin . Overexpression of the PIM kinases has been reported in several hematological and solid tumors (PIM 1), myeloma, lymphoma, leukemia (PIM 2) and adenocarcinomas (PIM 3). As such, the Pim kinases are a very attractive target for pharmacological inhibition in cancer therapy. Novel small molecule inhibitors of the human Pim kinases have been designed and are currently undergoing preclinical evaluation.

  3. Induction of viral, 7-methyl-guanosine cap-independent translation and oncolysis by mitogen-activated protein kinase-interacting kinase-mediated effects on the serine/arginine-rich protein kinase.

    Science.gov (United States)

    Brown, Michael C; Bryant, Jeffrey D; Dobrikova, Elena Y; Shveygert, Mayya; Bradrick, Shelton S; Chandramohan, Vidyalakshmi; Bigner, Darell D; Gromeier, Matthias

    2014-11-01

    Protein synthesis, the most energy-consuming process in cells, responds to changing physiologic priorities, e.g., upon mitogen- or stress-induced adaptations signaled through the mitogen-activated protein kinases (MAPKs). The prevailing status of protein synthesis machinery is a viral pathogenesis factor, particularly for plus-strand RNA viruses, where immediate translation of incoming viral RNAs shapes host-virus interactions. In this study, we unraveled signaling pathways centered on the ERK1/2 and p38α MAPK-interacting kinases MNK1/2 and their role in controlling 7-methyl-guanosine (m(7)G) "cap"-independent translation at enterovirus type 1 internal ribosomal entry sites (IRESs). Activation of Raf-MEK-ERK1/2 signals induced viral IRES-mediated translation in a manner dependent on MNK1/2. This effect was not due to MNK's known functions as eukaryotic initiation factor (eIF) 4G binding partner or eIF4E(S209) kinase. Rather, MNK catalytic activity enabled viral IRES-mediated translation/host cell cytotoxicity through negative regulation of the Ser/Arg (SR)-rich protein kinase (SRPK). Our investigations suggest that SRPK activity is a major determinant of type 1 IRES competency, host cell cytotoxicity, and viral proliferation in infected cells. We are targeting unfettered enterovirus IRES activity in cancer with PVSRIPO, the type 1 live-attenuated poliovirus (PV) (Sabin) vaccine containing a human rhinovirus type 2 (HRV2) IRES. A phase I clinical trial of PVSRIPO with intratumoral inoculation in patients with recurrent glioblastoma (GBM) is showing early promise. Viral translation proficiency in infected GBM cells is a core requirement for the antineoplastic efficacy of PVSRIPO. Therefore, it is critically important to understand the mechanisms controlling viral cap-independent translation in infected host cells. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  4. Td4IN2: A drought-responsive durum wheat (Triticum durum Desf.) gene coding for a resistance like protein with serine/threonine protein kinase, nucleotide binding site and leucine rich domains.

    Science.gov (United States)

    Rampino, Patrizia; De Pascali, Mariarosaria; De Caroli, Monica; Luvisi, Andrea; De Bellis, Luigi; Piro, Gabriella; Perrotta, Carla

    2017-11-01

    Wheat, the main food source for a third of world population, appears strongly under threat because of predicted increasing temperatures coupled to drought. Plant complex molecular response to drought stress relies on the gene network controlling cell reactions to abiotic stress. In the natural environment, plants are subjected to the combination of abiotic and biotic stresses. Also the response of plants to biotic stress, to cope with pathogens, involves the activation of a molecular network. Investigations on combination of abiotic and biotic stresses indicate the existence of cross-talk between the two networks and a kind of overlapping can be hypothesized. In this work we describe the isolation and characterization of a drought-related durum wheat (Triticum durum Desf.) gene, identified in a previous study, coding for a protein combining features of NBS-LRR type resistance protein with a S/TPK domain, involved in drought stress response. This is one of the few examples reported where all three domains are present in a single protein and, to our knowledge, it is the first report on a gene specifically induced by drought stress and drought-related conditions, with this particular structure. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  5. Rapid effector function of circulating CD4+T cells specific for immunodominant regions of the conserved serine/threonine kinase found in Streptococcus pneumoniae (StkP) in healthy adults

    Czech Academy of Sciences Publication Activity Database

    Aslam, A.; Mason, A.; Zemenides, S.; Chan, H.; Nováková, Linda; Branny, Pavel; Finn, A.; Chapel, H.; Ogg, G. S.

    2010-01-01

    Roč. 60, č. 2 (2010), s. 113-122 ISSN 0928-8244 R&D Projects: GA ČR GP204/07/P082; GA ČR GA204/08/0783 Institutional research plan: CEZ:AV0Z50200510 Keywords : epitopes * pneumococcus * ELISpot Subject RIV: EE - Microbiology, Virology Impact factor: 2.494, year: 2010

  6. Discrepancy between low levels of mTOR activity and high levels of p-S6 in primary central nervous system lymphoma may be explained by PAS domain-containing serine/threonine-protein kinase-mediated phosphorylation

    DEFF Research Database (Denmark)

    Marosvari, Dora; Nagy, Noemi; Kriston, Csilla

    2018-01-01

    The primary aim of this study was to determine mTOR-pathway activity in primary central nervous system lymphoma (PCNSL), which could be a potential target for therapy. After demonstrating that p-S6 positivity largely exceeded mTOR activity, we aimed to identify other pathways that may lead to S6...... phosphorylation. We measured mTOR activity with immunohistochemistry for p-mTOR and its downstream effectors p(T389)-p70S6K1, p-S6, and p-4EBP1 in 31 cases of PCNSL and 51 cases of systemic diffuse large B-cell lymphoma (DLBCL) and evaluated alternative S6 phosphorylation pathways with p-RSK, p(T229)-p70S6K1...... responsible for S6 phosphorylation, PASK proved to be positive in all cases of PCNSL and DLBCL. Inhibition of PASK resulted in reduced expression of p-S6 in BHD1-cells. This is the first study demonstrating an mTOR independent p-S6 activity in PCNSL and that PASK may contribute to the phosphorylation of S6...

  7. Regulation of glycogen synthase kinase-3{beta} (GSK-3{beta}) after ionizing radiation; Regulation der Glykogen Synthase Kinase-3{beta} (GSK-3{beta}) nach ionisierender Strahlung

    Energy Technology Data Exchange (ETDEWEB)

    Boehme, K.A.

    2006-12-15

    Glycogen Synthase Kinase-3{beta} (GSK-3{beta}) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3{beta} by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3{beta} at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3{beta} serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKB{beta}, is required for phosphorylation of GSK- 3{beta} at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3{beta} at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3{beta} in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer

  8. Comparative studies of a new subfamily of human Ste20-like kinases: homodimerization, subcellular localization, and selective activation of MKK3 and p38.

    Science.gov (United States)

    Yustein, Jason T; Xia, Liang; Kahlenburg, J Michelle; Robinson, Dan; Templeton, Dennis; Kung, Hsing-Jien

    2003-09-18

    The Sterile-20 or Ste20 family of serine/threonine kinases is a group of signaling molecules whose physiological roles within mammalian cells are just starting to be elucidated. Here, in this report we present the characterization of three human Ste20-like kinases with greater than 90% similarity within their catalytic domains that define a novel subfamily of Ste20s. Members of this kinase family include rat thousand and one (TAO1) and chicken KFC (kinase from chicken). For the lack of a consensus nomenclature in the literature, in this report, we shall call this family hKFC (for their homology to chicken KFC) and the three members hKFC-A, hKFC-B, and hKFC-C, respectively. These kinases have many similarities including an aminoterminal kinase domain, a serine-rich region, and a coiled-coil configuration within the C-terminus. All three kinases are able to activate the p38 MAP kinase pathway through the specific activation of the upstream MKK3 kinase. We also offer evidence, both theoretical and biochemical, showing that these kinases can undergo self-association. Despite these similarities, these kinases differ in tissue distribution, apparent subcellular localization, and feature structural differences largely within the carboxyl-terminal sequence.

  9. Overexpression of DJ-1/PARK7, the Parkinson's disease-related protein, improves mitochondrial function via Akt phosphorylation on threonine 308 in dopaminergic neuron-like cells.

    Science.gov (United States)

    Zhang, Yi; Gong, Xiao-Gang; Wang, Zhen-Zhen; Sun, Hong-Mei; Guo, Zhen-Yu; Hu, Jing-Hong; Ma, Ling; Li, Ping; Chen, Nai-Hong

    2016-05-01

    DJ-1/PARK7, the Parkinson's disease-related protein, plays an important role in mitochondrial function. However, the mechanisms by which DJ-1 affects mitochondrial function are not fully understood. Akt is a promoter of neuron survival and is partly involved in the neurodegenerative process. This research aimed at investigating a possible relationship between DJ-1 and Akt signalling in regulating mitochondrial function in the dopaminergic neuron-like cells SH-SY5Y and PC-12. Overexpression of DJ-1 was firstly validated at both the transcriptional and translational levels after transit transfection with plasmid pcDNA3-Flag-DJ-1. Confocal fluorescence microscopy demonstrated that overexpression of DJ-1 increased the mitochondrial mass, but did not disrupt the mitochondrial morphology. In addition, mitochondrial complex I activity was raised in DJ-1-overexpressing cells, and this rise occurred with an increase in cellular adenosine 5'-triphosphate content. Moreover, immunoblotting demonstrated that the levels of phosphoinositide 3-kinase and the total Akt were not altered in DJ-1-overexpressing cells, and nor was the Akt phosphorylation on serine 473 changed. By contrast, Akt phosphorylation on threonine 308 was significantly augmented by overexpression of DJ-1, and the expression of glycogen synthase kinase-3beta, a downstream effector of Akt, was suppressed. In summary, these results suggest that overexpression of DJ-1 improves the mitochondrial function, at least in part, through a mechanism involving Akt phosphorylation on threonine 308. © 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

  10. Fibronectin phosphorylation by ecto-protein kinase

    International Nuclear Information System (INIS)

    Imada, Sumi; Sugiyama, Yayoi; Imada, Masaru

    1988-01-01

    The presence of membrane-associated, extracellular protein kinase (ecto-protein kinase) and its substrate proteins was examined with serum-free cultures of Swiss 3T3 fibroblast. When cells were incubated with [γ- 32 ]ATP for 10 min at 37 degree C, four proteins with apparent molecular weights between 150 and 220 kDa were prominently phosphorylated. These proteins were also radiolabeled by lactoperoxidase catalyzed iodination and were sensitive to mild tryptic digestion, suggesting that they localized on the cell surface or in the extracellular matrix. Phosphorylation of extracellular proteins with [γ- 32 P]ATP in intact cell culture is consistent with the existence of ecto-protein kinase. Anti-fibronectin antibody immunoprecipitated one of the phosphoproteins which comigrated with a monomer and a dimer form of fibronectin under reducing and nonreducing conditions of electrophoresis, respectively. The protein had affinity for gelatin as demonstrated by retention with gelatin-conjugated agarose. This protein substrate of ecto-protein kinase was thus concluded to be fibronectin. The sites of phosphorylation by ecto-protein kinase were compared with those of intracellularly phosphorylated fibronectin by the analysis of radiolabeled amino acids and peptides. Ecto-protein kinase phosphorylated fibronectin at serine and threonine residues which were distinct from the sites of intracellular fibronectin phosphorylation

  11. The PIM kinases in hematological cancers.

    Science.gov (United States)

    Alvarado, Yesid; Giles, Francis J; Swords, Ronan T

    2012-02-01

    The PIM genes represent a family of proto-oncogenes that encode three different serine/threonine protein kinases (PIM1, PIM2 and PIM3) with essential roles in the regulation of signal transduction cascades, which promote cell survival, proliferation and drug resistance. PIM kinases are overexpressed in several hematopoietic tumors and support in vitro and in vivo malignant cell growth and survival, through cell cycle regulation and inhibition of apoptosis. PIM kinases do not have an identified regulatory domain, which means that these proteins are constitutively active once transcribed. They appear to be critical downstream effectors of important oncoproteins and, when overexpressed, can mediate drug resistance to available agents, such as rapamycin. Recent crystallography studies reveal that, unlike other kinases, they possess a hinge region, which creates a unique binding pocket for ATP, offering a target for an increasing number of potent small-molecule PIM kinase inhibitors. Preclinical studies in models of various hematologic cancers indicate that these novel agents show promising activity and some of them are currently being evaluated in a clinical setting. In this review, we profile the PIM kinases as targets for therapeutics in hematologic malignancies.

  12. Mass spectrometry and site-directed mutagenesis identify several autophosphorylated residues required for the activity of PrkC, a Ser/Thr kinase from Bacillus subtilis

    DEFF Research Database (Denmark)

    Madec, Edwige; Stensballe, Allan; Kjellström, Sven

    2003-01-01

    We have shown recently that PrkC, which is involved in developmental processes in Bacillus subtilis, is a Ser/Thr kinase with features of the receptor kinase family of eukaryotic Hanks kinases. In this study, we expressed and purified from Escherichia coli the cytoplasmic domain of PrkC containing...... the kinase and a short juxtamembrane region. This fragment, which we designate PrkCc, undergoes autophosphorylation in E.coli. PrkCc is further autophosphorylated in vitro, apparently through a trans-kinase, intermolecular reaction. PrkC also displays kinase activity with myelin basic protein. Using high...... mass accuracy electrospray tandem mass spectrometry (LC-MS/MS) and nanoelectrospray tandem mass spectrometry, we identified seven phosphorylated threonine and one serine residue in PrkCc. All the corresponding residues were replaced by systematic site-directed mutagenesis and the purified mutant...

  13. Effects of inhibitors of protein kinase C and NO-synthase on the radiation-induced cytogenetic adaptive response in Chinese hamster cells in culture

    International Nuclear Information System (INIS)

    Gil'yano, N.Ya.; Bondarev, G.N.; Bikineeva, E.G.; Krasotskaya, G.I.; Noskin, L.A.

    2001-01-01

    The effect of the serine-threonin kinase inhibitor - staurosporine and inhibitor of NO-synthase - L-NAME on the radiation-induced adaptive response were studied in fibroblasts of Chinese hamster in culture. It is shown that staurosporine and L-NAME inhibit cytogenetic adaptive response induced by β-particles in low doses. Inhibition is not connected with radiosensitizing effect of these agents. L-NAME decreases significantly the γ-rays-induced chromosome aberration yield also. Study confirms the role of protein kinase C in induction of the adaptive response and participation of NO-synthase in this process is noticed for the first time [ru

  14. STK33 kinase inhibitor BRD-8899 has no effect on KRAS-dependent cancer cell viability.

    Science.gov (United States)

    Luo, Tuoping; Masson, Kristina; Jaffe, Jacob D; Silkworth, Whitney; Ross, Nathan T; Scherer, Christina A; Scholl, Claudia; Fröhling, Stefan; Carr, Steven A; Stern, Andrew M; Schreiber, Stuart L; Golub, Todd R

    2012-02-21

    Approximately 30% of human cancers harbor oncogenic gain-of-function mutations in KRAS. Despite interest in KRAS as a therapeutic target, direct blockade of KRAS function with small molecules has yet to be demonstrated. Based on experiments that lower mRNA levels of protein kinases, KRAS-dependent cancer cells were proposed to have a unique requirement for the serine/threonine kinase STK33. Thus, it was suggested that small-molecule inhibitors of STK33 might have therapeutic benefit in these cancers. Here, we describe the development of selective, low nanomolar inhibitors of STK33's kinase activity. The most potent and selective of these, BRD8899, failed to kill KRAS-dependent cells. While several explanations for this result exist, our data are most consistent with the view that inhibition of STK33's kinase activity does not represent a promising anti-KRAS therapeutic strategy.

  15. Phosphorylation of human respiratory syncytial virus P protein at serine 54 regulates viral uncoating

    International Nuclear Information System (INIS)

    Asenjo, Ana; Gonzalez-Armas, Juan C.; Villanueva, Nieves

    2008-01-01

    The human respiratory syncytial virus (HRSV) structural P protein, phosphorylated at serine (S) and threonine (T) residues, is a co-factor of viral RNA polymerase. The phosphorylation of S54 is controlled by the coordinated action of two cellular enzymes: a lithium-sensitive kinase, probably glycogen synthetase kinase (GSK-3) β and protein phosphatase 2A (PP2A). Inhibition of lithium-sensitive kinase, soon after infection, blocks the viral growth cycle by inhibiting synthesis and/or accumulation of viral RNAs, proteins and extracellular particles. P protein phosphorylation at S54 is required to liberate viral ribonucleoproteins (RNPs) from M protein, during the uncoating process. Kinase inhibition, late in infection, produces a decrease in genomic RNA and infectious viral particles. LiCl, intranasally applied to mice infected with HRSV A2 strain, reduces the number of mice with virus in their lungs and the virus titre. Administration of LiCl to humans via aerosol should prevent HRSV infection, without secondary effects

  16. Regulation of glycogen synthase kinase-3β (GSK-3β) after ionizing radiation

    International Nuclear Information System (INIS)

    Boehme, K.A.

    2006-12-01

    Glycogen Synthase Kinase-3β (GSK-3β) phosphorylates the Mdm2 protein in the central domain. This phosphorylation is absolutely required for p53 degradation. Ionizing radiation inactivates GSK-3β by phosphorylation at serine 9 and in consequence prevents Mdm2 mediated p53 degradation. During the work for my PhD I identified Akt/PKB as the kinase that phosphorylates GSK-3β at serine 9 after ionizing radiation. Ionizing radiation leads to phosphorylation of Akt/PKB at threonine 308 and serine 473. The PI3 Kinase inhibitor LY294002 completely abolished Akt/PKB serine 473 phosphorylation and prevented the induction of GSK-3β serine 9 phosphorylation after ionizing radiation. Interestingly, the most significant activation of Akt/PKB after ionizing radiation occurred in the nucleus while cytoplasmic Akt/PKB was only weakly activated after radiation. By using siRNA, I showed that Akt1/PKBa, but not Akt2/PKBβ, is required for phosphorylation of GSK- 3β at serine 9 after ionizing radiation. Phosphorylation and activation of Akt/PKB after ionizing radiation depends on the DNA dependent protein kinase (DNA-PK), a member of the PI3 Kinase family, that is activated by free DNA ends. Both, in cells from SCID mice and after knockdown of the catalytic subunit of DNA-PK by siRNA in osteosarcoma cells, phosphorylation of Akt/PKB at serine 473 and of GSK-3β at serine 9 was completely abolished. Consistent with the principle that phosphorylation of GSK-3 at serine 9 contributes to p53 stabilization after radiation, the accumulation of p53 in response to ionizing radiation was largely prevented by downregulation of DNA-PK. From these results I conclude, that ionizing radiation induces a signaling cascade that leads to Akt1/PKBa activation mediated by DNA-PK dependent phosphorylation of serine 473. After activation Akt1/PKBa phosphorylates and inhibits GSK-3β in the nucleus. The resulting hypophosphorylated form of Mdm2 protein is no longer able to degrade p53 which in

  17. Inflammation kinase PKR phosphorylates α-synuclein and causes α-synuclein-dependent cell death

    DEFF Research Database (Denmark)

    Reimer, Lasse; Lund, Louise Buur; Betzer, Cristine

    2018-01-01

    , and acute brain slices), while overexpression of constitutively active PKR increases Ser129 α-syn phosphorylation. Treatment with pre-formed α-synuclein fibrils, proteostatic stress-promoting MG-132 and known PKR activators, herpes simplex virus-1-∆ICP34.5 and LPS, as well as PKR inducer, IFN-β-1b, lead...... on Ser129. Although the inflammation-associated serine-threonine kinase, PKR (EIF2AK2), promotes cellular protection against infection, we demonstrate a pro-degenerative role of activated PKR in an α-synuclein-dependent cell model of multiple system atrophy, where inhibition and silencing of PKR decrease...

  18. Three-Dimentional Structures of Autophosphorylation Complexes in Crystals of Protein Kinases

    KAUST Repository

    Dumbrack, Roland

    2016-01-26

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Several autophosphorylation complexes have been identified in crystals of protein kinases, with a known serine, threonine, or tyrosine autophosphorylation site of one kinase monomer sitting in the active site of another monomer of the same protein in the crystal. We utilized a structural bioinformatics method to identify all such autophosphorylation complexes in X-ray crystallographic structures in the Protein Data Bank (PDB) by generating all unique kinase/kinase interfaces within and between asymmetric units of each crystal and measuring the distance between the hydroxyl oxygen of potential autophosphorylation sites and the oxygen atoms of the active site aspartic acid residue side chain. We have identified 15 unique autophosphorylation complexes in the PDB, of which 5 complexes have not previously been described in the relevant publications on the crystal structures (N-terminal juxtamembrane regions of CSF1R and EPHA2, activation loop tyrosines of LCK and IGF1R, and a serine in a nuclear localization signal region of CLK2. Mutation of residues in the autophosphorylation complex interface of LCK either severely impaired autophosphorylation or increased it. Taking the autophosphorylation complexes as a whole and comparing them with peptide-substrate/kinase complexes, we observe a number of important features among them. The novel and previously observed autophosphorylation sites are conserved in many kinases, indicating that by homology we can extend the relevance of these complexes to many other clinically relevant drug targets.

  19. PAK4 crystal structures suggest unusual kinase conformational movements.

    Science.gov (United States)

    Zhang, Eric Y; Ha, Byung Hak; Boggon, Titus J

    2018-02-01

    In order for protein kinases to exchange nucleotide they must open and close their catalytic cleft. These motions are associated with rotations of the N-lobe, predominantly around the 'hinge region'. We conducted an analysis of 28 crystal structures of the serine-threonine kinase, p21-activated kinase 4 (PAK4), including three newly determined structures in complex with staurosporine, FRAX486, and fasudil (HA-1077). We find an unusual motion between the N-lobe and C-lobe of PAK4 that manifests as a partial unwinding of helix αC. Principal component analysis of the crystal structures rationalizes these movements into three major states, and analysis of the kinase hydrophobic spines indicates concerted movements that create an accessible back pocket cavity. The conformational changes that we observe for PAK4 differ from previous descriptions of kinase motions, and although we observe these differences in crystal structures there is the possibility that the movements observed may suggest a diversity of kinase conformational changes associated with regulation. Protein kinases are key signaling proteins, and are important drug targets, therefore understanding their regulation is important for both basic research and clinical points of view. In this study, we observe unusual conformational 'hinging' for protein kinases. Hinging, the opening and closing of the kinase sub-domains to allow nucleotide binding and release, is critical for proper kinase regulation and for targeted drug discovery. We determine new crystal structures of PAK4, an important Rho-effector kinase, and conduct analyses of these and previously determined structures. We find that PAK4 crystal structures can be classified into specific conformational groups, and that these groups are associated with previously unobserved hinging motions and an unusual conformation for the kinase hydrophobic core. Our findings therefore indicate that there may be a diversity of kinase hinging motions, and that these may

  20. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels

    NARCIS (Netherlands)

    van Tiel, Claudia M.; Westerman, Jan; Paasman, Marten A.; Hoebens, Martha M.; Wirtz, Karel W. A.; Snoek, Gerry T.

    2002-01-01

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165)

  1. Phosphorylation of the Fas associated factor FAF1 by protein kinase CK2 and identification of serines 289 and 291 as the in vitro phosphorylation sites

    DEFF Research Database (Denmark)

    Jensen, H H; Hjerrild, M; Guerra, B

    2001-01-01

    We previously identified the human Fas associated factor (FAF1) as one of the interacting partners of protein kinase CK2 beta subunit. Since FAF1 is a phosphoprotein we investigated whether it is a substrate for CK2. Here, we report the full length human FAF1 cDNA sequence, expression of FAF1...

  2. Protein Kinase C Enzymes in the Hematopoietic and Immune Systems.

    Science.gov (United States)

    Altman, Amnon; Kong, Kok-Fai

    2016-05-20

    The protein kinase C (PKC) family, discovered in the late 1970s, is composed of at least 10 serine/threonine kinases, divided into three groups based on their molecular architecture and cofactor requirements. PKC enzymes have been conserved throughout evolution and are expressed in virtually all cell types; they represent critical signal transducers regulating cell activation, differentiation, proliferation, death, and effector functions. PKC family members play important roles in a diverse array of hematopoietic and immune responses. This review covers the discovery and history of this enzyme family, discusses the roles of PKC enzymes in the development and effector functions of major hematopoietic and immune cell types, and points out gaps in our knowledge, which should ignite interest and further exploration, ultimately leading to better understanding of this enzyme family and, above all, its role in the many facets of the immune system.

  3. Importance of tyrosine phosphorylation in receptor kinase complexes.

    Science.gov (United States)

    Macho, Alberto P; Lozano-Durán, Rosa; Zipfel, Cyril

    2015-05-01

    Tyrosine phosphorylation is an important post-translational modification that is known to regulate receptor kinase (RK)-mediated signaling in animals. Plant RKs are annotated as serine/threonine kinases, but recent work has revealed that tyrosine phosphorylation is also crucial for the activation of RK-mediated signaling in plants. These initial observations have paved the way for subsequent detailed studies on the mechanism of activation of plant RKs and the biological relevance of tyrosine phosphorylation for plant growth and immunity. In this Opinion article we review recent reports on the contribution of RK tyrosine phosphorylation in plant growth and immunity; we propose that tyrosine phosphorylation plays a major regulatory role in the initiation and transduction of RK-mediated signaling in plants. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. 2-Aminopyridine-Based Mitogen-Activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Inhibitors: Assessment of Mechanism-Based Safety.

    Science.gov (United States)

    Dow, Robert L; Ammirati, Mark; Bagley, Scott W; Bhattacharya, Samit K; Buckbinder, Leonard; Cortes, Christian; El-Kattan, Ayman F; Ford, Kristen; Freeman, Gary B; Guimarães, Cristiano R W; Liu, Shenping; Niosi, Mark; Skoura, Athanasia; Tess, David

    2018-04-12

    Studies have linked the serine-threonine kinase MAP4K4 to the regulation of a number of biological processes and/or diseases, including diabetes, cancer, inflammation, and angiogenesis. With a majority of the members of our lead series (e.g., 1) suffering from time-dependent inhibition (TDI) of CYP3A4, we sought design avenues that would eliminate this risk. One such approach arose from the observation that carboxylic acid-based intermediates employed in our discovery efforts retained high MAP4K4 inhibitory potency and were devoid of the TDI risk. The medicinal chemistry effort that led to the discovery of this central nervous system-impaired inhibitor together with its preclinical safety profile is described.

  5. The DNA-dependent protein kinase: a multifunctional protein kinase with roles in DNA double strand break repair and mitosis

    Science.gov (United States)

    Jette, Nicholas; Lees-Miller, Susan P.

    2015-01-01

    The DNA-dependent protein kinase (DNA-PK) is a serine/threonine protein kinase composed of a large catalytic subunit (DNA-PKcs) and the Ku70/80 heterodimer. Over the past two decades, significant progress has been made in elucidating the role of DNA-PK in non-homologous end joining (NHEJ), the major pathway for repair of ionizing radiation-induced DNA double strand breaks in human cells and recently, additional roles for DNA-PK have been reported. In this review, we will describe the biochemistry, structure and function of DNA-PK, its roles in DNA double strand break repair and its newly described roles in mitosis and other cellular processes. PMID:25550082

  6. The wheat AGC kinase TaAGC1 is a positive contributor to host resistance to the necrotrophic pathogen Rhizoctonia cerealis.

    Science.gov (United States)

    Zhu, Xiuliang; Yang, Kun; Wei, Xuening; Zhang, Qiaofeng; Rong, Wei; Du, Lipu; Ye, Xingguo; Qi, Lin; Zhang, Zengyan

    2015-11-01

    Considerable progress has been made in understanding the roles of AGC kinases in mammalian systems. However, very little is known about the roles of AGC kinases in wheat (Triticum aestivum). The necrotrophic fungus Rhizoctonia cerealis is the major pathogen of the destructive disease sharp eyespot of wheat. In this study, the wheat AGC kinase gene TaAGC1, responding to R. cerealis infection, was isolated, and its properties and role in wheat defence were characterized. R. cerealis-resistant wheat lines expressed TaAGC1 at higher levels than susceptible wheat lines. Sequence and phylogenetic analyses showed that the TaAGC1 protein is a serine/threonine kinase belonging to the NDR (nuclear Dbf2-related) subgroup of AGC kinases. Kinase activity assays proved that TaAGC1 is a functional kinase and the Asp-239 residue located in the conserved serine/threonine kinase domain of TaAGC1 is required for the kinase activity. Subcellular localization assays indicated that TaAGC1 localized in the cytoplasm and nucleus. Virus-induced TaAGC1 silencing revealed that the down-regulation of TaAGC1 transcripts significantly impaired wheat resistance to R. cerealis. The molecular characterization and responses of TaAGC1 overexpressing transgenic wheat plants indicated that TaAGC1 overexpression significantly enhanced resistance to sharp eyespot and reduced the accumulation of reactive oxygen species (ROS) in wheat plants challenged with R. cerealis. Furthermore, ROS-scavenging and certain defence-associated genes were up-regulated in resistant plants overexpressing TaAGC1 but down-regulated in susceptible knock-down plants. These results suggested that the kinase TaAGC1 positively contributes to wheat immunity to R. cerealis through regulating expression of ROS-related and defence-associated genes. © The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.

  7. Protein kinase Cα phosphorylates a novel argininosuccinate synthase site at serine 328 during calcium-dependent stimulation of endothelial nitric-oxide synthase in vascular endothelial cells.

    Science.gov (United States)

    Haines, Ricci J; Corbin, Karen D; Pendleton, Laura C; Eichler, Duane C

    2012-07-27

    Endothelial nitric-oxide synthase (eNOS) utilizes l-arginine as its principal substrate, converting it to l-citrulline and nitric oxide (NO). l-Citrulline is recycled to l-arginine by two enzymes, argininosuccinate synthase (AS) and argininosuccinate lyase, providing the substrate arginine for eNOS and NO production in endothelial cells. Together, these three enzymes, eNOS, AS, and argininosuccinate lyase, make up the citrulline-NO cycle. Although AS catalyzes the rate-limiting step in NO production, little is known about the regulation of AS in endothelial cells beyond the level of transcription. In this study, we showed that AS Ser-328 phosphorylation was coordinately regulated with eNOS Ser-1179 phosphorylation when bovine aortic endothelial cells were stimulated by either a calcium ionophore or thapsigargin to produce NO. Furthermore, using in vitro kinase assay, kinase inhibition studies, as well as protein kinase Cα (PKCα) knockdown experiments, we demonstrate that the calcium-dependent phosphorylation of AS Ser-328 is mediated by PKCα. Collectively, these findings suggest that phosphorylation of AS at Ser-328 is regulated in accordance with the calcium-dependent regulation of eNOS under conditions that promote NO production and are in keeping with the rate-limiting role of AS in the citrulline-NO cycle of vascular endothelial cells.

  8. Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

    Directory of Open Access Journals (Sweden)

    Horacio Bach

    2018-01-01

    Full Text Available To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP, the causative agent of Johne’s disease, as the serine/threonine protein kinase G (PknG. In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

  9. Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

    Science.gov (United States)

    Montesano, M; Kõiv, V; Mäe, A; Palva, E T

    2001-11-01

    summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

  10. Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

    KAUST Repository

    Kim, Dongjin; Ntui, Valentine Otang; Zhang, Nianshu; Xiong, Liming

    2015-01-01

    Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

  11. Targeting Polo-Like Kinases: A Promising Therapeutic Approach for Cancer Treatment

    Directory of Open Access Journals (Sweden)

    Xiaoqi Liu

    2015-06-01

    Full Text Available Polo-like kinases (Plks are a family of serine-threonine kinases that regulate multiple intracellular processes including DNA replication, mitosis, and stress response. Plk1, the most well understood family member, regulates numerous stages of mitosis and is overexpressed in many cancers. Plk inhibitors are currently under clinical investigation, including phase III trials of volasertib, a Plk inhibitor, in acute myeloid leukemia and rigosertib, a dual inhibitor of Plk1/phosphoinositide 3-kinase signaling pathways, in myelodysplastic syndrome. Other Plk inhibitors, including the Plk1 inhibitors GSK461364A, TKM-080301, GW843682, purpurogallin, and poloxin and the Plk4 inhibitor CFI-400945 fumarate, are in earlier clinical development. This review discusses the biologic roles of Plks in cell cycle progression and cancer, and the mechanisms of action of Plk inhibitors currently in development as cancer therapies.

  12. Arabidopsis Yak1 protein (AtYak1) is a dual specificity protein kinase

    KAUST Repository

    Kim, Dongjin

    2015-10-09

    Yak1 is a member of dual-specificity Tyr phosphorylation-regulated kinases (DYRKs) that are evolutionarily conserved. The downstream targets of Yak1 and their functions are largely unknown. Here, a homologous protein AtYAK1 was identified in Arabidopsis thaliana and the phosphoprotein profiles of the wild type and an atyak1 mutant were compared on two-dimensional gel following Pro-Q Diamond phosphoprotein gel staining. Annexin1, Annexin2 and RBD were phosphorylated at serine/ threonine residues by the AtYak1 kinase. Annexin1, Annexin2 and Annexin4 were also phosphorylated at tyrosine residues. Our study demonstrated that AtYak1 is a dual specificity protein kinase in Arabidopsis that may regulate the phosphorylation status of the annexin family proteins.

  13. Genetic Disruption of Protein Kinase STK25 Ameliorates Metabolic Defects in a Diet-Induced Type 2 Diabetes Model

    DEFF Research Database (Denmark)

    Amrutkar, Manoj; Cansby, Emmelie; Chursa, Urszula

    2015-01-01

    Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator...... to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes....

  14. Serine 77 in the PDZ domain of PICK1 is a protein kinase Cα phosphorylation site regulated by lipid membrane binding

    DEFF Research Database (Denmark)

    Ammendrup-Johnsen, Ina; Thorsen, Thor Seneca; Gether, Ulrik

    2012-01-01

    PICK1 (protein interacting with C kinase 1) contains an N-terminal protein binding PDZ domain and a C-terminal lipid binding BAR domain. PICK1 plays a key role in several physiological processes, including synaptic plasticity. However, little is known about the cellular mechanisms governing the a...... lipid binding and/or polymerization capacity. We propose that PICK1 is phosphorylated at Ser77 by PKCα preferentially when bound to membrane vesicles and that this phosphorylation in turn modulates its cellular distribution....

  15. Purification and characterization of a casein kinase 2-type protein kinase from pea nuclei

    Science.gov (United States)

    Li, H.; Roux, S. J.

    1992-01-01

    Almost all the polyamine-stimulated protein kinase activity associated with the chromatin fraction of nuclei purified from etiolated pea (Pisum sativum L.) plumules is present in a single enzyme that can be extracted from chromatin by 0.35 molar NaCl. This protein kinase can be further purified over 2000-fold by salt fractionation and anion-exchange and casein-agarose column chromatography, after which it is more than 90% pure. The purified kinase has a specific activity of about 650 nanomoles per minute per milligram protein in the absence of polyamines, with either ATP or GTP as phosphoryl donor. Spermidine can stimulate its activity fourfold, with half-maximal activation at about 2 millimolar. Spermine and putrescine also stimulate activity, although somewhat less effectively. This kinase has a tetrameric alpha 2 beta 2 structure with a native molecular weight of 130,000, and subunit molecular weights of 36,000 for the catalytic subunit (alpha) and 29,000 for the regulatory subunit (beta). In western blot analyses, only the alpha subunit reacts strongly with polyclonal antibodies to a Drosophila casein kinase II. The pea kinase can use casein and phosvitin as artificial substrates, phosphorylating both the serine and threonine residues of casein. It has a pH optimum near 8.0, a Vmax of 1.5 micromoles per minute per milligram protein, and a Km for ATP of approximately 75 micromolar. Its activity can be almost completely inhibited by heparin at 5 micrograms per milliliter, but is relatively insensitive to concentrations of staurosporine, K252a, and chlorpromazine that strongly antagonize Ca(2+) -regulated protein kinases. These results are discussed in relation to recent findings that casein kinase 2-type kinases may phosphorylate trans-acting factors that bind to light-regulated promoters in plants.

  16. Heterologous activation of protein kinase C stimulates phosphorylation of delta-opioid receptor at serine 344, resulting in beta-arrestin- and clathrin-mediated receptor internalization

    DEFF Research Database (Denmark)

    Xiang, B; Yu, G H; Guo, J

    2001-01-01

    The purpose of the current study is to investigate the effect of opioid-independent, heterologous activation of protein kinase C (PKC) on the responsiveness of opioid receptor and the underlying molecular mechanisms. Our result showed that removing the C terminus of delta opioid receptor (DOR......) containing six Ser/Thr residues abolished both DPDPE- and phorbol 12-myristate 13-acetate (PMA)-induced DOR phosphorylation. The phosphorylation levels of DOR mutants T352A, T353A, and T358A/T361A/S363S were comparable to that of the wild-type DOR, whereas S344G substitution blocked PMA-induced receptor......, and ionomycin resulted in DOR internalization that required phosphorylation of Ser-344. Expression of dominant negative beta-arrestin and hypertonic sucrose treatment blocked PMA-induced DOR internalization, suggesting that PKC mediates DOR internalization via a beta-arrestin- and clathrin-dependent mechanism...

  17. Far-infrared radiation acutely increases nitric oxide production by increasing Ca(2+) mobilization and Ca(2+)/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179.

    Science.gov (United States)

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-07-12

    Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser(1179)) in a time-dependent manner (up to 40min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca(2+) levels. Treatment with KN-93, a selective inhibitor of Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser(1179) phosphorylation. This study suggests that FIR radiation increases NO production via increasing CaMKII-mediated eNOS-Ser(1179) phosphorylation but TRPV channels may not be involved in this pathway. Our results may provide the molecular mechanism by which FIR radiation improves endothelial function. Copyright © 2013 Elsevier Inc. All rights reserved.

  18. Phosphorylation sites of Arabidopsis MAP Kinase Substrate 1 (MKS1)

    DEFF Research Database (Denmark)

    Caspersen, M.B.; Qiu, J.-L.; Zhang, X.

    2007-01-01

    The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified by electrophore......The Arabidopsis MAP kinase 4 (MPK4) substrate MKS1 was expressed in Escherichia coli and purified, full-length, 6x histidine (His)-tagged MKS1 was phosphorylated in vitro by hemagglutinin (HA)-tagged MPK4 immuno-precipitated from plants. MKS1 phosphorylation was initially verified...... phosphopeptide detection. As MAP kinases generally phosphorylate serine or threonine followed by proline (Ser/Thr-Pro), theoretical masses of potentially phosphorylated peptides were calculated and mass spectrometric peaks matching these masses were fragmented and searched for a neutral-loss signal...... at approximately 98 Da indicative of phosphorylation. Additionally, mass spectrometric peaks present in the MPK4-treated MKS1, but not in the control peptide map of untreated MKS1, were fragmented. Fragmentation spectra were subjected to a MASCOT database search which identified three of the twelve Ser-Pro serine...

  19. Phenotypic Variability in a Family with Acrodysostosis Type 2 Caused by a Novel PDE4D Mutation Affecting the Serine Target of Protein Kinase-A Phosphorylation

    Science.gov (United States)

    Hoppmann, Julia; Gesing, Julia; Silve, Caroline; Leroy, Chrystel; Bertsche, Astrid; Hirsch, Franz Wolfgang; Kiess, Wieland; Pfäffle, Roland; Schuster, Volker

    2017-01-01

    Acrodysostosis is a very rare congenital multisystem condition characterized by skeletal dysplasia with severe brachydactyly, midfacial hypoplasia, and short stature, varying degrees of intellectual disability, and possible resistance to multiple G protein-coupled receptor signalling hormones. Two distinct subtypes are differentiated: acrodysostosis type 1 resulting from defects in protein kinase type 1-α regulatory subunit and acrodysostosis type 2 caused by mutations in phosphodiesterase 4D (PDE4D). Most cases are sporadic. We report on a rare multigenerational familial case of acrodysostosis type 2 due to a novel autosomal dominantly inherited PDE4D mutation. A 3.5-year-old boy presented with short stature, midfacial hypoplasia, severe brachydactyly, developmental delay, and behavioural problems. Laboratory investigations revealed mild thyrotropin resistance. His mother shared some characteristic features, such as midfacial hypoplasia and severe brachydactyly, but did not show short stature, intellectual disability or hormonal resistance. Genetic analysis identified the identical, novel heterozygous missense mutation of the PDE4D gene c.569C>T (p.Ser190Phe) in both patients. This case illustrates the significant phenotypic variability of acrodysostosis even within one family with identical mutations. Hence, a specific clinical diagnosis of acrodysostosis remains challenging because of great interindividual variability and a substantial overlap of the two subtypes as well as with other related Gsα-cAMP-signalling-linked disorders. PMID:28515031

  20. Peptide substrates for Rho-associated kinase 2 (Rho-kinase 2/ROCK2.

    Directory of Open Access Journals (Sweden)

    Jeong-Hun Kang

    Full Text Available Peptide substrates sensitive for a certain protein kinase could be important for new-drug development and to understand the mechanism of diseases. Rho-associated kinase (Rho-kinase/ROCK is a serine/threonine kinase, and plays an important part in cardiovascular disease, migration and invasion of tumor cells, and in neurological disorders. The purpose of this study was to find substrates with high affinity and sensitivity for ROCK2. We synthesized 136 peptide substrates from protein substrates for ROCK2 with different lengths and charged peptides. Incorporation of (32P [counts per minute (CPM] for each peptide substrate was determined by the radiolabel assay using [γ-(32P]ATP. When the top five peptide substrates showing high CPMs (R4, R22, R133, R134, and R135 were phosphorylated by other enzymes (PKA, PKCα, and ERK1, R22, R133, and R135 displayed the highest CPM level for ROCK2 compared with other enzymes, whereas R4 and R134 showed similar CPM levels for ROCK2 and PKCα. We hypothesize that R22, R133, and R135 can be useful peptide substrates for ROCK2.

  1. A chemical genetic approach to engineer phototropin kinases for substrate labeling.

    Science.gov (United States)

    Schnabel, Jonathan; Hombach, Peter; Waksman, Thomas; Giuriani, Giovanni; Petersen, Jan; Christie, John M

    2018-04-13

    Protein kinases (PKs) control many aspects of plant physiology by regulating signaling networks through protein phosphorylation. Phototropins (phots) are plasma membrane-associated serine/threonine PKs that control a range of physiological processes that collectively serve to optimize photosynthetic efficiency in plants. These include phototropism, leaf positioning and flattening, chloroplast movement, and stomatal opening. Despite their identification over two decades ago, only a handful of substrates have been identified for these PKs. Progress in this area has been hampered by the lack of a convenient means to confirm the identity of potential substrate candidates. Here we demonstrate that the kinase domain of Arabidopsis phot1 and phot2 can be successfully engineered to accommodate non-natural ATP analogues by substituting the bulky gatekeeper residue threonine for glycine. This approach circumvents the need for radioactivity to track phot kinase activity and follow light-induced receptor autophosphorylation in vitro by incorporating thiophosphate from N 6 -benzyl-ATPγS. Consequently, thiophosphorylation of phot substrate candidates can be readily monitored when added or co-expressed with phots in vitro Furthermore, gatekeeper-modified phot1 retained its functionality and its ability to accommodate N 6 -benzyl-ATPγS as a phosphodonor when expressed in Arabidopsis We therefore anticipate that this chemical genetic approach will provide new opportunities for labeling and identifying substrates for phots and other related AGC kinases under in vitro and near-native in vivo conditions. © 2018 Schnabel et al.

  2. The Golgi localization of phosphatidylinositol transfer protein beta requires the protein kinase C-dependent phosphorylation of serine 262 and is essential for maintaining plasma membrane sphingomyelin levels.

    Science.gov (United States)

    van Tiel, Claudia M; Westerman, Jan; Paasman, Marten A; Hoebens, Martha M; Wirtz, Karel W A; Snoek, Gerry T

    2002-06-21

    Recombinant mouse phosphatidylinositol transfer protein (PI-TP)beta is a substrate for protein kinase C (PKC)-dependent phosphorylation in vitro. Based on site-directed mutagenesis and two-dimensional tryptic peptide mapping, Ser(262) was identified as the major site of phosphorylation and Ser(165) as a minor phosphorylation site. The phospholipid transfer activities of wild-type PI-TP beta and PI-TP beta(S262A) were identical, whereas PI-TP beta(S165A) was completely inactive. PKC-dependent phosphorylation of Ser(262) also had no effect on the transfer activity of PI-TP beta. To investigate the role of Ser(262) in the functioning of PI-TP beta, wtPI-TP beta and PI-TP beta(S262A) were overexpressed in NIH3T3 fibroblast cells. Two-dimensional PAGE analysis of cell lysates was used to separate PI-TP beta from its phosphorylated form. After Western blotting, wtPI-TP beta was found to be 85% phosphorylated, whereas PI-TP beta(S262A) was not phosphorylated. In the presence of the PKC inhibitor GF 109203X, the phosphorylated form of wtPI-TP beta was strongly reduced. Immunolocalization showed that wtPI-TP beta was predominantly associated with the Golgi membranes. In the presence of the PKC inhibitor, wtPI-TP beta was distributed throughout the cell similar to what was observed for PI-TP beta(S262A). In contrast to wtPI-TP beta overexpressors, cells overexpressing PI-TP beta(S262A) were unable to rapidly replenish sphingomyelin in the plasma membrane upon degradation by sphingomyelinase. This implies that PKC-dependent association with the Golgi complex is a prerequisite for PI-TP beta to express its effect on sphingomyelin metabolism.

  3. Protein kinase C activation decreases cell surface expression of the GLT-1 subtype of glutamate transporter. Requirement of a carboxyl-terminal domain and partial dependence on serine 486.

    Science.gov (United States)

    Kalandadze, Avtandil; Wu, Ying; Robinson, Michael B

    2002-11-29

    Na(+)-dependent glutamate transporters are required for the clearance of extracellular glutamate and influence both physiological and pathological effects of this excitatory amino acid. In the present study, the effects of a protein kinase C (PKC) activator on the cell surface expression and activity of the GLT-1 subtype of glutamate transporter were examined in two model systems, primary co-cultures of neurons and astrocytes that endogenously express GLT-1 and C6 glioma cells transfected with GLT-1. In both systems, activation of PKC with phorbol ester caused a decrease in GLT-1 cell surface expression. This effect is opposite to the one observed for the EAAC1 subtype of glutamate transporter (Davis, K. E., Straff, D. J., Weinstein, E. A., Bannerman, P. G., Correale, D. M., Rothstein, J. D., and Robinson, M. B. (1998) J. Neurosci. 18, 2475-2485). Several recombinant chimeric proteins between GLT-1 and EAAC1 transporter subtypes were generated to identify domains required for the subtype-specific redistribution of GLT-1. We identified a carboxyl-terminal domain consisting of 43 amino acids (amino acids 475-517) that is required for PKC-induced GLT-1 redistribution. Mutation of a non-conserved serine residue at position 486 partially attenuated but did not completely abolish the PKC-dependent redistribution of GLT-1. Although we observed a phorbol ester-dependent incorporation of (32)P into immunoprecipitable GLT-1, mutation of serine 486 did not reduce this signal. We also found that chimeras containing the first 446 amino acids of GLT-1 were not functional unless amino acids 475-517 of GLT-1 were also present. These non-functional transporters were not as efficiently expressed on the cell surface and migrated to a smaller molecular weight, suggesting that a subtype-specific interaction is required for the formation of functional transporters. These studies demonstrate a novel effect of PKC on GLT-1 activity and define a unique carboxyl-terminal domain as an

  4. Chloride sensing by WNK1 kinase involves inhibition of autophosphorylation

    Science.gov (United States)

    Piala, Alexander T.; Moon, Thomas M.; Akella, Radha; He, Haixia; Cobb, Melanie H.; Goldsmith, Elizabeth J.

    2014-01-01

    WNK1 [with no lysine (K)] is a serine-threonine kinase associated with a form of familial hypertension. WNK1 is at the top of a kinase cascade leading to phosphorylation of several cotransporters, in particular those transporting sodium, potassium, and chloride (NKCC), sodium and chloride (NCC), and potassium and chloride (KCC). The responsiveness of NKCC, NCC, and KCC to changes in extracellular chloride parallels their phosphorylation state, provoking the proposal that these transporters are controlled by a chloride-sensitive protein kinase. Here, we found that chloride stabilizes the inactive conformation of WNK1, preventing kinase autophosphorylation and activation. Crystallographic studies of inactive WNK1 in the presence of chloride revealed that chloride binds directly to the catalytic site, providing a basis for the unique position of the catalytic lysine. Mutagenesis of the chloride binding site rendered the kinase less sensitive to inhibition of autophosphorylation by chloride, validating the binding site. Thus, these data suggest that WNK1 functions as a chloride sensor through direct binding of a regulatory chloride ion to the active site, which inhibits autophosphorylation. PMID:24803536

  5. Interactions between Casein kinase Iepsilon (CKIepsilon and two substrates from disparate signaling pathways reveal mechanisms for substrate-kinase specificity.

    Directory of Open Access Journals (Sweden)

    Caroline Lund Dahlberg

    Full Text Available Members of the Casein Kinase I (CKI family of serine/threonine kinases regulate diverse biological pathways. The seven mammalian CKI isoforms contain a highly conserved kinase domain and divergent amino- and carboxy-termini. Although they share a preferred target recognition sequence and have overlapping expression patterns, individual isoforms often have specific substrates. In an effort to determine how substrates recognize differences between CKI isoforms, we have examined the interaction between CKIepsilon and two substrates from different signaling pathways.CKIepsilon, but not CKIalpha, binds to and phosphorylates two proteins: Period, a transcriptional regulator of the circadian rhythms pathway, and Disheveled, an activator of the planar cell polarity pathway. We use GST-pull-down assays data to show that two key residues in CKIalpha's kinase domain prevent Disheveled and Period from binding. We also show that the unique C-terminus of CKIepsilon does not determine Dishevelled's and Period's preference for CKIepsilon nor is it essential for binding, but instead plays an auxillary role in stabilizing the interactions of CKIepsilon with its substrates. We demonstrate that autophosphorylation of CKIepsilon's C-terminal tail prevents substrate binding, and use mass spectrometry and chemical crosslinking to reveal how a phosphorylation-dependent interaction between the C-terminal tail and the kinase domain prevents substrate phosphorylation and binding.The biochemical interactions between CKIepsilon and Disheveled, Period, and its own C-terminus lead to models that explain CKIepsilon's specificity and regulation.

  6. Far-infrared radiation acutely increases nitric oxide production by increasing Ca2+ mobilization and Ca2+/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    International Nuclear Information System (INIS)

    Park, Jung-Hyun; Lee, Sangmi; Cho, Du-Hyong; Park, Young Mi; Kang, Duk-Hee; Jo, Inho

    2013-01-01

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser 1179 phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser 1179 phosphorylation. •FIR increases intracellular Ca 2+ levels. •Thermo-sensitive TRPV Ca 2+ channels are unlikely to be involved in the FIR-mediated eNOS-Ser 1179 phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser 1179 ) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca 2+ levels. Treatment with KN-93, a selective inhibitor of Ca 2+ /calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser 1179 phosphorylation. This study suggests that FIR radiation increases NO

  7. Far-infrared radiation acutely increases nitric oxide production by increasing Ca{sup 2+} mobilization and Ca{sup 2+}/calmodulin-dependent protein kinase II-mediated phosphorylation of endothelial nitric oxide synthase at serine 1179

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jung-Hyun; Lee, Sangmi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Cho, Du-Hyong [Department of Neuroscience, School of Medicine, Konkuk University, Seoul 143-701 (Korea, Republic of); Park, Young Mi [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Kang, Duk-Hee [Division of Nephrology, Department of Internal Medicine, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of); Jo, Inho, E-mail: inhojo@ewha.ac.kr [Department of Molecular Medicine and Ewha Medical Research Institute, Ewha Womans University Medical School, Seoul 158-710 (Korea, Republic of)

    2013-07-12

    Highlights: •Far-infrared (FIR) radiation increases eNOS-Ser{sup 1179} phosphorylation and NO production in BAEC. •CaMKII and PKA mediate FIR-stimulated increases in eNOS-Ser{sup 1179} phosphorylation. •FIR increases intracellular Ca{sup 2+} levels. •Thermo-sensitive TRPV Ca{sup 2+} channels are unlikely to be involved in the FIR-mediated eNOS-Ser{sup 1179} phosphorylation pathway. -- Abstract: Repeated thermal therapy manifested by far-infrared (FIR) radiation improves vascular function in both patients and mouse model with coronary heart disease, but its underlying mechanism is not fully understood. Using FIR as a thermal therapy agent, we investigate the molecular mechanism of its effect on endothelial nitric oxide synthase (eNOS) activity and NO production. FIR increased the phosphorylation of eNOS at serine 1179 (eNOS-Ser{sup 1179}) in a time-dependent manner (up to 40 min of FIR radiation) in bovine aortic endothelial cells (BAEC) without alterations in eNOS expression. This increase was accompanied by increases in NO production and intracellular Ca{sup 2+} levels. Treatment with KN-93, a selective inhibitor of Ca{sup 2+}/calmodulin-dependent protein kinase II (CaMKII) and H-89, a protein kinase A inhibitor, inhibited FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. FIR radiation itself also increased the temperature of culture medium. As transient receptors potential vanilloid (TRPV) ion channels are known to be temperature-sensitive calcium channels, we explore whether TRPV channels mediate these observed effects. Reverse transcription-PCR assay revealed two TRPV isoforms in BAEC, TRPV2 and TRPV4. Although ruthenium red, a pan-TRPV inhibitor, completely reversed the observed effect of FIR radiation, a partial attenuation (∼20%) was found in cells treated with Tranilast, TRPV2 inhibitor. However, ectopic expression of siRNA of TRPV2 showed no significant alteration in FIR radiation-stimulated eNOS-Ser{sup 1179} phosphorylation. This

  8. The mitogen-activated protein kinase (MAPK pathway: role in immune evasion by trypanosomatids

    Directory of Open Access Journals (Sweden)

    Mercedes Carolina Soares-Silva

    2016-02-01

    Full Text Available Leishmania spp and Trypanosoma cruzi are the causative agents of leishmaniasis and Chagas' disease, respectively, two neglected tropical diseases that affect about 25 million people worldwide. These parasites belong to the family Trypanosomatidae and are both obligate intracellular parasites that manipulate host signaling pathways to establish the infection, and also subvert the host innate immune system. Mitogen-activated protein kinases (MAPKs are serine and threonine protein kinases, highly conserved in eukaryotes, and are involved in signal transduction pathways that are related to modulation of physiological and pathophysiological cell responses. This mini-review highlights the current knowledge about the mechanisms that Leishmania spp and T. cruzi have evolved to target host MAPK signaling pathway, highjack immune response, and in this manner, promote parasite maintenance in the host.

  9. Functional Roles of p38 Mitogen-Activated Protein Kinase in Macrophage-Mediated Inflammatory Responses

    Directory of Open Access Journals (Sweden)

    Yanyan Yang

    2014-01-01

    Full Text Available Inflammation is a natural host defensive process that is largely regulated by macrophages during the innate immune response. Mitogen-activated protein kinases (MAPKs are proline-directed serine and threonine protein kinases that regulate many physiological and pathophysiological cell responses. p38 MAPKs are key MAPKs involved in the production of inflammatory mediators, including tumor necrosis factor-α (TNF-α and cyclooxygenase-2 (COX-2. p38 MAPK signaling plays an essential role in regulating cellular processes, especially inflammation. In this paper, we summarize the characteristics of p38 signaling in macrophage-mediated inflammation. In addition, we discuss the potential of using inhibitors targeting p38 expression in macrophages to treat inflammatory diseases.

  10. The AMP-activated protein kinase beta 1 subunit modulates erythrocyte integrity.

    Science.gov (United States)

    Cambridge, Emma L; McIntyre, Zoe; Clare, Simon; Arends, Mark J; Goulding, David; Isherwood, Christopher; Caetano, Susana S; Reviriego, Carmen Ballesteros; Swiatkowska, Agnieszka; Kane, Leanne; Harcourt, Katherine; Adams, David J; White, Jacqueline K; Speak, Anneliese O

    2017-01-01

    Failure to maintain a normal in vivo erythrocyte half-life results in the development of hemolytic anemia. Half-life is affected by numerous factors, including energy balance, electrolyte gradients, reactive oxygen species, and membrane plasticity. The heterotrimeric AMP-activated protein kinase (AMPK) is an evolutionarily conserved serine/threonine kinase that acts as a critical regulator of cellular energy balance. Previous roles for the alpha 1 and gamma 1 subunits in the control of erythrocyte survival have been reported. In the work described here, we studied the role of the beta 1 subunit in erythrocytes and observed microcytic anemia with compensatory extramedullary hematopoiesis together with splenomegaly and increased osmotic resistance. Copyright © 2016 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.

  11. Glycogen synthase kinase 3 alpha phosphorylates and regulates the osteogenic activity of Osterix.

    Science.gov (United States)

    Li, Hongyan; Jeong, Hyung Min; Choi, You Hee; Lee, Sung Ho; Jeong, Hye Gwang; Jeong, Tae Cheon; Lee, Kwang Youl

    2013-05-10

    Osteoblast-specific transcription factor Osterix is a zinc-finger transcription factor that required for osteoblast differentiation and new bone formation. The function of Osterix can be modulated by post-translational modification. Glycogen synthase kinase 3 alpha (GSK3α) is a multifunctional serine/threonine protein kinase that plays a role in the Wnt signaling pathways and is implicated in the control of several regulatory proteins and transcription factors. In the present study, we investigated how GSK3α regulates Osterix during osteoblast differentiation. Wide type GSK3α up-regulated the protein level, protein stability and transcriptional activity of Osterix. These results suggest that GSK3α regulates osteogenic activity of Osterix. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Phosphorylation of histone H3 at threonine 11 establishes a novel chromatin mark for transcriptional regulation

    OpenAIRE

    Metzger, Eric; Yin, Na; Wissmann, Melanie; Kunowska, Natalia; Fischer, Kristin; Friedrichs, Nicolaus; Patnaik, Debasis; Higgins, Jonathan M.G.; Potier, Noelle; Scheidtmann, Karl-Heinz; Buettner, Reinhard; Schüle, Roland

    2007-01-01

    Posttranslational modifications of histones such as methylation, acetylation, and phosphorylation regulate chromatin structure and gene expression. Here we show that protein kinase C-related kinase 1 (PRK1) phosphorylates histone H3 at threonine 11 (H3T11) upon ligand-dependent recruitment to androgen receptor (AR) target genes. PRK1 is pivotal to AR function since PRK1 knockdown or inhibition impedes AR-dependent transcription. Blocking PRK1 function abrogates androgen-induced H3T11 phosphor...

  13. Inhibitory effects of homeodomain-interacting protein kinase 2 on the aorta-gonad-mapharsen hematopoiesis

    International Nuclear Information System (INIS)

    Ohtsu, Naoki; Nobuhisa, Ikuo; Mochita, Miyuki; Taga, Tetsuya

    2007-01-01

    Definitive hematopoiesis starts in the aorta-gonad-mesonephros (AGM) region of the mouse embryo. Our previous studies revealed that STAT3, a gp130 downstream transcription factor, is required for AGM hematopoiesis and that homeodomain-interacting protein kinase 2 (HIPK2) phosphorylates serine-727 of STAT3. HIPK2 is a serine/threonine kinase known to be involved in transcriptional repression and apoptosis. In the present study, we examined the role of HIPK2 in hematopoiesis in mouse embryo. HIPK2 transcripts were found in fetal hematopoietic tissues such as the mouse AGM region and fetal liver. In cultured AGM cells, HIPK2 protein was detected in adherent cells. Functional analyses of HIPK2 were carried out by introducing wild-type and mutant HIPK2 constructs into AGM cultures. Production of CD45 + hematopoietic cells was suppressed by forced expression of HIPK2 in AGM cultures. This suppression required the kinase domain and nuclear localization signals of HIPK2, but the kinase activity was dispensable. HIPK2-overexpressing AGM-derived nonadherent cells did not form cobblestone-like colonies in cultures with stromal cells. Furthermore, overexpression of HIPK2 in AGM cultures impeded the expansion of CD45 low c-Kit + cells, which exhibit the immature hematopoietic progenitor phenotype. These data indicate that HIPK2 plays a negative regulatory role in AGM hematopoiesis in the mouse embryo

  14. An HTRF® Assay for the Protein Kinase ATM.

    Science.gov (United States)

    Adams, Phillip; Clark, Jonathan; Hawdon, Simon; Hill, Jennifer; Plater, Andrew

    2017-01-01

    Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase that plays a key role in the regulation of DNA damage pathways and checkpoint arrest. In recent years, there has been growing interest in ATM as a therapeutic target due to its association with cancer cell survival following genotoxic stress such as radio- and chemotherapy. Large-scale targeted drug screening campaigns have been hampered, however, by technical issues associated with the production of sufficient quantities of purified ATM and the availability of a suitable high-throughput assay. Using a purified, functionally active recombinant ATM and one of its physiological substrates, p53, we have developed an in vitro FRET-based activity assay that is suitable for high-throughput drug screening.

  15. Drug design with Cdc7 kinase: a potential novel cancer therapy target

    Directory of Open Access Journals (Sweden)

    Masaaki Sawa

    2008-11-01

    Full Text Available Masaaki Sawa1, Hisao Masai21Carna Biosciences, Inc., Kobe, Japan; 2Genome Dynamics Project, Tokyo Metropolitan Institute of Medical Science, Tokyo, JapanAbstract: Identification of novel molecular targets is critical in development of new and efficient cancer therapies. Kinases are one of the most common drug targets with a potential for cancer therapy. Cell cycle progression is regulated by a number of kinases, some of which are being developed to treat cancer. Cdc7 is a serine-threonine kinase originally discovered in budding yeast, which has been shown to be necessary to initiate the S phase. Inhibition of Cdc7 in cancer cells retards the progression of the S phase, accumulates DNA damage, and induces p53-independent cell death, but the same treatment in normal cells does not significantly affect viability. Low-molecular-weight compounds that inhibit Cdc7 kinase with an IC50 of less than 10 nM have been identified, and shown to be effective in the inhibition of tumor growth in animal models. Thus Cdc7 kinase can be recognized as a novel molecular target for cancer therapy.Keywords: Cdc7 kinase, cell cycle, replication fork, genome stability, DNA damages, ATP-binding pocket, kinase inhibitor

  16. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis*

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V.; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-01-01

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK−/− and LOK+/− lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. PMID:26945071

  17. Caspase Cleavages of the Lymphocyte-oriented Kinase Prevent Ezrin, Radixin, and Moesin Phosphorylation during Apoptosis.

    Science.gov (United States)

    Leroy, Catherine; Belkina, Natalya V; Long, Thavy; Deruy, Emeric; Dissous, Colette; Shaw, Stephen; Tulasne, David

    2016-05-06

    The lymphocyte-oriented kinase (LOK), also called serine threonine kinase 10 (STK10), is synthesized mainly in lymphocytes. It is involved in lymphocyte migration and polarization and can phosphorylate ezrin, radixin, and moesin (the ERM proteins). In a T lymphocyte cell line and in purified human lymphocytes, we found LOK to be cleaved by caspases during apoptosis. The first cleavage occurs at aspartic residue 332, located between the kinase domain and the coiled-coil regulation domain. This cleavage generates an N-terminal fragment, p50 N-LOK, containing the kinase domain and a C-terminal fragment, which is further cleaved during apoptosis. Although these cleavages preserve the entire kinase domain, p50 N-LOK displays no kinase activity. In apoptotic lymphocytes, caspase cleavages of LOK are concomitant with a decrease in ERM phosphorylation. When non-apoptotic lymphocytes from mice with homozygous and heterozygous LOK knockout were compared, the latter showed a higher level of ERM phosphorylation, but when apoptosis was induced, LOK(-/-) and LOK(+/-) lymphocytes showed the same low level, confirming in vivo that LOK-induced ERM phosphorylation is prevented during lymphocyte apoptosis. Our results demonstrate that cleavage of LOK during apoptosis abolishes its kinase activity, causing a decrease in ERM phosphorylation, crucial to the role of the ERM proteins in linking the plasma membrane to actin filaments. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

  18. Identifying three-dimensional structures of autophosphorylation complexes in crystals of protein kinases

    Science.gov (United States)

    Xu, Qifang; Malecka, Kimberly L.; Fink, Lauren; Jordan, E. Joseph; Duffy, Erin; Kolander, Samuel; Peterson, Jeffrey; Dunbrack, Roland L.

    2016-01-01

    Protein kinase autophosphorylation is a common regulatory mechanism in cell signaling pathways. Crystal structures of several homomeric protein kinase complexes have a serine, threonine, or tyrosine autophosphorylation site of one kinase monomer located in the active site of another monomer, a structural complex that we call an “autophosphorylation complex.” We developed and applied a structural bioinformatics method to identify all such autophosphorylation kinase complexes in X-ray crystallographic structures in the Protein Data Bank (PDB). We identified 15 autophosphorylation complexes in the PDB, of which 5 complexes had not previously been described in the publications describing the crystal structures. These 5 consist of tyrosine residues in the N-terminal juxtamembrane regions of colony stimulating factor 1 receptor (CSF1R, Tyr561) and EPH receptor A2 (EPHA2, Tyr594), tyrosine residues in the activation loops of the SRC kinase family member LCK (Tyr394) and insulin-like growth factor 1 receptor (IGF1R, Tyr1166), and a serine in a nuclear localization signal region of CDC-like kinase 2 (CLK2, Ser142). Mutations in the complex interface may alter autophosphorylation activity and contribute to disease; therefore we mutated residues in the autophosphorylation complex interface of LCK and found that two mutations impaired autophosphorylation (T445V and N446A) and mutation of Pro447 to Ala, Gly, or Leu increased autophosphorylation. The identified autophosphorylation sites are conserved in many kinases, suggesting that, by homology, these complexes may provide insight into autophosphorylation complex interfaces of kinases that are relevant drug targets. PMID:26628682

  19. LRRK2 Kinase Activity and Biology are Not Uniformly Predicted by its Autophosphorylation and Cellular Phosphorylation Site Status

    Directory of Open Access Journals (Sweden)

    April eReynolds

    2014-06-01

    Full Text Available Missense mutations in the Leucine Rich Repeat protein Kinase 2 (LRRK2 gene are the most common genetic predisposition to develop Parkinson’s disease (PD LRRK2 is a large multi-domain phosphoprotein with a GTPase domain and a serine/threonine protein kinase domain whose activity is implicated in neuronal toxicity; however the precise mechanism is unknown. LRRK2 autophosphorylates on several serine/threonine residues across the enzyme and is found constitutively phosphorylated on Ser910, Ser935, Ser955 and Ser973, which are proposed to be regulated by upstream kinases. Here we investigate the phosphoregulation at these sites by analyzing the effects of disease-associated mutations Arg1441Cys, Arg1441Gly, Ala1442Pro, Tyr1699Cys, Ile2012Thr, Gly2019Ser, and Ile2020Thr. We also studied alanine substitutions of phosphosite serines 910, 935, 955 and 973 and specific LRRK2 inhibition on autophosphorylation of LRRK2 Ser1292, Thr1491, Thr2483 and phosphorylation at the cellular sites. We found that mutants in the Roc-COR domains, including Arg1441Cys, Arg1441His, Ala1442Pro and Tyr1699Cys, can positively enhance LRRK2 kinase activity while concomitantly inducing the dephosphorylation of the cellular sites. Mutation of the cellular sites individually did not affect LRRK2 intrinsic kinase activity; however, Ser910/935/955/973Ala mutations trended toward increased kinase activity of LRRK2. Increased cAMP levels did not lead to increased LRRK2 cellular site phosphorylation, 14-3-3 binding or kinase activity. In cells, inhibition of LRRK2 kinase activity leads to dephosphorylation of Ser1292 by Calyculin A and okadaic acid sensitive phosphatases, while the cellular sites are dephosphorylated by Calyculin A sensitive phosphatases. These findings indicate that comparative analysis of both Ser1292 and Ser910/935/955/973 phosphorylation sites will provide important and distinct measures of LRRK2 kinase and biological activity in vitro and in vivo.

  20. Functional intersection of ATM and DNA-dependent protein kinase catalytic subunit in coding end joining during V(D)J recombination

    DEFF Research Database (Denmark)

    Lee, Baeck-Seung; Gapud, Eric J; Zhang, Shichuan

    2013-01-01

    V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA-PKcs) ar......V(D)J recombination is initiated by the RAG endonuclease, which introduces DNA double-strand breaks (DSBs) at the border between two recombining gene segments, generating two hairpin-sealed coding ends and two blunt signal ends. ATM and DNA-dependent protein kinase catalytic subunit (DNA......-PKcs) are serine-threonine kinases that orchestrate the cellular responses to DNA DSBs. During V(D)J recombination, ATM and DNA-PKcs have unique functions in the repair of coding DNA ends. ATM deficiency leads to instability of postcleavage complexes and the loss of coding ends from these complexes. DNA...... when ATM is present and its kinase activity is intact. The ability of ATM to compensate for DNA-PKcs kinase activity depends on the integrity of three threonines in DNA-PKcs that are phosphorylation targets of ATM, suggesting that ATM can modulate DNA-PKcs activity through direct phosphorylation of DNA...

  1. Protein Kinase C-Related Kinase (PKN/PRK). Potential Key-Role for PKN1 in Protection of Hypoxic Neurons.

    Science.gov (United States)

    Thauerer, Bettina; Zur Nedden, Stephanie; Baier-Bitterlich, Gabriele

    2014-05-01

    Serine/threonine protein kinase C-related kinase (PKN/PRK) is a family of three isoenzymes (PKN1, PKN2, PKN3), which are widely distributed in eukaryotic organisms and share the same overall domain structure. The Nterminal region encompasses a conserved repeated domain, termed HR1a-c as well as a HR2/C2 domain. The serine/threonine kinase domain is found in the C-terminal region of the protein and shows high sequence homology to other members of the PKC superfamily. In neurons, PKN1 is the most abundant isoform and has been implicated in a variety of functions including cytoskeletal organization and neuronal differentiation and its deregulation may contribute to neuropathological processes such as amyotrophic lateral sclerosis and Alzheimer's disease. We have recently identified a candidate role of PKN1 in the regulation of neuroprotective processes during hypoxic stress. Our key findings were that: 1) the activity of PKN1 was significantly increased by hypoxia (1% O2) and neurotrophins (nerve growth factor and purine nucleosides); 2) Neuronal cells, deficient of PKN1 showed a decrease of cell viability and neurite formation along with a disturbance of the F-actinassociated cytoskeleton; 3) Purine nucleoside-mediated neuroprotection during hypoxia was severely hampered in PKN1 deficient neuronal cells, altogether suggesting a potentially critical role of PKN1 in neuroprotective processes. This review gives an up-to-date overview of the PKN family with a special focus on the neuroprotective role of PKN1 in hypoxia.

  2. The scaffold protein calcium/calmodulin-dependent serine protein kinase controls ATP release in sensory ganglia upon P2X3 receptor activation and is part of an ATP keeper complex.

    Science.gov (United States)

    Bele, Tanja; Fabbretti, Elsa

    2016-08-01

    P2X3 receptors, gated by extracellular ATP, are expressed by sensory neurons and are involved in peripheral nociception and pain sensitization. The ability of P2X3 receptors to transduce extracellular stimuli into neuronal signals critically depends on the dynamic molecular partnership with the calcium/calmodulin-dependent serine protein kinase (CASK). The present work used trigeminal sensory neurons to study the impact that activation of P2X3 receptors (evoked by the agonist α,β-meATP) has on the release of endogenous ATP and how CASK modulates this phenomenon. P2X3 receptor function was followed by ATP efflux via Pannexin1 (Panx1) hemichannels, a mechanism that was blocked by the P2X3 receptor antagonist A-317491, and by P2X3 silencing. ATP efflux was enhanced by nerve growth factor, a treatment known to potentiate P2X3 receptor function. Basal ATP efflux was not controlled by CASK, and carbenoxolone or Pannexin silencing reduced ATP release upon P2X3 receptor function. CASK-controlled ATP efflux followed P2X3 receptor activity, but not depolarization-evoked ATP release. Molecular biology experiments showed that CASK was essential for the transactivation of Panx1 upon P2X3 receptor activation. These data suggest that P2X3 receptor function controls a new type of feed-forward purinergic signaling on surrounding cells, with consequences at peripheral and spinal cord level. Thus, P2X3 receptor-mediated ATP efflux may be considered for the future development of pharmacological strategies aimed at containing neuronal sensitization. P2X3 receptors are involved in sensory transduction and associate to CASK. We have studied in primary sensory neurons the molecular mechanisms downstream P2X3 receptor activation, namely ATP release and partnership with CASK or Panx1. Our data suggest that CASK and P2X3 receptors are part of an ATP keeper complex, with important feed-forward consequences at peripheral and central level. © 2016 International Society for Neurochemistry.

  3. Novel receptor-like kinases in cacao contain PR-1 extracellular domains.

    Science.gov (United States)

    Teixeira, Paulo José Pereira Lima; Costa, Gustavo Gilson Lacerda; Fiorin, Gabriel Lorencini; Pereira, Gonçalo Amarante Guimarães; Mondego, Jorge Maurício Costa

    2013-08-01

    Members of the pathogenesis-related protein 1 (PR-1) family are well-known markers of plant defence responses, forming part of the arsenal of the secreted proteins produced on pathogen recognition. Here, we report the identification of two cacao (Theobroma cacao L.) PR-1s that are fused to transmembrane regions and serine/threonine kinase domains, in a manner characteristic of receptor-like kinases (RLKs). These proteins (TcPR-1f and TcPR-1g) were named PR-1 receptor kinases (PR-1RKs). Phylogenetic analysis of RLKs and PR-1 proteins from cacao indicated that PR-1RKs originated from a fusion between sequences encoding PR-1 and the kinase domain of a LecRLK (Lectin Receptor-Like Kinase). Retrotransposition marks surround TcPR-1f, suggesting that retrotransposition was involved in the origin of PR-1RKs. Genes with a similar domain architecture to cacao PR-1RKs were found in rice (Oryza sativa), barrel medic (Medicago truncatula) and a nonphototrophic bacterium (Herpetosiphon aurantiacus). However, their kinase domains differed from those found in LecRLKs, indicating the occurrence of convergent evolution. TcPR-1g expression was up-regulated in the biotrophic stage of witches' broom disease, suggesting a role for PR-1RKs during cacao defence responses. We hypothesize that PR-1RKs transduce a defence signal by interacting with a PR-1 ligand. © 2013 BSPP AND JOHN WILEY & SONS LTD.

  4. Serine biosynthesis and transport defects.

    Science.gov (United States)

    El-Hattab, Ayman W

    2016-07-01

    l-serine is a non-essential amino acid that is biosynthesized via the enzymes phosphoglycerate dehydrogenase (PGDH), phosphoserine aminotransferase (PSAT), and phosphoserine phosphatase (PSP). Besides its role in protein synthesis, l-serine is a potent neurotrophic factor and a precursor of a number of essential compounds including phosphatidylserine, sphingomyelin, glycine, and d-serine. Serine biosynthesis defects result from impairments of PGDH, PSAT, or PSP leading to systemic serine deficiency. Serine biosynthesis defects present in a broad phenotypic spectrum that includes, at the severe end, Neu-Laxova syndrome, a lethal multiple congenital anomaly disease, intermediately, infantile serine biosynthesis defects with severe neurological manifestations and growth deficiency, and at the mild end, the childhood disease with intellectual disability. A serine transport defect resulting from deficiency of the ASCT1, the main transporter for serine in the central nervous system, has been recently described in children with neurological manifestations that overlap with those observed in serine biosynthesis defects. l-serine therapy may be beneficial in preventing or ameliorating symptoms in serine biosynthesis and transport defects, if started before neurological damage occurs. Herein, we review serine metabolism and transport, the clinical, biochemical, and molecular aspects of serine biosynthesis and transport defects, the mechanisms of these diseases, and the potential role of serine therapy. Copyright © 2016 Elsevier Inc. All rights reserved.

  5. Targeting the Pim kinases in multiple myeloma.

    LENUS (Irish Health Repository)

    Keane, N A

    2015-07-17

    Multiple myeloma (MM) is a plasma cell malignancy that remains incurable. Novel treatment strategies to improve survival are urgently required. The Pims are a small family of serine\\/threonine kinases with increased expression across the hematological malignancies. Pim-2 shows highest expression in MM and constitutes a promising therapeutic target. It is upregulated by the bone marrow microenvironment to mediate proliferation and promote MM survival. Pim-2 also has a key role in the bone destruction typically seen in MM. Additional putative roles of the Pim kinases in MM include trafficking of malignant cells, promoting oncogenic signaling in the hypoxic bone marrow microenvironment and mediating resistance to therapy. A number of Pim inhibitors are now under development with lead compounds entering the clinic. The ATP-competitive Pim inhibitor LGH447 has recently been reported to have single agent activity in MM. It is anticipated that Pim inhibition will be of clinical benefit in combination with standard treatments and\\/or with novel drugs targeting other survival pathways in MM.

  6. Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1).

    Science.gov (United States)

    Zhu, Xiang-Yu; Liu, Ning; Liu, Wei; Song, Shao-Wei; Guo, Ke-Jian

    2012-04-01

    Integrin-linked kinase (ILK) is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1) cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

  7. Silencing of the integrin-linked kinase gene suppresses the proliferation, migration and invasion of pancreatic cancer cells (Panc-1

    Directory of Open Access Journals (Sweden)

    Xiang-Yu Zhu

    2012-01-01

    Full Text Available Integrin-linked kinase (ILK is an ankyrin repeat-containing serine-threonine protein kinase that is involved in the regulation of integrin-mediated processes such as cancer cell proliferation, migration and invasion. In this study, we examined the effect of a lentivirus-mediated knockdown of ILK on the proliferation, migration and invasion of pancreatic cancer (Panc-1 cells. Immunohistochemical staining showed that ILK expression was enhanced in pancreatic cancer tissue. The silencing of ILK in human Panc-1 cells led to cell cycle arrest in the G0/G1 phase and delayed cell proliferation, in addition to down-regulating cell migration and invasion. The latter effects were mediated by up-regulating the expression of E-cadherin, a key protein in cell adhesion. These findings indicate that ILK may be a new diagnostic marker for pancreatic cancer and that silencing ILK could be a potentially useful therapeutic approach for treating pancreatic cancer.

  8. The Emerging Role of Polo-Like Kinase 1 in Epithelial-Mesenchymal Transition and Tumor Metastasis

    Directory of Open Access Journals (Sweden)

    Zheng Fu

    2017-09-01

    Full Text Available Polo-like kinase 1 (PLK1 is a serine/threonine kinase that plays a key role in the regulation of the cell cycle. PLK1 is overexpressed in a variety of human tumors, and its expression level often correlates with increased cellular proliferation and poor prognosis in cancer patients. It has been suggested that PLK1 controls cancer development through multiple mechanisms that include canonical regulation of mitosis and cytokinesis, modulation of DNA replication, and cell survival. However, emerging evidence suggests novel and previously unanticipated roles for PLK1 during tumor development. In this review, we will summarize the recent advancements in our understanding of the oncogenic functions of PLK1, with a focus on its role in epithelial-mesenchymal transition and tumor invasion. We will further discuss the therapeutic potential of these functions.

  9. A novel non-canonical mechanism of regulation of MST3 (mammalian Sterile20-related kinase 3).

    Science.gov (United States)

    Fuller, Stephen J; McGuffin, Liam J; Marshall, Andrew K; Giraldo, Alejandro; Pikkarainen, Sampsa; Clerk, Angela; Sugden, Peter H

    2012-03-15

    The canonical pathway of regulation of the GCK (germinal centre kinase) III subgroup member, MST3 (mammalian Sterile20-related kinase 3), involves a caspase-mediated cleavage between N-terminal catalytic and C-terminal regulatory domains with possible concurrent autophosphorylation of the activation loop MST3(Thr(178)), induction of serine/threonine protein kinase activity and nuclear localization. We identified an alternative 'non-canonical' pathway of MST3 activation (regulated primarily through dephosphorylation) which may also be applicable to other GCKIII (and GCKVI) subgroup members. In the basal state, inactive MST3 co-immunoprecipitated with the Golgi protein GOLGA2/gm130 (golgin A2/Golgi matrix protein 130). Activation of MST3 by calyculin A (a protein serine/threonine phosphatase 1/2A inhibitor) stimulated (auto)phosphorylation of MST3(Thr(178)) in the catalytic domain with essentially simultaneous cis-autophosphorylation of MST3(Thr(328)) in the regulatory domain, an event also requiring the MST3(341-376) sequence which acts as a putative docking domain. MST3(Thr(178)) phosphorylation increased MST3 kinase activity, but this activity was independent of MST3(Thr(328)) phosphorylation. Interestingly, MST3(Thr(328)) lies immediately C-terminal to a STRAD (Sterile20-related adaptor) pseudokinase-like site identified recently as being involved in binding of GCKIII/GCKVI members to MO25 scaffolding proteins. MST3(Thr(178)/Thr(328)) phosphorylation was concurrent with dissociation of MST3 from GOLGA2/gm130 and association of MST3 with MO25, and MST3(Thr(328)) phosphorylation was necessary for formation of the activated MST3-MO25 holocomplex.

  10. The GCKIII Kinase Sps1 and the 14-3-3 Isoforms, Bmh1 and Bmh2, Cooperate to Ensure Proper Sporulation in Saccharomyces cerevisiae

    Science.gov (United States)

    Slubowski, Christian J.; Paulissen, Scott M.; Huang, Linda S.

    2014-01-01

    Sporulation in the budding yeast Saccharomyces cerevisiae is a developmental program initiated in response to nutritional deprivation. Sps1, a serine/threonine kinase, is required for sporulation, but relatively little is known about the molecular mechanisms through which it regulates this process. Here we show that SPS1 encodes a bona-fide member of the GCKIII subfamily of STE20 kinases, both through phylogenetic analysis of the kinase domain and examination of its C-terminal regulatory domain. Within the regulatory domain, we find Sps1 contains an invariant ExxxPG region conserved from plant to human GCKIIIs that we call the EPG motif; we show this EPG motif is important for SPS1 function. We also find that Sps1 is phosphorylated near its N-terminus on Threonine 12, and that this phosphorylation is required for the efficient production of spores. In Sps1, Threonine 12 lies within a 14-3-3 consensus binding sequence, and we show that the S. cerevisiae 14-3-3 proteins Bmh1 and Bmh2 bind Sps1 in a Threonine 12-dependent fashion. This interaction is significant, as BMH1 and BMH2 are required during sporulation and genetically interact with SPS1 in sporulating cells. Finally, we observe that Sps1, Bmh1 and Bmh2 are present in both the nucleus and cytoplasm during sporulation. We identify a nuclear localization sequence in Sps1 at amino acids 411–415, and show that this sequence is necessary and sufficient for nuclear localization. Taken together, these data identify regions within Sps1 critical for its function and indicate that SPS1 and 14-3-3s act together to promote proper sporulation in S. cerevisiae. PMID:25409301

  11. Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

    Directory of Open Access Journals (Sweden)

    Annika Sommerfeld

    2015-05-01

    Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

  12. Molecular Imaging of the ATM Kinase Activity

    Energy Technology Data Exchange (ETDEWEB)

    Williams, Terence M. [Department of Radiation Oncology, Ohio State University, Columbus, Ohio (United States); Nyati, Shyam [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Ross, Brian D. [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Rehemtulla, Alnawaz, E-mail: alnawaz@umich.edu [Department of Radiation Oncology, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Center for Molecular Imaging, University of Michigan Medical Center, Ann Arbor, Michigan (United States); Department of Radiology, University of Michigan Medical Center, Ann Arbor, Michigan (United States)

    2013-08-01

    Purpose: Ataxia telangiectasia mutated (ATM) is a serine/threonine kinase critical to the cellular DNA-damage response, including from DNA double-strand breaks. ATM activation results in the initiation of a complex cascade of events including DNA damage repair, cell cycle checkpoint control, and survival. We sought to create a bioluminescent reporter that dynamically and noninvasively measures ATM kinase activity in living cells and subjects. Methods and Materials: Using the split luciferase technology, we constructed a hybrid cDNA, ATM-reporter (ATMR), coding for a protein that quantitatively reports on changes in ATM kinase activity through changes in bioluminescence. Results: Treatment of ATMR-expressing cells with ATM inhibitors resulted in a dose-dependent increase in bioluminescence activity. In contrast, induction of ATM kinase activity upon irradiation resulted in a decrease in reporter activity that correlated with ATM and Chk2 activation by immunoblotting in a time-dependent fashion. Nuclear targeting improved ATMR sensitivity to both ATM inhibitors and radiation, whereas a mutant ATMR (lacking the target phosphorylation site) displayed a muted response. Treatment with ATM inhibitors and small interfering (si)RNA-targeted knockdown of ATM confirm the specificity of the reporter. Using reporter expressing xenografted tumors demonstrated the ability of ATMR to report in ATM activity in mouse models that correlated in a time-dependent fashion with changes in Chk2 activity. Conclusions: We describe the development and validation of a novel, specific, noninvasive bioluminescent reporter that enables monitoring of ATM activity in real time, in vitro and in vivo. Potential applications of this reporter include the identification and development of novel ATM inhibitors or ATM-interacting partners through high-throughput screens and in vivo pharmacokinetic/pharmacodynamic studies of ATM inhibitors in preclinical models.

  13. AGC kinases, mechanisms of regulation ‎and innovative drug development.

    Science.gov (United States)

    Leroux, Alejandro E; Schulze, Jörg O; Biondi, Ricardo M

    2018-02-01

    The group of AGC kinases consists of 63 evolutionarily related serine/threonine protein kinases comprising PDK1, PKB/Akt, SGK, PKC, PRK/PKN, MSK, RSK, S6K, PKA, PKG, DMPK, MRCK, ROCK, NDR, LATS, CRIK, MAST, GRK, Sgk494, and YANK, while two other families, Aurora and PLK, are the most closely related to the group. Eight of these families are physiologically activated downstream of growth factor signalling, while other AGC kinases are downstream effectors of a wide range of signals. The different AGC kinase families share aspects of their mechanisms of inhibition and activation. In the present review, we update the knowledge of the mechanisms of regulation of different AGC kinases. The conformation of the catalytic domain of many AGC kinases is regulated allosterically through the modulation of the conformation of a regulatory site on the small lobe of the kinase domain, the PIF-pocket. The PIF-pocket acts like an ON-OFF switch in AGC kinases with different modes of regulation, i.e. PDK1, PKB/Akt, LATS and Aurora kinases. In this review, we make emphasis on how the knowledge of the molecular mechanisms of regulation can guide the discovery and development of small allosteric modulators. Molecular probes stabilizing the PIF-pocket in the active conformation are activators, while compounds stabilizing the disrupted site are allosteric inhibitors. One challenge for the rational development of allosteric modulators is the lack of complete structural information of the inhibited forms of full-length AGC kinases. On the other hand, we suggest that the available information derived from molecular biology and biochemical studies can already guide screening strategies for the identification of innovative mode of action molecular probes and the development of selective allosteric drugs for the treatment of human diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  14. TRESK background K(+ channel is inhibited by PAR-1/MARK microtubule affinity-regulating kinases in Xenopus oocytes.

    Directory of Open Access Journals (Sweden)

    Gabriella Braun

    Full Text Available TRESK (TWIK-related spinal cord K(+ channel, KCNK18 is a major background K(+ channel of sensory neurons. Dominant-negative mutation of TRESK is linked to familial migraine. This important two-pore domain K(+ channel is uniquely activated by calcineurin. The calcium/calmodulin-dependent protein phosphatase directly binds to the channel and activates TRESK current several-fold in Xenopus oocytes and HEK293 cells. We have recently shown that the kinase, which is responsible for the basal inhibition of the K(+ current, is sensitive to the adaptor protein 14-3-3. Therefore we have examined the effect of the 14-3-3-inhibited PAR-1/MARK, microtubule-associated-protein/microtubule affinity-regulating kinase on TRESK in the Xenopus oocyte expression system. MARK1, MARK2 and MARK3 accelerated the return of TRESK current to the resting state after the calcium-dependent activation. Several other serine-threonine kinase types, generally involved in the modulation of other ion channels, failed to influence TRESK current recovery. MARK2 phosphorylated the primary determinant of regulation, the cluster of three adjacent serine residues (S274, 276 and 279 in the intracellular loop of mouse TRESK. In contrast, serine 264, the 14-3-3-binding site of TRESK, was not phosphorylated by the kinase. Thus MARK2 selectively inhibits TRESK activity via the S274/276/279 cluster, but does not affect the direct recruitment of 14-3-3 to the channel. TRESK is the first example of an ion channel phosphorylated by the dynamically membrane-localized MARK kinases, also known as general determinants of cellular polarity. These results raise the possibility that microtubule dynamics is coupled to the regulation of excitability in the neurons, which express TRESK background potassium channel.

  15. Cdc7 kinase - a new target for drug development.

    Science.gov (United States)

    Swords, Ronan; Mahalingam, Devalingam; O'Dwyer, Michael; Santocanale, Corrado; Kelly, Kevin; Carew, Jennifer; Giles, Francis

    2010-01-01

    The cell division cycle 7 (Cdc7) is a serine threonine kinase that is of critical importance in the regulation of normal cell cycle progression. Cdc7 kinase is highly conserved during evolution and much has been learned about its biological roles in humans through the study of lower eukaryotes, particularly yeasts. Two important regulator proteins, Dbf4 and Drf1, bind to and modulate the kinase activity of human Cdc7 which phosphorylates several sites on Mcm2 (minichromosome maintenance protein 2), one of the six subunits of the replicative DNA helicase needed for duplication of the genome. Through regulation of both DNA synthesis and DNA damage response, both key functions in the survival of tumour cells, Cdc7 becomes an attractive target for pharmacological inhibition. There are much data available on the pre-clinical anti-cancer effects of Cdc7 depletion and although there are no available Cdc7 inhibitors in clinical trials as yet, several lead compounds are being optimised for this purpose. In this review, we will address the current status of Cdc7 as an important target for new drug development.

  16. Glycogen synthase kinase 3: more than a namesake.

    Science.gov (United States)

    Rayasam, Geetha Vani; Tulasi, Vamshi Krishna; Sodhi, Reena; Davis, Joseph Alex; Ray, Abhijit

    2009-03-01

    Glycogen synthase kinase 3 (GSK3), a constitutively acting multi-functional serine threonine kinase is involved in diverse physiological pathways ranging from metabolism, cell cycle, gene expression, development and oncogenesis to neuroprotection. These diverse multiple functions attributed to GSK3 can be explained by variety of substrates like glycogen synthase, tau protein and beta catenin that are phosphorylated leading to their inactivation. GSK3 has been implicated in various diseases such as diabetes, inflammation, cancer, Alzheimer's and bipolar disorder. GSK3 negatively regulates insulin-mediated glycogen synthesis and glucose homeostasis, and increased expression and activity of GSK3 has been reported in type II diabetics and obese animal models. Consequently, inhibitors of GSK3 have been demonstrated to have anti-diabetic effects in vitro and in animal models. However, inhibition of GSK3 poses a challenge as achieving selectivity of an over achieving kinase involved in various pathways with multiple substrates may lead to side effects and toxicity. The primary concern is developing inhibitors of GSK3 that are anti-diabetic but do not lead to up-regulation of oncogenes. The focus of this review is the recent advances and the challenges surrounding GSK3 as an anti-diabetic therapeutic target.

  17. Cloning and Sequencing of Protein Kinase cDNA from Harbor Seal (Phoca vitulina Lymphocytes

    Directory of Open Access Journals (Sweden)

    Jennifer C. C. Neale

    2004-01-01

    Full Text Available Protein kinases (PKs play critical roles in signal transduction and activation of lymphocytes. The identification of PK genes provides a tool for understanding mechanisms of immunotoxic xenobiotics. As part of a larger study investigating persistent organic pollutants in the harbor seal and their possible immunomodulatory actions, we sequenced harbor seal cDNA fragments encoding PKs. The procedure, using degenerate primers based on conserved motifs of human protein tyrosine kinases (PTKs, successfully amplified nine phocid PK gene fragments with high homology to human and rodent orthologs. We identified eight PTKs and one dual (serine/threonine and tyrosine kinase. Among these were several PKs important in early signaling events through the B- and T-cell receptors (FYN, LYN, ITK and SYK and a MAP kinase involved in downstream signal transduction. V-FGR, RET and DDR2 were also expressed. Sequential activation of protein kinases ultimately induces gene transcription leading to the proliferation and differentiation of lymphocytes critical to adaptive immunity. PKs are potential targets of bioactive xenobiotics, including persistent organic pollutants of the marine environment; characterization of these molecules in the harbor seal provides a foundation for further research illuminating mechanisms of action of contaminants speculated to contribute to large-scale die-offs of marine mammals via immunosuppression.

  18. Protein kinase N2 regulates AMP kinase signaling and insulin responsiveness of glucose metabolism in skeletal muscle.

    Science.gov (United States)

    Ruby, Maxwell A; Riedl, Isabelle; Massart, Julie; Åhlin, Marcus; Zierath, Juleen R

    2017-10-01

    Insulin resistance is central to the development of type 2 diabetes and related metabolic disorders. Because skeletal muscle is responsible for the majority of whole body insulin-stimulated glucose uptake, regulation of glucose metabolism in this tissue is of particular importance. Although Rho GTPases and many of their affecters influence skeletal muscle metabolism, there is a paucity of information on the protein kinase N (PKN) family of serine/threonine protein kinases. We investigated the impact of PKN2 on insulin signaling and glucose metabolism in primary human skeletal muscle cells in vitro and mouse tibialis anterior muscle in vivo. PKN2 knockdown in vitro decreased insulin-stimulated glucose uptake, incorporation into glycogen, and oxidation. PKN2 siRNA increased 5'-adenosine monophosphate-activated protein kinase (AMPK) signaling while stimulating fatty acid oxidation and incorporation into triglycerides and decreasing protein synthesis. At the transcriptional level, PKN2 knockdown increased expression of PGC-1α and SREBP-1c and their target genes. In mature skeletal muscle, in vivo PKN2 knockdown decreased glucose uptake and increased AMPK phosphorylation. Thus, PKN2 alters key signaling pathways and transcriptional networks to regulate glucose and lipid metabolism. Identification of PKN2 as a novel regulator of insulin and AMPK signaling may provide an avenue for manipulation of skeletal muscle metabolism. Copyright © 2017 the American Physiological Society.

  19. Phospholipid composition and a polybasic motif determine D6 PROTEIN KINASE polar association with the plasma membrane and tropic responses.

    Science.gov (United States)

    Barbosa, Inês C R; Shikata, Hiromasa; Zourelidou, Melina; Heilmann, Mareike; Heilmann, Ingo; Schwechheimer, Claus

    2016-12-15

    Polar transport of the phytohormone auxin through PIN-FORMED (PIN) auxin efflux carriers is essential for the spatiotemporal control of plant development. The Arabidopsis thaliana serine/threonine kinase D6 PROTEIN KINASE (D6PK) is polarly localized at the plasma membrane of many cells where it colocalizes with PINs and activates PIN-mediated auxin efflux. Here, we show that the association of D6PK with the basal plasma membrane and PINs is dependent on the phospholipid composition of the plasma membrane as well as on the phosphatidylinositol phosphate 5-kinases PIP5K1 and PIP5K2 in epidermis cells of the primary root. We further show that D6PK directly binds polyacidic phospholipids through a polybasic lysine-rich motif in the middle domain of the kinase. The lysine-rich motif is required for proper PIN3 phosphorylation and for auxin transport-dependent tropic growth. Polybasic motifs are also present at a conserved position in other D6PK-related kinases and required for membrane and phospholipid binding. Thus, phospholipid-dependent recruitment to membranes through polybasic motifs might not only be required for D6PK-mediated auxin transport but also other processes regulated by these, as yet, functionally uncharacterized kinases. © 2016. Published by The Company of Biologists Ltd.

  20. cAMP-dependent protein kinase A (PKA) regulates angiogenesis by modulating tip cell behavior in a Notch-independent manner.

    Science.gov (United States)

    Nedvetsky, Pavel I; Zhao, Xiaocheng; Mathivet, Thomas; Aspalter, Irene M; Stanchi, Fabio; Metzger, Ross J; Mostov, Keith E; Gerhardt, Holger

    2016-10-01

    cAMP-dependent protein kinase A (PKA) is a ubiquitously expressed serine/threonine kinase that regulates a variety of cellular functions. Here, we demonstrate that endothelial PKA activity is essential for vascular development, specifically regulating the transition from sprouting to stabilization of nascent vessels. Inhibition of endothelial PKA by endothelial cell-specific expression of dominant-negative PKA in mice led to perturbed vascular development, hemorrhage and embryonic lethality at mid-gestation. During perinatal retinal angiogenesis, inhibition of PKA resulted in hypersprouting as a result of increased numbers of tip cells. In zebrafish, cell autonomous PKA inhibition also increased and sustained endothelial cell motility, driving cells to become tip cells. Although these effects of PKA inhibition were highly reminiscent of Notch inhibition effects, our data demonstrate that PKA and Notch independently regulate tip and stalk cell formation and behavior. © 2016. Published by The Company of Biologists Ltd.

  1. Regulation of taurine homeostasis by protein kinase CK2 in mouse fibroblasts

    DEFF Research Database (Denmark)

    Hansen, Daniel Bloch; Guerra, Barbara; Jacobsen, Jack Hummeland

    2011-01-01

    Increased expression of the ubiquitous serine/threonine protein kinase CK2 has been associated with increased proliferative capacity and increased resistance towards apoptosis. Taurine is the primary organic osmolyte involved in cell volume control in mammalian cells, and shift in cell volume...... is a critical step in cell proliferation, differentiation and induction of apoptosis. In the present study, we use mouse NIH3T3 fibroblasts and Ehrlich Lettré ascites tumour cells with different CK2 expression levels. Taurine uptake via the Na(+) dependent transporter TauT and taurine release are increased...... and reduced, respectively, following pharmacological CK2 inhibition. The effect of CK2 inhibition on TauT involves modulation of transport kinetics, whereas the effect on the taurine release pathway involves reduction in the open-probability of the efflux pathway. Stimulation of PLA(2) activity, exposure...

  2. The Aurora B kinase in chromosome biorientation and spindle checkpoint signalling

    Directory of Open Access Journals (Sweden)

    Veronica eKrenn

    2015-10-01

    Full Text Available Aurora B, a member of the Aurora family of serine/threonine protein kinases, is a key player in chromosome segregation. As part of a macromolecular complex known as the chromosome passenger complex, Aurora B concentrates early during mitosis in the proximity of centromeres and kinetochores, the sites of attachment of chromosomes to spindle microtubules. There, it contributes to a number of processes that impart fidelity to cell division, including kinetochore stabilization, kinetochore-microtubule attachment, and the regulation of a surveillance mechanism named the spindle assembly checkpoint. In the regulation of these processes, Aurora B is the fulcrum of a remarkably complex network of interactions that feed back on its localization and activation state. In this review we discuss the multiple roles of Aurora B during mitosis, focusing in particular on its role at centromeres and kinetochores. Many details of the network of interactions at these locations remain poorly understood, and we focus here on several crucial outstanding questions.

  3. Protein kinase C isozymes, novel phorbol ester receptors and cancer chemotherapy.

    LENUS (Irish Health Repository)

    Barry, O P

    2012-02-03

    Recent years have seen extensive growth in the understanding of the role(s) of the various PKC isozymes and novel receptors for the phorbol ester tumor promoters. The PKC family of serine-threonine kinases is an important regulator of signaling cascades that control cell proliferation and death, and therefore represent targets for cancer therapy. While past interests have focused on PKC-selective inhibitors, more recently, intensive research has been underway for selective activators and inhibitors for each individual PKC isozyme. In the past few years a large number of PKC activators and inhibitors with potential as anticancer agents have been developed. A number of these compounds are already in Phase II clinical testing. As a new generation of cancer chemotherapeutic agents are designed, developed and put through a series of rigorous clinical trials, we can anticipate achieving exquisite control over PKC-mediated regulatory pathways, leading ultimately to a greater understanding of different cancers.

  4. LeCPK1, a Calcium-Dependent Protein Kinase from Tomato. Plasma Membrane Targeting and Biochemical Characterization1

    Science.gov (United States)

    Rutschmann, Frank; Stalder, Urs; Piotrowski, Markus; Oecking, Claudia; Schaller, Andreas

    2002-01-01

    The cDNA of LeCPK1, a calcium-dependent protein kinase, was cloned from tomato (Lycopersicon esculentum Mill.). LeCPK1 was expressed in Escherichia coli and purified from bacterial extracts. The recombinant protein was shown to be a functional protein kinase using a synthetic peptide as the substrate (syntide-2, Km = 85 μm). Autophosphorylation of LeCPK1 was observed on threonine and serine residues, one of which was identified as serine-439. Kinase activity was shown to be Ca2+ dependent and required the C-terminal, calmodulin-like domain of LeCPK1. Two classes of high- and low-affinity Ca2+-binding sites were observed, exhibiting dissociation constants of 0.6 and 55 μm, respectively. LeCPK1 was found to phosphorylate the regulatory C-terminal domain of the plasma membrane H+-ATPase in vitro. A potential role in the regulation of proton pump activity is corroborated by the apparent colocalization of the plasma membrane H+-ATPase and LeCPK1 in vivo. Upon transient expression in suspension-cultured cells, a C-terminal fusion of LeCPK1 with the green fluorescent protein was targeted to the plasma membrane. Myristoylation of the LeCPK1 N terminus was found to be required for plasma membrane targeting. PMID:12011347

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

    Directory of Open Access Journals (Sweden)

    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.

  6. Serine/threonine/tyrosine phosphorylation regulates DNA binding of bacterial transcriptional regulators

    DEFF Research Database (Denmark)

    Kalantari, Aida; Derouiche, Abderahmane; Shi, Lei

    2015-01-01

    Reversible phosphorylation of bacterial transcriptional regulators (TRs) belonging to the family of two-component systems (TCSs) is a well-established mechanism for regulating gene expression. Recent evidence points to the fact that reversible phosphorylation of bacterial TRs on other types...

  7. Convergent signaling pathways – interaction between methionine oxidation and serine/threonine/tyrosine O-phosphorylation

    Science.gov (United States)

    Oxidation of Methionine (Met) to Met sulfoxide (MetSO) is a frequently found reversible post-translational modification. It has been presumed that the major functional role for oxidation-labile Met residues is to protect proteins/cells from oxidative stress. However, Met oxidation has been establi...

  8. Anti-apoptotic effect of heat shock protein 90 on hypoxia-mediated cardiomyocyte damage is mediated via the phosphatidylinositol 3-kinase/AKT pathway.

    Science.gov (United States)

    Wang, Wei; Peng, Yizhi; Wang, Yuanyuan; Zhao, Xiaohui; Yuan, Zhiqiang

    2009-09-01

    1. Hypoxia-induced cardiomyocyte apoptosis contributes significantly to cardiac dysfunction following trauma, shock and burn injury. There is evidence that heat shock protein (HSP) 90 is anti-apoptotic in cardiomyocytes subjected to a variety of apoptotic stimuli. Because HSP90 acts as an upstream regulator of the serine/threonine protein kinase Akt survival pathway during cellular stress, we hypothesized that HSP90 exerts a cardioprotective effect via the phosphatidylinositol 3-kinase (PI3-K)/Akt pathway. 2. Neonatal rat cardiomyocytes were subjected to normoxia or hypoxia in the absence or presence of the HSP90 inhibitor geldanamycin (1 μg/mL). Cardiomyocyte apoptosis was assessed by release of lactate dehydrogenase (LDH), terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end-labelling (TUNEL) staining and caspase 3 activity. Expression of HSP90, Akt, Bad and cytochrome c release was determined by western blot analysis. 3. Following exposure of cells to hypoxia, HSP90 was markedly elevated in a time-dependent manner, reaching a peak at 6 h (eightfold increase). Geldanamycin significantly increased hypoxia-induced release of LDH by 114%, the percentage of apoptotic cardiomyocytes by 102% and caspase 3 activity by 78%. Pretreatment of cells with geldanamycin also suppressed phosphorylation of both Akt and its downstream target Bad, but promoted the mitochondrial release of cytochrome c. 4. In conclusion, HSP90 activity is enhanced in cardiomyocytes following hypoxic insult. The anti-apoptotic effect of HSP90 on cardiomyocytes subjected to hypoxia is mediated, at least in part, by the PI3-K/Akt pathway. Key words: apoptosis, cardiomyocyte, heart failure, heat shock protein 90, hypoxia, phosphatidylinositol 3-kinase/Akt signalling pathway, serine/threonine protein kinase Akt.

  9. Molecular Dynamics Simulation of Replacing of Conservate Glycine by Serine in a G-Loop for a Mutant {\\it cdc28-srm} Yeast Using a Crystal Lattice of Kinase CDK2 of the Human

    CERN Document Server

    Kholmurodov, Kh T; Koltovaya, N A; Kretov, D A

    2006-01-01

    and the structural conformations are shown to mostly differ in those regions that play a key role in the kinase function. Based on the computer simulation results the influence of the structuralconformational changes on the kinase activity and the regulation of phosphorylation are discussed.

  10. Identification of a highly reactive threonine residue at the active site of γ-glutamyl transpeptidase

    International Nuclear Information System (INIS)

    Stole, E.; Seddon, A.P.; Wellner, D.; Meister, A.

    1990-01-01

    γ-Glutamyl transpeptidase an enzyme of major importance in glutathione metabolism, was inactivated by treating it with L-(αS,5S)-α-amino-3-chloro-4,5-dihydro-5-[3- 14 C]isoxazoleacetic acid. This selective reagent binds stoichiometrically to the enzyme; more than 90% of the label was bound to its light subunit. Enzymatic digestion of the light subunit gave a 14 C-labeled peptide that corresponds to amino acid residues 517-527 of the enzyme and two incomplete digestion products that contain this labeled peptide moiety. The radioactivity associated with this peptide was released with threonine-523 during sequencing by the automated gas-phase Edman method. The light subunit contains 14 other threonine residues and a total of 19 serine residues; these were not labeled. Threonine-523 is situated in the enzyme in an environment that greatly increases its reactivity, indicating that other amino acid residues of the enzyme must also participate in the active-site chemistry of the enzyme

  11. Preparative resolution of D,L-threonine catalyzed by immobilized phosphatase.

    Science.gov (United States)

    Scollar, M P; Sigal, G; Klibanov, A M

    1985-03-01

    Hydrolysis of L- and D-O-phosphothreonines catalyzed by four different phosphatases, alkaline phosphatases from calf intestine and E. coli and acid phosphatases from wheat germ and potato, has been kinetically studied. Alkaline phosphatases were found to have comparable reactivities towards the optical isomers. On the other hand, both acid phosphatases displayed a marked stereoselectivity, hydrolyzing the L-ester much faster than its D counterpart. Wheat germ acid phosphatase was the most stereoselective enzyme: V(L)/V(D) = 24 and K(m,L)/K(m,D) = 0.17. This enzyme was immobilized (in k-carrageenan gel, followed by crosslinking with glutaraldehyde) and used for the preparative resolution of D,L-threonine: the latter was first chemically O-phosphorylated and then asymmetrically hydrolyzed by the immobilized phosphatase. As a result, gram quantities of L-threonine of high optical purity and O-phospho-D-threonine were prepared. Immobilized wheat germ phosphatase has been tested for the resolution of other racemic alcohols: serine, 2-amino-1-butanol, 1-amino-2-propanol, 2-octanol, and menthol. In all those cases, the enzyme was either not sufficiently stereoselective or too slow for preparative resolutions.

  12. Sphingoid bases and the serine catabolic enzyme CHA1 define a novel feedforward/feedback mechanism in the response to serine availability.

    Science.gov (United States)

    Montefusco, David J; Newcomb, Benjamin; Gandy, Jason L; Brice, Sarah E; Matmati, Nabil; Cowart, L Ashley; Hannun, Yusuf A

    2012-03-16

    Targets of bioactive sphingolipids in Saccharomyces cerevisiae were previously identified using microarray experiments focused on sphingolipid-dependent responses to heat stress. One of these heat-induced genes is the serine deamidase/dehydratase Cha1 known to be regulated by increased serine availability. This study investigated the hypothesis that sphingolipids may mediate the induction of Cha1 in response to serine availability. The results showed that inhibition of de novo synthesis of sphingolipids, pharmacologically or genetically, prevented the induction of Cha1 in response to increased serine availability. Additional studies implicated the sphingoid bases phytosphingosine and dihydrosphingosine as the likely mediators of Cha1 up-regulation. The yeast protein kinases Pkh1 and Pkh2, known sphingoid base effectors, were found to mediate CHA1 up-regulation via the transcription factor Cha4. Because the results disclosed a role for sphingolipids in negative feedback regulation of serine metabolism, we investigated the effects of disrupting this mechanism on sphingolipid levels and on cell growth. Intriguingly, exposure of the cha1Δ strain to high serine resulted in hyperaccumulation of endogenous serine and in turn a significant accumulation of sphingoid bases and ceramides. Under these conditions, the cha1Δ strain displayed a significant growth defect that was sphingolipid-dependent. Together, this work reveals a feedforward/feedback loop whereby the sphingoid bases serve as sensors of serine availability and mediate up-regulation of Cha1 in response to serine availability, which in turn regulates sphingolipid levels by limiting serine accumulation.

  13. Phosphorylation of mouse serine racemase regulates D-serine synthesis

    DEFF Research Database (Denmark)

    Foltyn, Veronika N; Zehl, Martin; Dikopoltsev, Elena

    2010-01-01

    Serine racemase (SR) catalyses the synthesis of the transmitter/neuromodulator D-serine, which plays a major role in synaptic plasticity and N-methyl D-aspartate receptor neurotoxicity. We now report that SR is phosphorylated at Thr71 and Thr227 as revealed by mass spectrometric analysis and in v...... with a phosphorylation-deficient SR mutant indicate that Thr71 phosphorylation increases SR activity, suggesting a novel mechanism for regulating D-serine production....

  14. Identification of Phosphorylation Consensus Sequences and Endogenous Neuronal Substrates of the Psychiatric Risk Kinase TNIK.

    Science.gov (United States)

    Wang, Qi; Amato, Stephen P; Rubitski, David M; Hayward, Matthew M; Kormos, Bethany L; Verhoest, Patrick R; Xu, Lan; Brandon, Nicholas J; Ehlers, Michael D

    2016-02-01

    Traf2- and Nck-interacting kinase (TNIK) is a serine/threonine kinase highly expressed in the brain and enriched in the postsynaptic density of glutamatergic synapses in the mammalian brain. Accumulating genetic evidence and functional data have implicated TNIK as a risk factor for psychiatric disorders. However, the endogenous substrates of TNIK in neurons are unknown. Here, we describe a novel selective small molecule inhibitor of the TNIK kinase family. Using this inhibitor, we report the identification of endogenous neuronal TNIK substrates by immunoprecipitation with a phosphomotif antibody followed by mass spectrometry. Phosphorylation consensus sequences were defined by phosphopeptide sequence analysis. Among the identified substrates were members of the delta-catenin family including p120-catenin, δ-catenin, and armadillo repeat gene deleted in velo-cardio-facial syndrome (ARVCF), each of which is linked to psychiatric or neurologic disorders. Using p120-catenin as a representative substrate, we show TNIK-induced p120-catenin phosphorylation in cells requires intact kinase activity and phosphorylation of TNIK at T181 and T187 in the activation loop. Addition of the small molecule TNIK inhibitor or knocking down TNIK by two shRNAs reduced endogenous p120-catenin phosphorylation in cells. Together, using a TNIK inhibitor and phosphomotif antibody, we identify endogenous substrates of TNIK in neurons, define consensus sequences for TNIK, and suggest signaling pathways by which TNIK influences synaptic development and function linked to psychiatric and neurologic disorders. Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.

  15. Insulin treatment promotes tyrosine phosphorylation of PKR and inhibits polyIC induced PKR threonine phosphorylation.

    Science.gov (United States)

    Swetha, Medchalmi; Ramaiah, Kolluru V A

    2015-11-01

    Tyrosine phosphorylation of insulin receptor beta (IRβ) in insulin treated HepG2 cells is inversely correlated to ser(51) phosphorylation in the alpha-subunit of eukaryotic initiation factor 2 (eIF2α) that regulates protein synthesis. Insulin stimulates interaction between IRβ and PKR, double stranded RNA-dependent protein kinase, also known as EIF2AK2, and phosphorylation of tyrosine residues in PKR, as analyzed by immunoprecipitation and pull down assays using anti-IRβ and anti-phosphotyrosine antibodies, recombinant IRβ and immunopurified PKR. Further polyIC or synthetic double stranded RNA-induced threonine phosphorylation or activation of immunopurified and cellular PKR is suppressed in the presence of insulin treated purified IRβ and cell extracts. Acute, but not chronic, insulin treatment enhances tyrosine phosphorylation of IRβ, its interaction with PKR and tyrosine phosphorylation of PKR. In contrast, lipopolysaccharide that stimulates threonine phosphorylation of PKR and eIF2α phosphorylation and AG 1024, an inhibitor of the tyrosine kinase activity of IRβ, reduces PKR association with the receptor, IRβ in HepG2 cells. These findings therefore may suggest that tyrosine phosphorylated PKR plays a role in the regulation of insulin induced protein synthesis and in maintaining insulin sensitivity, whereas, suppression of polyIC-mediated threonine phosphorylation of PKR by insulin compromises its ability to fight against virus infection in host cells. Copyright © 2015 Elsevier Inc. All rights reserved.

  16. Protein kinase C activation and myosin light chain phosphorylation in 32P-labeled arterial smooth muscle

    International Nuclear Information System (INIS)

    Singer, H.A.

    1990-01-01

    Experiments using 32P-labeled strips of swine carotid artery medial smooth muscle were performed to define the relative contribution of myosin light chain (MLC) phosphorylation as an activation mechanism mediating contractile responses stimulated by phorbol dibutyrate (PDB). Tryptic phosphopeptide mapping of phosphorylated MLC indicated that near-maximal force responses were associated with increases in functional MLC phosphorylation of less than 10% of the total MLC content following tonic (45 min) stimulation by PDB. Significant phosphorylation of MLC residues, consistent with the specificity of protein kinase C, occurred in response to high concentrations of PDB (greater than 0.1 microM). Histamine (10 microM)-induced MLC phosphorylation after 2 min (72.5% of total MLC) or 45 min (61.7%) was restricted to serine residues on peptides thought to contain serine19. Although agonist (histamine)-induced responses were eliminated under conditions of Ca2+ depletion, near-maximal force in response to 10 microM PDB (89.4% of a standard KCl response) was associated with monophosphorylation of less than 9% of the total MLC on peptides interpreted as containing serine19. A substantial fraction of this was localized to threonine residues. The quantitative analysis of the relation between PDB-stimulated force and the residues in MLC phosphorylated supports the concept that PDB stimulation results in activation of arterial smooth muscle cross bridges by MLC-phosphorylation-independent mechanisms

  17. Amaryllidaceae Alkaloids as Potential Glycogen Synthase Kinase-3β Inhibitors

    Directory of Open Access Journals (Sweden)

    Daniela Hulcová

    2018-03-01

    Full Text Available Glycogen synthase kinase-3β (GSK-3β is a multifunctional serine/threonine protein kinase that was originally identified as an enzyme involved in the control of glycogen metabolism. It plays a key role in diverse physiological processes including metabolism, the cell cycle, and gene expression by regulating a wide variety of well-known substances like glycogen synthase, tau-protein, and β-catenin. Recent studies have identified GSK-3β as a potential therapeutic target in Alzheimer´s disease, bipolar disorder, stroke, more than 15 types of cancer, and diabetes. GSK-3β is one of the most attractive targets for medicinal chemists in the discovery, design, and synthesis of new selective potent inhibitors. In the current study, twenty-eight Amaryllidaceae alkaloids of various structural types were studied for their potency to inhibit GSK-3β. Promising results have been demonstrated by alkaloids of the homolycorine-{9-O-demethylhomolycorine (IC50 = 30.00 ± 0.71 µM, masonine (IC50 = 27.81 ± 0.01 μM}, and lycorine-types {caranine (IC50 = 30.75 ± 0.04 μM}.

  18. Protein Kinase C-ε Promotes EMT in Breast Cancer

    Science.gov (United States)

    Jain, Kirti; Basu, Alakananda

    2014-01-01

    Protein kinase C (PKC), a family of serine/threonine kinases, plays critical roles in signal transduction and cell regulation. PKCε, a member of the novel PKC family, is known to be a transforming oncogene and a tumor biomarker for aggressive breast cancers. In this study, we examined the involvement of PKCε in epithelial to mesenchymal transition (EMT), the process that leads the way to metastasis. Overexpression of PKCε was sufficient to induce a mesenchymal phenotype in non-tumorigenic mammary epithelial MCF-10 A cells. This was accompanied by a decrease in the epithelial markers, such as E-cadherin, zonula occludens (ZO)-1, and claudin-1, and an increase in mesenchymal marker vimentin. Transforming growth factor β (TGFβ) induced Snail expression and mesenchymal morphology in MCF-10 A cells, and these effects were partially reversed by the PKCε knockdown. PKCε also mediated cell migration and anoikis resistance, which are hallmarks of EMT. Thus, our study demonstrates that PKCε is an important mediator of EMT in breast cancer. PMID:24701121

  19. Myotonic dystrophy protein kinase (DMPK) induces actin cytoskeletal reorganization and apoptotic-like blebbing in lens cells

    Science.gov (United States)

    Jin, S.; Shimizu, M.; Balasubramanyam, A.; Epstein, H. F.

    2000-01-01

    DMPK, the product of the DM locus, is a member of the same family of serine-threonine protein kinases as the Rho-associated enzymes. In DM, membrane inclusions accumulate in lens fiber cells producing cataracts. Overexpression of DMPK in cultured lens epithelial cells led to apoptotic-like blebbing of the plasma membrane and reorganization of the actin cytoskeleton. Enzymatically active DMPK was necessary for both effects; inactive mutant DMPK protein did not produce either effect. Active RhoA but not constitutive GDP-state mutant protein produced similar effects as DMPK. The similar actions of DMPK and RhoA suggest that they may function in the same regulatory network. The observed effects of DMPK may be relevant to the removal of membrane organelles during normal lens differentiation and the retention of intracellular membranes in DM lenses. Copyright 2000 Wiley-Liss, Inc.

  20. AMP-Activated Protein Kinase Directly Phosphorylates and Destabilizes Hedgehog Pathway Transcription Factor GLI1 in Medulloblastoma

    Directory of Open Access Journals (Sweden)

    Yen-Hsing Li

    2015-07-01

    Full Text Available The Hedgehog (Hh pathway regulates cell differentiation and proliferation during development by controlling the Gli transcription factors. Cell fate decisions and progression toward organ and tissue maturity must be coordinated, and how an energy sensor regulates the Hh pathway is not clear. AMP-activated protein kinase (AMPK is an important sensor of energy stores and controls protein synthesis and other energy-intensive processes. AMPK is directly responsive to intracellular AMP levels, inhibiting a wide range of cell activities if ATP is low and AMP is high. Thus, AMPK can affect development by influencing protein synthesis and other processes needed for growth and differentiation. Activation of AMPK reduces GLI1 protein levels and stability, thus blocking Sonic-hedgehog-induced transcriptional activity. AMPK phosphorylates GLI1 at serines 102 and 408 and threonine 1074. Mutation of these three sites into alanine prevents phosphorylation by AMPK. This leads to increased GLI1 protein stability, transcriptional activity, and oncogenic potency.

  1. Glycogen synthase kinase-3 (GSK3) regulates TNF production and haemocyte phagocytosis in the immune response of Chinese mitten crab Eriocheir sinensis.

    Science.gov (United States)

    Li, Xiaowei; Jia, Zhihao; Wang, Weilin; Wang, Lingling; Liu, Zhaoqun; Yang, Bin; Jia, Yunke; Song, Xiaorui; Yi, Qilin; Qiu, Limei; Song, Linsheng

    2017-08-01

    Glycogen synthase kinase-3 (GSK3) is a serine/threonine protein kinase firstly identified as a regulator of glycogen synthesis. Recently, it has been proved to be a key regulator of the immune reaction. In the present study, a GSK3 homolog gene (designated as EsGSK3) was cloned from Chinese mitten crab, Eriocheir sinensis. The open reading frame (ORF) was 1824 bp, which encoded a predicted polypeptide of 607 amino acids. There was a conserved Serine/Threonine Kinase domain and a DNA binding domain found in EsGSK3. Phylogenetic analysis showed that EsGSK3 was firstly clustered with GSK3-β from oriental river prawn Macrobrachium nipponense in the invertebrate branch, while GSK3s from vertebrates formed the other distinct branch. EsGSK3 mRNA transcripts could be detected in all tested tissues of the crab including haepatopancreas, eyestalk, muscle, gonad, haemocytes and haematopoietic tissue with the highest expression level in haepatopancreas. And EsGSK3 protein was mostly detected in the cytoplasm of haemocyte by immunofluorescence analysis. The expression levels of EsGSK3 mRNA increased significantly at 6 h after Aeromonas hydrophila challenge (p level at 48 h (p > 0.05). The mRNA expression of lipopolysaccharide-induced tumor necrosis factor (TNF)-α factor (EsLITAF) was also induced by A. hydrophila challenge. However, the mRNA expression of EsLITAF and TNF-α production was significantly suppressed after EsGSK3 was blocked in vivo with specific inhibitor lithium, while the phagocytosis of crab haemocytes was significantly promoted. These results collectively demonstrated that EsGSK3 could regulate the innate immune responses of E. sinensis by promoting TNF-α production and inhibiting haemocyte phagocytosis. Copyright © 2017 Elsevier Ltd. All rights reserved.

  2. Phosphorylation of Krüppel-like factor 3 (KLF3/BKLF) and C-terminal binding protein 2 (CtBP2) by homeodomain-interacting protein kinase 2 (HIPK2) modulates KLF3 DNA binding and activity.

    Science.gov (United States)

    Dewi, Vitri; Kwok, Alister; Lee, Stella; Lee, Ming Min; Tan, Yee Mun; Nicholas, Hannah R; Isono, Kyo-ichi; Wienert, Beeke; Mak, Ka Sin; Knights, Alexander J; Quinlan, Kate G R; Cordwell, Stuart J; Funnell, Alister P W; Pearson, Richard C M; Crossley, Merlin

    2015-03-27

    Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3. © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  3. Sibiriline, a new small chemical inhibitor of receptor-interacting protein kinase 1, prevents immune-dependent hepatitis.

    Science.gov (United States)

    Le Cann, Fabienne; Delehouzé, Claire; Leverrier-Penna, Sabrina; Filliol, Aveline; Comte, Arnaud; Delalande, Olivier; Desban, Nathalie; Baratte, Blandine; Gallais, Isabelle; Piquet-Pellorce, Claire; Faurez, Florence; Bonnet, Marion; Mettey, Yvette; Goekjian, Peter; Samson, Michel; Vandenabeele, Peter; Bach, Stéphane; Dimanche-Boitrel, Marie-Thérèse

    2017-09-01

    Necroptosis is a regulated form of cell death involved in several disease models including in particular liver diseases. Receptor-interacting protein kinases, RIPK1 and RIPK3, are the main serine/threonine kinases driving this cell death pathway. We screened a noncommercial, kinase-focused chemical library which allowed us to identify Sibiriline as a new inhibitor of necroptosis induced by tumor necrosis factor (TNF) in Fas-associated protein with death domain (FADD)-deficient Jurkat cells. Moreover, Sib inhibits necroptotic cell death induced by various death ligands in human or mouse cells while not protecting from caspase-dependent apoptosis. By using competition binding assay and recombinant kinase assays, we demonstrated that Sib is a rather specific competitive RIPK1 inhibitor. Molecular docking analysis shows that Sib is trapped closed to human RIPK1 adenosine triphosphate-binding site in a relatively hydrophobic pocket locking RIPK1 in an inactive conformation. In agreement with its RIPK1 inhibitory property, Sib inhibits both TNF-induced RIPK1-dependent necroptosis and RIPK1-dependent apoptosis. Finally, Sib protects mice from concanavalin A-induced hepatitis. These results reveal the small-molecule Sib as a new RIPK1 inhibitor potentially of interest for the treatment of immune-dependent hepatitis. © 2017 Federation of European Biochemical Societies.

  4. A chemical-genetic strategy reveals distinct temporal requirements for SAD-1 kinase in neuronal polarization and synapse formation

    Directory of Open Access Journals (Sweden)

    Shokat Kevan M

    2008-09-01

    Full Text Available Abstract Background Neurons assemble into a functional network through a sequence of developmental processes including neuronal polarization and synapse formation. In Caenorhabditis elegans, the serine/threonine SAD-1 kinase is essential for proper neuronal polarity and synaptic organization. To determine if SAD-1 activity regulates the establishment or maintenance of these neuronal structures, we examined its temporal requirements using a chemical-genetic method that allows for selective and reversible inactivation of its kinase activity in vivo. Results We generated a PP1 analog-sensitive variant of SAD-1. Through temporal inhibition of SAD-1 kinase activity we show that its activity is required for the establishment of both neuronal polarity and synaptic organization. However, while SAD-1 activity is needed strictly when neurons are polarizing, the temporal requirement for SAD-1 is less stringent in synaptic organization, which can also be re-established during maintenance. Conclusion This study reports the first temporal analysis of a neural kinase activity using the chemical-genetic system. It reveals that neuronal polarity and synaptic organization have distinct temporal requirements for SAD-1.

  5. Expression and purification of functional JNK2beta2: perspectives on high-level production of recombinant MAP kinases.

    Science.gov (United States)

    Savopoulos, John W; Dowd, Stephen; Armour, Carolyn; Carter, Paul S; Greenwood, Catherine J; Mills, David; Powell, David; Pettman, Gary R; Jenkins, Owen; Walsh, Frank S; Philpott, Karen L

    2002-02-01

    The mitogen-activated protein (MAP) kinases are a group of serine/threonine kinases that mediate intracellular signal transduction in response to environmental stimuli including stress, growth factors, and various cytokines. Of this family, the c-Jun N-terminal kinases (JNKs) are members which, depending on cell type, have been shown to activate the transcription of genes involved in the inflammatory response, apoptosis, and hypertrophy. Here we report the use Baculovirus/Sf9 cells to produce milligram quantities of recombinant JNK2beta2 substrate which could be purified to >90% as judged by SDS-PAGE. In addition, we report a novel method for the site-specific biotinylation for this enzyme and demonstrate that the biotinylated product is an authentic substrate of the upstream kinases MKK4 and 7 and can phosphorylate a downstream target, ATF-2. We also show that the phosphorylated product can be captured efficiently on streptavidin-coated beads for use in scintillation proximity assays. Copyright 2002 Elsevier Science (USA).

  6. Identification of a novel receptor-like protein kinase that interacts with a geminivirus nuclear shuttle protein

    International Nuclear Information System (INIS)

    Mariano, Andrea C.; Andrade, Maxuel O.; Santos, Anesia A.; Carolino, Sonia M.B.; Oliveira, Marli L.; Baracat-Pereira, Maria Cristina; Brommonshenkel, Sergio H.; Fontes, Elizabeth P.B.

    2004-01-01

    Despite extensive studies in plant virus-host interactions, the molecular mechanisms of geminivirus movement and interactions with host components remain largely unknown. A tomato kinase protein and its soybean homolog were found to interact specifically with the nuclear shuttle protein (NSP) of Tomato golden mosaic virus (TGMV) and Tomato crinkle leaf yellows virus (TCrLYV) through yeast two-hybrid screening and in vitro protein binding assays. These proteins, designated LeNIK (Lycopersicon esculentum NSP-Interacting Kinase) and GmNIK (Glycine max NIK), belong to the LRR-RLK (leucine rich-repeat receptor-like kinase) family that is involved in plant developmental processes and/or resistance response. As such, NIK is structurally organized into characteristic domains, including a serine/threonine kinase domain with a nucleotide binding site at the C-terminal region, an internal transmembrane segment and leucine-rich repeats (LRR) at the N-terminal portion. The potential significance of the NSP-NIK interaction is discussed

  7. An integrated chemical biology approach identifies specific vulnerability of Ewing's sarcoma to combined inhibition of Aurora kinases A and B.

    Science.gov (United States)

    Winter, Georg E; Rix, Uwe; Lissat, Andrej; Stukalov, Alexey; Müllner, Markus K; Bennett, Keiryn L; Colinge, Jacques; Nijman, Sebastian M; Kubicek, Stefan; Kovar, Heinrich; Kontny, Udo; Superti-Furga, Giulio

    2011-10-01

    Ewing's sarcoma is a pediatric cancer of the bone that is characterized by the expression of the chimeric transcription factor EWS-FLI1 that confers a highly malignant phenotype and results from the chromosomal translocation t(11;22)(q24;q12). Poor overall survival and pronounced long-term side effects associated with traditional chemotherapy necessitate the development of novel, targeted, therapeutic strategies. We therefore conducted a focused viability screen with 200 small molecule kinase inhibitors in 2 different Ewing's sarcoma cell lines. This resulted in the identification of several potential molecular intervention points. Most notably, tozasertib (VX-680, MK-0457) displayed unique nanomolar efficacy, which extended to other cell lines, but was specific for Ewing's sarcoma. Furthermore, tozasertib showed strong synergies with the chemotherapeutic drugs etoposide and doxorubicin, the current standard agents for Ewing's sarcoma. To identify the relevant targets underlying the specific vulnerability toward tozasertib, we determined its cellular target profile by chemical proteomics. We identified 20 known and unknown serine/threonine and tyrosine protein kinase targets. Additional target deconvolution and functional validation by RNAi showed simultaneous inhibition of Aurora kinases A and B to be responsible for the observed tozasertib sensitivity, thereby revealing a new mechanism for targeting Ewing's sarcoma. We further corroborated our cellular observations with xenograft mouse models. In summary, the multilayered chemical biology approach presented here identified a specific vulnerability of Ewing's sarcoma to concomitant inhibition of Aurora kinases A and B by tozasertib and danusertib, which has the potential to become a new therapeutic option.

  8. Characterization and enzymatic properties of protein kinase ACR4 from Arabidopsis thaliana.

    Science.gov (United States)

    Zhao, Yu; Liu, Xuehe; Xu, Ziyan; Yang, Hui; Li, Jixi

    2017-07-22

    Serine/threonine-protein kinase-like protein ARABIDOPSIS CRINKLY4 (ACR4), a transmembrane protein of Arabidopsis thaliana, plays important roles in cell division and differentiation. Although accumulating studies shed light on the function of ACR4, the structure and catalytic mechanism of ACR4 remain to be elucidated. Here, we report the purification and enzymatic properties of the intracellular kinase domain (residues 464-799) of ACR4 (ACR4 IKD ). Through Ni-affinity chromatography and gel filter chromatography methods, we successfully obtain high-purity ACR4 IKD protein from Escherichia coli. Dynamic light scattering and gel-filtration methods reveal that ACR4 IKD distributes with high homogeneity and exists as a monomer in solution. In addition, the ACR4 IKD protein has typical kinase activity with myelin basic protein (MBP) as the substrate. Our study may lay the foundation for structure determination of ACR4 IKD and further functional research, for example, screening significant substrates of ACR4 in Arabidopsis thaliana. Copyright © 2017 Elsevier Inc. All rights reserved.

  9. Genomic organization, expression, and chromosome localization of a third aurora-related kinase gene, Aie1.

    Science.gov (United States)

    Hu, H M; Chuang, C K; Lee, M J; Tseng, T C; Tang, T K

    2000-11-01

    We previously reported two novel testis-specific serine/threonine kinases, Aie1 (mouse) and AIE2 (human), that share high amino acid identities with the kinase domains of fly aurora and yeast Ipl1. Here, we report the entire intron-exon organization of the Aie1 gene and analyze the expression patterns of Aie1 mRNA during testis development. The mouse Aie1 gene spans approximately 14 kb and contains seven exons. The sequences of the exon-intron boundaries of the Aie1 gene conform to the consensus sequences (GT/AG) of the splicing donor and acceptor sites of most eukaryotic genes. Comparative genomic sequencing revealed that the gene structure is highly conserved between mouse Aie1 and human AIE2. However, much less homology was found in the sequence outside the kinase-coding domains. The Aie1 locus was mapped to mouse chromosome 7A2-A3 by fluorescent in situ hybridization. Northern blot analysis indicates that Aie1 mRNA likely is expressed at a low level on day 14 and reaches its plateau on day 21 in the developing postnatal testis. RNA in situ hybridization indicated that the expression of the Aie1 transcript was restricted to meiotically active germ cells, with the highest levels detected in spermatocytes at the late pachytene stage. These findings suggest that Aie1 plays a role in spermatogenesis.

  10. Xanthene derivatives increase glucose utilization through activation of LKB1-dependent AMP-activated protein kinase.

    Directory of Open Access Journals (Sweden)

    Yonghoon Kwon

    Full Text Available 5' AMP-activated protein kinase (AMPK is a highly conserved serine-threonine kinase that regulates energy expenditure by activating catabolic metabolism and suppressing anabolic pathways to increase cellular energy levels. Therefore AMPK activators are considered to be drug targets for treatment of metabolic diseases such as diabetes mellitus. To identify novel AMPK activators, we screened xanthene derivatives. We determined that the AMPK activators 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-nitro-phenyl-thioureido]-ethyl}-amide (Xn and 9H-xanthene-9-carboxylic acid {2,2,2-trichloro-1-[3-(3-cyano-phenyl-thioureido]-ethyl}-amide (Xc elevated glucose uptake in L6 myotubes by stimulating translocation of glucose transporter type 4 (GLUT4. Treatment with the chemical AMPK inhibitor compound C and infection with dominant-negative AMPKa2-virus inhibited AMPK phosphorylation and glucose uptake in myotubes induced by either Xn or Xc. Of the two major upstream kinases of AMPK, we found that Xn and Xc showed LKB1 dependency by knockdown of STK11, an ortholog of human LKB1. Single intravenous administration of Xn and Xc to high-fat diet-induced diabetic mice stimulated AMPK phosphorylation of skeletal muscle and improved glucose tolerance. Taken together, these results suggest that Xn and Xc regulate glucose homeostasis through LKB1-dependent AMPK activation and that the compounds are potential candidate drugs for the treatment of type 2 diabetes mellitus.

  11. STK33 kinase activity is nonessential in KRAS-dependent cancer cells.

    Science.gov (United States)

    Babij, Carol; Zhang, Yihong; Kurzeja, Robert J; Munzli, Anke; Shehabeldin, Amro; Fernando, Manory; Quon, Kim; Kassner, Paul D; Ruefli-Brasse, Astrid A; Watson, Vivienne J; Fajardo, Flordeliza; Jackson, Angela; Zondlo, James; Sun, Yu; Ellison, Aaron R; Plewa, Cherylene A; San, Miguel Tisha; Robinson, John; McCarter, John; Schwandner, Ralf; Judd, Ted; Carnahan, Josette; Dussault, Isabelle

    2011-09-01

    Despite the prevalence of KRAS mutations in human cancers, there remain no targeted therapies for treatment. The serine-threonine kinase STK33 has been proposed to be required for the survival of mutant KRAS-dependent cell lines, suggesting that small molecule kinase inhibitors of STK33 may be useful to treat KRAS-dependent tumors. In this study, we investigated the role of STK33 in mutant KRAS human cancer cells using RNA interference, dominant mutant overexpression, and small molecule inhibitors. As expected, KRAS downregulation decreased the survival of KRAS-dependent cells. In contrast, STK33 downregulation or dominant mutant overexpression had no effect on KRAS signaling or survival of these cells. Similarly, a synthetic lethal siRNA screen conducted in a broad panel of KRAS wild-type or mutant cells identified KRAS but not STK33 as essential for survival. We also obtained similar negative results using small molecule inhibitors of the STK33 kinase identified by high-throughput screening. Taken together, our findings refute earlier proposals that STK33 inhibition may be a useful therapeutic approach to target human KRAS mutant tumors. ©2011 AACR.

  12. Pim kinase inhibition sensitizes FLT3-ITD acute myeloid leukemia cells to topoisomerase 2 inhibitors through increased DNA damage and oxidative stress

    Science.gov (United States)

    Doshi, Kshama A.; Trotta, Rossana; Natarajan, Karthika; Rassool, Feyruz V.; Tron, Adriana E.; Huszar, Dennis; Perrotti, Danilo; Baer, Maria R.

    2016-01-01

    Internal tandem duplication of fms-like tyrosine kinase-3 (FLT3-ITD) is frequent (30 percent) in acute myeloid leukemia (AML), and is associated with short disease-free survival following chemotherapy. The serine threonine kinase Pim-1 is a pro-survival oncogene transcriptionally upregulated by FLT3-ITD that also promotes its signaling in a positive feedback loop. Thus inhibiting Pim-1 represents an attractive approach in targeting FLT3-ITD cells. Indeed, co-treatment with the pan-Pim kinase inhibitor AZD1208 or expression of a kinase-dead Pim-1 mutant sensitized FLT3-ITD cell lines to apoptosis triggered by chemotherapy drugs including the topoisomerase 2 inhibitors daunorubicin, etoposide and mitoxantrone, but not the nucleoside analog cytarabine. AZD1208 sensitized primary AML cells with FLT3-ITD to topoisomerase 2 inhibitors, but did not sensitize AML cells with wild-type FLT3 or remission bone marrow cells, supporting a favorable therapeutic index. Mechanistically, the enhanced apoptosis observed with AZD1208 and topoisomerase 2 inhibitor combination treatment was associated with increased DNA double-strand breaks and increased levels of reactive oxygen species (ROS), and co-treatment with the ROS scavenger N-acetyl cysteine rescued FLT3-ITD cells from AZD1208 sensitization to topoisomerase 2 inhibitors. Our data support testing of Pim kinase inhibitors with topoisomerase 2 inhibitors, but not with cytarabine, to improve treatment outcomes in AML with FLT3-ITD. PMID:27374090

  13. Threonine and lysine requirements for maintenance in chickens ...

    African Journals Online (AJOL)

    The maintenance requirement for threonine and lysine were estimated in two different experiments by measuring the nitrogen balance of adult male cockerels. Measured amounts of a diet first-limiting in threonine or lysine were fed by intubation each day for 4 d to give a range of intakes (unbalanced series) of from 0 to 239 ...

  14. Identification of the kinase that activates a nonmetazoan STAT gives insights into the evolution of phosphotyrosine-SH2 domain signaling.

    Science.gov (United States)

    Araki, Tsuyoshi; Kawata, Takefumi; Williams, Jeffrey G

    2012-07-10

    SH2 domains are integral to many animal signaling pathways. By interacting with specific phosphotyrosine residues, they provide regulatable protein-protein interaction domains. Dictyostelium is the only nonmetazoan with functionally characterized SH2 domains, but the cognate tyrosine kinases are unknown. There are no orthologs of the animal tyrosine kinases, but there are very many tyrosine kinase-like kinases (TKLs), a group of kinases which, despite their family name, are classified mainly as serine-threonine kinases. STATs are transcription factors that dimerize via phosphotyrosine-SH2 domain interactions. STATc is activated by phosphorylation on Tyr922 when cells are exposed to the prestalk inducer differentiation inducing factor (DIF-1), a chlorinated hexaphenone. We show that in a null mutant for Pyk2, a tyrosine-specific TKL, exposure to DIF-1 does not activate STATc. Conversely, overexpression of Pyk2 causes constitutive STATc activation. Pyk2 phosphorylates STATc on Tyr922 in vitro and complexes with STATc both in vitro and in vivo. This demonstration that a TKL directly activates a STAT has significant implications for understanding the evolutionary origins of SH2 domain-phosphotyrosine signaling. It also has mechanistic implications. Our previous work suggested that a predicted constitutive STATc tyrosine kinase activity is counterbalanced in vivo by the DIF-1-regulated activity of PTP3, a Tyr922 phosphatase. Here we show that the STATc-Pyk2 complex is formed constitutively by an interaction between the STATc SH2 domain and phosphotyrosine residues on Pyk2 that are generated by autophosphorylation. Also, as predicted, Pyk2 is constitutively active as a STATc kinase. This observation provides further evidence for this highly atypical, possibly ancestral, STAT regulation mechanism.

  15. Development of Pharmacophore Model for Indeno[1,2-b]indoles as Human Protein Kinase CK2 Inhibitors and Database Mining

    Directory of Open Access Journals (Sweden)

    Samer Haidar

    2017-01-01

    Full Text Available Protein kinase CK2, initially designated as casein kinase 2, is an ubiquitously expressed serine/threonine kinase. This enzyme, implicated in many cellular processes, is highly expressed and active in many tumor cells. A large number of compounds has been developed as inhibitors comprising different backbones. Beside others, structures with an indeno[1,2-b]indole scaffold turned out to be potent new leads. With the aim of developing new inhibitors of human protein kinase CK2, we report here on the generation of common feature pharmacophore model to further explain the binding requirements for human CK2 inhibitors. Nine common chemical features of indeno[1,2-b]indole-type CK2 inhibitors were determined using MOE software (Chemical Computing Group, Montreal, Canada. This pharmacophore model was used for database mining with the aim to identify novel scaffolds for developing new potent and selective CK2 inhibitors. Using this strategy several structures were selected by searching inside the ZINC compound database. One of the selected compounds was bikaverin (6,11-dihydroxy-3,8-dimethoxy-1-methylbenzo[b]xanthene-7,10,12-trione, a natural compound which is produced by several kinds of fungi. This compound was tested on human recombinant CK2 and turned out to be an active inhibitor with an IC50 value of 1.24 µM.

  16. DNA-dependent protein kinase (DAN-PK), a key enzyme in the re-ligation of DNA double-strand breaks

    International Nuclear Information System (INIS)

    Hennequin, C.; Averbeck, D.

    1999-01-01

    Repair pathways of DNA are now defined and some important findings have been discovered in the last few years. DNA non-homologous end-joining (NEH) is a crucial process in the repair of radiation-induced double-strand breaks (DSBs). NHEj implies at least three steps: the DNA free-ends must get closer, preparation of the free-ends by exonucleases and then a transient hybridization in a region of DNA with weak homology. DNA-dependent protein kinase (DNA-PK) is the key enzyme in this process. DNA-PK is a nuclear serine/threonine kinase that comprises three components: a catalytic subunit (DNA-PK cs ) and two regulatory subunits, DNA-binding proteins, Ku80 and Ku70. The severe combined immuno-deficient (scid) mice are deficient in DNA-PK cs : this protein is involved both in DNA repair and in the V(D)J recombination of immunoglobulin and T-cell receptor genes. It is a protein-kinase of the P13-kinase family and which can phosphorylate Ku proteins, p53 and probably some other proteins still unknown. DNA-PK is an important actor of DSBs repair (induced by ionising radiations or by drugs like etoposide), but obviously it is not the only mechanism existing in the cell for this function. Some others, like homologous recombination, seem also to have a great importance for cell survival. (authors)

  17. The cytotoxic effects of regorafenib in combination with protein kinase D inhibition in human colorectal cancer cells

    Science.gov (United States)

    Wei, Ning; Chu, Edward; Wu, Shao-yu; Wipf, Peter; Schmitz, John C.

    2015-01-01

    Metastatic colorectal cancer (mCRC) remains a major public health problem, and diagnosis of metastatic disease is usually associated with poor prognosis. The multi-kinase inhibitor regorafenib was approved in 2013 in the U.S. for the treatment of mCRC patients who progressed after standard therapies. However, the clinical efficacy of regorafenib is quite limited. One potential strategy to improve mCRC therapy is to combine agents that target key cellular signaling pathways, which may lead to synergistic enhancement of antitumor efficacy and overcome cellular drug resistance. Protein kinase D (PKD), a family of serine/threonine kinases, mediates key signaling pathways implicated in multiple cellular processes. Herein, we evaluated the combination of regorafenib with a PKD inhibitor in several human CRC cells. Using the Chou-Talalay model, the combination index values for this combination treatment demonstrated synergistic effects on inhibition of cell proliferation and clonal formation. This drug combination resulted in induction of apoptosis as determined by flow cytometry, increased PARP cleavage, and decreased activation of the anti-apoptotic protein HSP27. This combination also yielded enhanced inhibition of ERK, AKT, and NF-κB signaling. Taken together, PKD inhibition in combination with regorafenib appears to be a promising strategy for the treatment of mCRC. PMID:25544765

  18. Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

    Science.gov (United States)

    Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

    2005-01-01

    The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

  19. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Directory of Open Access Journals (Sweden)

    Neil Arvin Bretaña

    Full Text Available Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase

  20. Identifying protein phosphorylation sites with kinase substrate specificity on human viruses.

    Science.gov (United States)

    Bretaña, Neil Arvin; Lu, Cheng-Tsung; Chiang, Chiu-Yun; Su, Min-Gang; Huang, Kai-Yao; Lee, Tzong-Yi; Weng, Shun-Long

    2012-01-01

    Viruses infect humans and progress inside the body leading to various diseases and complications. The phosphorylation of viral proteins catalyzed by host kinases plays crucial regulatory roles in enhancing replication and inhibition of normal host-cell functions. Due to its biological importance, there is a desire to identify the protein phosphorylation sites on human viruses. However, the use of mass spectrometry-based experiments is proven to be expensive and labor-intensive. Furthermore, previous studies which have identified phosphorylation sites in human viruses do not include the investigation of the responsible kinases. Thus, we are motivated to propose a new method to identify protein phosphorylation sites with its kinase substrate specificity on human viruses. The experimentally verified phosphorylation data were extracted from virPTM--a database containing 301 experimentally verified phosphorylation data on 104 human kinase-phosphorylated virus proteins. In an attempt to investigate kinase substrate specificities in viral protein phosphorylation sites, maximal dependence decomposition (MDD) is employed to cluster a large set of phosphorylation data into subgroups containing significantly conserved motifs. The experimental human phosphorylation sites are collected from Phospho.ELM, grouped according to its kinase annotation, and compared with the virus MDD clusters. This investigation identifies human kinases such as CK2, PKB, CDK, and MAPK as potential kinases for catalyzing virus protein substrates as confirmed by published literature. Profile hidden Markov model is then applied to learn a predictive model for each subgroup. A five-fold cross validation evaluation on the MDD-clustered HMMs yields an average accuracy of 84.93% for Serine, and 78.05% for Threonine. Furthermore, an independent testing data collected from UniProtKB and Phospho.ELM is used to make a comparison of predictive performance on three popular kinase-specific phosphorylation site

  1. Crystal structure and characterization of a novel L-serine ammonia-lyase from Rhizomucor miehei

    Energy Technology Data Exchange (ETDEWEB)

    Qin, Zhen [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China); Yan, Qiaojuan [College of Engineering, China Agricultural University, Beijing 100083 (China); Ma, Qingjun [Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071 (China); Jiang, Zhengqiang, E-mail: zhqjiang@cau.edu.cn [College of Food Science and Nutritional Engineering, Beijing Advanced Innovation Center of Food Nutrition and Human Health, China Agricultural University, Beijing 100083 (China)

    2015-10-23

    L-serine ammonia-lyase, as a member of the β-family of pyridoxal-5′-phosphate (PLP) dependent enzymes, catalyzes the conversion of L-serine (L-threonine) to pyruvate (α-ketobutyrate) and ammonia. The crystal structure of L-serine ammonia-lyase from Rhizomucor miehei (RmSDH) was solved at 1.76 Å resolution by X-ray diffraction method. The overall structure of RmSDH had the characteristic β-family PLP dependent enzyme fold. It consisted of two distinct domains, both of which show the typical open twisted α/β structure. A PLP cofactor was located in the crevice between the two domains, which was attached to Lys52 by a Schiff-base linkage. Unique residue substitutions (Gly78, Pro79, Ser146, Ser147 and Thr312) were discovered at the catalytic site of RmSDH by comparison of structures of RmSDH and other reported eukaryotic L-serine ammonia-lyases. Optimal pH and temperature of the purified RmSDH were 7.5 and 40 °C, respectively. It was stable in the pH range of 7.0–9.0 and at temperatures below 40 °C. This is the first crystal structure of a fungal L-serine ammonia-lyase. It will be useful to study the catalytic mechanism of β-elimination enzymes and will provide a basis for further enzyme engineering. - Highlights: • The crystal structure of a fungal L-serine ammonia-lyase (RmSDH) was solved. • Five unique residue substitutions are found at the catalytic site of RmSDH. • RmSDH was expressed in Pichia. pastoris and biochemically characterized. • RmSDH has potential application in splitting D/L-serine.

  2. Individual responses of growing pigs to threonine intake

    Directory of Open Access Journals (Sweden)

    Renan Di Giovanni Isola

    2018-02-01

    Full Text Available ABSTRACT A nitrogen balance test was performed to evaluate the individual responses of growing pigs to threonine intake. Eight commercial barrows were used (body weight ranging from 15 to 20 kg. A dose-response study was performed, in which the threonine supply increased in seven equidistant steps (the seven dietary threonine levels ranged from 50 to 120% of the requirements every three days for each pig. The levels of all other amino acids were 20% higher than the tested amino acid. Nitrogen retention as a function of threonine intake was calculated per individual and per group (NLIN and NLMixed, respectively using a linear plateau model. The highest break point value was 42.42 g of threonine intake (the most demanding individual, whereas the lowest value was 34.16 g (the least demanding individual, corresponding to a difference of 19%. In terms of N retention, the highest plateau value was 66.71 g and the lowest was 49.48 g, with a difference of 25%. There was no significant correlation between slope and plateau values or between slope and break point values. When using the model in which all parameters were random effects, the variations in threonine intake and nitrogen retention were 1.68±1.30 and 0.01±0.10 g, respectively, and no variance in the slope of the curve was detected. The average daily threonine intake values for the maximum response obtained in the group, as calculated by the NLIN and NLMixed procedures, were 13.96 and 14.02 g/day, respectively. The threonine intake for the maximum N retention between individuals ranged from 34.16 to 42.42 g, corresponding to a difference of 19%. The current recommended intake to optimize N retention is 14.02 g/day. The group responses obtained by the NLMixed procedures are very similar to those estimated by the NLIN procedure (all individuals.

  3. The Aspergillus fumigatus SchASCH9 kinase modulates SakAHOG1 MAP kinase activity and it is essential for virulence.

    Science.gov (United States)

    Alves de Castro, Patrícia; Dos Reis, Thaila Fernanda; Dolan, Stephen K; Oliveira Manfiolli, Adriana; Brown, Neil Andrew; Jones, Gary W; Doyle, Sean; Riaño-Pachón, Diego M; Squina, Fábio Márcio; Caldana, Camila; Singh, Ashutosh; Del Poeta, Maurizio; Hagiwara, Daisuke; Silva-Rocha, Rafael; Goldman, Gustavo H

    2016-11-01

    The serine-threonine kinase TOR, the Target of Rapamycin, is an important regulator of nutrient, energy and stress signaling in eukaryotes. Sch9, a Ser/Thr kinase of AGC family (the cAMP-dependent PKA, cGMP- dependent protein kinase G and phospholipid-dependent protein kinase C family), is a substrate of TOR. Here, we characterized the fungal opportunistic pathogen Aspergillus fumigatus Sch9 homologue (SchA). The schA null mutant was sensitive to rapamycin, high concentrations of calcium, hyperosmotic stress and SchA was involved in iron metabolism. The ΔschA null mutant showed increased phosphorylation of SakA, the A. fumigatus Hog1 homologue. The schA null mutant has increased and decreased trehalose and glycerol accumulation, respectively, suggesting SchA performs different roles for glycerol and trehalose accumulation during osmotic stress. The schA was transcriptionally regulated by osmotic stress and this response was dependent on SakA and MpkC. The double ΔschA ΔsakA and ΔschA ΔmpkC mutants were more sensitive to osmotic stress than the corresponding parental strains. Transcriptomics and proteomics identified direct and indirect targets of SchA post-exposure to hyperosmotic stress. Finally, ΔschA was avirulent in a low dose murine infection model. Our results suggest there is a complex network of interactions amongst the A. fumigatus TOR, SakA and SchA pathways. © 2016 John Wiley & Sons Ltd.

  4. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    Energy Technology Data Exchange (ETDEWEB)

    Simarro, Maria [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Gimenez-Cassina, Alfredo [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Kedersha, Nancy [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A. [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Rhee, Kirsten [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States); Tisdale, Sarah; Danial, Nika [Department of Cancer Biology at Dana Farber Institute, Boston, MA 02115 (United States); Benarafa, Charaf [Theodor Kocher Institute, University of Bern, 3012 Bern (Switzerland); Orduna, Anonio [Unidad de Investigacion, Hospital Clinico Universitario de Valladolid, 47005 Valladolid (Spain); Anderson, Paul, E-mail: panderson@rics.bwh.harvard.edu [Division of Rheumatology, Immunology and Allergy, Brigham and Women' s Hospital, and Department of Medicine, Harvard Medical School, Boston, MA 02115 (United States)

    2010-10-22

    Research highlights: {yields} Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. {yields} The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. {yields} Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  5. Protein kinase Cɛ inhibition restores megakaryocytic differentiation of hematopoietic progenitors from primary myelofibrosis patients.

    Science.gov (United States)

    Masselli, E; Carubbi, C; Gobbi, G; Mirandola, P; Galli, D; Martini, S; Bonomini, S; Crugnola, M; Craviotto, L; Aversa, F; Vitale, M

    2015-11-01

    Among the three classic Philadelphia chromosome-negative myeloproliferative neoplasms, primary myelofibrosis (PMF) is the most severe in terms of disease biology, survival and quality of life. Abnormalities in the process of differentiation of PMF megakaryocytes (MKs) are a hallmark of the disease. Nevertheless, the molecular events that lead to aberrant megakaryocytopoiesis have yet to be clarified. Protein kinase Cɛ (PKCɛ) is a novel serine/threonine kinase that is overexpressed in a variety of cancers, promoting aggressive phenotype, invasiveness and drug resistance. Our previous findings on the role of PKCɛ in normal (erythroid and megakaryocytic commitment) and malignant (acute myeloid leukemia) hematopoiesis prompted us to investigate whether it could be involved in the pathogenesis of PMF MK-impaired differentiation. We demonstrate that PMF megakaryocytic cultures express higher levels of PKCɛ than healthy donors, which correlate with higher disease burden but not with JAK2V617F mutation. Inhibition of PKCɛ function (by a negative regulator of PKCɛ translocation) or translation (by target small hairpin RNA) leads to reduction in PMF cell growth, restoration of PMF MK differentiation and inhibition of PKCɛ-related anti-apoptotic signaling (Bcl-xL). Our data suggest that targeting PKCɛ directly affects the PMF neoplastic clone and represent a proof-of-concept for PKCɛ inhibition as a novel therapeutic strategy in PMF.

  6. Akt1/protein kinase Bα is critical for ischemic and VEGF-mediated angiogenesis

    Science.gov (United States)

    Ackah, Eric; Yu, Jun; Zoellner, Stefan; Iwakiri, Yasuko; Skurk, Carsten; Shibata, Rei; Ouchi, Noriyuki; Easton, Rachael M.; Galasso, Gennaro; Birnbaum, Morris J.; Walsh, Kenneth; Sessa, William C.

    2005-01-01

    Akt, or protein kinase B, is a multifunctional serine-threonine protein kinase implicated in a diverse range of cellular functions including cell metabolism, survival, migration, and gene expression. However, the in vivo roles and effectors of individual Akt isoforms in signaling are not explicitly clear. Here we show that the genetic loss of Akt1, but not Akt2, in mice results in defective ischemia and VEGF-induced angiogenesis as well as severe peripheral vascular disease. Akt1 knockout (Akt1–/–) mice also have reduced endothelial progenitor cell (EPC) mobilization in response to ischemia, and reintroduction of WT EPCs, but not EPCs isolated from Akt1–/– mice, into WT mice improves limb blood flow after ischemia. Mechanistically, the loss of Akt1 reduces the basal phosphorylation of several Akt substrates, the migration of fibroblasts and ECs, and NO release. Reconstitution of Akt1–/– ECs with Akt1 rescues the defects in substrate phosphorylation, cell migration, and NO release. Thus, the Akt1 isoform exerts an essential role in blood flow control, cellular migration, and NO synthesis during postnatal angiogenesis. PMID:16075056

  7. Trans-kingdom mimicry underlies ribosome customization by a poxvirus kinase.

    Science.gov (United States)

    Jha, Sujata; Rollins, Madeline G; Fuchs, Gabriele; Procter, Dean J; Hall, Elizabeth A; Cozzolino, Kira; Sarnow, Peter; Savas, Jeffrey N; Walsh, Derek

    2017-06-29

    Ribosomes have the capacity to selectively control translation through changes in their composition that enable recognition of specific RNA elements. However, beyond differential subunit expression during development, evidence for regulated ribosome specification within individual cells has remained elusive. Here we report that a poxvirus kinase phosphorylates serine/threonine residues in the human small ribosomal subunit protein, receptor for activated C kinase (RACK1), that are not phosphorylated in uninfected cells or cells infected by other viruses. These modified residues cluster in an extended loop in RACK1, phosphorylation of which selects for translation of viral or reporter mRNAs with 5' untranslated regions that contain adenosine repeats, so-called polyA-leaders. Structural and phylogenetic analyses revealed that although RACK1 is highly conserved, this loop is variable and contains negatively charged amino acids in plants, in which these leaders act as translational enhancers. Phosphomimetics and inter-species chimaeras have shown that negative charge in the RACK1 loop dictates ribosome selectivity towards viral RNAs. By converting human RACK1 to a charged, plant-like state, poxviruses remodel host ribosomes so that adenosine repeats erroneously generated by slippage of the viral RNA polymerase confer a translational advantage. Our findings provide insight into ribosome customization through trans-kingdom mimicry and the mechanics of species-specific leader activity that underlie poxvirus polyA-leaders.

  8. The role of DNA dependent protein kinase in synapsis of DNA ends.

    Science.gov (United States)

    Weterings, Eric; Verkaik, Nicole S; Brüggenwirth, Hennie T; Hoeijmakers, Jan H J; van Gent, Dik C

    2003-12-15

    DNA dependent protein kinase (DNA-PK) plays a central role in the non-homologous end-joining pathway of DNA double strand break repair. Its catalytic subunit (DNA-PK(CS)) functions as a serine/threonine protein kinase. We show that DNA-PK forms a stable complex at DNA termini that blocks the action of exonucleases and ligases. The DNA termini become accessible after autophosphorylation of DNA-PK(CS), which we demonstrate to require synapsis of DNA ends. Interestingly, the presence of DNA-PK prevents ligation of the two synapsed termini, but allows ligation to another DNA molecule. This alteration of the ligation route is independent of the type of ligase that we used, indicating that the intrinsic architecture of the DNA-PK complex itself is not able to support ligation of the synapsed DNA termini. We present a working model in which DNA-PK creates a stable molecular bridge between two DNA ends that is remodeled after DNA-PK autophosphorylation in such a way that the extreme termini become accessible without disrupting synapsis. We infer that joining of synapsed DNA termini would require an additional protein factor.

  9. Aberrant glycogen synthase kinase 3β in the development of pancreatic cancer

    Directory of Open Access Journals (Sweden)

    Takeo Shimasaki

    2012-01-01

    Full Text Available Development and progression of pancreatic cancer involves general metabolic disorder, local chronic inflammation, and multistep activation of distinct oncogenic molecular pathways. These pathologic processes result in a highly invasive and metastatic tumor phenotype that is a major obstacle to curative surgical intervention, infusional gemcitabine-based chemotherapy, and radiation therapy. Many clinical trials with chemical compounds and therapeutic antibodies targeting growth factors, angiogenic factors, and matrix metalloproteinases have failed to demonstrate definitive therapeutic benefits to refractory pancreatic cancer patients. Glycogen synthase kinase 3β (GSK3β, a serine/threonine protein kinase, has emerged as a therapeutic target in common chronic and progressive diseases, including cancer. Here we review accumulating evidence for a pathologic role of GSK3β in promoting tumor cell survival, proliferation, invasion, and resistance to chemotherapy and radiation in pancreatic cancer. We also discuss the putative involvement of GSK3β in mediating metabolic disorder, local inflammation, and molecular alteration leading to pancreatic cancer development. Taken together, we highlight potential therapeutic as well as preventive effects of GSK3β inhibition in pancreatic cancer.

  10. PI3 kinase inhibition improves vascular malformations in mouse models of hereditary haemorrhagic telangiectasia.

    Science.gov (United States)

    Ola, Roxana; Dubrac, Alexandre; Han, Jinah; Zhang, Feng; Fang, Jennifer S; Larrivée, Bruno; Lee, Monica; Urarte, Ana A; Kraehling, Jan R; Genet, Gael; Hirschi, Karen K; Sessa, William C; Canals, Francesc V; Graupera, Mariona; Yan, Minhong; Young, Lawrence H; Oh, Paul S; Eichmann, Anne

    2016-11-29

    Activin receptor-like kinase 1 (ALK1) is an endothelial serine-threonine kinase receptor for bone morphogenetic proteins (BMPs) 9 and 10. Inactivating mutations in the ALK1 gene cause hereditary haemorrhagic telangiectasia type 2 (HHT2), a disabling disease characterized by excessive angiogenesis with arteriovenous malformations (AVMs). Here we show that inducible, endothelial-specific homozygous Alk1 inactivation and BMP9/10 ligand blockade both lead to AVM formation in postnatal retinal vessels and internal organs including the gastrointestinal (GI) tract in mice. VEGF and PI3K/AKT signalling are increased on Alk1 deletion and BMP9/10 ligand blockade. Genetic deletion of the signal-transducing Vegfr2 receptor prevents excessive angiogenesis but does not fully revert AVM formation. In contrast, pharmacological PI3K inhibition efficiently prevents AVM formation and reverts established AVMs. Thus, Alk1 deletion leads to increased endothelial PI3K pathway activation that may be a novel target for the treatment of vascular lesions in HHT2.

  11. Fast kinase domain-containing protein 3 is a mitochondrial protein essential for cellular respiration

    International Nuclear Information System (INIS)

    Simarro, Maria; Gimenez-Cassina, Alfredo; Kedersha, Nancy; Lazaro, Jean-Bernard; Adelmant, Guillaume O.; Marto, Jarrod A.; Rhee, Kirsten; Tisdale, Sarah; Danial, Nika; Benarafa, Charaf; Orduna, Anonio; Anderson, Paul

    2010-01-01

    Research highlights: → Five members of the FAST kinase domain-containing proteins are localized to mitochondria in mammalian cells. → The FASTKD3 interactome includes proteins involved in various aspects of mitochondrial metabolism. → Targeted knockdown of FASTKD3 significantly reduces basal and maximal mitochondrial oxygen consumption. -- Abstract: Fas-activated serine/threonine phosphoprotein (FAST) is the founding member of the FAST kinase domain-containing protein (FASTKD) family that includes FASTKD1-5. FAST is a sensor of mitochondrial stress that modulates protein translation to promote the survival of cells exposed to adverse conditions. Mutations in FASTKD2 have been linked to a mitochondrial encephalomyopathy that is associated with reduced cytochrome c oxidase activity, an essential component of the mitochondrial electron transport chain. We have confirmed the mitochondrial localization of FASTKD2 and shown that all FASTKD family members are found in mitochondria. Although human and mouse FASTKD1-5 genes are expressed ubiquitously, some of them are most abundantly expressed in mitochondria-enriched tissues. We have found that RNA interference-mediated knockdown of FASTKD3 severely blunts basal and stress-induced mitochondrial oxygen consumption without disrupting the assembly of respiratory chain complexes. Tandem affinity purification reveals that FASTKD3 interacts with components of mitochondrial respiratory and translation machineries. Our results introduce FASTKD3 as an essential component of mitochondrial respiration that may modulate energy balance in cells exposed to adverse conditions by functionally coupling mitochondrial protein synthesis to respiration.

  12. Substrate analysis of the Pneumocystis carinii protein kinases PcCbk1 and PcSte20 using yeast proteome microarrays provides a novel method for Pneumocystis signalling biology.

    Science.gov (United States)

    Kottom, Theodore J; Limper, Andrew H

    2011-10-01

    Pneumocystis carinii (Pc) undergoes morphological transitions between cysts and trophic forms. We have previously described two Pc serine/threonine kinases, termed PcCbk1 and PcSte20, with PcSte20 belonging to a family of kinases involved in yeast mating, while PcCbk1 is a member of a group of protein kinases involved in regulation of cell cycle, shape, and proliferation. As Pc remains genetically intractable, knowledge on specific substrates phosphorylated by these kinases remains limited. Utilizing the phylogenetic relatedness of Pc to Saccharomyces cerevisiae, we interrogated a yeast proteome microarray containing >4000 purified protein based peptides, leading to the identification of 18 potential PcCbk1 and 15 PcSte20 substrates (Z-score > 3.0). A number of these potential protein substrates are involved in bud site selection, polarized growth, and response to mating α factor and pseudohyphal and invasive growth. Full-length open reading frames suggested by the PcCbk1 and PcSte20 protoarrays were amplified and expressed. These five proteins were used as substrates for PcCbk1 or PcSte20, with each being highly phosphorylated by the respective kinase. Finally, to demonstrate the utility of this method to identify novel PcCbk1 and PcSte20 substrates, we analysed DNA sequence data from the partially complete Pc genome database and detected partial sequence information of potential PcCbk1 kinase substrates PcPxl1 and PcInt1. We additionally identified the potential PcSte20 kinase substrate PcBdf2. Full-length Pc substrates were cloned and expressed in yeast, and shown to be phosphorylated by the respective Pc kinases. In conclusion, the yeast protein microarray represents a novel crossover technique for identifying unique potential Pc kinase substrates. Copyright © 2011 John Wiley & Sons, Ltd.

  13. Increasing L-threonine production in Escherichia coli by engineering the glyoxylate shunt and the L-threonine biosynthesis pathway.

    Science.gov (United States)

    Zhao, Hui; Fang, Yu; Wang, Xiaoyuan; Zhao, Lei; Wang, Jianli; Li, Ye

    2018-04-30

    L-threonine is an important amino acid that can be added in food, medicine, or feed. Here, the influence of glyoxylate shunt on an L-threonine producing strain Escherichia coli TWF001 has been studied. The gene iclR was deleted, and the native promoter of the aceBA operon was replaced by the trc promoter in the chromosome of TWF001, the resulting strainTWF004 could produce 0.39 g L-threonine from1 g glucose after 36-h flask cultivation. Further replacing the native promoter of aspC by the trc promoter in the chromosome of TWF004 resulted in the strain TWF006. TWF006 could produce 0.42 g L-threonine from 1 g glucose after 36-h flask cultivation. Three key genes in the biosynthetic pathway of L-threonine, thrA * (a mutated thrA), thrB, and thrC were overexpressed in TWF006, resulting the strain TWF006/pFW01-thrA * BC. TWF006/pFW01-thrA * BC could produce 0.49 g L-threonine from 1 g glucose after 36-h flask cultivation. Next, the genes asd, rhtA, rhtC, or thrE were inserted into the plasmid TWF006/pFW01-thrA * BC, and TWF006 was transformed with these plasmids, resulting the strains TWF006/pFW01-thrA * BC-asd, TWF006/pFW01-thrA * BC-rhtA, TWF006/pFW01-thrA * BC-rhtC, and TWF006/pFW01-thrA * BC-thrE, respectively. These four strains could produce more L-threonine than the control strain, and the highest yield was produced by TWF006/pFW01-thrA * BC-asd; after 36-h flask cultivation, TWF006/pFW01-thrA * BC-asd could produce 15.85 g/l L-threonine, i.e., 0.53 g L-threonine per 1 g glucose, which is a 70% increase relative to the control strain TWF001. The results suggested that the combined engineering of glyoxylate shunt and L-threonine biosynthesis pathway could significantly increase the L-threonine production in E. coli.

  14. Involvement of the Eukaryote-Like Kinase-Phosphatase System and a Protein That Interacts with Penicillin-Binding Protein 5 in Emergence of Cephalosporin Resistance in Cephalosporin-Sensitive Class A Penicillin-Binding Protein Mutants in Enterococcus faecium

    Directory of Open Access Journals (Sweden)

    Charlene Desbonnet

    2016-04-01

    Full Text Available The intrinsic resistance of Enterococcus faecium to ceftriaxone and cefepime (here referred to as “cephalosporins” is reliant on the presence of class A penicillin-binding proteins (Pbps PbpF and PonA. Mutants lacking these Pbps exhibit cephalosporin susceptibility that is reversible by exposure to penicillin and by selection on cephalosporin-containing medium. We selected two cephalosporin-resistant mutants (Cro1 and Cro2 of class A Pbp-deficient E. faecium CV598. Genome analysis revealed changes in the serine-threonine kinase Stk in Cro1 and a truncation in the associated phosphatase StpA in Cro2 whose respective involvements in resistance were confirmed in separate complementation experiments. In an additional effort to identify proteins linked to cephalosporin resistance, we performed tandem affinity purification using Pbp5 as bait in penicillin-exposed E. faecium; these experiments yielded a protein designated Pbp5-associated protein (P5AP. Transcription of the P5AP gene was increased after exposure to penicillin in wild-type strains and in Cro2 and suppressed in Cro2 complemented with the wild-type stpA. Transformation of class A Pbp-deficient strains with the plasmid-carried P5AP gene conferred cephalosporin resistance. These data suggest that Pbp5-associated cephalosporin resistance in E. faecium devoid of typical class A Pbps is related to the presence of P5AP, whose expression is influenced by the activity of the serine-threonine phosphatase/kinase system.

  15. Functional, genetic and bioinformatic characterization of a calcium/calmodulin kinase gene in Sporothrix schenckii

    Directory of Open Access Journals (Sweden)

    Rodriguez-del Valle Nuri

    2007-11-01

    Full Text Available Abstract Background Sporothrix schenckii is a pathogenic, dimorphic fungus, the etiological agent of sporotrichosis, a subcutaneous lymphatic mycosis. Dimorphism in S. schenckii responds to second messengers such as cAMP and calcium, suggesting the possible involvement of a calcium/calmodulin kinase in its regulation. In this study we describe a novel calcium/calmodulin-dependent protein kinase gene in S. schenckii, sscmk1, and the effects of inhibitors of calmodulin and calcium/calmodulin kinases on the yeast to mycelium transition and the yeast cell cycle. Results Using the PCR homology approach a new member of the calcium/calmodulin kinase family, SSCMK1, was identified in this fungus. The cDNA sequence of sscmk1 revealed an open reading frame of 1,221 nucleotides encoding a 407 amino acid protein with a predicted molecular weight of 45.6 kDa. The genomic sequence of sscmk1 revealed the same ORF interrupted by five introns. Bioinformatic analyses of SSCMK1 showed that this protein had the distinctive features that characterize a calcium/calmodulin protein kinase: a serine/threonine protein kinase domain and a calmodulin-binding domain. When compared to homologues from seven species of filamentous fungi, SSCMK1 showed substantial similarities, except for a large and highly variable region that encompasses positions 330 – 380 of the multiple sequence alignment. Inhibition studies using calmodulin inhibitor W-7, and calcium/calmodulin kinase inhibitors, KN-62 and lavendustin C, were found to inhibit budding by cells induced to re-enter the yeast cell cycle and to favor the yeast to mycelium transition. Conclusion This study constitutes the first evidence of the presence of a calcium/calmodulin kinase-encoding gene in S. schenckii and its possible involvement as an effector of dimorphism in this fungus. These results suggest that a calcium/calmodulin dependent signaling pathway could be involved in the regulation of dimorphism in this fungus

  16. Decreased levels of free D-aspartic acid in the forebrain of serine racemase (Srr) knock-out mice.

    Science.gov (United States)

    Horio, Mao; Ishima, Tamaki; Fujita, Yuko; Inoue, Ran; Mori, Hisashi; Hashimoto, Kenji

    2013-05-01

    d-Serine, an endogenous co-agonist of the N-methyl-d-aspartate (NMDA) receptor is synthesized from l-serine by serine racemase (SRR). A previous study of Srr knockout (Srr-KO) mice showed that levels of d-serine in forebrain regions, such as frontal cortex, hippocampus, and striatum, but not cerebellum, of mutant mice are significantly lower than those of wild-type (WT) mice, suggesting that SRR is responsible for d-serine production in the forebrain. In this study, we attempted to determine whether SRR affects the level of other amino acids in brain tissue. We found that tissue levels of d-aspartic acid in the forebrains (frontal cortex, hippocampus and striatum) of Srr-KO mice were significantly lower than in WT mice, whereas levels of d-aspartic acid in the cerebellum were not altered. Levels of d-alanine, l-alanine, l-aspartic acid, taurine, asparagine, arginine, threonine, γ-amino butyric acid (GABA) and methionine, remained the same in frontal cortex, hippocampus, striatum and cerebellum of WT and mutant mice. Furthermore, no differences in d-aspartate oxidase (DDO) activity were detected in the forebrains of WT and Srr-KO mice. These results suggest that SRR and/or d-serine may be involved in the production of d-aspartic acid in mouse forebrains, although further detailed studies will be necessary to confirm this finding. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells

    KAUST Repository

    Suen, K.M.

    2017-12-06

    Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.

  18. Phosphorylation of threonine residues on Shc promotes ligand binding and mediates crosstalk between MAPK and Akt pathways in breast cancer cells

    KAUST Repository

    Suen, K.M.; Lin, C.C.; Seiler, C.; George, R.; Poncet-Montange, G.; Biter, A.B.; Ahmed, Z.; Arold, Stefan T.; Ladbury, J.E.

    2017-01-01

    Scaffold proteins play important roles in regulating signalling network fidelity, the absence of which is often the basis for diseases such as cancer. In the present work, we show that the prototypical scaffold protein Shc is phosphorylated by the extracellular signal-regulated kinase, Erk. In addition, Shc threonine phosphorylation is specifically up-regulated in two selected triple-negative breast cancer (TNBC) cell lines. To explore how Erk-mediated threonine phosphorylation on Shc might play a role in the dysregulation of signalling events, we investigated how Shc affects pathways downstream of EGF receptor. Using an in vitro model and biophysical analysis, we show that Shc threonine phosphorylation is responsible for elevated Akt and Erk signalling, potentially through the recruitment of the 14-3-3 ζ and Pin-1 proteins.

  19. Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae

    OpenAIRE

    Elbing, Karin; McCartney, Rhonda R.; Schmidt, Martin C.

    2006-01-01

    Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerpr...

  20. Extracellular signal regulated kinase 5 mediates signals triggered by the novel tumor promoter palytoxin

    International Nuclear Information System (INIS)

    Charlson, Aaron T.; Zeliadt, Nicholette A.; Wattenberg, Elizabeth V.

    2009-01-01

    Palytoxin is classified as a non-12-O-tetradecanoylphorbol-13-acetate (TPA)-type skin tumor because it does not bind to or activate protein kinase C. Palytoxin is thus a novel tool for investigating alternative signaling pathways that may affect carcinogenesis. We previously showed that palytoxin activates three major members of the mitogen activated protein kinase (MAPK) family, extracellular signal regulated kinase 1 and 2 (ERK1/2), c-Jun N-terminal kinase (JNK), and p38. Here we report that palytoxin also activates another MAPK family member, called ERK5, in HeLa cells and in keratinocytes derived from initiated mouse skin (308 cells). By contrast, TPA does not activate ERK5 in these cell lines. The major cell surface receptor for palytoxin is the Na+,K+-ATPase. Accordingly, ouabain blocked the ability of palytoxin to activate ERK5. Ouabain alone did not activate ERK5. ERK5 thus represents a divergence in the signaling pathways activated by these two agents that bind to the Na+,K+-ATPase. Cycloheximide, okadaic acid, and sodium orthovanadate did not mimic the effect of palytoxin on ERK5. These results indicate that the stimulation of ERK5 by palytoxin is not simply due to inhibition of protein synthesis or inhibition of serine/threonine or tyrosine phosphatases. Therefore, the mechanism by which palytoxin activates ERK5 differs from that by which it activates ERK1/2, JNK, and p38. Finally, studies that used pharmacological inhibitors and shRNA to block ERK5 action indicate that ERK5 contributes to palytoxin-stimulated c-Fos gene expression. These results suggest that ERK5 can act as an alternative mediator for transmitting diverse tumor promoter-stimulated signals.

  1. Myosin Light Chain Kinase and the Role of Myosin Light Chain Phosphorylation in Skeletal Muscle

    Science.gov (United States)

    Stull, James T.; Kamm, Kristine E.; Vandenboom, Rene

    2011-01-01

    Skeletal muscle myosin light chain kinase (skMLCK) is a dedicated Ca2+/calmodulin-dependent serine-threonine protein kinase that phosphorylates the regulatory light chain (RLC) of sarcomeric myosin. It is expressed from the MYLK2 gene specifically in skeletal muscle fibers with most abundance in fast contracting muscles. Biochemically, activation occurs with Ca2+ binding to calmodulin forming a (Ca2+)4•calmodulin complex sufficient for activation with a diffusion limited, stoichiometic binding and displacement of a regulatory segment from skMLCK catalytic core. The N-terminal sequence of RLC then extends through the exposed catalytic cleft for Ser15 phosphorylation. Removal of Ca2+ results in the slow dissociation of calmodulin and inactivation of skMLCK. Combined biochemical properties provide unique features for the physiological responsiveness of RLC phosphorylation, including (1) rapid activation of MLCK by Ca2+/calmodulin, (2) limiting kinase activity so phosphorylation is slower than contraction, (3) slow MLCK inactivation after relaxation and (4) much greater kinase activity relative to myosin light chain phosphatase (MLCP). SkMLCK phosphorylation of myosin RLC modulates mechanical aspects of vertebrate skeletal muscle function. In permeabilized skeletal muscle fibers, phosphorylation-mediated alterations in myosin structure increase the rate of force-generation by myosin cross bridges to increase Ca2+-sensitivity of the contractile apparatus. Stimulation-induced increases in RLC phosphorylation in intact muscle produces isometric and concentric force potentiation to enhance dynamic aspects of muscle work and power in unfatigued or fatigued muscle. Moreover, RLC phosphorylation-mediated enhancements may interact with neural strategies for human skeletal muscle activation to ameliorate either central or peripheral aspects of fatigue. PMID:21284933

  2. Non-opsonic phagocytosis of Legionella pneumophila by macrophages is mediated by phosphatidylinositol 3-kinase.

    Directory of Open Access Journals (Sweden)

    Souvenir D Tachado

    Full Text Available BACKGROUND: Legionella pneumophila, is an intracellular pathogen that causes Legionnaires' disease in humans, a potentially lethal pneumonia. L. pneumophila has the ability to enter and replicate in the host and is essential for pathogenesis. METHODOLOGY/PRINCIPAL FINDINGS: Phagocytosis was measured by cell invasion assays. Construction of PI3K mutant by PCR cloning and expression of dominant negative mutant was detected by Western blot. PI3K activity was measured by 32P labeling and detection of phospholipids products by thin layer chromatography. Infection of macrophages with virulent L. pneumophila stimulated the formation of phosphatidylinositol 3-phosphate (PIP3, a phosphorylated lipid product of PI3K whereas two structurally distinct phosphatidylinositol 3 kinase (PI3K inhibitors, wortmannin and LY294002, reduced L. pneumophila entry into macrophages in a dose-dependent fashion. Furthermore, PI3K activation led to Akt stimulation, a serine/threonine kinase, which was also inhibited by wortmannin and LY294002. In contrast, PI3K and protein kinase B (PKB/Akt activities were lower in macrophages infected with an avirulent bacterial strain. Only virulent L. pneumophila increased lipid kinase activity present in immunoprecipitates of the p85alpha subunit of class I PI3K and tyrosine phosphorylated proteins. In addition, macrophages expressing a specific dominant negative mutant of PI3K reduced L. pneumophila entry into these cells. CONCLUSION/SIGNIFICANCE: Entry of L. pneumophila is mediated by PI3K/Akt signaling pathway. These results suggest an important role for PI3K and Akt in the L. pneumophila infection process. They point to possible novel strategies for undermining L. pneumophila host uptake and reducing pathogenesis of Legionnaires' disease.

  3. Protein kinase C (PKC) isoforms in cancer, tumor promotion and tumor suppression.

    Science.gov (United States)

    Isakov, Noah

    2018-02-01

    The AGC family of serine/threonine kinases (PKA, PKG, PKC) includes more than 60 members that are critical regulators of numerous cellular functions, including cell cycle and differentiation, morphogenesis, and cell survival and death. Mutation and/or dysregulation of AGC kinases can lead to malignant cell transformation and contribute to the pathogenesis of many human diseases. Members of one subgroup of AGC kinases, the protein kinase C (PKC), have been singled out as critical players in carcinogenesis, following their identification as the intracellular receptors of phorbol esters, which exhibit tumor-promoting activities. This observation attracted the attention of researchers worldwide and led to intense investigations on the role of PKC in cell transformation and the potential use of PKC as therapeutic drug targets in cancer diseases. Studies demonstrated that many cancers had altered expression and/or mutation of specific PKC genes. However, the causal relationships between the changes in PKC gene expression and/or mutation and the direct cause of cancer remain elusive. Independent studies in normal cells demonstrated that activation of PKC is essential for the induction of cell activation and proliferation, differentiation, motility, and survival. Based on these observations and the general assumption that PKC isoforms play a positive role in cell transformation and/or cancer progression, many PKC inhibitors have entered clinical trials but the numerous attempts to target PKC in cancer has so far yielded only very limited success. More recent studies demonstrated that PKC function as tumor suppressors, and suggested that future clinical efforts should focus on restoring, rather than inhibiting, PKC activity. The present manuscript provides some historical perspectives on the tumor promoting function of PKC, reviewing some of the observations linking PKC to cancer progression, and discusses the role of PKC in the pathogenesis of cancer diseases and its

  4. Novel mutations in cyclin-dependent kinase-like 5 (CDKL5) gene in Indian cases of Rett syndrome.

    Science.gov (United States)

    Das, Dhanjit Kumar; Mehta, Bhakti; Menon, Shyla R; Raha, Sarbani; Udani, Vrajesh

    2013-03-01

    Rett syndrome is a severe neurodevelopmental disorder, almost exclusively affecting females and characterized by a wide spectrum of clinical manifestations. Both the classic and atypical forms of Rett syndrome are primarily due to mutations in the methyl-CpG-binding protein 2 (MECP2) gene. Mutations in the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene have been identified in patients with atypical Rett syndrome, X-linked infantile spasms sharing common features of generally early-onset seizures and mental retardation. CDKL5 is known as serine/threonine protein kinase 9 (STK9) and is mapped to the Xp22 region. It has a conserved serine/threonine kinase domain within its amino terminus and a large C-terminal region. Disease-causing mutations are distributed in both the amino terminal domain and in the large C-terminal domain. We have screened the CDKL5 gene in 44 patients with atypical Rett syndrome who had tested negative for MECP2 gene mutations and have identified 6 sequence variants, out of which three were novel and three known mutations. Two of these novel mutations p.V966I and p.A1011V were missense and p.H589H a silent mutation. Other known mutations identified were p.V999M, p.Q791P and p.T734A. Sequence homology for all the mutations revealed that the two mutations (p.Q791P and p.T734A) were conserved across species. This indicated the importance of these residues in structure and function of the protein. The damaging effects of these mutations were analysed in silico using PolyPhen-2 online software. The PolyPhen-2 scores of p.Q791P and p.T734A were 0.998 and 0.48, revealing that these mutations could be deleterious and might have potential functional effect. All other mutations had a low score suggesting that they might not alter the activity of CDKL5. We have also analysed the position of the mutations in the CDKL5 protein and found that all the mutations were present in the C-terminal domain of the protein. The C-terminal domain is required for

  5. Isolation and characterization of human cDNA clones encoding the α and the α' subunits of casein kinase II

    International Nuclear Information System (INIS)

    Lozeman, F.J.; Litchfield, D.W.; Piening, C.; Takio, Koji; Walsh, K.A.; Krebs, E.G.

    1990-01-01

    Casein kinase II is a widely distributed protein serine/threonine kinase. The holoenzyme appears to be a tetramer, containing two α or α' subunits (or one of each) and two β subunits. Complementary DNA clones encoding the subunits of casein kinase II were isolated from a human T-cell λgt 10 library using cDNA clones isolated from Drosophila melanogasten. One of the human cDNA clones (hT4.1) was 2.2 kb long, including a coding region of 1176 bp preceded by 156 bp (5' untranslated region) and followed by 871 bp (3' untranslated region). The hT4.1 close was nearly identical in size and sequence with a cDNA clone from HepG2 human hepatoma cultured cells. Another of the human T-cell cDNA clones (hT9.1) was 1.8 kb long, containing a coding region of 1053 bp preceded by 171 by (5' untranslated region) and followed by 550 bp (3' untranslated region). Amino acid sequences deduced from these two cDNA clones were about 85% identical. Most of the difference between the two encoded polypeptides was in the carboxy-terminal region, but heterogeneity was distributed throughout the molecules. Partial amino acid sequence was determined in a mixture of α and α' subunits from bovine lung casein kinase II. The bovine sequences aligned with the 2 human cDNA-encoded polypeptides with only 2 discrepancies out of 535 amino acid positions. This confirmed that the two human T-cell cDNA clones encoded the α and α' subunits of casein kinase II. These studies show that there are two distinct catalytic subunits for casein II (α and α') and that the sequence of these subunits is largely conserved between the bovine and the human

  6. Leucine-rich repeat kinase-1 regulates osteoclast function by modulating RAC1/Cdc42 Small GTPase phosphorylation and activation.

    Science.gov (United States)

    Zeng, Canjun; Goodluck, Helen; Qin, Xuezhong; Liu, Bo; Mohan, Subburaman; Xing, Weirong

    2016-10-01

    Leucine-rich repeat kinase-1 (Lrrk1) consists of ankyrin repeats (ANK), leucine-rich repeats (LRR), a GTPase-like domain of Roc (ROC), a COR domain, a serine/threonine kinase domain (KD), and WD40 repeats (WD40). Previous studies have revealed that knockout (KO) of Lrrk1 in mice causes severe osteopetrosis, and a human mutation of Lrrk1 leads to osteosclerotic metaphysial dysplasia. The molecular mechanism by which Lrrk1 regulates osteoclast function is unknown. In this study, we generated a series of Lrrk1 mutants and evaluated their ability to rescue defective bone resorption in Lrrk1-deficient osteoclasts by use of pit formation assays. Overexpression of Lrrk1 or LRR-truncated Lrrk1, but not ANK-truncated Lrrk1, WD40-truncated Lrrk1, Lrrk1-KD, or K651A mutant Lrrk1, rescued bone resorption function of Lrrk1 KO osteoclasts. We next examined whether RAC1/Cdc42 small GTPases are direct substrates of Lrrk1 in osteoclasts. Western blot and pull-down assays revealed that Lrrk1 deficiency in osteoclasts resulted in reduced phosphorylation and activation of RAC1/Cdc42. In vitro kinase assays confirmed that recombinant Lrrk1 phosphorylated RAC1-GST protein, and immunoprecipitation showed that the interaction of Lrrk1 with RAC1 occurred within 10 min after RANKL treatment. Overexpression of constitutively active Q61L RAC1 partially rescued the resorptive function of Lrrk1-deficient osteoclasts. Furthermore, lack of Lrrk1 in osteoclasts led to reduced autophosphorylation of p21 protein-activated kinase-1 at Ser 144 , catalyzed by RAC1/Cdc42 binding and activation. Our data indicate that Lrrk1 regulates osteoclast function by directly modulating phosphorylation and activation of small GTPase RAC1/Cdc42 and that its function depends on ANK, ROC, WD40, and kinase domains. Copyright © 2016 the American Physiological Society.

  7. Crystal Structure of Human Dual-Specificity Tyrosine-Regulated Kinase 3 Reveals New Structural Features and Insights into its Auto-phosphorylation.

    Science.gov (United States)

    Kim, Kuglae; Cha, Jeong Seok; Cho, Yong-Soon; Kim, Hoyoung; Chang, Nienping; Kim, Hye-Jung; Cho, Hyun-Soo

    2018-04-07

    Dual-specificity tyrosine-regulated kinases (DYRKs) auto-phosphorylate a critical tyrosine residue in their activation loop and phosphorylate their substrate on serine and threonine residues. The auto-phosphorylation occurs intramolecularly and is a one-off event. DYRK3 is selectively expressed at a high level in hematopoietic cells and attenuates erythroblast development, leading to anemia. In the present study, we determined the crystal structure of the mature form of human DYRK3 in complex with harmine, an ATP competitive inhibitor. The crystal structure revealed a phosphorylation site, residue S350, whose phosphorylation increases the stability of DYRK3 and enhances its kinase activity. In addition, our structural and biochemical assays suggest that the N-terminal auto-phosphorylation accessory domain stabilizes the DYRK3 protein, followed by auto-phosphorylation of the tyrosine of the activation loop, which is important for kinase activity. Finally, our docking analysis provides information for the design of novel and potent therapeutics to treat anemia. Copyright © 2018 Elsevier Ltd. All rights reserved.

  8. FHA domains as phospho-threonine binding modules in cell signaling.

    Science.gov (United States)

    Hammet, Andrew; Pike, Brietta L; McNees, Carolyn J; Conlan, Lindus A; Tenis, Nora; Heierhorst, Jörg

    2003-01-01

    Forkhead-associated (FHA) domains are present in >200 diverse proteins in all phyla from bacteria to mammals and seem to be particularly prevalent in proteins with cell cycle control functions. Recent work from several laboratories has considerably improved our understanding of the structure and function of these domains that were virtually unknown a few years ago, and the first disease associations of FHA domains have now emerged. FHA domains form 11-stranded beta-sandwiches that contain some 100-180 amino acid residues with a high degree of sequence diversity. FHA domains act as phosphorylation-dependent protein-protein interaction modules that preferentially bind to phospho-threonine residues in their targets. Interestingly, point mutations in the human CHK2 gene that lead to single-residue amino acid substitutions in the FHA domain of this cell cycle checkpoint kinase have been found to cause a subset of cases of the Li-Fraumeni multi-cancer syndrome.

  9. Quantitative partition of threonine oxidation in pigs: Effect of dietary threonine

    International Nuclear Information System (INIS)

    Ballevre, O.; Cadenhead, A.; Calder, A.G.; Rees, W.D.; Lobley, G.E.; Fuller, M.F.; Garlick, P.J.

    1990-01-01

    Kinetic aspects of threonine (Thr) metabolism were examined in eight pigs fed hourly with a diet containing either 0.68% (LT group) or 0.81% (HT group) of Thr (wt/wt), corresponding to 10 and 30% Thr excess, respectively, compared with an ideal diet. Primary production (PR) and disposal (DR) rates were obtained for Thr, glycine (Gly), and 2-keto-butyrate (KB) after a 12-h continuous infusion of L-[U-14C]-Thr together with [1-13C]Gly and a 6-h continuous infusion of [1-14C]KB. Transfer of Thr into secondary pools was also monitored, and from these the rates of Thr oxidation through the catabolic pathways of L-Thr 3-dehydrogenase (DR(Thr-Gly)) and threonine dehydratase (DR(Thr-KB)) were estimated. For the LT group the results were (mumol.kg-1.h-1) PR(Thr) 314 +/- 3, PR(Gly) 551 +/- 24, PR(KB) 41 +/- 3, DR(Thr-Gly) 22 +/- 2, and DR(Thr-KB) 7 +/- 1. For the HT group they were PR(Thr) 301 +/- 23, PR(Gly) 598 +/- 55, PR(KB) 39 +/- 4, DR(Thr-Gly) 32 +/- 2, and DR(Thr-KB) 8 +/- 1. The increase in Thr intake (14 mumol.kg-1.h-1, P less than 0.01) induced a commensurate increase in the sum of DR(Thr-Gly) and DR(Thr-KB) (14 mumol.kg-1.h-1, P less than 0.001) when liver was used as the precursor pool. This was mainly due to the increased DR(Thr-Gly) (13 mumol.kg-1.h-1, P less than 0.01); the change in DR(Thr-KB) was not statistically significant. By comparison of intracellular-to-plasma ratios of specific activities (or enrichments) for different tissues with each type of infusion, liver was shown to be the major site of production of Gly and KB from Thr. These data suggest that in fed growing pigs a 30% excess of Thr in the diet does not alter the partition of Thr oxidation, since 80% of Thr oxidation occurs through the L-Thr 3-dehydrogenase pathway for both LT and HT groups

  10. ATM-mediated Snail Serine 100 phosphorylation regulates cellular radiosensitivity

    International Nuclear Information System (INIS)

    Boohaker, Rebecca J.; Cui, Xiaoli; Stackhouse, Murray; Xu, Bo

    2013-01-01

    Purpose: Activation of the DNA damage responsive protein kinase ATM is a critical step for cellular survival in response to ionizing irradiation (IR). Direct targets of ATM regulating radiosensitivity remain to be fully investigated. We have recently reported that ATM phosphorylates the transcriptional repressor Snail on Serine 100. We aimed to further study the functional significance of ATM-mediated Snail phosphorylation in response to IR. Material and methods: We transfected vector-only, wild-type, the Serine 100 to alanine (S100A) or to glutamic acid (S100E) substitution of Snail into various cell lines. We assessed colony formation, γ-H2AX focus formation and the invasion index in the cells treated with or without IR. Results: We found that over-expression of the S100A mutant Snail in HeLa cells significantly increased radiosensitivity. Meanwhile the expression of S100E, a phospho-mimicking mutation, resulted in enhanced radio-resistance. Interestingly, S100E could rescue the radiosensitive phenotype in ATM-deficient cells. We also found that expression of S100E increased γ-H2AX focus formation and compromised inhibition of invasion in response to IR independent of cell survival. Conclusion: ATM-mediated Snail Serine 100 phosphorylation in response to IR plays an important part in the regulation of radiosensitivity

  11. Kin3 protein, a NIMA-related kinase of Saccharomyces cerevisiae, is involved in DNA adduct damage response.

    Science.gov (United States)

    Moura, Dinara J; Castilhos, Bruna; Immich, Bruna F; Cañedo, Andrés D; Henriques, João A P; Lenz, Guido; Saffi, Jenifer

    2010-06-01

    Kin3 is a nonessential serine/threonine protein kinase of the budding yeast Saccharomyces cerevisiae with unknown cellular role. It is an ortholog of the Aspergillus nidulans protein kinase NIMA (Never-In Mitosis, gene A), which is involved in the regulation of G2/M phase progression, DNA damage response and mitosis. The aim of this study was to determine whether Kin3 is required for proper checkpoint activation and DNA repair. Here we show that KIN3 gene deficient cells present sensitivity and fail to arrest properly at G2/M-phase checkpoint in response to the DNA damage inducing agents MMS, cisplatin, doxorubicin and nitrogen mustard, suggesting that Kin3 can be involved in DNA strand breaks recognition or signaling. In addition, there is an increase in KIN3 gene expression in response to the mutagenic treatment, which was confirmed by the increase of Kin3 protein. We also showed that co-treatment with caffeine induces a slight increase in the susceptibility to genotoxic agents in kin3 cells and abolishes KIN3 gene expression in wild-type strain, suggesting that Kin3p can play a role in Tel1/Mec1-dependent pathway activation induced after genotoxic stress. These data provide the first evidence of the involvement of S. cerevisiae Kin3 in the DNA damage response.

  12. Glycogen synthase kinase-3 inhibition by 3-anilino-4-phenylmaleimides: insights from 3D-QSAR and docking

    Science.gov (United States)

    Prasanna, Sivaprakasam; Daga, Pankaj R.; Xie, Aihua; Doerksen, Robert J.

    2009-02-01

    Glycogen synthase kinase-3, a serine/threonine kinase, has been implicated in a wide variety of pathological conditions such as diabetes, Alzheimer's disease, stroke, bipolar disorder, malaria and cancer. Herein we report 3D-QSAR analyses using CoMFA and CoMSIA and molecular docking studies on 3-anilino-4-phenylmaleimides as GSK-3α inhibitors, in order to better understand the mechanism of action and structure-activity relationship of these compounds. Comparison of the active site residues of GSK-3α and GSK-3β isoforms shows that all the key amino acids involved in polar interactions with the maleimides for the β isoform are the same in the α isoform, except that Asp133 in the β isoform is replaced by Glu196 in the α isoform. We prepared a homology model for GSK-3α, and showed that the change from Asp to Glu should not affect maleimide binding significantly. Docking studies revealed the binding poses of three subclasses of these ligands, namely anilino, N-methylanilino and indoline derivatives, within the active site of the β isoform, and helped to explain the difference in their inhibitory activity.

  13. Anti-diabetes drug pioglitazone ameliorates synaptic defects in AD transgenic mice by inhibiting cyclin-dependent kinase5 activity.

    Directory of Open Access Journals (Sweden)

    Jinan Chen

    Full Text Available Cyclin-dependent kinase 5 (Cdk5 is a serine/threonine kinase that is activated by the neuron specific activators p35/p39 and plays many important roles in neuronal development. However, aberrant activation of Cdk5 is believed to be associated with the pathogenesis of several neurodegenerative diseases, including Alzheimer's disease (AD and Parkinson's disease (PD. Here in the present study, enhanced Cdk5 activity was observed in mouse models of AD; whereas soluble amyloid-β oligomers (Aβ, which contribute to synaptic failures during AD pathogenesis, induced Cdk5 hyperactivation in cultured hippocampal neurons. Inhibition of Cdk5 activity by pharmacological or genetic approaches reversed dendritic spine loss caused by soluble amyloid-β oligomers (Aβ treatment. Interestingly, we found that the anti-diabetes drug pioglitazone could inhibit Cdk5 activity by decreasing p35 protein level. More importantly, pioglitazone treatment corrected long-term potentiation (LTP deficit caused by Aβ exposure in cultured slices and pioglitazone administration rescued impaired LTP and spatial memory in AD mouse models. Taken together, our study describes an unanticipated role of pioglitazone in alleviating AD and reveals a potential therapeutic drug for AD curing.

  14. Inhibition of casein kinase 2 modulates XBP1-GRP78 arm of unfolded protein responses in cultured glial cells.

    Directory of Open Access Journals (Sweden)

    Toru Hosoi

    Full Text Available Stress signals cause abnormal proteins to accumulate in the endoplasmic reticulum (ER. Such stress is known as ER stress, which has been suggested to be involved in neurodegenerative diseases, diabetes, obesity and cancer. ER stress activates the unfolded protein response (UPR to reduce levels of abnormal proteins by inducing the production of chaperon proteins such as GRP78, and to attenuate translation through the phosphorylation of eIF2α. However, excessive stress leads to apoptosis by generating transcription factors such as CHOP. Casein kinase 2 (CK2 is a serine/threonine kinase involved in regulating neoplasia, cell survival and viral infections. In the present study, we investigated a possible linkage between CK2 and ER stress using mouse primary cultured glial cells. 4,5,6,7-tetrabromobenzotriazole (TBB, a CK2-specific inhibitor, attenuated ER stress-induced XBP-1 splicing and subsequent induction of GRP78 expression, but was ineffective against ER stress-induced eIF2α phosphorylation and CHOP expression. Similar results were obtained when endogenous CK2 expression was knocked-down by siRNA. Immunohistochemical analysis suggested that CK2 was present at the ER. These results indicate CK2 to be linked with UPR and to resist ER stress by activating the XBP-1-GRP78 arm of UPR.

  15. LKB1 mediates the development of conventional and innate T cells via AMP-dependent kinase autonomous pathways.

    Directory of Open Access Journals (Sweden)

    Marouan Zarrouk

    Full Text Available The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK. The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.

  16. Hepatitis C Virus Particle Assembly Involves Phosphorylation of NS5A by the c-Abl Tyrosine Kinase.

    Science.gov (United States)

    Yamauchi, Shota; Takeuchi, Kenji; Chihara, Kazuyasu; Sun, Xuedong; Honjoh, Chisato; Yoshiki, Hatsumi; Hotta, Hak; Sada, Kiyonao

    2015-09-04

    Hepatitis C virus (HCV) nonstructural protein 5A (NS5A) is thought to regulate the replication of viral RNA and the assembly of virus particles in a serine/threonine phosphorylation-dependent manner. However, the host kinases that phosphorylate NS5A have not been fully identified. Here, we show that HCV particle assembly involves the phosphorylation of NS5A by the c-Abl tyrosine kinase. Pharmacological inhibition or knockdown of c-Abl reduces the production of infectious HCV (J6/JFH1) particles in Huh-7.5 cells without markedly affecting viral RNA translation and replication. NS5A is tyrosine-phosphorylated in HCV-infected cells, and this phosphorylation is also reduced by the knockdown of c-Abl. Mutational analysis reveals that NS5A tyrosine phosphorylation is dependent, at least in part, on Tyr(330) (Tyr(2306) in polyprotein numbering). Mutation of this residue to phenylalanine reduces the production of infectious HCV particles but does not affect the replication of the JFH1 subgenomic replicon. These findings suggest that c-Abl promotes HCV particle assembly by phosphorylating NS5A at Tyr(330). © 2015 by The American Society for Biochemistry and Molecular Biology, Inc.

  17. Anticancer screening of medicinal plant phytochemicals against Cyclin-Dependent Kinase-2 (CDK2: An in-silico approach

    Directory of Open Access Journals (Sweden)

    Wajahat Khan

    2017-08-01

    Full Text Available Background: Cyclin-Dependent Kinase-2 (CDK2 is a member of serine/threonine protein kinases family and plays an important role in regulation of various eukaryotic cell division events. Over-expression of CDK2 during cell cycle may lead to several cellular functional aberrations including diverse types of cancers (lung cancer, primary colorectal carcinoma, ovarian cancer, melanoma and pancreatic carcinoma in humans. Medicinal plants phytochemicals which have anticancer potential can be used as an alternative drug resource. Methods: This study was designed to find out anticancer phytochemicals from medicinal plants which could inhibit CDK2 with the help of molecular docking technique. Molecular Operating Environment (MOE v2009 software was used to dock 2300 phytochemicals in this study. Results: The outcome of this study shows that four phytochemicals Kushenol T, Remangiflavanone B, Neocalyxins A and Elenoside showed the lowest S-score (-17.83, -17.57, -17.26, -17.17 respectively and binds strongly with all eight active residues Tyr15, Lys33, Ileu52, Lys56, Leu78, phe80, Asp145 and Phe146 of CDK2 binding site. These phytochemicals could successfully inhibit the CDK2. Conclusion: These phytochemicals can be considered as potential anticancer agents and used in drug development against CDK2. We anticipate that this study would pave way for phytochemical based novel small molecules as more efficacious and selective anti-cancer therapeutic compounds.

  18. Activation of the LRR Receptor-Like Kinase PSY1R Requires Transphosphorylation of Residues in the Activation Loop

    Directory of Open Access Journals (Sweden)

    Christian B. Oehlenschlæger

    2017-11-01

    Full Text Available PSY1R is a leucine-rich repeat (LRR receptor-like kinase (RLK previously shown to act as receptor for the plant peptide hormone PSY1 (peptide containing sulfated tyrosine 1 and to regulate cell expansion. PSY1R phosphorylates and thereby regulates the activity of plasma membrane-localized H+-ATPases. While this mechanism has been studied in detail, little is known about how PSY1R itself is activated. Here we studied the activation mechanism of PSY1R. We show that full-length PSY1R interacts with members of the SERK co-receptor family in planta. We identified seven in vitro autophosphorylation sites on serine and threonine residues within the kinase domain of PSY1R using mass spectrometry. We furthermore show that PSY1R autophosphorylation occurs in trans and that the initial transphosphorylation takes place within the activation loop at residues Ser951, Thr959, and Thr963. While Thr959 and Thr963 are conserved among other related plant LRR RLKs, Ser951 is unique to PSY1R. Based on homology modeling we propose that phosphorylation of Ser951 stabilize the inactive conformation of PSY1R.

  19. Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.

    Science.gov (United States)

    Ku, H; Meier, K E

    2000-04-14

    Intracellular signals can regulate cell adhesion via several mechanisms in a process referred to as "inside-out" signaling. In phorbol ester-sensitive EL4 thymoma cells, phorbol-12-myristate 13-acetate (PMA) induces activation of extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases and promotes cell adhesion. In this study, clonal EL4 cell lines with varying abilities to activate ERKs in response to PMA were used to examine signaling events occurring downstream of ERK activation. Paxillin, a multifunctional docking protein involved in cell adhesion, was phosphorylated on serine/threonine residues in response to PMA treatment. This response was correlated with the extent and time course of ERK activation. PMA-induced phosphorylation of paxillin was inhibited by compounds that block the ERK activation pathway in EL4 cells, primary murine thymocytes, and primary murine splenocytes. Paxillin was phosphorylated in vitro by purified active ERK2. Two-dimensional electrophoresis revealed that PMA treatment generated a complex pattern of phosphorylated paxillin species in intact cells, some of which were generated by ERK-mediated phosphorylation in vitro. An ERK pathway inhibitor interfered with PMA-induced adhesion of sensitive EL4 cells to substrate. These findings describe a novel inside-out signaling pathway by which the ERK cascade may regulate events involved in adhesion.

  20. Inhibition of protein phosphatase 2A induces serine/threonine phosphorylation, subcellular redistribution, and functional inhibition of STAT3

    DEFF Research Database (Denmark)

    Woetmann, A; Nielsen, M; Christensen, S T

    1999-01-01

    Signal transducers and activators of transcription (STATs) are rapidly phosphorylated on tyrosine residues in response to cytokine and growth factor stimulation of cell surface receptors. STATs hereafter are translocated to the nucleus where they act as transcription factors. Recent reports suggest...

  1. The threonine protease activity of testes-specific protease 50 (TSP50 is essential for its function in cell proliferation.

    Directory of Open Access Journals (Sweden)

    Yu-Yin Li

    Full Text Available BACKGROUND: Testes-specific protease 50 (TSP50, a newly discovered threonine enzyme, has similar amino acid sequences and enzymatic structures to those of many serine proteases. It may be an oncogene. TSP50 is up-regulated in breast cancer epithelial cells, and ectopic expression of TSP50 in TSP50-deficient Chinese hamster ovary (CHO cells has been found to promote cell proliferation. However, the mechanisms by which TSP50 exerts its growth-promoting effects are not yet fully understood. METHODOLOGY/PRINCIPAL FINDINGS: To delineate whether the threonine protease activity of TSP50 is essential to its function in cell proliferation, we constructed and characterized a mutant TSP50, called TSP50 T310A, which was identified as a protease-dead mutant of TSP50. By a series of proliferation analyses, colony formation assays and apoptosis analyses, we showed that T310A mutation significantly depresses TSP50-induced cell proliferation in vitro. Next, the CHO stable cell line expressing either wild-type or T310A mutant TSP50 was injected subcutaneously into nude mice. We found that the T310A mutation could abolish the tumorigenicity of TSP50 in vivo. A mechanism investigation revealed that the T310A mutation prevented interaction between TSP50 and the NF-κBIκBα complex, which is necessary for TSP50 to perform its function in cell proliferation. CONCLUSION: Our data highlight the importance of threonine 310, the most critical protease catalytic site in TSP50, to TSP50-induced cell proliferation and tumor formation.

  2. Effects of dietary L-threonine and Saccharomyces cerevisiae on ...

    African Journals Online (AJOL)

    threonine (0, 2.5, 5 and 7.5 g/kg) with or without Saccharomyces cerevisiae (SC) on performance, carcass characteristics, intestinal morphology and immune system of broiler chickens. A total of 360 1-d-old male broiler chicks were randomly ...

  3. Glycogen Synthase Kinase-3β

    DEFF Research Database (Denmark)

    Munkholm, Klaus; Lenskjold, Toke; Jacoby, Anne Sophie

    2016-01-01

    cells were quantitated using enzyme immunometric assays. The activity of GSK-3β (serine-9-phosphorylated GSK-3β/total GSK-3β) was lower at baseline compared with follow-up. No significant mean change over time was observed in levels of total GSK-3β and serine-9-phosphorylated GSK-3β. Exploratory......Evidence indicates a role for glycogen synthase kinase-3β (GSK-3β) in the pathophysiology of mood disorders and in cognitive disturbances; however, the natural variation in GSK-3β activity over time is unknown. We aimed to investigate GSK-3β activity over time and its possible correlation...... with emotional lability, subjective mood fluctuations and cognitive function in healthy individuals. Thirty-seven healthy subjects were evaluated with neuropsychological tests and blood samples at baseline and 12-week follow-up. Total GSK-3β and serine-9-phosphorylated GSK-3β in peripheral blood mononuclear...

  4. Anks3 alters the sub-cellular localization of the Nek7 kinase

    Energy Technology Data Exchange (ETDEWEB)

    Ramachandran, Haribaskar; Engel, Christina; Müller, Barbara [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Dengjel, Jörn [Department of Dermatology, University Freiburg Medical Center and Center of Biological Systems Analysis, Habsburgerstr. 49, 79104 Freiburg (Germany); Walz, Gerd [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany); Center for Biological Signaling Studies (BIOSS), Albertstr. 19, 79104 Freiburg (Germany); Yakulov, Toma A., E-mail: toma.antonov.yakulov@uniklinik-freiburg.de [Renal Division, Department of Medicine, University Freiburg Medical Center, Hugstetter Str. 55, 79106 Freiburg (Germany)

    2015-08-28

    Nephronophthisis (NPH) is an autosomal recessive cystic kidney disease, and a frequent cause of end-stage renal failure in children. To date, 17 NPH-associated gene products (NPHPs) have been identified. Most NPHPs participate in large multi-protein complexes that localize to the cilium and/or basal body; however, the precise composition of these complexes and their biological function remain largely unknown. We recently observed that the ankyrin repeat protein Anks3 interacts with the NPH family member Anks6. Both Anks3 and Anks6 form complexes with multiple other NPHPs, suggesting that both proteins function in similar or overlapping signaling pathways. Here, we show that Anks3, but not Anks6 interacted with the NIMA-related kinase Nek7, and was heavily modified in the presence of Nek7, resulting in an approximately 20 kD increase in molecular weight. Although mass spectrometry revealed increased serine and threonine phosphorylation of Anks3 primarily within the N-terminal ankyrin repeats also required for Nek7 interaction, the molecular weight increase occurred even in the presence of a kinase-dead Nek7 mutant, indicating that this modification was not caused by Nek7-dependent Anks3 phosphorylation. Furthermore, the Anks3 modification was specific for Nek7, and did not occur in the presence of Nek8. Importantly, Anks3 retained Nek7 in the cytoplasm, suggesting that, Nek7 triggers the modification of Anks3, which in turn prevents the nuclear localization of Nek7. - Highlights: • Anks3 interacted with Nek7 kinase, and was heavily modified in the presence of Nek7. • Anks3 N-terminal ankyrin repeats, but not SAM domain required for Nek7 interaction. • Nek7 increased Ser/Thr phosphorylation of Anks3 primarily within ankyrin domain. • Interaction with Anks3 led to cytoplasmic retention and nuclear exclusion of Nek7.

  5. Functional characterization of a constitutively active kinase variant of Arabidopsis phototropin 1.

    Science.gov (United States)

    Petersen, Jan; Inoue, Shin-Ichiro; Kelly, Sharon M; Sullivan, Stuart; Kinoshita, Toshinori; Christie, John M

    2017-08-18

    Phototropins (phots) are plasma membrane-associated serine/threonine kinases that coordinate a range of processes linked to optimizing photosynthetic efficiency in plants. These photoreceptors contain two light-, oxygen-, or voltage-sensing (LOV) domains within their N terminus, with each binding one molecule of flavin mononucleotide as a UV/blue light-absorbing chromophore. Although phots contain two LOV domains, light-induced activation of the C-terminal kinase domain and subsequent receptor autophosphorylation is controlled primarily by the A'α-LOV2-Jα photosensory module. Mutations that disrupt interactions between the LOV2 core and its flanking helical segments can uncouple this mode of light regulation. However, the impact of these mutations on phot function in Arabidopsis has not been explored. Here we report that histidine substitution of Arg-472 located within the A'α-helix of Arabidopsis phot1 constitutively activates phot1 kinase activity in vitro without affecting LOV2 photochemistry. Expression analysis of phot1 R472H in the phot-deficient mutant confirmed that it is autophosphorylated in darkness in vivo but unable to initiate phot1 signaling in the absence of light. Instead, we found that phot1 R472H is poorly functional under low-light conditions but can restore phototropism, chloroplast accumulation, stomatal opening, and leaf positioning and expansion at higher light intensities. Our findings suggest that Arabidopsis can adapt to the elevated phosphorylation status of the phot1 R472H mutant in part by reducing its stability, whereas the activity of the mutant under high-light conditions can be attributed to additional increases in LOV2-mediated photoreceptor autophosphorylation. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Roles of protein kinase R in cancer: Potential as a therapeutic target.

    Science.gov (United States)

    Watanabe, Takao; Imamura, Takeshi; Hiasa, Yoichi

    2018-04-01

    Double-stranded (ds) RNA-dependent protein kinase (PKR) is a ubiquitously expressed serine/threonine protein kinase. It was initially identified as an innate immune antiviral protein induced by interferon (IFN) and activated by dsRNA. PKR is recognized as a key executor of antiviral host defense. Moreover, it contributes to inflammation and immune regulation through several signaling pathways. In addition to IFN and dsRNA, PKR is activated by multiple stimuli and regulates various signaling pathways including the mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells pathways. PKR was initially thought to be a tumor suppressor as a result of its ability to suppress cell growth and interact with major tumor suppressor genes. However, in several types of malignant disease, such as colon and breast cancers, its role remains controversial. In hepatocellular carcinoma, hepatitis C virus (HCV) is the main cause of liver cancer, and PKR inhibits HCV replication, indicating its role as a tumor suppressor. However, PKR is overexpressed in cirrhotic patients, and acts as a tumor promoter through enhancement of cancer cell growth by mediating MAPK or signal transducer and activator of transcription pathways. Moreover, PKR is reportedly required for the activation of inflammasomes and influences metabolic disorders. In the present review, we introduce the multifaceted roles of PKR such as antiviral function, tumor cell growth, regulation of inflammatory immune responses, and maintaining metabolic homeostasis; and discuss future perspectives on PKR biology including its potential as a therapeutic target for liver cancer. © 2018 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  7. Protein kinase G confers survival advantage to Mycobacterium tuberculosis during latency-like conditions.

    Science.gov (United States)

    Khan, Mehak Zahoor; Bhaskar, Ashima; Upadhyay, Sandeep; Kumari, Pooja; Rajmani, Raju S; Jain, Preeti; Singh, Amit; Kumar, Dhiraj; Bhavesh, Neel Sarovar; Nandicoori, Vinay Kumar

    2017-09-29

    Protein kinase G (PknG), a thioredoxin-fold-containing eukaryotic-like serine/threonine protein kinase, is a virulence factor in Mycobacterium tuberculosis , required for inhibition of phagolysosomal fusion. Here, we unraveled novel functional facets of PknG during latency-like conditions. We found that PknG mediates persistence under stressful conditions like hypoxia and abets drug tolerance. PknG mutant displayed minimal growth in nutrient-limited conditions, suggesting its role in modulating cellular metabolism. Intracellular metabolic profiling revealed that PknG is necessary for efficient metabolic adaptation during hypoxia. Notably, the PknG mutant exhibited a reductive shift in mycothiol redox potential and compromised stress response. Exposure to antibiotics and hypoxic environment resulted in higher oxidative shift in mycothiol redox potential of PknG mutant compared with the wild type. Persistence during latency-like conditions required kinase activity and thioredoxin motifs of PknG and is mediated through phosphorylation of a central metabolic regulator GarA. Finally, using a guinea pig model of infection, we assessed the in vivo role of PknG in manifestation of disease pathology and established a role for PknG in the formation of stable granuloma, hallmark structures of latent tuberculosis. Taken together, PknG-mediated GarA phosphorylation is important for maintenance of both mycobacterial physiology and redox poise, an axis that is dispensable for survival under normoxic conditions but is critical for non-replicating persistence of mycobacteria. In conclusion, we propose that PknG probably acts as a modulator of latency-associated signals. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  8. Role of myotonic dystrophy protein kinase (DMPK in glucose homeostasis and muscle insulin action.

    Directory of Open Access Journals (Sweden)

    Esther Llagostera

    2007-11-01

    Full Text Available Myotonic dystrophy 1 (DM1 is caused by a CTG expansion in the 3'-unstranslated region of the DMPK gene, which encodes a serine/threonine protein kinase. One of the common clinical features of DM1 patients is insulin resistance, which has been associated with a pathogenic effect of the repeat expansions. Here we show that DMPK itself is a positive modulator of insulin action. DMPK-deficient (dmpk-/- mice exhibit impaired insulin signaling in muscle tissues but not in adipocytes and liver, tissues in which DMPK is not expressed. Dmpk-/- mice display metabolic derangements such as abnormal glucose tolerance, reduced glucose uptake and impaired insulin-dependent GLUT4 trafficking in muscle. Using DMPK mutants, we show that DMPK is required for a correct intracellular trafficking of insulin and IGF-1 receptors, providing a mechanism to explain the molecular and metabolic phenotype of dmpk-/- mice. Taken together, these findings indicate that reduced DMPK expression may directly influence the onset of insulin-resistance in DM1 patients and point to dmpk as a new candidate gene for susceptibility to type 2-diabetes.

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

    Science.gov (United States)

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

    2017-08-15

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

  10. The Ser/Thr Protein Kinase Protein-Protein Interaction Map of M. tuberculosis.

    Science.gov (United States)

    Wu, Fan-Lin; Liu, Yin; Jiang, He-Wei; Luan, Yi-Zhao; Zhang, Hai-Nan; He, Xiang; Xu, Zhao-Wei; Hou, Jing-Li; Ji, Li-Yun; Xie, Zhi; Czajkowsky, Daniel M; Yan, Wei; Deng, Jiao-Yu; Bi, Li-Jun; Zhang, Xian-En; Tao, Sheng-Ce

    2017-08-01

    Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis, the leading cause of death among all infectious diseases. There are 11 eukaryotic-like serine/threonine protein kinases (STPKs) in Mtb, which are thought to play pivotal roles in cell growth, signal transduction and pathogenesis. However, their underlying mechanisms of action remain largely uncharacterized. In this study, using a Mtb proteome microarray, we have globally identified the binding proteins in Mtb for all of the STPKs, and constructed the first STPK protein interaction (KPI) map that includes 492 binding proteins and 1,027 interactions. Bioinformatics analysis showed that the interacting proteins reflect diverse functions, including roles in two-component system, transcription, protein degradation, and cell wall integrity. Functional investigations confirmed that PknG regulates cell wall integrity through key components of peptidoglycan (PG) biosynthesis, e.g. MurC. The global STPK-KPIs network constructed here is expected to serve as a rich resource for understanding the key signaling pathways in Mtb, thus facilitating drug development and effective control of Mtb. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  11. Rho Kinase (ROCK) collaborates with Pak to Regulate Actin Polymerization and Contraction in Airway Smooth Muscle.

    Science.gov (United States)

    Zhang, Wenwu; Bhetwal, Bhupal P; Gunst, Susan J

    2018-05-10

    The mechanisms by which Rho kinase (ROCK) regulates airway smooth muscle contraction were determined in tracheal smooth muscle tissues. ROCK may mediate smooth muscle contraction by inhibiting myosin regulatory light chain (RLC) phosphatase. ROCK can also regulate F-actin dynamics during cell migration, and actin polymerization is critical for airway smooth muscle contraction. Our results show that ROCK does not regulate airway smooth muscle contraction by inhibiting myosin RLC phosphatase or by stimulating myosin RLC phosphorylation. We find that ROCK regulates airway smooth muscle contraction by activating the serine-threonine kinase Pak, which mediates the activation of Cdc42 and Neuronal-Wiskott-Aldrich Syndrome protein (N-WASp). N-WASP transmits signals from cdc42 to the Arp2/3 complex for the nucleation of actin filaments. These results demonstrate a novel molecular function for ROCK in the regulation of Pak and cdc42 activation that is critical for the processes of actin polymerization and contractility in airway smooth muscle. Rho kinase (ROCK), a RhoA GTPase effector, can regulate the contraction of airway and other smooth muscle tissues. In some tissues, ROCK can inhibit myosin regulatory light chain (RLC) phosphatase, which increases the phosphorylation of myosin RLC and promotes smooth muscle contraction. ROCK can also regulate cell motility and migration by affecting F-actin dynamics. Actin polymerization is stimulated by contractile agonists in airway smooth muscle tissues and is required for contractile tension development in addition to myosin RLC phosphorylation. We investigated the mechanisms by which ROCK regulates the contractility of tracheal smooth muscle tissues by expressing a kinase inactive mutant of ROCK, ROCK-K121G, in the tissues or by treating them with the ROCK inhibitor, H-1152P. Our results show no role for ROCK in the regulation of non-muscle or smooth muscle myosin RLC phosphorylation during contractile stimulation in this tissue

  12. Characterization of a rabbit polyclonal antibody against threonine-AMPylation

    Science.gov (United States)

    Hao, Yi-Heng; Chuang, Trinette; Ball, Haydn L.; Luong, Phi; Li, Yan; Flores-Saaib, Ruben D.; Orth, Kim

    2014-01-01

    An antibody against the posttranslational modification AMPylation was produced using a peptide corresponding to human Rac1 switch I region with AMPylated threonine-35 residue as an antigen. The resulting rabbit antiserum was tested for its abilities to recognize AMPylated proteins by western blot and immunoprecipitation. The antiserum is highly specific for threonine-AMPylated proteins and weakly recognizes tyrosine-AMPylated proteins. Depletion of serum with modified protein abolished its activity against tyrosine-AMPylated proteins. The antiserum also recognized native proteins with modification in an immunoprecipitation experiment. Interactions of the antiserum could be inhibited by competition with AMP but not with GMP or UMP. This antiserum had potential utility for the identification of unknown AMPylated proteins. PMID:21185336

  13. Computational-experimental approach to drug-target interaction mapping: A case study on kinase inhibitors.

    Directory of Open Access Journals (Sweden)

    Anna Cichonska

    2017-08-01

    Full Text Available Due to relatively high costs and labor required for experimental profiling of the full target space of chemical compounds, various machine learning models have been proposed as cost-effective means to advance this process in terms of predicting the most potent compound-target interactions for subsequent verification. However, most of the model predictions lack direct experimental validation in the laboratory, making their practical benefits for drug discovery or repurposing applications largely unknown. Here, we therefore introduce and carefully test a systematic computational-experimental framework for the prediction and pre-clinical verification of drug-target interactions using a well-established kernel-based regression algorithm as the prediction model. To evaluate its performance, we first predicted unmeasured binding affinities in a large-scale kinase inhibitor profiling study, and then experimentally tested 100 compound-kinase pairs. The relatively high correlation of 0.77 (p < 0.0001 between the predicted and measured bioactivities supports the potential of the model for filling the experimental gaps in existing compound-target interaction maps. Further, we subjected the model to a more challenging task of predicting target interactions for such a new candidate drug compound that lacks prior binding profile information. As a specific case study, we used tivozanib, an investigational VEGF receptor inhibitor with currently unknown off-target profile. Among 7 kinases with high predicted affinity, we experimentally validated 4 new off-targets of tivozanib, namely the Src-family kinases FRK and FYN A, the non-receptor tyrosine kinase ABL1, and the serine/threonine kinase SLK. Our sub-sequent experimental validation protocol effectively avoids any possible information leakage between the training and validation data, and therefore enables rigorous model validation for practical applications. These results demonstrate that the kernel

  14. AMP-activated protein kinase regulates lymphocyte responses to metabolic stress but is largely dispensable for immune cell development and function.

    Science.gov (United States)

    Mayer, Alice; Denanglaire, Sébastien; Viollet, Benoit; Leo, Oberdan; Andris, Fabienne

    2008-04-01

    AMP-activated protein kinase (AMPK), a phylogenetically conserved serine/threonine protein kinase, represents an energy sensor able to adapt cellular metabolism in response to nutritional environmental variations. TCR stimulation activates AMPK, a regulatory event that is known to stimulate ATP-producing processes, possibly in anticipation of the increased energetic needs associated with cell division and expression of effector function. Taking advantage of the selective expression of the AMPKalpha1 catalytic subunit in lymphoid cells, we have analyzed the in vitro and in vivo capacity of lymphocytes lacking AMPK activity (AMPKalpha1-KO cells) to respond to metabolic stress and to initiate and sustain an immune response. AMPKalpha1-KO cells displayed increasing sensitivity to energetic stress in vitro, and were found unable to maintain adequate ATP levels in response to ATP synthase inhibition. These cells were, however, able to respond to antigen stimulation in vitro, as shown by optimal proliferation and cytokine production. Similarly, AMPKalpha1-KO mice were fully immunocompetent in vivo and displayed normal cell proliferation, humoral, cytotoxic and delayed-type hypersensitivity (DTH) responses following antigen injection. In conclusion, AMPK represents an important enzyme allowing lymphocytes to resist a mild energy crisis in vitro, but is largely dispensable for activation and expression of effector function in response to antigen stimulation.

  15. In Silico Exploration of 1,7-Diazacarbazole Analogs as Checkpoint Kinase 1 Inhibitors by Using 3D QSAR, Molecular Docking Study, and Molecular Dynamics Simulations

    Directory of Open Access Journals (Sweden)

    Xiaodong Gao

    2016-05-01

    Full Text Available Checkpoint kinase 1 (Chk1 is an important serine/threonine kinase with a self-protection function. The combination of Chk1 inhibitors and anti-cancer drugs can enhance the selectivity of tumor therapy. In this work, a set of 1,7-diazacarbazole analogs were identified as potent Chk1 inhibitors through a series of computer-aided drug design processes, including three-dimensional quantitative structure–activity relationship (3D-QSAR modeling, molecular docking, and molecular dynamics simulations. The optimal QSAR models showed significant cross-validated correlation q2 values (0.531, 0.726, fitted correlation r2 coefficients (higher than 0.90, and standard error of prediction (less than 0.250. These results suggested that the developed models possess good predictive ability. Moreover, molecular docking and molecular dynamics simulations were applied to highlight the important interactions between the ligand and the Chk1 receptor protein. This study shows that hydrogen bonding and electrostatic forces are key interactions that confer bioactivity.

  16. The Role of Unfolded Protein Response and Mitogen-Activated Protein Kinase Signaling in Neurodegenerative Diseases with Special Focus on Prion Diseases

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    Lifeng Yang

    2017-05-01

    Full Text Available Prion diseases are neurodegenerative pathologies characterized by the accumulation of a protease-resistant form of the cellular prion protein named prion protein scrapie (PrPSc in the brain. PrPSc accumulation in the endoplasmic reticulum (ER result in a dysregulated calcium (Ca2+ homeostasis and subsequent initiation of unfolded protein response (UPR leading to neuronal dysfunction and apoptosis. The molecular mechanisms for the transition between adaptation to ER stress and ER stress-induced apoptosis are still unclear. Mitogen-activated protein kinases (MAPKs are serine/threonine protein kinases that rule the signaling of many extracellular stimuli from plasma membrane to the nucleus. However the identification of numerous points of cross talk between the UPR and MAPK signaling pathways may contribute to our understanding of the consequences of ER stress in prion diseases. Indeed the MAPK signaling network is known to regulate cell cycle progression and cell survival or death responses following a variety of stresses including misfolded protein response stress. In this article, we review the UPR signaling in prion diseases and discuss the triad of MAPK signaling pathways. We also describe the role played by MAPK signaling cascades in Alzheimer’s (AD and Parkinson’s disease (PD. We will also overview the mechanisms of cell death and the role of MAPK signaling in prion disease progression and highlight potential avenues for therapeutic intervention.

  17. Alternative splicing of the porcine glycogen synthase kinase 3β (GSK-3β gene with differential expression patterns and regulatory functions.

    Directory of Open Access Journals (Sweden)

    Linjie Wang

    Full Text Available Glycogen synthase kinase 3 (GSK3α and GSK3β are serine/threonine kinases involved in numerous cellular processes and diverse diseases including mood disorders, Alzheimer's disease, diabetes, and cancer. However, in pigs, the information on GSK3 is very limited. Identification and characterization of pig GSK3 are not only important for pig genetic improvement, but also contribute to the understanding and development of porcine models for human disease prevention and treatment.Five different isoforms of GSK3β were identified in porcine different tissues, in which three isoforms are novel. These isoforms had differential expression patterns in the fetal and adult of the porcine different tissues. The mRNA expression level of GSK3β isoforms was differentially regulated during the course of the insulin treatment, suggesting that different GSK3β isoforms may have different roles in insulin signaling pathway. Moreover, GSK3β5 had a different role on regulating the glycogen synthase activity, phosphorylation and the expression of porcine GYS1 and GYS2 gene compared to other GSK3β isoforms.We are the first to report five different isoforms of GSK3β identified from the porcine different tissues. Splice variants of GSK3β exhibit differential activity towards glycogen synthase. These results provide new insight into roles of the GSK3β on regulating glycogen metabolism.

  18. PF-4708671, a specific inhibitor of p70 ribosomal S6 kinase 1, activates Nrf2 by promoting p62-dependent autophagic degradation of Keap1

    Energy Technology Data Exchange (ETDEWEB)

    Park, Jeong Su [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Kang, Dong Hoon [Department of Life Science and Ewha Research Center for Systems Biology (Korea, Republic of); The Research Center for Cell Homeostasis, Ewha Womans University, Seoul 127-750 (Korea, Republic of); Lee, Da Hyun [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of); Bae, Soo Han, E-mail: soohanbae@yuhs.ac [Severance Biomedical Science Institute (Korea, Republic of); Yonsei Biomedical Research Institute, Yonsei University College of Medicine, 50 Yonsei-ro, Seodaemun-gu, Seoul 120-752 (Korea, Republic of)

    2015-10-23

    p70 ribosomal S6 kinase 1 (S6K1) is an important serine/threonine kinase and downstream target of the mechanistic target of rapamycin complex 1 (mTORC1) signaling pathway. PF-4708671 is a specific inhibitor of S6K1, and prevents S6K1-mediated phosphorylation of the S6 protein. PF-4708671 treatment often leads to apoptotic cell death. However, the protective mechanism against PF-4708671-induced cell death has not been elucidated. The nuclear factor erythroid 2-related factor 2 (Nrf2)-Kelch-like ECH-associated protein 1 (Keap1) pathway is essential for protecting cells against oxidative stress. p62, an adaptor protein in the autophagic process, enhances Nrf2 activation through the impairment of Keap1 activity. In this study, we showed that PF-4708671 induces autophagic Keap1 degradation-mediated Nrf2 activation in p62-dependent manner. Furthermore, p62-dependent Nrf2 activation plays a crucial role in protecting cells from PF-4708671-mediated apoptosis. - Highlights: • PF-4708671, a S6K1-specific inhibitor, prevents S6K1-mediated S6 phosphorylation. • However, PF-4708671 treatment often leads to apoptotic cell death. • Protective mechanism against PF-4708671-induced cell death remains to be elucidated. • PF-4708671 induced p62-dependent, autophagic Keap1 degradation-mediated Nrf2 activation. • p62-dependent Nrf2 activation protects cells from PF-4708671-mediated apoptosis.

  19. The discovery and the structural basis of an imidazo[4,5-b]pyridine-based p21-activated kinase 4 inhibitor.

    Science.gov (United States)

    Park, Jeung Kuk; Kim, Sunmin; Han, Yu Jin; Kim, Seong Hwan; Kang, Nam Sook; Lee, Hyuk; Park, SangYoun

    2016-06-01

    p21-Activated kinases (PAKs) which belong to the family of ste20 serine/threonine protein kinases regulate cytoskeletal reorganization, cell motility, cell proliferation, and oncogenic transformation which are all related to the cellular functions during cancer induction and metastasis. The fact that PAK mutations are detected in multiple tumor tissues makes PAKs a novel therapeutic drug target. In this study, an imidazo[4,5-b]pyridine-based PAK4 inhibitor, KY-04045 (6-Bromo-2-(3-isopropyl-1-methyl-1H-pyrazol-4-yl)-1H-imidazo[4,5-b]pyridine), was discovered using a virtual site-directed fragment-based drug design and was validated using an inhibition assay. Although PAK4 affinity to KY-04045 seems much weaker than that of the reported PAK4 inhibitors, the location of KY-04045 is clearly defined in the structure of PAK4 co-crystallized with KY-04045. The crystal structure illustrates that the pyrazole and imidazopyridine rings of KY-04045 are sufficient for mediating PAK4 hinge loop interaction. Hence, we believe that KY-04045 can be exploited as a basic building block in designing novel imidazo[4,5-b]pyridine-based PAK4 inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

  20. Imoxin attenuates high fructose-induced oxidative stress and apoptosis in renal epithelial cells via downregulation of protein kinase R pathway.

    Science.gov (United States)

    Kalra, Jaspreet; Mangali, Suresh Babu; Bhat, Audesh; Dhar, Indu; Udumula, Mary Priyanka; Dhar, Arti

    2018-02-11

    Double-stranded RNA (dsRNA)-activated protein kinase R (PKR), a ubiquitously expressed serine/threonine kinase, is a key inducer of inflammation, insulin resistance, and glucose homeostasis in obesity. Recent studies have demonstrated that PKR can respond to metabolic stress in mice as well as in humans. However, the underlying molecular mechanism is not fully understood. The aim of this study was to examine the effect of high fructose (HF) in cultured renal tubular epithelial cells (NRK-52E) derived from rat kidney and to investigate whether inhibition of PKR could prevent any deleterious effects of HF in these cells. PKR expression was determined by immunofluorescence staining and Western blotting. Oxidative damage and apoptosis were measured by flow cytometry. HF-treated renal cells developed a significant increase in PKR expression. A significant increase in reactive oxygen species generation and apoptosis was also observed in HF-treated cultured renal epithelial cells. All these effects of HF were attenuated by a selective PKR inhibitor, imoxin (C16). In conclusion, our study demonstrates PKR induces oxidative stress and apoptosis, is a significant contributor involved in vascular complications and is a possible mediator of HF-induced hypertension. Inhibition of PKR pathway can be used as a therapeutic strategy for the treatment of cardiovascular and metabolic disorders. © 2018 Société Française de Pharmacologie et de Thérapeutique.

  1. The Kinase STK3 Interacts with the Viral Structural Protein VP1 and Inhibits Foot-and-Mouth Disease Virus Replication

    Science.gov (United States)

    Xue, Qiao

    2017-01-01

    Foot-and-mouth disease virus (FMDV) is the etiological agent of FMD, which affects domestic and wild cloven-hoofed animals. The structural protein VP1 plays an important role in FMDV pathogenesis. However, the interacting partners of VP1 in host cells and the effects of these interactions in FMDV replication remain incompletely elucidated. Here, we identified a porcine cell protein, serine/threonine kinase 3 (STK3), which interacts with FMDV VP1 using the yeast two-hybrid system. The VP1-STK3 interaction was further confirmed by coimmunoprecipitation experiments in human embryonic kidney 293T and porcine kidney 15 (PK-15) cells. The carboxyl-terminal region (amino acids 180–214) of VP1 was essential for its interaction with STK3. The effects of overexpression and underexpressing of STK3 in PK-15 cells were assessed, and the results indicated that STK3 significantly inhibited FMDV replication. Our data expand the role of STK3 during viral infection, provide new information regarding the host cell kinases that are involved in viral replication, and identify potential targets for future antiviral strategies. PMID:29226127

  2. Mitogen activated protein kinase 6 and MAP kinase phosphatase 1 are involved in the response of Arabidopsis roots to L-glutamate.

    Science.gov (United States)

    López-Bucio, Jesús Salvador; Raya-González, Javier; Ravelo-Ortega, Gustavo; Ruiz-Herrera, León Francisco; Ramos-Vega, Maricela; León, Patricia; López-Bucio, José; Guevara-García, Ángel Arturo

    2018-03-01

    The function and components of L-glutamate signaling pathways in plants have just begun to be elucidated. Here, using a combination of genetic and biochemical strategies, we demonstrated that a MAPK module is involved in the control of root developmental responses to this amino acid. Root system architecture plays an essential role in plant adaptation to biotic and abiotic factors via adjusting signal transduction and gene expression. L-Glutamate (L-Glu), an amino acid with neurotransmitter functions in animals, inhibits root growth, but the underlying genetic mechanisms are poorly understood. Through a combination of genetic analysis, in-gel kinase assays, detailed cell elongation and division measurements and confocal analysis of expression of auxin, quiescent center and stem cell niche related genes, the critical roles of L-Glu in primary root growth acting through the mitogen-activated protein kinase 6 (MPK6) and the dual specificity serine-threonine-tyrosine phosphatase MKP1 could be revealed. In-gel phosphorylation assays revealed a rapid and dose-dependent induction of MPK6 and MPK3 activities in wild-type Arabidopsis seedlings in response to L-Glu. Mutations in MPK6 or MKP1 reduced or increased root cell division and elongation in response to L-Glu, possibly modulating auxin transport and/or response, but in a PLETHORA1 and 2 independent manner. Our data highlight MPK6 and MKP1 as components of an L-Glu pathway linking the auxin response, and cell division for primary root growth.

  3. Protein kinase C signaling and cell cycle regulation

    Directory of Open Access Journals (Sweden)

    Adrian R Black

    2013-01-01

    Full Text Available A link between T cell proliferation and the protein kinase C (PKC family of serine/threonine kinases has been recognized for about thirty years. However, despite the wealth of information on PKC-mediated control of T cell activation, understanding of the effects of PKCs on the cell cycle machinery in this cell type remains limited. Studies in other systems have revealed important cell cycle-specific effects of PKC signaling that can either positively or negatively impact proliferation. The outcome of PKC activation is highly context-dependent, with the precise cell cycle target(s and overall effects determined by the specific isozyme involved, the timing of PKC activation, the cell type, and the signaling environment. Although PKCs can regulate all stages of the cell cycle, they appear to predominantly affect G0/G1 and G2. PKCs can modulate multiple cell cycle regulatory molecules, including cyclins, cyclin-dependent kinases (cdks, cdk inhibitors and cdc25 phosphatases; however, evidence points to Cip/Kip cdk inhibitors and D-type cyclins as key mediators of PKC-regulated cell cycle-specific effects. Several PKC isozymes can target Cip/Kip proteins to control G0/G1→S and/or G2→M transit, while effects on D-type cyclins regulate entry into and progression through G1. Analysis of PKC signaling in T cells has largely focused on its roles in T cell activation; thus, observed cell cycle effects are mainly positive. A prominent role is emerging for PKCθ, with non-redundant functions of other isozymes also described. Additional evidence points to PKCδ as a negative regulator of the cell cycle in these cells. As in other cell types, context-dependent effects of individual isozymes have been noted in T cells, and Cip/Kip cdk inhibitors and D-type cyclins appear to be major PKC targets. Future studies are anticipated to take advantage of the similarities between these various systems to enhance understanding of PKC-mediated cell cycle regulation in

  4. NCBI nr-aa BLAST: CBRC-ACAR-01-1202 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-ACAR-01-1202 ref|ZP_01461017.1| serine/threonine-protein kinase Pkn6 [Stigmate...lla aurantiaca DW4/3-1] gb|EAU68211.1| serine/threonine-protein kinase Pkn6 [Stigmatella aurantiaca DW4/3-1] ZP_01461017.1 1.9 31% ...

  5. NCBI nr-aa BLAST: CBRC-OSAT-05-0034 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-05-0034 gb|AAM20520.1| serine/threonine protein kinase isolog [Arabidopsi...s thaliana] gb|AAO30076.1| serine/threonine protein kinase isolog [Arabidopsis thaliana] AAM20520.1 1e-156 49% ...

  6. NCBI nr-aa BLAST: CBRC-OSAT-01-0062 [SEVENS

    Lifescience Database Archive (English)

    Full Text Available CBRC-OSAT-01-0062 gb|AAM20520.1| serine/threonine protein kinase isolog [Arabidopsi...s thaliana] gb|AAO30076.1| serine/threonine protein kinase isolog [Arabidopsis thaliana] AAM20520.1 1e-152 47% ...

  7. Glycine serine interconversion in the rooster

    International Nuclear Information System (INIS)

    Sugahara, Michihiro; Kandatsu, Makoto

    1976-01-01

    Serine was isolated by the column chromatography from the hydrolyzates of proteins of the serum, the liver and the pectoral muscle which were obtained from the roosters fed a diet containing 2- 14 C glycine for 16 - 17 days. The carbon chain of serine was cut off by treating with sodium periodate. The specific activity of each carbon (as barium carbonate) was estimated. Carboxyl carbon had little radioactivity. The specific activity of hydroxymethyl carbon was 10 - 19% of that of methylene carbon. Glycine isolated from the same hydrolyzates was degraded by ninhydrin oxidation. Formaldehyde produced from 2-C was oxidized to carbon dioxide by treating with mercuric chloride. Carboxyl carbon had little radioactivity. The specific activities of 2-C of glycine and 2-C of serine in the same tissue protein were compared. The ratio of serine 2-C/glycine 2-C was between 0.7 - 1.5. These results seem to indicate that glycine directly converts to serine in the rooster. The quantitative significance of the pathways of glycine (serine) biosynthesis is discussed. (auth.)

  8. D-serine increases adult hippocampal neurogenesis

    Directory of Open Access Journals (Sweden)

    Sebastien eSultan

    2013-08-01

    Full Text Available Adult hippocampal neurogenesis results in the continuous formation of new neurons and is a process of brain plasticity involved in learning and memory. The neurogenic niche regulates the stem cell proliferation and the differentiation and survival of new neurons and a major contributor to the neurogenic niche are astrocytes. Among the molecules secreted by astrocytes, D-serine is an important gliotransmitter and is a co-agonist of the glutamate, N-methyl-D-aspartate (NMDA receptor. D-serine has been shown to enhance the proliferation of neural stem cells in vitro, but its effect on adult neurogenesis in vivo is unknown. Here, we tested the effect of exogenous administration of D-serine on adult neurogenesis in the mouse dentate gyrus. We found that 1 week of treatment with D-serine increased cell proliferation in vivo and in vitro and increased the density of neural stem cells and transit amplifying progenitors. Furthermore, D-serine increased the survival of newborn neurons. Together, these results indicate that D-serine treatment resulted in the improvement of several steps of adult neurogenesis in vivo.

  9. Potential role of p21 Activated Kinase 1 (PAK1) in the invasion and motility of oral cancer cells

    International Nuclear Information System (INIS)

    Parvathy, Muraleedharan; Sreeja, Sreeharshan; Kumar, Rakesh; Pillai, Madhavan Radhakrishna

    2016-01-01

    Oral cancer malignancy consists of uncontrolled division of cells primarily in and around the floor of the oral cavity, gingiva, oropharynx, lower lip and base of the tongue. According to GLOBOCAN 2012 report, oral cancer is one of the most common cancers among males and females in India. Even though significant advancements have been made in the field of oral cancer treatment modalities, the overall prognosis for the patients has not improved in the past few decades and hence, this demands a new thrust for the identification of novel therapeutic targets in oral cancer. p21 Activated Kinases (PAKs) are potential therapeutic targets that are involved in numerous physiological functions. PAKs are serine-threonine kinases and they serve as important regulators of cytoskeletal dynamics and cell motility, transcription through MAP kinase cascades, death and survival signalling, and cell-cycle progression. Although PAKs are known to play crucial roles in cancer progression, the role and clinical significance of PAKs in oral cancer remains poorly understood. Our results suggest that PAK1 is over-expressed in oral cancer cell lines. Stimulation of Oral Squamous Cell Carcinoma (OSCC) cells with serum growth factors leads to PAK1 re-localization and might cause a profound cytoskeletal remodelling. PAK1 was also found to be involved in the invasion, migration and cytoskeletal remodelling of OSCC cells. Our study revealed that PAK1 may play a crucial role in the progression of OSCC. Studying the role of PAK1 and its substrates is likely to enhance our understanding of oral carcinogenesis and potential therapeutic value of PAKs in oral cancer. The online version of this article (doi:10.1186/s12885-016-2263-8) contains supplementary material, which is available to authorized users

  10. Toward a Comprehensive Phylogenetic Reconstruction of the Evolutionary History of Mitogen-Activated Protein Kinases in the Plant Kingdom

    OpenAIRE

    Janitza, Philipp; Ullrich, Kristian Karsten; Quint, Marcel

    2012-01-01

    The mitogen-activated protein kinase (MAPK) pathway is a three-tier signaling cascade that transmits cellular information from the plasma membrane to the cytoplasm where it triggers downstream responses. The MAPKs represent the last step in this cascade and are activated when both tyrosine and threonine residues in a conserved TxY motif are phosphorylated by MAPK kinases, which in turn are themselves activated by phosphorylation by MAPK kinase kinases. To understand the molecular evolution of...

  11. Macrophage Liver Kinase B1 Inhibits Foam Cell Formation and Atherosclerosis.

    Science.gov (United States)

    Liu, Zhaoyu; Zhu, Huaiping; Dai, Xiaoyan; Wang, Cheng; Ding, Ye; Song, Ping; Zou, Ming-Hui

    2017-10-13

    LKB1 (liver kinase B1) is a serine/threonine kinase and tumor suppressor, which regulates the homeostasis of hematopoietic cells and immune responses. Macrophages transform into foam cells upon taking-in lipids. No role for LKB1 in foam cell formation has previously been reported. We sought to establish the role of LKB1 in atherosclerotic foam cell formation. LKB1 expression was examined in human carotid atherosclerotic plaques and in western diet-fed atherosclerosis-prone Ldlr -/- and ApoE -/- mice. LKB1 expression was markedly reduced in human plaques when compared with nonatherosclerotic vessels. Consistently, time-dependent reduction of LKB1 levels occurred in atherosclerotic lesions in western diet-fed Ldlr -/- and ApoE -/- mice. Exposure of macrophages to oxidized low-density lipoprotein downregulated LKB1 in vitro. Furthermore, LKB1 deficiency in macrophages significantly increased the expression of SRA (scavenger receptor A), modified low-density lipoprotein uptake and foam cell formation, all of which were abolished by blocking SRA. Further, we found LKB1 phosphorylates SRA resulting in its lysosome degradation. To further investigate the role of macrophage LKB1 in vivo, ApoE -/- LKB1 fl/fl LysM cre and ApoE -/- LKB1 fl/fl mice were fed with western diet for 16 weeks. Compared with ApoE -/- LKB1 fl/fl wild-type control, ApoE -/- LKB1 fl/fl LysM cre mice developed more atherosclerotic lesions in whole aorta and aortic root area, with markedly increased SRA expression in aortic root lesions. We conclude that macrophage LKB1 reduction caused by oxidized low-density lipoprotein promotes foam cell formation and the progression of atherosclerosis. © 2017 American Heart Association, Inc.

  12. Dysfunction of G Protein-Coupled Receptor Kinases in Alzheimer’'s Disease

    Directory of Open Access Journals (Sweden)

    William Z. Suo

    2010-01-01

    Full Text Available Although mutations and variations of several genes have been identified to be involved in Alzheimer's disease (AD, the efforts towards understanding the pathogenic mechanisms of the disease still have a long journey to go. One such effort is to identify the signal transduction deficits, for which previous studies have suggested that the central problems appear to be at the interface between G proteins and their coupled receptors. G protein-coupled receptor kinases (GRKs are a small family of serine/threonine protein kinases primarily acting at the “receptor-G protein interface””. Recent studies have indicated the possible involvement of GRK, primarily GRK2 and GRK5, dysfunction in the pathogenesis of AD. It seems that mild, soluble, β-amyloid accumulation can lead to a reduced membrane (functional and an elevated cytosolic GRK2/5. The increased cytosolic GRK2 appears to be colocalized with damaged mitochondria and neurofibrillary tangles. Moreover, the total levels of GRK2, not only in the brain, but also in peripheral blood samples, are increased in a manner inversely correlated with the patient's cognitive levels. The deficiency of GRK5, on the other hand, impairs presynaptic M2 autoreceptor desensitization, which leads to a reduced acetylcholine release, axonal/synaptic degenerative changes, and associated amnestic, mild cognitive impairment. It also promotes an evil cycle to further increase Beta-amyloid accumulation and exaggerates brain inflammation, possibly even the basal forebrain cholinergic degeneration. Therefore, continuous efforts in this direction are necessary before translating the knowledge to any therapeutic strategies.

  13. Development of diacyltetrol lipids as activators for the C1 domain of protein kinase C.

    Science.gov (United States)

    Mamidi, Narsimha; Gorai, Sukhamoy; Mukherjee, Rakesh; Manna, Debasis

    2012-04-01

    The protein kinase C (PKC) family of serine/threonine kinases is an attractive drug target for the treatment of cancer and other diseases. Diacylglycerol (DAG), phorbol esters and others act as ligands for the C1 domain of PKC isoforms. Inspection of the crystal structure of the PKCδ C1b subdomain in complex with phorbol-13-O-acetate shows that one carbonyl group and two hydroxyl groups play pivotal roles in recognition of the C1 domain. To understand the importance of two hydroxyl groups of phorbol esters in PKC binding and to develop effective PKC activators, we synthesized DAG like diacyltetrols (DATs) and studied binding affinities with C1b subdomains of PKCδ and PKCθ. DATs, with the stereochemistry of natural DAGs at the sn-2 position, were synthesized from (+)-diethyl L-tartrate in four to seven steps as single isomers. The calculated EC(50) values for the short and long chain DATs varied in the range of 3-6 μM. Furthermore, the fluorescence anisotropy values of the proteins were increased in the presence of DATs in a similar manner to that of DAGs. Molecular docking of DATs (1b-4b) with PKCδ C1b showed that the DATs form hydrogen bonds with the polar residues and backbone of the protein, at the same binding site, as that of DAG and phorbol esters. Our findings reveal that DATs represent an attractive group of C1 domain ligands that can be used as research tools or further structurally modified for potential drug development.

  14. Phosphoinositide protein kinase PDPK1 is a crucial cell signaling mediator in multiple myeloma.

    Science.gov (United States)

    Chinen, Yoshiaki; Kuroda, Junya; Shimura, Yuji; Nagoshi, Hisao; Kiyota, Miki; Yamamoto-Sugitani, Mio; Mizutani, Shinsuke; Sakamoto, Natsumi; Ri, Masaki; Kawata, Eri; Kobayashi, Tsutomu; Matsumoto, Yosuke; Horiike, Shigeo; Iida, Shinsuke; Taniwaki, Masafumi

    2014-12-15

    Multiple myeloma is a cytogenetically/molecularly heterogeneous hematologic malignancy that remains mostly incurable, and the identification of a universal and relevant therapeutic target molecule is essential for the further development of therapeutic strategy. Herein, we identified that 3-phosphoinositide-dependent protein kinase 1 (PDPK1), a serine threonine kinase, is expressed and active in all eleven multiple myeloma-derived cell lines examined regardless of the type of cytogenetic abnormality, the mutation state of RAS and FGFR3 genes, or the activation state of ERK and AKT. Our results revealed that PDPK1 is a pivotal regulator of molecules that are essential for myelomagenesis, such as RSK2, AKT, c-MYC, IRF4, or cyclin Ds, and that PDPK1 inhibition caused the growth inhibition and the induction of apoptosis with the activation of BIM and BAD, and augmented the in vitro cytotoxic effects of antimyeloma agents in myeloma cells. In the clinical setting, PDPK1 was active in myeloma cells of approximately 90% of symptomatic patients at diagnosis, and the smaller population of patients with multiple myeloma exhibiting myeloma cells without active PDPK1 showed a significantly less frequent proportion of the disease stage III by the International Staging System and a significantly more favorable prognosis, including the longer overall survival period and the longer progression-free survival period by bortezomib treatment, than patients with active PDPK1, suggesting that PDPK1 activation accelerates the disease progression and the resistance to treatment in multiple myeloma. Our study demonstrates that PDPK1 is a potent and a universally targetable signaling mediator in multiple myeloma regardless of the types of cytogenetic/molecular profiles. ©2014 American Association for Cancer Research.

  15. Protein kinase B/Akt1 inhibits autophagy by down-regulating UVRAG expression

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Wonseok; Ju, Ji-hyun; Lee, Kyung-min; Nam, KeeSoo; Oh, Sunhwa [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of); Shin, Incheol, E-mail: incheol@hanyang.ac.kr [Department of Life Science, College of Natural Science, Hanyang University, 17 Haengdang-dong, Seongdong-gu, Seoul 133-791 (Korea, Republic of)

    2013-02-01

    Autophagy, or autophagocytosis, is a selective intracellular degradative process involving the cell's own lysosomal apparatus. An essential component in cell development, homeostasis, repair and resistance to stress, autophagy may result in either cell death or survival. The targeted region of the cell is sequestered within a membrane structure, the autophagosome, for regulation of the catabolic process. A key factor in both autophagosome formation and autophagosome maturation is a protein encoded by the ultraviolet irradiation resistance-associated gene (UVRAG). Conversely, the serine/threonine-specific protein kinase B (PKB, also known as Akt), which regulates survival in various cancers, inhibits autophagy through mTOR activation. We found that Akt1 may also directly inhibit autophagy by down-regulating UVRAG both in a 293T transient transfection system and breast cancer cells stably expressing Akt1. The UVRAG with mutations at putative Akt1-phosphorylation sites were still inhibited by Akt1, and dominant-negative Akt1 also inhibited UVRAG expression, suggesting that Akt1 down-regulates UVRAG by a kinase activity-independent mechanism. We showed that Akt1 overexpression in MDA-MB-231 breast cancer cells down-regulated UVRAG transcription. Cells over-expressing Akt1 were more resistant than control cells to ultraviolet light-induced autophagy and exhibited the associated reduction in cell viability. Levels of the autophagosome indicator protein LC3B-II and mRFP-GFP-LC3 were reduced in cells that over-expressing Akt1. Inhibiting Akt1 by siRNA or reintroducing UVRAG gene rescued the level of LC3B-II in UV-irradiation. Altogether, these data suggest that Akt1 may inhibit autophagy by decreasing UVRAG expression, which also sensitizes cancer cells to UV irradiation.

  16. Alterations in protein kinase C activity and processing during zinc-deficiency-induced cell death.

    Science.gov (United States)

    Chou, Susan S; Clegg, Michael S; Momma, Tony Y; Niles, Brad J; Duffy, Jodie Y; Daston, George P; Keen, Carl L

    2004-10-01

    Protein kinases C (PKCs) are a family of serine/threonine kinases that are critical for signal transduction pathways involved in growth, differentiation and cell death. All PKC isoforms have four conserved domains, C1-C4. The C1 domain contains cysteine-rich finger-like motifs, which bind two zinc atoms. The zinc-finger motifs modulate diacylglycerol binding; thus, intracellular zinc concentrations could influence the activity and localization of PKC family members. 3T3 cells were cultured in zinc-deficient or zinc-supplemented medium for up to 32 h. Cells cultured in zinc-deficient medium had decreased zinc content, lowered cytosolic classical PKC activity, increased caspase-3 processing and activity, and reduced cell number. Zinc-deficient cytosols had decreased activity and expression levels of PKC-alpha, whereas PKC-alpha phosphorylation was not altered. Inhibition of PKC-alpha with Gö6976 had no effect on cell number in the zinc-deficient group. Proteolysis of the novel PKC family member, PKC-delta, to its 40-kDa catalytic fragment occurred in cells cultured in the zinc-deficient medium. Occurrence of the PKC-delta fragment in mitochondria was co-incident with caspase-3 activation. Addition of the PKC-delta inhibitor, rottlerin, or zinc to deficient medium reduced or eliminated proteolysis of PKC-delta, activated caspase-3 and restored cell number. Inhibition of caspase-3 processing by Z-DQMD-FMK (Z-Asp-Gln-Met-Asp-fluoromethylketone) did not restore cell number in the zinc-deficient group, but resulted in processing of full-length PKC-delta to a 56-kDa fragment. These results support the concept that intracellular zinc concentrations influence PKC activity and processing, and that zinc-deficiency-induced apoptosis occurs in part through PKC-dependent pathways.

  17. Up-regulation of DNA-dependent protein kinase correlates with radiation resistance in oral squamous cell carcinoma

    International Nuclear Information System (INIS)

    Shintani, Satoru; Mihara, Mariko; Li, Chunnan; Nakahara Yuuji; Hino, Satoshi; Nakashiro, Koh-ichi; Hamakawa, Hiroyuki

    2003-01-01

    DNA-PK is a nuclear protein with serine/threonine kinase activity and forms a complex consisting of the DNA-PKcs and a heterodimer of Ku70 and Ku80 proteins. Recent laboratory experiments have demonstrated that the DNA-PK complex formation is one of the major pathways by which mammalian cells respond to DNA double-strand breaks induced by ionizing radiation. In this study, we evaluated the relationship between expression levels of DNA-PKcs, Ku70 and Ku80 proteins and radiation sensitivity in oral squamous cell carcinoma (OSCC) cell lines and in OSCC patients treated with preoperative radiation therapy. The OSCC cell lines greatly differed in their response to irradiation, as assessed by a standard colony formation assay. However, the expression levels of the DNA-PK complex proteins were all similar, and there was no association between the magnitude of their expression and the tumor radiation sensitivity. Expression of DNA-PK complex proteins increased after radiation treatment, and the increased values correlated with the tumor radiation resistance. Expression of DNA-PKcs and Ku70 after irradiation was increased in the surviving cells of OSCC tissues irradiated preoperatively. These results suggest that up-regulation of DNA-PK complex protein, especially DNA-PKcs, after radiation treatment correlates to radiation resistance. DNA-PKcs might be a molecular target for a novel radiation sensitization therapy of OSCC. (author)

  18. PASTA repeats of the protein kinase StkP interconnect cell constriction and separation of Streptococcus pneumoniae.

    Science.gov (United States)

    Zucchini, Laure; Mercy, Chryslène; Garcia, Pierre Simon; Cluzel, Caroline; Gueguen-Chaignon, Virginie; Galisson, Frédéric; Freton, Céline; Guiral, Sébastien; Brochier-Armanet, Céline; Gouet, Patrice; Grangeasse, Christophe

    2018-02-01

    Eukaryotic-like serine/threonine kinases (eSTKs) with extracellular PASTA repeats are key membrane regulators of bacterial cell division. How PASTA repeats govern eSTK activation and function remains elusive. Using evolution- and structural-guided approaches combined with cell imaging, we disentangle the role of each PASTA repeat of the eSTK StkP from Streptococcus pneumoniae. While the three membrane-proximal PASTA repeats behave as interchangeable modules required for the activation of StkP independently of cell wall binding, they also control the septal cell wall thickness. In contrast, the fourth and membrane-distal PASTA repeat directs StkP localization at the division septum and encompasses a specific motif that is critical for final cell separation through interaction with the cell wall hydrolase LytB. We propose a model in which the extracellular four-PASTA domain of StkP plays a dual function in interconnecting the phosphorylation of StkP endogenous targets along with septal cell wall remodelling to allow cell division of the pneumococcus.

  19. Cyclin-Dependent Kinase 5/p35/p39: A Novel and Imminent Therapeutic Target for Diabetes Mellitus

    Directory of Open Access Journals (Sweden)

    Danish Ahmed

    2011-01-01

    Full Text Available Present therapies to minify hyperglycaemia and insulin resistance mainly target ATP-sensitive K+ channels (KATP of pancreatic cells and PPAR-γ to enhance the insulin secretion and potential for GLUT expression, respectively. These current approaches are frequently associated with the various side effects such as hypoglycaemia and cardiovascular adverse events. CDK5 is a serine/threonine protein kinase, which forms active complexes with p35 or p39 found principally in neurons and in pancreatic β cells. Pieces of evidence from recent studies recommend the vital role of CDK5 in physiological functions in nonneuronal cells such as glucose-stimulated insulin secretion in pancreatic cells. Inhibition of CDK5 averts the decrease of insulin gene expression through the inhibition of nuclear translocation of PDX-1 which is a transcription factor for the insulin gene. The present pieces of evidence designate that CDK5 might be a potential drug target for the regulation of glucose-stimulated insulin secretion in the treatment of diabetes mellitus.

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

    Science.gov (United States)

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

    2007-01-01

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

  1. A historical overview of protein kinases and their targeted small molecule inhibitors.

    Science.gov (United States)

    Roskoski, Robert

    2015-10-01

    Protein kinases play a predominant regulatory role in nearly every aspect of cell biology and they can modify the function of a protein in almost every conceivable way. Protein phosphorylation can increase or decrease enzyme activity and it can alter other biological activities such as transcription and translation. Moreover, some phosphorylation sites on a given protein are stimulatory while others are inhibitory. The human protein kinase gene family consists of 518 members along with 106 pseudogenes. Furthermore, about 50 of the 518 gene products lack important catalytic residues and are called protein pseudokinases. The non-catalytic allosteric interaction of protein kinases and pseudokinases with other proteins has added an important regulatory feature to the biochemistry and cell biology of the protein kinase superfamily. With rare exceptions, a divalent cation such as Mg2+ is required for the reaction. All protein kinases exist in a basal state and are activated only as necessary by divergent regulatory stimuli. The mechanisms for switching between dormant and active protein kinases can be intricate. Phosphorylase kinase was the first protein kinase to be characterized biochemically and the mechanism of its regulation led to the discovery of cAMP-dependent protein kinase (protein kinase A, or PKA), which catalyzes the phosphorylation and activation of phosphorylase kinase. This was the first protein kinase cascade or signaling module to be elucidated. The epidermal growth factor receptor-Ras-Raf-MEK-ERK signaling module contains protein-tyrosine, protein-serine/threonine, and dual specificity protein kinases. PKA has served as a prototype of this enzyme family and more is known about this enzyme than any other protein kinase. The inactive PKA holoenzyme consists of two regulatory and two catalytic subunits. After binding four molecules of cAMP, the holoenzyme dissociates into a regulatory subunit dimer (each monomer binds two cAMP) and two free and active

  2. Serine protease inhibitors of parasitic helminths.

    Science.gov (United States)

    Molehin, Adebayo J; Gobert, Geoffrey N; McManus, Donald P

    2012-05-01

    Serine protease inhibitors (serpins) are a superfamily of structurally conserved proteins that inhibit serine proteases and play key physiological roles in numerous biological systems such as blood coagulation, complement activation and inflammation. A number of serpins have now been identified in parasitic helminths with putative involvement in immune regulation and in parasite survival through interference with the host immune response. This review describes the serpins and smapins (small serine protease inhibitors) that have been identified in Ascaris spp., Brugia malayi, Ancylostoma caninum Onchocerca volvulus, Haemonchus contortus, Trichinella spiralis, Trichostrongylus vitrinus, Anisakis simplex, Trichuris suis, Schistosoma spp., Clonorchis sinensis, Paragonimus westermani and Echinococcus spp. and discusses their possible biological functions, including roles in host-parasite interplay and their evolutionary relationships.

  3. Serine:glyoxylate aminotransferase mutant of barley

    International Nuclear Information System (INIS)

    Blackwell, R.; Murray, A.; Joy, K.; Lea, P.

    1987-01-01

    A photorespiratory mutant of barley (LaPr 85/84), deficient in both of the major peaks of serine:glyoxylate aminotransferase activity detected in the wild type, also lacks serine:pyruvate and asparagine:glyoxylate aminotransferase activities. Genetic analysis of the mutation demonstrated that these three activities are all carried on the same enzyme. The mutant, when placed in air, accumulated a large pool of serine, showed the expected rate (50%) of ammonia release during photorespiration but produced CO 2 at twice the wild type rate when it was fed [ 14 C] glyoxylate. Compared with the wild type, LaPr 85/84 exhibited abnormal transient changes in chlorophyll a fluorescence when the CO 2 concentration of the air was altered, indicating that the rates of the fluorescence quenching mechanisms were affected in vivo by the lack of this enzyme

  4. A natural product from Cannabis sativa subsp. sativa inhibits homeodomain-interacting protein kinase 2 (HIPK2), attenuating MPP+-induced apoptosis in human neuroblastoma SH-SY5Y cells.

    Science.gov (United States)

    Wang, Guan; Zhu, Lingjuan; Zhao, Yuqian; Gao, Suyu; Sun, Dejuan; Yuan, Jingquan; Huang, Yuxin; Zhang, Xue; Yao, Xinsheng

    2017-06-01

    Homeodomain-interacting protein kinase 2 (HIPK2) is a conserved serine/threonine kinase, which regulate transcription, cell differentiation, proliferation and apoptosis. Previous evidences indicated that HIPK2 could be involved in the pathogenesis of neurodegenerative diseases, suggesting as a novel target for Parkinson's disease (PD) therapeutic development. Herein, gene microarray analysis was performed to verify the key regulatory function of HIPK2 in PD. (Z)-methylp-hydroxycinnamate (ZMHC, 7) with other eighteen compounds were isolated from Cannabis sativa subsp. sativa, growing in Bama Yao Autonomous County, one of the five largest longevity regions of the world. Intriguingly, ZMHC was identified to bind HIPK2 with high affinity through molecular modeling and molecular dynamics (MD) simulations. Moreover, cell morphology, flow cytometry and western blot assay suggested that ZMHC inhibited HIPK2, which attenuated MPP + -induced apoptosis in SH-SY5Y cells. In conclusion, these findings discovered a natural product that inhibited HIPK2, and highlighted that ZMHC could be a potential precursor agent for future PD therapy. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Large-scale label-free comparative proteomics analysis of polo-like kinase 1 inhibition via the small-molecule inhibitor BI 6727 (Volasertib) in BRAF(V600E) mutant melanoma cells.

    Science.gov (United States)

    Cholewa, Brian D; Pellitteri-Hahn, Molly C; Scarlett, Cameron O; Ahmad, Nihal

    2014-11-07

    Polo-like kinase 1 (Plk1) is a serine/threonine kinase that plays a key role during the cell cycle by regulating mitotic entry, progression, and exit. Plk1 is overexpressed in a variety of human cancers and is essential to sustained oncogenic proliferation, thus making Plk1 an attractive therapeutic target. However, the clinical efficacy of Plk1 inhibition has not emulated the preclinical success, stressing an urgent need for a better understanding of Plk1 signaling. This study addresses that need by utilizing a quantitative proteomics strategy to compare the proteome of BRAF(V600E) mutant melanoma cells following treatment with the Plk1-specific inhibitor BI 6727. Employing label-free nano-LC-MS/MS technology on a Q-exactive followed by SIEVE processing, we identified more than 20 proteins of interest, many of which have not been previously associated with Plk1 signaling. Here we report the down-regulation of multiple metabolic proteins with an associated decrease in cellular metabolism, as assessed by lactate and NAD levels. Furthermore, we have also identified the down-regulation of multiple proteasomal subunits, resulting in a significant decrease in 20S proteasome activity. Additionally, we have identified a novel association between Plk1 and p53 through heterogeneous ribonucleoprotein C1/C2 (hnRNPC), thus providing valuable insight into Plk1's role in cancer cell survival.

  6. Structure Determination of Mycobacterium tuberculosis Serine Protease Hip1 (Rv2224c)

    Energy Technology Data Exchange (ETDEWEB)

    Naffin-Olivos, Jacqueline L.; Daab, Andrew; White, Andre; Goldfarb, Nathan E.; Milne, Amy C.; Liu, Dali; Baikovitz, Jacqueline; Dunn, Ben M.; Rengarajan, Jyothi; Petsko, Gregory A.; Ringe, Dagmar

    2017-04-07

    The Mycobacterium tuberculosis (Mtb) serine protease Hip1 (hydrolase important for pathogenesis; Rv2224c) promotes tuberculosis (TB) pathogenesis by impairing host immune responses through proteolysis of a protein substrate, Mtb GroEL2. The cell surface localization of Hip1 and its immunomodulatory functions make Hip1 a good drug target for new adjunctive immune therapies for TB. Here, we report the crystal structure of Hip1 to a resolution of 2.6 Å and the kinetic studies of the enzyme against model substrates and the protein GroEL2. The structure shows a two-domain protein, one of which contains the catalytic residues that are the signature of a serine protease. Surprisingly, a threonine is located within the active site close enough to hydrogen bond with the catalytic residues Asp463 and His490. Mutation of this residue, Thr466, to alanine established its importance for function. Our studies provide insights into the structure of a member of a novel family of proteases. Knowledge of the Hip1 structure will aid in designing inhibitors that could block Hip1 activity

  7. Evolutionary relationships of Aurora kinases: Implications for model organism studies and the development of anti-cancer drugs

    Directory of Open Access Journals (Sweden)

    Patrick Denis R

    2004-10-01

    Full Text Available Abstract Background As key regulators of mitotic chromosome segregation, the Aurora family of serine/threonine kinases play an important role in cell division. Abnormalities in Aurora kinases have been strongly linked with cancer, which has lead to the recent development of new classes of anti-cancer drugs that specifically target the ATP-binding domain of these kinases. From an evolutionary perspective, the species distribution of the Aurora kinase family is complex. Mammals uniquely have three Aurora kinases, Aurora-A, Aurora-B, and Aurora-C, while for other metazoans, including the frog, fruitfly and nematode, only Aurora-A and Aurora-B kinases are known. The fungi have a single Aurora-like homolog. Based on the tacit assumption of orthology to human counterparts, model organism studies have been central to the functional characterization of Aurora kinases. However, the ortholog and paralog relationships of these kinases across various species have not been rigorously examined. Here, we present comprehensive evolutionary analyses of the Aurora kinase family. Results Phylogenetic trees suggest that all three vertebrate Auroras evolved from a single urochordate ancestor. Specifically, Aurora-A is an orthologous lineage in cold-blooded vertebrates and mammals, while structurally similar Aurora-B and Aurora-C evolved more recently in mammals from a duplication of an ancestral Aurora-B/C gene found in cold-blooded vertebrates. All so-called Aurora-A and Aurora-B kinases of non-chordates are ancestral to the clade of chordate Auroras and, therefore, are not strictly orthologous to vertebrate counterparts. Comparisons of human Aurora-B and Aurora-C sequences to the resolved 3D structure of human Aurora-A lends further support to the evolutionary scenario that vertebrate Aurora-B and Aurora-C are closely related paralogs. Of the 26 residues lining the ATP-binding active site, only three were variant and all were specific to Aurora-A. Conclusions In

  8. Fragmentation of Protein Kinase N (PKN) in the Hydrocephalic Rat Brain

    International Nuclear Information System (INIS)

    Okii, Norifumi; Amano, Taku; Seki, Takahiro; Matsubayashi, Hiroaki; Mukai, Hideyuki; Ono, Yoshitaka; Kurisu, Kaoru; Sakai, Norio

    2007-01-01

    PKN (protein kinase N; also called protein kinase C-related kinase (PRK-1)), is a serine/threonine protein kinase that is ubiquitously expressed in several organs, including the brain. PKN has a molecular mass of 120 kDa and has two domains, a regulatory and a catalytic domain, in its amino-terminals and carboxyl-terminus, respectively. Although the role of PKN has not been fully elucidated, previous studies have revealed that PKN is cleaved to a constitutively active catalytic fragment of 55 kDa in response to apoptotic signals. Hydrocephalus is a pathological condition caused by insufficient cerebrospinal fluid (CSF) circulation and subsequent excess of CSF in the brain. In this study, in order to elucidate the role of PKN in the pathophysiology of hydrocephalus, we examined PKN fragmentation in hydrocephalic model rats. Hydrocephalus was induced in rats by injecting kaolin into the cisterna magna. Kaolin-induced rats (n=60) were divided into three groups according to the observation period after treatment (group 1: 3–6 weeks, group 2: 7–12 weeks, and group 3: 13–18 weeks). Sham-treated control rats, injected with sterile saline (n=20), were similarly divided into three groups. Spatial learning ability was estimated by a modified water maze test. Thereafter, brains were cut into slices and ventricular dilatation was estimated. Fragmentation of PKN was observed by Western blotting in samples collected from the parietal cortex, striatum, septal nucleus, hippocampus, and periaqueductal gray matter. All kaolin-induced rats showed ventricular dilatation. Most of them showed less spatial learning ability than those of sham-treated controls. In most regions, fragmentation of PKN had occurred in a biphasic manner more frequently than that in controls. The appearance of PKN fragmentation in periaqueductal gray matter was correlated with the extent of ventricular dilation and spatial learning disability. These results revealed that PKN fragmentation was observed in

  9. Efficient Reassignment of a Frequent Serine Codon in Wild-Type Escherichia coli.

    Science.gov (United States)

    Ho, Joanne M; Reynolds, Noah M; Rivera, Keith; Connolly, Morgan; Guo, Li-Tao; Ling, Jiqiang; Pappin, Darryl J; Church, George M; Söll, Dieter

    2016-02-19

    Expansion of the genetic code through engineering the translation machinery has greatly increased the chemical repertoire of the proteome. This has been accomplished mainly by read-through of UAG or UGA stop codons by the noncanonical aminoacyl-tRNA of choice. While stop codon read-through involves competition with the translation release factors, sense codon reassignment entails competition with a large pool of endogenous tRNAs. We used an engineered pyrrolysyl-tRNA synthetase to incorporate 3-iodo-l-phenylalanine (3-I-Phe) at a number of different serine and leucine codons in wild-type Escherichia coli. Quantitative LC-MS/MS measurements of amino acid incorporation yields carried out in a selected reaction monitoring experiment revealed that the 3-I-Phe abundance at the Ser208AGU codon in superfolder GFP was 65 ± 17%. This method also allowed quantification of other amino acids (serine, 33 ± 17%; phenylalanine, 1 ± 1%; threonine, 1 ± 1%) that compete with 3-I-Phe at both the aminoacylation and decoding steps of translation for incorporation at the same codon position. Reassignments of different serine (AGU, AGC, UCG) and leucine (CUG) codons with the matching tRNA(Pyl) anticodon variants were met with varying success, and our findings provide a guideline for the choice of sense codons to be reassigned. Our results indicate that the 3-iodo-l-phenylalanyl-tRNA synthetase (IFRS)/tRNA(Pyl) pair can efficiently outcompete the cellular machinery to reassign select sense codons in wild-type E. coli.

  10. Therapeutic Value of PLK1 Knockdown in Combination with Prostate Cancer Drugs in PIM-1 Overexpressing Prostate Cancer Cells

    Science.gov (United States)

    2014-11-13

    targeting a total of 570 genes involved in key cancer relevant pathways (111 cell cycle, 318 apoptosis, 87 serine- threonine kinase and 54 tyrosine kinase...siRNAs that target genes encoding selected serine/threonine kinases, tyrosine kinases, cell cycle protein and apoptosis proteins to identify genes...PIM1 (Santa Cruz, sc-13513), PLK1 (Santa Cruz, sc-17783), phospho-PLK1 (Thr 210; Cell Signaling, #5472), phospho-histone H3 (Upstate, #06- 570 ), cleaved

  11. Evaluating acetaldehyde synthesis from L-14C(U)] threonine by Streptococcus thermophilus and Lactobacillus bulgaricus

    International Nuclear Information System (INIS)

    Wilkins, D.W.; Schmidt, R.H.; Shireman, R.B.; Smith, K.L.; Jezeski, J.J.

    1986-01-01

    To evaluate the synthesis of acetaldehyde from threonine during growth of yogurt cultures, Streptococcus thermophilus MS1 and Lactobacillus bulgaricus MR1 were grown in defined medium in which 10% of the total threonine was composed of L-[carbon-14(U)]threonine. Acetaldehyde production was monitored by formation of 2,4-dinitrophenylhydrazone followed by separation and analysis using high performance liquid chromatography. After growth for 8 h at 42 0 C, approximately 2.0% of the total acetaldehyde (780.4 nmol) produced was from L-[carbon-14]threonine. Threonine aldolase activity was determined in cell-free extracts from S. thermophilus and L. bulgaricus grown in Elliker broth. Increasing incubation temperature from 30 to 42 0 C decreased threonine aldolase activity in cells of the streptococcus harvested after 8 h of incubation. Effect of incubation temperature was more dramatic in cells harvested after 18 h where the activity of cells grown at 48 0 C was 89% lower than that of cells grown at 30 0 C. Cell extracts from S. thermophilus MS1 possessed higher threonine aldolase activity than did those from L. bulgaricus MR1. Increased assay temperature from 30 to 42 0 C increased threonine aldolase activity in S. thermophilus MS1

  12. AKT phosphorylates H3-threonine 45 to facilitate termination of gene transcription in response to DNA damage.

    Science.gov (United States)

    Lee, Jong-Hyuk; Kang, Byung-Hee; Jang, Hyonchol; Kim, Tae Wan; Choi, Jinmi; Kwak, Sojung; Han, Jungwon; Cho, Eun-Jung; Youn, Hong-Duk

    2015-05-19

    Post-translational modifications of core histones affect various cellular processes, primarily through transcription. However, their relationship with the termination of transcription has remained largely unknown. In this study, we show that DNA damage-activated AKT phosphorylates threonine 45 of core histone H3 (H3-T45). By genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis, H3-T45 phosphorylation was distributed throughout DNA damage-responsive gene loci, particularly immediately after the transcription termination site. H3-T45 phosphorylation pattern showed close-resemblance to that of RNA polymerase II C-terminal domain (CTD) serine 2 phosphorylation, which establishes the transcription termination signal. AKT1 was more effective than AKT2 in phosphorylating H3-T45. Blocking H3-T45 phosphorylation by inhibiting AKT or through amino acid substitution limited RNA decay downstream of mRNA cleavage sites and decreased RNA polymerase II release from chromatin. Our findings suggest that AKT-mediated phosphorylation of H3-T45 regulates the processing of the 3' end of DNA damage-activated genes to facilitate transcriptional termination. © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.

  13. Eukaryote-like Ser/Thr protein kinase PrkA modulates sporulation via regulating the transcriptional factor σ(K) in Bacillus subtilis.

    Science.gov (United States)

    Yan, Jinyuan; Zou, Wei; Fang, Juan; Huang, Xiaowei; Gao, Feng; He, Zeying; Zhang, Keqin; Zhao, Ninghui

    2015-01-01

    Protein kinase A (PrkA), also known as AMP-activated protein kinase, functions as a serine/threonine protein kinase (STPK), has been shown to be involved in a variety of important biologic processes, including pathogenesis of many important diseases in mammals. However, the biological functions of PrkA are less known in prokaryote cells. Here, we explored the function of PrkA as well as its underlying molecular mechanisms using the model bacterium Bacillus subtilis168. When PrkA is inhibited by 9-β-D-arabinofuranosyladenine (ara-A) in the wild type strain or deleted in the ΔprkA mutant strain, we observed sporulation defects in B. subtilis 168, suggesting that PrkA functions as a sporulation-related protein. Transcriptional analysis using the lacZ reporter gene demonstrated that deletion of prkA significantly reduced the expression of the transcriptional factor σ(K) and its downstream genes. Complementation of sigK gene in prkA knockout mutant partially rescued the phenotype of ΔprkA, further supporting the hypothesis that the decreased σ(K) expression should be one of the reasons for the sporulation defect resulting from prkA disruption. Finally, our data confirmed that Hpr (ScoC) negatively controlled the expression of transcriptional factor σ(K), and thus PrkA accelerated sporulation and the expression of σ(K) by suppression of Hpr (ScoC). Taken together, our study discovered a novel function of the eukaryotic-like STPK PrkA in spore development as well as its underlying molecular mechanism in B. subtilis.

  14. Identification of Ser/Thr kinase and forkhead associated domains in Mycobacterium ulcerans: characterization of novel association between protein kinase Q and MupFHA.

    Directory of Open Access Journals (Sweden)

    Gunjan Arora

    2014-11-01

    Full Text Available Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK PknQ.In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c, a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c, which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA.Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain -pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes.

  15. Increased tolerance towards serine obtained by adaptive laboratory evolution

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Seoane, Jose Miguel; Koza, Anna

    2014-01-01

    The amino acid serine has previously been identified as one of the top 30 candidates of value added chemicals, making the production of serine from glucose attractive. Production of serine have previously been attempted in E. coli and C. glutamicum, however, titers sufficient for commercial...... by glyA), the conversion of serine to pyruvate (encoded by sdaA, sdaB and tdcG) was also deleted. As expected, the resulting strain turned out to be susceptible to even low concentrations of serine in the media. In order to improve the tolerance of the strain towards serine, adaptive laboratory evolution....... During the evolution experiment, the serine tolerance was increased substantially. Genome re-sequencing was subsequently used to analyze the genotype of a number of selected strains. These results reveal insights towards the adaptation process as well as the mechanism of serine tolerance....

  16. Nutritional requirements of digestible threonine for growing meat-type quails

    Directory of Open Access Journals (Sweden)

    Ana Paula Silva Ton

    2013-07-01

    Full Text Available The objective of this study was to estimate the nutrient requirements of digestible threonine for meat-type quails (Coturnix coturnix sp in the growth phase. A total of 1350 not sexed meat-type quails were distributed in a completely randomized design with six threonine levels (10.80, 11.50, 12.20, 12.90, 13.60 and 14.30 g/kg as fed, five replications and 45 quails per experimental unit, from 1 to 14 days of age. The threonine levels in the diet had a quadratic effect on body weight, feed intake and weight gain. Estimates for highest body weight (79.41 g, feed intake (128.96 g/bird and weight gain (70.73 g were observed with diets containing 12.60 g/kg of digestible threonine. According to the LRP model, the threonine intake was estimated at 13.40 g/kg of digestible threonine. Protein deposition rate and energy retained in the carcass showed quadratic effect, with estimated digestible threonine levels of 11.80 and 12.00 g/kg in the diet for maximum protein deposition rate (2.00 g/bird and retained energy in the carcass (15.88 kcal/g, respectively. There was a linear effect on feed cost per kg of live weight gain as threonine levels were increased. Nutritional requirement of digestible threonine for meat-type quails for maximum growth is 12.60 g/kg, corresponding to a digestible threonine:digestible lysine ratio of 67.02.

  17. Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

    International Nuclear Information System (INIS)

    Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin; Fu, Xinlu; Shen, Feihai; Huang, Zhiying

    2016-01-01

    Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

  18. The Rho Kinases: Critical Mediators of Multiple Profibrotic Processes and Rational Targets for New Therapies for Pulmonary Fibrosis

    Science.gov (United States)

    Knipe, Rachel S.; Tager, Andrew M.

    2015-01-01

    Idiopathic pulmonary fibrosis (IPF) is characterized by progressive lung scarring, short median survival, and limited therapeutic options, creating great need for new pharmacologic therapies. IPF is thought to result from repetitive environmental injury to the lung epithelium, in the context of aberrant host wound healing responses. Tissue responses to injury fundamentally involve reorganization of the actin cytoskeleton of participating cells, including epithelial cells, fibroblasts, endothelial cells, and macrophages. Actin filament assembly and actomyosin contraction are directed by the Rho-associated coiled-coil forming protein kinase (ROCK) family of serine/threonine kinases (ROCK1 and ROCK2). As would therefore be expected, lung ROCK activation has been demonstrated in humans with IPF and in animal models of this disease. ROCK inhibitors can prevent fibrosis in these models, and more importantly, induce the regression of already established fibrosis. Here we review ROCK structure and function, upstream activators and downstream targets of ROCKs in pulmonary fibrosis, contributions of ROCKs to profibrotic cellular responses to lung injury, ROCK inhibitors and their efficacy in animal models of pulmonary fibrosis, and potential toxicities of ROCK inhibitors in humans, as well as involvement of ROCKs in fibrosis in other organs. As we discuss, ROCK activation is required for multiple profibrotic responses, in the lung and multiple other organs, suggesting ROCK participation in fundamental pathways that contribute to the pathogenesis of a broad array of fibrotic diseases. Multiple lines of evidence therefore indicate that ROCK inhibition has great potential to be a powerful therapeutic tool in the treatment of fibrosis, both in the lung and beyond. PMID:25395505

  19. Inhibition of glycogen synthase kinase 3beta ameliorates triptolide-induced acute cardiac injury by desensitizing mitochondrial permeability transition

    Energy Technology Data Exchange (ETDEWEB)

    Wang, Wenwen; Yang, Yanqin; Xiong, Zhewen; Kong, Jiamin [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Fu, Xinlu [Laboratory Animals Center, Sun Yat-sen University, Guangzhou 510006 (China); Shen, Feihai, E-mail: shenfh3@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China); Huang, Zhiying, E-mail: hzhiying@mail.sysu.edu.cn [School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006 (China)

    2016-12-15

    Triptolide (TP), a diterpene triepoxide, is a major active component of Tripterygium wilfordii extracts, which are prepared as tablets and has been used clinically for the treatment of inflammation and autoimmune disorders. However, TP's therapeutic potential is limited by severe adverse effects. In a previous study, we reported that TP induced mitochondria dependent apoptosis in cardiomyocytes. Glycogen synthase kinase-3β (GSK-3β) is a multifunctional serine/threonine kinase that plays important roles in the necrosis and apoptosis of cardiomyocytes. Our study aimed to investigate the role of GSK-3β in TP-induced cardiotoxicity. Inhibition of GSK-3β activity by SB 216763, a potent and selective GSK-3 inhibitor, prominently ameliorated the detrimental effects in C57BL/6J mice with TP administration, which was associated with a correction of GSK-3β overactivity. Consistently, in TP-treated H9c2 cells, SB 216763 treatment counteracted GSK-3β overactivity, improved cell viability, and prevented apoptosis by modulating the expression of Bcl-2 family proteins. Mechanistically, GSK-3β interacted with and phosphorylated cyclophilin F (Cyp-F), a key regulator of mitochondrial permeability transition pore (mPTP). GSK-3β inhibition prevented the phosphorylation and activation of Cyp-F, and desensitized mPTP. Our findings suggest that pharmacological targeting of GSK-3β could represent a promising therapeutic strategy for protecting against cardiotoxicity induced by TP. - Highlights: • GSK-3β inhibition ameliorates TP-induced cardiotoxicity in vitro and in vivo. • GSK-3β controls Cyp-F activation, and regulates mPTP and apoptosis in H9c2 cells. • The protective effect is attributed to GSK-3β activity rather than to protein level. • GSK-3β may be a promising target against TP-induced cardiotoxicity.

  20. G protein-coupled receptor kinase-2-deficient mice are protected from dextran sodium sulfate-induced acute colitis.

    Science.gov (United States)

    Steury, Michael D; Kang, Ho Jun; Lee, Taehyung; Lucas, Peter C; McCabe, Laura R; Parameswaran, Narayanan

    2018-06-01

    G protein-coupled receptor kinase 2 (GRK2) is a serine/threonine kinase and plays a key role in different disease processes. Previously, we showed that GRK2 knockdown enhances wound healing in colonic epithelial cells. Therefore, we hypothesized that ablation of GRK2 would protect mice from dextran sodium sulfate (DSS)-induced acute colitis. To test this, we administered DSS to wild-type (GRK2 +/+ ) and GRK2 heterozygous (GRK +/- ) mice in their drinking water for 7 days. As predicted, GRK2 +/- mice were protected from colitis as demonstrated by decreased weight loss (20% loss in GRK2 +/+ vs. 11% loss in GRK2 +/- ). lower disease activity index (GRK2 +/+ 9.1 vs GRK2 +/- 4.1), and increased colon lengths (GRK2 +/+ 4.7 cm vs GRK2 +/- 5.3 cm). To examine the mechanisms by which GRK2 +/- mice are protected from colitis, we investigated expression of inflammatory genes in the colon as well as immune cell profiles in colonic lamina propria, mesenteric lymph node, and in bone marrow. Our results did not reveal differences in immune cell profiles between the two genotypes. However, expression of inflammatory genes was significantly decreased in DSS-treated GRK2 +/- mice compared with GRK2 +/+ . To understand the mechanisms, we generated myeloid-specific GRK2 knockout mice and subjected them to DSS-induced colitis. Similar to whole body GRK2 heterozygous knockout mice, myeloid-specific knockout of GRK2 was sufficient for the protection from DSS-induced colitis. Together our results indicate that deficiency of GRK2 protects mice from DSS-induced colitis and further suggests that the mechanism of this effect is likely via GRK2 regulation of inflammatory genes in the myeloid cells.

  1. The consequences of long-term glycogen synthase kinase-3 inhibition on normal and insulin resistant rat hearts.

    Science.gov (United States)

    Flepisi, T B; Lochner, Amanda; Huisamen, Barbara

    2013-10-01

    Glycogen synthase kinase-3 (GSK-3) is a serine-threonine protein kinase, discovered as a regulator of glycogen synthase. GSK-3 may regulate the expression of SERCA-2a potentially affecting myocardial contractility. It is known to phosphorylate and inhibit IRS-1, thus disrupting insulin signalling. This study aimed to determine whether myocardial GSK-3 protein and its substrate proteins are dysregulated in obesity and insulin resistance, and whether chronic GSK-3 inhibition can prevent or reverse this. Weight matched male Wistar rats were rendered obese by hyperphagia using a special diet (DIO) for 16 weeks and compared to chow fed controls. Half of each group was treated with the GSK-3 inhibitor CHIR118637 (30 mg/kg/day) from week 12 to16 of the diet period. Biometric and biochemical parameters were measured and protein expression determined by Western blotting and specific antibodies. Ca(2+)ATPase activity was determined spectrophotometrically. Cardiomyocytes were prepared by collagenase perfusion and insulin stimulated 2-deoxy-glucose uptake determined. DIO rats were significantly heavier than controls, associated with increased intra-peritoneal fat and insulin resistance. GSK-3 inhibition did not affect weight but improved insulin resistance, also on cellular level. It had no effect on GSK-3 expression but elevated its phospho/total ratio and elevated IRS-2 expression. Obesity lowered SERCA-2a expression and activity while GSK-3 inhibition alleviated this. The phospho/total ratio of phospholamban underscored inhibition of SERCA-2a in obesity. In addition, signs of myocardial hypertrophy were observed in treated control rats. GSK-3 inhibition could not reverse all the detrimental effects of obesity but may be harmful in normal rat hearts. It regulates IRS-2, SERCA-2a and phospholamban expression but not IRS-1.

  2. Inhibition of polo-like kinase 1 leads to the suppression of osteosarcoma cell growth in vitro and in vivo.

    Science.gov (United States)

    Liu, Xianzhe; Choy, Edwin; Harmon, David; Yang, Shuhua; Yang, Cao; Mankin, Henry; Hornicek, Francis J; Duan, Zhenfeng

    2011-06-01

    Osteosarcoma is the most common type of primary bone cancer in children and adolescents. Treatment options for osteosarcoma may include surgery, chemotherapy, and radiotherapy. Unfortunately, many patients eventually relapse, resulting in an unsatisfactory outcome. The serine/threonine-specific polo-like kinase 1 (PLK1) is a kinase that plays an important role in mitosis and the maintenance of genomic stability. PLK1 has been found to be highly expressed in the malignant cells of osteosarcoma. Here, we describe the in-vitro and in-vivo effects of BI 2536, a small-molecule inhibitor of PLK1, which through inhibiting PLK1 enzymatic activity, causes mitotic arrest and eventually induces cancer cell apoptosis. In this study, we show that the PLK1 inhibitor, BI 2536, inhibits proliferation and induces apoptosis in two-dimensional and three-dimensional cultures of osteosarcoma cell lines, KHOS and U-2OS. A proliferation assay performed both in two-dimensional and three-dimensional culture showed that the growth of both cell lines was inhibited by BI 2536. Cell cycle analysis showed that the cells treated with BI 2536 were mainly arrested in the G2/M phase. Immunofluorescence and western blotting analysis confirmed that the administration of BI 2536 led to significant decrease of PLK1 and Mcl-1 protein expression levels in dose-dependent and time-dependent manners. Furthermore, BI 2536-induced apoptosis in the osteosarcoma cell lines was shown by poly (ADP-ribose) polymerase cleavage and caspase assay. Finally, in mouse osteosarcoma xenografts, BI 2536-treated mice had significantly smaller tumors compared with the control mice. These findings offer evidence of the potential role for targeting PLK1 in osteosarcoma therapy.

  3. Glycogen synthase kinase-3 inhibition attenuates fibroblast activation and development of fibrosis following renal ischemia-reperfusion in mice

    Directory of Open Access Journals (Sweden)

    Shailendra P. Singh

    2015-08-01

    Full Text Available Glycogen synthase kinase-3β (GSK3β is a serine/threonine protein kinase that plays an important role in renal tubular injury and regeneration in acute kidney injury. However, its role in the development of renal fibrosis, often a long-term consequence of acute kidney injury, is unknown. Using a mouse model of renal fibrosis induced by ischemia-reperfusion injury, we demonstrate increased GSK3β expression and activity in fibrotic kidneys, and its presence in myofibroblasts in addition to tubular epithelial cells. Pharmacological inhibition of GSK3 using TDZD-8 starting before or after ischemia-reperfusion significantly suppressed renal fibrosis by reducing the myofibroblast population, collagen-1 and fibronectin deposition, inflammatory cytokines, and macrophage infiltration. GSK3 inhibition in vivo reduced TGF-β1, SMAD3 activation and plasminogen activator inhibitor-1 levels. Consistently in vitro, TGF-β1 treatment increased GSK3β expression and GSK3 inhibition abolished TGF-β1-induced SMAD3 activation and α-smooth muscle actin (α-SMA expression in cultured renal fibroblasts. Importantly, overexpression of constitutively active GSK3β stimulated α-SMA expression even in the absence of TGF-β1 treatment. These results suggest that TGF-β regulates GSK3β, which in turn is important for TGF-β–SMAD3 signaling and fibroblast-to-myofibroblast differentiation. Overall, these studies demonstrate that GSK3 could promote renal fibrosis by activation of TGF-β signaling and the use of GSK3 inhibitors might represent a novel therapeutic approach for progressive renal fibrosis that develops as a consequence of acute kidney injury.

  4. Casein kinase 1 alpha regulates chromosome congression and separation during mouse oocyte meiotic maturation and early embryo development.

    Directory of Open Access Journals (Sweden)

    Lu Wang

    Full Text Available Casein kinase I alpha (CK1α is a member of serine/threonine protein kinase, generally present in all eukaryotes. In mammals, CK1α regulates the transition from interphase to metaphase in mitosis. However, little is known about its role in meiosis. Here we examined Ck1α mRNA and protein expression, as well as its subcellular localization in mouse oocytes from germinal vesicle to the late 1-cell stage. Our results showed that the expression level of CK1α was increased in metaphase. Immunostaining results showed that CK1α colocalized with condensed chromosomes during oocyte meiotic maturation and early embryo development. We used the loss-of-function approach by employing CK1α specific morpholino injection to block the function of CK1α. This functional blocking leads to failure of polar body 1 (PB1 extrusion, chromosome misalignment and MII plate incrassation. We further found that D4476, a specific and efficient CK1 inhibitor, decreased the rate of PB1 extrusion. Moreover, D4476 resulted in giant polar body extrusion, oocyte pro-MI arrest, chromosome congression failure and impairment of embryo developmental potential. In addition, we employed pyrvinium pamoate (PP, an allosteric activator of CK1α, to enhance CK1α activity in oocytes. Supplementation of PP induced oocyte meiotic maturation failure, severe congression abnormalities and misalignment of chromosomes. Taken together, our study for the first time demonstrates that CK1α is required for chromosome alignment and segregation during oocyte meiotic maturation and early embryo development.

  5. Two amino acid residues confer different binding affinities of Abelson family kinase SRC homology 2 domains for phosphorylated cortactin.

    Science.gov (United States)

    Gifford, Stacey M; Liu, Weizhi; Mader, Christopher C; Halo, Tiffany L; Machida, Kazuya; Boggon, Titus J; Koleske, Anthony J

    2014-07-11

    The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. Protein kinase C-dependent signaling controls the midgut epithelial barrier to malaria parasite infection in anopheline mosquitoes.

    Directory of Open Access Journals (Sweden)

    Nazzy Pakpour

    Full Text Available Anopheline mosquitoes are the primary vectors of parasites in the genus Plasmodium, the causative agents of malaria. Malaria parasites undergo a series of complex transformations upon ingestion by the mosquito host. During this process, the physical barrier of the midgut epithelium, along with innate immune defenses, functionally restrict parasite development. Although these defenses have been studied for some time, the regulatory factors that control them are poorly understood. The protein kinase C (PKC gene family consists of serine/threonine kinases that serve as central signaling molecules and regulators of a broad spectrum of cellular processes including epithelial barrier function and immunity. Indeed, PKCs are highly conserved, ranging from 7 isoforms in Drosophila to 16 isoforms in mammals, yet none have been identified in mosquitoes. Despite conservation of the PKC gene family and their potential as targets for transmission-blocking strategies for malaria, no direct connections between PKCs, the mosquito immune response or epithelial barrier integrity are known. Here, we identify and characterize six PKC gene family members--PKCδ, PKCε, PKCζ, PKD, PKN, and an indeterminate conventional PKC--in Anopheles gambiae and Anopheles stephensi. Sequence and phylogenetic analyses of the anopheline PKCs support most subfamily assignments. All six PKCs are expressed in the midgut epithelia of A. gambiae and A. stephensi post-blood feeding, indicating availability for signaling in a tissue that is critical for malaria parasite development. Although inhibition of PKC enzymatic activity decreased NF-κB-regulated anti-microbial peptide expression in mosquito cells in vitro, PKC inhibition had no effect on expression of a panel of immune genes in the midgut epithelium in vivo. PKC inhibition did, however, significantly increase midgut barrier integrity and decrease development of P. falciparum oocysts in A. stephensi, suggesting that PKC

  7. Activated type I TGFbeta receptor (Alk5) kinase confers enhancedsurvival to mammary epithelial cells and accelerates mammary tumorprogression

    Energy Technology Data Exchange (ETDEWEB)

    Muraoka-Cook, Rebecca S.; Shin, Incheol; Yi, Jae Youn; Easterly,Evangeline; Barcellos-Hoff, Mary Helen; Yingling, Jonathan M.; Zent, Roy; Arteaga, Carlos L.

    2005-01-02

    The transforming growth factor-betas (TGF{beta}s) are members of a large superfamily of pleiotropic cytokines that also includes the activins and the bone morphogenetic proteins (BMPs). Members of the TGF{beta} family regulate complex physiological processes such cell proliferation, differentiation, adhesion, cell-cell and cell-matrix interactions, motility, and cell death, among others (Massague, 1998). Dysregulation of TGF{beta} signaling contributes to several pathological processes including cancer, fibrosis, and auto-immune disorders (Massague et al., 2000). The TGF{beta}s elicit their biological effects by binding to type II and type I transmembrane receptor serine-threonine kinases (T{beta}RII and T{beta}RI) which, in turn, phosphorylated Smad 2 and Smad 3. Phosphorylated Smad 2/3 associate with Smad 4 and, as a heteromeric complex, translocate to the nucleus where they regulate gene transcription. The inhibitory Smad7 down regulates TGF{beta} signaling by binding to activated T{beta}RI and interfering with its ability to phosphorylate Smad 2/3 (Derynck and Zhang, 2003; Shi and Massague, 2003). Signaling is also regulated by Smad proteolysis. TGF{beta} receptor-mediated activation results in multi-ubiquitination of Smad 2 in the nucleus and subsequent degradation of Smad 2 by the proteasome (Lo and Massague, 1999). Activation of TGF{beta} receptors also induces mobilization of a Smad 7-Smurf complex from the nucleus to the cytoplasm; this complex recognizes the activated receptors and mediates their ubiquitination and internalization via caveolin-rich vesicles, leading to termination of TGF{beta} signaling (Di Guglielmo et al., 2003). Other signal transducers/pathways have been implicated in TGF{beta} actions. These include the extracellular signal-regulated kinase (Erk), c-Jun N-terminal kinase (Jnk), p38 mitogen-activated protein kinase (MAPK), protein phosphatase PP2A, phosphatidylinositol-3 kinase (PI3K), and the family of Rho GTPases [reviewed in

  8. Temporal and SUMO-specific SUMOylation contribute to the dynamics of Polo-like kinase 1 (PLK1) and spindle integrity during mouse oocyte meiosis.

    Science.gov (United States)

    Feitosa, Weber Beringui; Hwang, KeumSil; Morris, Patricia L

    2018-02-15

    During mammalian meiosis, Polo-like kinase 1 (PLK1) is essential during cell cycle progression. In oocyte maturation, PLK1 expression is well characterized but timing of posttranslational modifications regulating its activity and subcellular localization are less clear. Small ubiquitin-related modifier (SUMO) posttranslational modifier proteins have been detected in mammalian gametes but their precise function during gametogenesis is largely unknown. In the present paper we report for mouse oocytes that both PLK1 and phosphorylated PLK1 undergo SUMOylation in meiosis II (MII) oocytes using immunocytochemistry, immunoprecipitation and in vitro SUMOylation assays. At MII, PLK1 is phosphorylated at threonine-210 and serine-137. MII oocyte PLK1 and phosphorylated PLK1 undergo SUMOylation by SUMO-1, -2 and -3 as shown by individual in vitro assays. Using these assays, forms of phosphorylated PLK1 normalized to PLK1 increased significantly and correlated with SUMOylated PLK1 levels. During meiotic progression and maturation, SUMO-1-SUMOylation of PLK1 is involved in spindle formation whereas SUMO-2/3-SUMOylation may regulate PLK1 activity at kinetochore-spindle attachment sites. Microtubule integrity is required for PLK1 localization with SUMO-1 but not with SUMO-2/3. Inhibition of SUMOylation disrupts proper meiotic bipolar spindle organization and spindle-kinetochore attachment. The data show that both temporal and SUMO-specific-SUMOylation play important roles in orchestrating functional dynamics of PLK1 during mouse oocyte meiosis, including subcellular compartmentalization. Copyright © 2018 Elsevier Inc. All rights reserved.

  9. Method for the production of l-serine using genetically engineered microorganisms deficient in serine degradation pathways

    DEFF Research Database (Denmark)

    2016-01-01

    The present invention generally relates to the microbiological industry, and specifically to the production of L-serine using genetically modified bacteria. The present invention provides genetically modified microorganisms, such as bacteria, wherein the expression of genes encoding for enzymes...... concentrations of serine. The present invention also provides methods for the production of L-serine or L-serine derivative using such genetically modified microorganisms....

  10. Digestible Threonine Levels in the Starter Diet of Broilers Derived from Breeders of Different Ages

    Directory of Open Access Journals (Sweden)

    CBGS Tanure

    2015-12-01

    Full Text Available ABSTRACT The aim of this study was to evaluate the effect of digestible threonine supplementation in the starter diet on the performance, intestinal parameters, and nutrient metabolism of broilers derived from breeders of different ages. In total, 480 one-day-old Cobb chicks, derived from 38-or 49-week-oldbreeders, were housed in experimental battery cages until 21 days of age and fed four different threonine levels (800, 900, 1,000, or 1,100 mg/kg in the starter feed. A completely randomized experimental design in a 2x4 factorial arrangement (breeder age x threonine levels was applied, totaling eight treatments with five replicates of 12 birds each. Broilers from older breeders fed 800 mg digestible threonine/kg of diet presented higher weight gain, with a positive linear effect. There was also an interaction between breeder age and threonine levels for the weight gain of 21-d-old broilers supplemented at maximum level of 1,003 mg Thr/kg diet during the starter phase. There was no effect of breeder age or threonine levels on nutrient metabolism during the period of 17-21 days. There was no influence of breeder age or threonine levels in the starter diet on intestinal morphometric measurements, absorption area, or percentage of goblet cells.

  11. DETERMINATION OF OPTIMUM THREONINE REQUIREMENTS OF JAPANESE QUAIL (Coturnix coturnix japonica CHICKS REARED UNDER TROPICAL ENVIRONMENT

    Directory of Open Access Journals (Sweden)

    Isaac Samuel

    2017-12-01

    Full Text Available Threonine, like most amino acids, is traditionally noted for its role in protein synthesis. However, dietary threonine concentration required for the maximum performance of Japanese quails is yet to be determined, therefore, a study was conducted to determine the optimum threonine requirement of Japanese quail chicks in the tropical environment. A total of four hundred and fifty, two weeks old quail chicks (mixed sexes were randomly allocated to five dietary treatments (0.67, 0.81, 0.95, 1.08 and 1.22 % total threonine and replicated thrice in a completely randomized design (CRD. There were no significant differences (P>0.05 in average weight gain, average feed intake, feed conversion ratio and age at first lay of quails fed the dietary treatments. Weight of first egg laid was significantly (P0.05 similar for all treatments. There were significant differences (P0.05. It was concluded that the performance of birds were not influenced by the dietary treatments up to the highest dietary level of threonine studied (1.22%. Therefore, it is possible that the optimum dietary level of threonine may be above 1.22 %. Further studies will be necessary to establish higher doses of dietary threonine requirement of Japanese quails reared under tropical climatic environment.

  12. Estimation of L-threonine requirements for Longyan laying ducks

    Directory of Open Access Journals (Sweden)

    A. M. Fouad

    2017-02-01

    Full Text Available Objective A study was conducted to test six threonine (Thr levels (0.39%, 0.44%, 0.49%, 0.54%, 0.59%, and 0.64% to estimate the optimal dietary Thr requirements for Longyan laying ducks from 17 to 45 wk of age. Methods Nine hundred Longyan ducks aged 17 wk were assigned randomly to the six dietary treatments, where each treatment comprised six replicate pens with 25 ducks per pen. Results Increasing the Thr level enhanced egg production, egg weight, egg mass, and the feed conversion ratio (FCR (linearly or quadratically; p<0.05. The Haugh unit score, yolk color, albumen height, and the weight, percentage, thickness, and breaking strength of the eggshell did not response to increases in the Thr levels, but the albumen weight and its proportion increased significantly (p<0.05, whereas the yolk weight and its proportion decreased significantly as the Thr levels increased. Conclusion According to a regression model, the optimal Thr requirement for egg production, egg mass, and FCR in Longyan ducks is 0.57%, while 0.58% is the optimal level for egg weight from 17 to 45 wk of age.

  13. Protein kinase CK2 modulates IL-6 expression in inflammatory breast cancer

    Energy Technology Data Exchange (ETDEWEB)

    Drygin, Denis, E-mail: ddrygin@cylenepharma.com; Ho, Caroline B.; Omori, Mayuko; Bliesath, Joshua; Proffitt, Chris; Rice, Rachel; Siddiqui-Jain, Adam; O' Brien, Sean; Padgett, Claire; Lim, John K.C.; Anderes, Kenna; Rice, William G.; Ryckman, David

    2011-11-11

    Highlights: Black-Right-Pointing-Pointer We examine the potential cross-talk between CK2 and IL-6. Black-Right-Pointing-Pointer Inhibition of CK2 by siRNA or CX-4945 inhibits expression of IL-6 in models of IBC. Black-Right-Pointing-Pointer Treatment of IBC patient in the clinic with CX-4945 reduces her IL-6 plasma levels. Black-Right-Pointing-Pointer We demonstrate that CK2 is a potential therapeutic target for IL-6 driven diseases. -- Abstract: Inflammatory breast cancer is driven by pro-angiogenic and pro-inflammatory cytokines. One of them Interleukin-6 (IL-6) is implicated in cancer cell proliferation and survival, and promotes angiogenesis, inflammation and metastasis. While IL-6 has been shown to be upregulated by several oncogenes, the mechanism behind this phenomenon is not well characterized. Here we demonstrate that the pleotropic Serine/Threonine kinase CK2 is implicated in the regulation of IL-6 expression in a model of inflammatory breast cancer. We used siRNAs targeted toward CK2 and a selective small molecule inhibitor of CK2, CX-4945, to inhibit the expression and thus suppress the secretion of IL-6 in in vitro as well as in vivo models. Moreover, we report that in a clinical trial, CX-4945 was able to dramatically reduce IL-6 levels in plasma of an inflammatory breast cancer patient. Our data shed a new light on the regulation of IL-6 expression and position CX-4945 and potentially other inhibitors of CK2, for the treatment of IL-6-driven cancers and possibly other diseases where IL-6 is instrumental, including rheumatoid arthritis.

  14. Downregulation of an Aim-1 Kinase Couples with Megakaryocytic Polyploidization of Human Hematopoietic Cells

    Science.gov (United States)

    Kawasaki, Akira; Matsumura, Itaru; Miyagawa, Jun-ichiro; Ezoe, Sachiko; Tanaka, Hirokazu; Terada, Yasuhiko; Tatsuka, Masaaki; Machii, Takashi; Miyazaki, Hiroshi; Furukawa, Yusuke; Kanakura, Yuzuru

    2001-01-01

    During the late phase of megakaryopoiesis, megakaryocytes undergo polyploidization, which is characterized by DNA duplication without concomitant cell division. However, it remains unknown by which mechanisms this process occurs. AIM-1 and STK15 belong to the Aurora/increase-in-ploidy (Ipl)1 serine/threonine kinase family and play key roles in mitosis. In a human interleukin-3–dependent cell line, F-36P, the expressions of AIM-1 and STK15 mRNA were specifically observed at G2/M phase of the cell cycle during proliferation. In contrast, the expressions of AIM-1 and STK15 were continuously repressed during megakaryocytic polyploidization of human erythro/megakaryocytic cell lines (F-36P, K562, and CMK) treated with thrombopoietin, activated ras (H-rasG12V), or phorbol ester. Furthermore, their expressions were suppressed during thrombopoietin-induced polyploidization of normal human megakaryocytes. Activation of AIM-1 by the induced expression of AIM-1(wild-type) canceled TPA-induced polyploidization of K562 cells significantly, whereas that of STK15 did not. Moreover, suppression of AIM-1 by the induced expression of AIM-1 (K/R, dominant-negative type) led to polyploidization in 25% of K562 cells, whereas STK15(K/R) showed no effect. Also, the induced expression of AIM-1(K/R) in CMK cells provoked polyploidization up to 32N. These results suggested that downregulation of AIM-1 at M phase may be involved in abortive mitosis and polyploid formation of megakaryocytes. PMID:11266445

  15. Akt kinases in breast cancer and the results of adjuvant therapy

    International Nuclear Information System (INIS)

    Stål, Olle; Pérez-Tenorio, Gizeh; Åkerberg, Linda; Olsson, Birgit; Nordenskjöld, Bo; Skoog, Lambert; Rutqvist, Lars Erik

    2003-01-01

    The serine/threonine kinase Akt, or protein kinase B, has recently been a focus of interest because of its activity to inhibit apoptosis. It mediates cell survival by acting as a transducer of signals from growth factor receptors that activate phosphatidylinositol 3-kinase. We analysed the expression of the isoforms Akt1 and Akt2 as well as phosphorylated Akt (pAkt) by immunohistochemistry in frozen tumour samples from 280 postmenopausal patients who participated in a randomised trial comparing cyclophosphamide–methotrexate–5-fluorouracil chemotherapy and postoperative radiotherapy. The patients were simultaneously randomised to tamoxifen or to no endocrine treatment. Marked staining was found in 24% of the tumours for Akt1, but in only 4% for Akt2. A low frequency of Akt2-positive cells (1–10%) was observed in another 26% of the tumours. pAkt was significantly associated with both Akt1 and Akt2 expression. Overexpression of erbB2 correlated significantly with pAkt (P = 0.0028). The benefit from tamoxifen was analysed in oestrogen receptor (ER)-positive patients. Patients with a negative status of Akt (no overexpression of Akt1, Akt2 or pAkt) showed significant benefit from tamoxifen. The relative rate of distant recurrence, with versus without tamoxifen, was 0.44 (95% confidence interval [CI], 0.25–0.79) for ER+/Akt1- patients, while it was 0.72 (95% CI, 0.34–1.53) for ER+/Akt1+ patients. The difference in rate ratio did not reach statistical significance. The rate of locoregional recurrence was significantly decreased with radiotherapy versus chemotherapy for Akt-negative patients (rate ratio, 0.23; 95% CI, 0.08–0.67; P = 0.0074), while no benefit was evident for the Akt-positive subgroup (rate ratio, 0.77; 95% CI, 0.31–1.9; P = 0.58). The interaction between Akt and the efficacy of radiotherapy was significant in multivariate analysis (P = 0.042). Activation of the Akt pathway is correlated with erbB2 overexpression in breast cancer. The results

  16. Regulation of Adrenal Aldosterone Production by Serine Protease Prostasin

    Directory of Open Access Journals (Sweden)

    Takehiro Ko

    2010-01-01

    Full Text Available A serine protease prostasin has been demonstrated to have a pivotal role in the activation of the epithelial sodium channel. Systemic administration of adenovirus carrying human prostasin gene in rats resulted in an increase in plasma prostasin and aldosterone levels. However, the mechanism by which the elevation of prostasin levels in the systemic circulation stimulated the plasma aldosterone levels remains unknown. Therefore, we examined if prostasin increases the aldosterone synthesis in a human adrenocortical cell line (H295R cells. Luciferase assay using CYP11B2 promoter revealed that prostasin significantly increased the transcriptional activity of CYP11B2. Prostasin significantly increased both CYP11B2 mRNA expression and aldosterone production in a dose-dependent manner. Surprisingly, treatment with camostat mesilate, a potent prostasin inhibitor, had no effect on the aldosterone synthesis by prostasin and also a protease-dead mutant of prostasin significantly stimulated the aldosterone production. A T-type/L-type calcium channel blocker and a protein kinase C (PKC inhibitor significantly reduced the aldosterone synthesis by prostasin. Our findings suggest a stimulatory effect of prostasin on the aldosterone synthesis by adrenal gland through the nonproteolytic action and indicate a new role of prostasin in the systemic circulation.

  17. Aurora-B Mediated ATM Serine 1403 Phosphorylation Is Required For Mitotic ATM Activation and the Spindle Checkpoint

    OpenAIRE

    Yang, Chunying; Tang, Xi; Guo, Xiaojing; Niikura, Yohei; Kitagawa, Katsumi; Cui, Kemi; Wong, Stephen T.C.; Fu, Li; Xu, Bo

    2011-01-01

    The ATM kinase plays a critical role in the maintenance of genetic stability. ATM is activated in response to DNA damage and is essential for cell cycle checkpoints. Here, we report that ATM is activated in mitosis in the absence of DNA damage. We demonstrate that mitotic ATM activation is dependent on the Aurora-B kinase and that Aurora-B phosphorylates ATM on serine 1403. This phosphorylation event is required for mitotic ATM activation. Further, we show that loss of ATM function results in...

  18. Differential binding of RhoA, RhoB, and RhoC to protein kinase C-related kinase (PRK) isoforms PRK1, PRK2, and PRK3: PRKs have the highest affinity for RhoB.

    Science.gov (United States)

    Hutchinson, Catherine L; Lowe, Peter N; McLaughlin, Stephen H; Mott, Helen R; Owen, Darerca

    2013-11-12

    Protein kinase C-related kinases (PRKs) are members of the protein kinase C superfamily of serine-threonine kinases and can be activated by binding to members of the Rho family of GTPases via a Rho-binding motif known as an HR1 domain. Three tandem HR1 domains reside at the N-terminus of the PRKs. We have assessed the ability of the HR1a and HR1b domains from the three PRK isoforms (PRK1, PRK2, and PRK3) to interact with the three Rho isoforms (RhoA, RhoB, and RhoC). The affinities of RhoA and RhoC for a construct encompassing both PRK1 HR1 domains were similar to those for the HR1a domain alone, suggesting that these interactions are mediated solely by the HR1a domain. The affinities of RhoB for both the PRK1 HR1a domain and the HR1ab didomain were higher than those of RhoA or RhoC. RhoB also bound more tightly to the didomain than to the HR1a domain alone, implicating the HR1b domain in the interaction. As compared with PRK1 HR1 domains, PRK2 and PRK3 domains bind less well to all Rho isoforms. Uniquely, however, the PRK3 domains display a specificity for RhoB that requires both the C-terminus of RhoB and the PRK3 HR1b domain. The thermal stability of the HR1a and HR1b domains was also investigated. The PRK2 HR1a domain was found to be the most thermally stable, while PRK2 HR1b, PRK3 HR1a, and PRK3 HR1b domains all exhibited lower melting temperatures, similar to that of the PRK1 HR1a domain. The lower thermal stability of the PRK2 and PRK3 HR1b domains may impart greater flexibility, driving their ability to interact with Rho isoforms.

  19. Tyrosine-610 in the Receptor Kinase BAK1 Does Not Play a Major Role in Brassinosteroid Signaling or Innate Immunity

    Directory of Open Access Journals (Sweden)

    Vijayata Singh

    2017-08-01

    Full Text Available The plasma membrane-localized BRI1-ASSOCIATED KINASE1 (BAK1 functions as a co-receptor with several receptor kinases including the brassinosteroid (BR receptor BRASSINOSTEROID-INSENSITIVE 1 (BRI1, which is involved in growth, and the receptors for bacterial flagellin and EF-Tu, FLAGELLIN-SENSING 2 (FLS2 and EF-TU RECEPTOR (EFR, respectively, which are involved in immunity. BAK1 is a dual specificity protein kinase that can autophosphorylate on serine, threonine and tyrosine residues. It was previously reported that phosphorylation of Tyr-610 in the carboxy-terminal domain of BAK1 is required for its function in BR signaling and immunity. However, the functional role of Tyr-610 in vivo has recently come under scrutiny. Therefore, we have generated new BAK1 (Y610F transgenic plants for functional studies. We first produced transgenic Arabidopsis lines expressing BAK1 (Y610F-Flag in the homozygous bak1-4 bkk1-1 double null background. In a complementary approach, we expressed untagged BAK1 and BAK1 (Y610F in the bak1-4 null mutant. Neither BAK1 (Y610F transgenic line had any obvious growth phenotype when compared to wild-type BAK1 expressed in the same background. In addition, the BAK1 (Y610F-Flag plants responded similarly to plants expressing BAK1-Flag in terms of brassinolide (BL inhibition of root elongation, and there were only minor changes in gene expression between the two transgenic lines as monitored by microarray analysis and quantitative real-time PCR. In terms of plant immunity, there were no significant differences between plants expressing BAK1 (Y610F-Flag and BAK1-Flag in the growth of the non-pathogenic hrpA- mutant of Pseudomonas syringae pv. tomato DC3000. Furthermore, untagged BAK1 (Y610F transgenic plants were as responsive as plants expressing BAK1 (in the bak1-4 background and wild-type Col-0 plants toward treatment with the EF-Tu- and flagellin-derived peptide epitopes elf18- and flg22, respectively, as measured by reactive

  20. Phosphorylation of Threonine 794 on Tie1 by Rac1/PAK1 Reveals a Novel Angiogenesis Regulatory Pathway.

    Directory of Open Access Journals (Sweden)

    Jessica L Reinardy

    Full Text Available The endothelial receptor tyrosine kinase (RTK Tie1 was discovered over 20 years ago, yet its precise function and mode of action remain enigmatic. To shed light on Tie1's role in endothelial cell biology, we investigated a potential threonine phosphorylation site within the juxtamembrane domain of Tie1. Expression of a non-phosphorylatable mutant of this site (T794A in zebrafish (Danio rerio significantly disrupted vascular development, resulting in fish with stunted and poorly branched intersomitic vessels. Similarly, T794A-expressing human umbilical vein endothelial cells formed significantly shorter tubes with fewer branches in three-dimensional Matrigel cultures. However, mutation of T794 did not alter Tie1 or Tie2 tyrosine phosphorylation or downstream signaling in any detectable way, suggesting that T794 phosphorylation may regulate a Tie1 function independent of its RTK properties. Although T794 is within a consensus Akt phosphorylation site, we were unable to identify a physiological activator of Akt that could induce T794 phosphorylation, suggesting that Akt is not the physiological Tie1-T794 kinase. However, the small GTPase Ras-related C3 botulinum toxin substrate 1 (Rac1, which is required for angiogenesis and capillary morphogenesis, was found to associate with phospho-T794 but not the non-phosphorylatable T794A mutant. Pharmacological activation of Rac1 induced downstream activation of p21-activated kinase (PAK1 and T794 phosphorylation in vitro, and inhibition of PAK1 abrogated T794 phosphorylation. Our results provide the first demonstration of a signaling pathway mediated by Tie1 in endothelial cells, and they suggest that a novel feedback loop involving Rac1/PAK1 mediated phosphorylation of Tie1 on T794 is required for proper angiogenesis.

  1. IRS-1 serine phosphorylation and insulin resistance in skeletal muscle from pancreas tranplant recipient

    DEFF Research Database (Denmark)

    Bouzakri, K; Karlsson, HRK; Vestergaard, Henrik

    2006-01-01

    Insulin-dependent diabetic recipients of successful pancreas allografts achieve self-regulatory insulin secretion and discontinue exogenous insulin therapy; however, chronic hyperinsulinemia and impaired insulin sensitivity generally develop. To determine whether insulin resistance is accompanied....... In conclusion, peripheral insulin resistance in pancreas-kidney transplant recipients may arise from a negative feedback regulation of the canonical insulin-signaling cascade from excessive serine phosphorylation of IRS-1, possibly as a consequence of immunosuppressive therapy and hyperinsulinemia....... insulin receptor substrate (IRS)-1 Ser (312) and Ser (616) phosphorylation, IRS-1-associated phosphatidylinositol 3-kinase activity, and extracellular signal-regulated kinase (ERK)-1/2 phosphorylation were elevated in pancreas-kidney transplant recipients, coincident with fasting hyperinsulinemia. Basal...

  2. Serine proteinases and their inhibitors in fertilization

    Czech Academy of Sciences Publication Activity Database

    Jonáková, Věra; Jelínková-Slavíčková, Petra

    2004-01-01

    Roč. 8, 3,4 (2004), s. 108-110 ISSN 1211-8869. [Central European Conference on Human Tumor Markers /5./. Praha, 01.10.2004-03.10.2004] R&D Projects: GA ČR GA303/02/0433; GA ČR GP303/02/P069; GA ČR GP303/04/P070; GA MZd NJ7463 Institutional research plan: CEZ:AV0Z5052915 Keywords : serine proteinase * proteinase inhibitors * fertilization Subject RIV: CE - Biochemistry

  3. Study of the affinity between the protein kinase PKA and homoarginine-containing peptides derived from kemptide: Free energy perturbation (FEP) calculations.

    Science.gov (United States)

    Mena-Ulecia, Karel; Gonzalez-Norambuena, Fabian; Vergara-Jaque, Ariela; Poblete, Horacio; Tiznado, William; Caballero, Julio

    2018-02-05

    Protein kinases (PKs) discriminate between closely related sequences that contain serine, threonine, and/or tyrosine residues. Such specificity is defined by the amino acid sequence surrounding the phosphorylatable residue, so that it is possible to identify an optimal recognition motif (ORM) for each PK. The ORM for the protein kinase A (PKA), a well-known member of the PK family, is the sequence RRX(S/T)X, where arginines at the -3 and -2 positions play a key role with respect to the primed phosphorylation site. In this work, differential affinities of PKA for the peptide substrate Kemptide (LRRASLG) and mutants that substitute the arginine residues by the unnatural peptide homoarginine were evaluated through molecular dynamics (MD) and free energy perturbation (FEP) calculations. The FEP study for the homoarginine mutants required previous elaboration of a CHARMM "arginine to homoarginine" (R2B) hybrid topology file which is available in this manuscript as Supporting Information. Mutants substituting the arginine residues by alanine, lysine, and histidine were also considered in the comparison by using the same protocol. FEP calculations allowed estimating the free energy changes from the free PKA to PKA-substrate complex (ΔΔG E→ES ) when Kemptide structure was mutated. Both ΔΔG S→ES values for homoarginine mutants were predicted with a difference below 1 kcal/mol. In addition, FEP correctly predicted that all the studied mutations decrease the catalytic efficiency of Kemptide for PKA. © 2018 Wiley Periodicals, Inc. © 2018 Wiley Periodicals, Inc.

  4. Herpesvirus gB-induced fusion between the virion envelope and outer nuclear membrane during virus egress is regulated by the viral US3 kinase.

    Science.gov (United States)

    Wisner, Todd W; Wright, Catherine C; Kato, Akihisa; Kawaguchi, Yasushi; Mou, Fan; Baines, Joel D; Roller, Richard J; Johnson, David C

    2009-04-01

    Herpesvirus capsids collect along the inner surface of the nuclear envelope and bud into the perinuclear space. Enveloped virions then fuse with the outer nuclear membrane (NM). We previously showed that herpes simplex virus (HSV) glycoproteins gB and gH act in a redundant fashion to promote fusion between the virion envelope and the outer NM. HSV mutants lacking both gB and gH accumulate enveloped virions in herniations, vesicles that bulge into the nucleoplasm. Earlier studies had shown that HSV mutants lacking the viral serine/threonine kinase US3 also accumulate herniations. Here, we demonstrate that HSV gB is phosphorylated in a US3-dependent manner in HSV-infected cells, especially in a crude nuclear fraction. Moreover, US3 directly phosphorylated the gB cytoplasmic (CT) domain in in vitro assays. Deletion of gB in the context of a US3-null virus did not add substantially to defects in nuclear egress. The majority of the US3-dependent phosphorylation of gB involved the CT domain and amino acid T887, a residue present in a motif similar to that recognized by US3 in other proteins. HSV recombinants lacking gH and expressing either gB substitution mutation T887A or a gB truncated at residue 886 displayed substantial defects in nuclear egress. We concluded that phosphorylation of the gB CT domain is important for gB-mediated fusion with the outer NM. This suggested a model in which the US3 kinase is incorporated into the tegument layer (between the capsid and envelope) in HSV virions present in the perinuclear space. By this packaging, US3 might be brought close to the gB CT tail, leading to phosphorylation and triggering fusion between the virion envelope and the outer NM.

  5. Concurrent Inhibition of Pim and FLT3 Kinases Enhances Apoptosis of FLT3-ITD Acute Myeloid Leukemia Cells through Increased Mcl-1 Proteasomal Degradation.

    Science.gov (United States)

    Kapoor, Shivani; Natarajan, Karthika; Baldwin, Patrick R; Doshi, Kshama A; Lapidus, Rena G; Mathias, Trevor J; Scarpa, Mario; Trotta, Rossana; Davila, Eduardo; Kraus, Manfred; Huszar, Dennis; Tron, Adriana E; Perrotti, Danilo; Baer, Maria R

    2018-01-01

    Purpose: fms -like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) is present in 30% of acute myeloid leukemia (AML), and these patients have short disease-free survival. FLT3 inhibitors have limited and transient clinical activity, and concurrent treatment with inhibitors of parallel or downstream signaling may improve responses. The oncogenic serine/threonine kinase Pim-1 is upregulated downstream of FLT3-ITD and also promotes its signaling in a positive feedback loop, suggesting benefit of combined Pim and FLT3 inhibition. Experimental Design: Combinations of clinically active Pim and FLT3 inhibitors were studied in vitro and in vivo Results: Concurrent treatment with the pan-Pim inhibitor AZD1208 and FLT3 inhibitors at clinically applicable concentrations abrogated in vitro growth of FLT3-ITD, but not wild-type FLT3 (FLT3-WT), cell lines. AZD1208 cotreatment increased FLT3 inhibitor-induced apoptosis of FLT3-ITD, but not FLT3-WT, cells measured by sub-G 1 fraction, annexin V labeling, mitochondrial membrane potential, and PARP and caspase-3 cleavage. Concurrent treatment with AZD1208 and the FLT3 inhibitor quizartinib decreased growth of MV4-11 cells, with FLT3-ITD, in mouse xenografts, and prolonged survival, enhanced apoptosis of FLT3-ITD primary AML blasts, but not FLT3-WT blasts or remission marrow cells, and decreased FLT3-ITD AML blast colony formation. Mechanistically, AZD1208 and quizartinib cotreatment decreased expression of the antiapoptotic protein Mcl-1. Decrease in Mcl-1 protein expression was abrogated by treatment with the proteasome inhibitor MG132, and was preceded by downregulation of the Mcl-1 deubiquitinase USP9X, a novel mechanism of Mcl-1 regulation in AML. Conclusions: The data support clinical testing of Pim and FLT3 inhibitor combination therapy for FLT3-ITD AML. Clin Cancer Res; 24(1); 234-47. ©2017 AACR . ©2017 American Association for Cancer Research.

  6. 3D-QSAR and molecular docking studies on derivatives of MK-0457, GSK1070916 and SNS-314 as inhibitors against Aurora B kinase.

    Science.gov (United States)

    Zhang, Baidong; Li, Yan; Zhang, Huixiao; Ai, Chunzhi

    2010-11-02

    Development of anticancer drugs targeting Aurora B, an important member of the serine/threonine kinases family, has been extensively focused on in recent years. In this work, by applying an integrated computational method, including comparative molecular field analysis (CoMFA), comparative molecular similarity indices analysis (CoMSIA), homology modeling and molecular docking, we investigated the structural determinants of Aurora B inhibitors based on three different series of derivatives of 108 molecules. The resultant optimum 3D-QSAR models exhibited (q(2) = 0.605, r(2) (pred) = 0.826), (q(2) = 0.52, r(2) (pred) = 0.798) and (q(2) = 0.582, r(2) (pred) = 0.971) for MK-0457, GSK1070916 and SNS-314 classes, respectively, and the 3D contour maps generated from these models were analyzed individually. The contour map analysis for the MK-0457 model revealed the relative importance of steric and electrostatic effects for Aurora B inhibition, whereas, the electronegative groups with hydrogen bond donating capacity showed a great impact on the inhibitory activity for the derivatives of GSK1070916. Additionally, the predictive model of the SNS-314 class revealed the great importance of hydrophobic favorable contour, since hydrophobic favorable substituents added to this region bind to a deep and narrow hydrophobic pocket composed of residues that are hydrophobic in nature and thus enhanced the inhibitory activity. Moreover, based on the docking study, a further comparison of the binding modes was accomplished to identify a set of critical residues that play a key role in stabilizing the drug-target interactions. Overall, the high level of consistency between the 3D contour maps and the topographical features of binding sites led to our identification of several key structural requirements for more potency inhibitors. Taken together, the results will serve as a basis for future drug development of inhibitors against Aurora B kinase for various tumors.

  7. 3D-QSAR and Molecular Docking Studies on Derivatives of MK-0457, GSK1070916 and SNS-314 as Inhibitors against Aurora B Kinase

    Directory of Open Access Journals (Sweden)

    Chunzhi Ai

    2010-11-01

    Full Text Available Development of anticancer drugs targeting Aurora B, an important member of the serine/threonine kinases family, has been extensively focused on in recent years. In this work, by applying an integrated computational method, including comparative molecular field analysis (CoMFA, comparative molecular similarity indices analysis (CoMSIA, homology modeling and molecular docking, we investigated the structural determinants of Aurora B inhibitors based on three different series of derivatives of 108 molecules. The resultant optimum 3D-QSAR models exhibited (q2 = 0.605, r2pred = 0.826, (q2 = 0.52, r2pred = 0.798 and (q2 = 0.582, r2pred = 0.971 for MK-0457, GSK1070916 and SNS-314 classes, respectively, and the 3D contour maps generated from these models were analyzed individually. The contour map analysis for the MK-0457 model revealed the relative importance of steric and electrostatic effects for Aurora B inhibition, whereas, the electronegative groups with hydrogen bond donating capacity showed a great impact on the inhibitory activity for the derivatives of GSK1070916. Additionally, the predictive model of the SNS-314 class revealed the great importance of hydrophobic favorable contour, since hydrophobic favorable substituents added to this region bind to a deep and narrow hydrophobic pocket composed of residues that are hydrophobic in nature and thus enhanced the inhibitory activity. Moreover, based on the docking study, a further comparison of the binding modes was accomplished to identify a set of critical residues that play a key role in stabilizing the drug-target interactions. Overall, the high level of consistency between the 3D contour maps and the topographical features of binding sites led to our identification of several key structural requirements for more potency inhibitors. Taken together, the results will serve as a basis for future drug development of inhibitors against Aurora B kinase for various tumors.

  8. Involvement of adenosine monophosphate-activated protein kinase in the influence of timed high-fat evening diet on the hepatic clock and lipogenic gene expression in mice.

    Science.gov (United States)

    Huang, Yan; Zhu, Zengyan; Xie, Meilin; Xue, Jie

    2015-09-01

    A high-fat diet may result in changes in hepatic clock gene expression, but potential mechanisms are not yet elucidated. Adenosine monophosphate-activated protein kinase (AMPK) is a serine/threonine protein kinase that is recognized as a key regulator of energy metabolism and certain clock genes. Therefore, we hypothesized that AMPK may be involved in the alteration of hepatic clock gene expression under a high-fat environment. This study aimed to examine the effects of timed high-fat evening diet on the activity of hepatic AMPK, clock genes, and lipogenic genes. Mice with hyperlipidemic fatty livers were induced by orally administering high-fat milk via gavage every evening (19:00-20:00) for 6 weeks. Results showed that timed high-fat diet in the evening not only decreased the hepatic AMPK protein expression and activity but also disturbed its circadian rhythm. Accordingly, the hepatic clock genes, including clock, brain-muscle-Arnt-like 1, cryptochrome 2, and period 2, exhibited prominent changes in their expression rhythms and/or amplitudes. The diurnal rhythms of the messenger RNA expression of peroxisome proliferator-activated receptorα, acetyl-CoA carboxylase 1α, and carnitine palmitoyltransferase 1 were also disrupted; the amplitude of peroxisome proliferator-activated receptorγcoactivator 1α was significantly decreased at 3 time points, and fatty liver was observed. These findings demonstrate that timed high-fat diet at night can change hepatic AMPK protein levels, activity, and circadian rhythm, which may subsequently alter the circadian expression of several hepatic clock genes and finally result in the disorder of hepatic lipogenic gene expression and the formation of fatty liver. Copyright © 2015 Elsevier Inc. All rights reserved.

  9. TANK-Binding Kinase 1 (TBK1 Isoforms Negatively Regulate Type I Interferon Induction by Inhibiting TBK1-IRF3 Interaction and IRF3 Phosphorylation

    Directory of Open Access Journals (Sweden)

    Yi Wei Hu

    2018-01-01

    Full Text Available TANK-binding kinase 1 (TBK1 is an important serine/threonine-protein kinase that mediates phosphorylation and nuclear translocation of IRF3, which contributes to induction of type I interferons (IFNs in the innate antiviral response. In mammals, TBK1 spliced isoform negatively regulates the virus-triggered IFN-β signaling pathway by disrupting the interaction between retinoic acid-inducible gene I (RIG-I and mitochondria antiviral-signaling protein (MAVS. However, it is still unclear whether alternative splicing patterns and the function of TBK1 isoform(s exist in teleost fish. In this study, we identify two alternatively spliced isoforms of TBK1 from zebrafish, termed TBK1_tv1 and TBK1_tv2. Both TBK1_tv1 and TBK1_tv2 contain an incomplete STKc_TBK1 domain. Moreover, the UBL_TBK1_like domain is also missing for TBK1_tv2. TBK1_tv1 and TBK1_tv2 are expressed in zebrafish larvae. Overexpression of TBK1_tv1 and TBK1_tv2 inhibits RIG-I-, MAVS-, TBK1-, and IRF3-mediated activation of IFN promoters in response to spring viremia of carp virus infection. Also, TBK1_tv1 and TBK1_tv2 inhibit expression of IFNs and IFN-stimulated genes induced by MAVS and TBK1. Mechanistically, TBK1_tv1 and TBK1_tv2 competitively associate with TBK1 and IRF3 to disrupt the formation of a functional TBK1-IRF3 complex, impeding the phosphorylation of IRF3 mediated by TBK1. Collectively, these results demonstrate that TBK1 spliced isoforms are dominant negative regulators in the RIG-I/MAVS/TBK1/IRF3 antiviral pathway by targeting the functional TBK1-IRF3 complex formation. Identification and functional characterization of piscine TBK1 spliced isoforms may contribute to understanding the role of TBK1 expression in innate antiviral response.

  10. The importance of regulation of blood glucose levels through activation of peripheral 5'-AMP-activated protein kinase on ischemic neuronal damage.

    Science.gov (United States)

    Harada, Shinichi; Fujita-Hamabe, Wakako; Tokuyama, Shogo

    2010-09-10

    5'-AMP-activated protein kinase (AMPK) is a serine/threonine kinase that plays a key role in energy homeostasis. Recently, it was reported that centrally activated AMPK is involved in the development of ischemic neuronal damage, while the effect of peripherally activated AMPK on ischemic neuronal damage is not known. In addition, we have previously reported that the development of post-ischemic glucose intolerance could be one of the triggers for the aggravation of neuronal damage. In this study, we focused on effect of activation of peripheral or central AMPK on the development of ischemic neuronal damage. Male ddY mice were subjected to 2 h of middle cerebral artery occlusion (MCAO). Neuronal damage was estimated by histological and behavioral analysis after MCAO. In the liver and skeletal muscle, AMPK activity was not affected by MCAO. But, application of intraperitoneal metformin (250 mg/kg), an AMPK activator, significantly suppressed the development of post-ischemic glucose intolerance and ischemic neuronal damage without alteration of central AMPK activity. On the other hand, application of intracerebroventricular metformin (25, 100 microg/mouse) significantly exacerbated the development of neuronal damage observed on day 1 after MCAO, in a dose-dependent manner. These effects were significantly blocked by compound C, a specific AMPK inhibitor. These results suggest that central AMPK was activated by ischemic stress per se, however, peripheral AMPK was not altered. Furthermore, the regulation of post-ischemic glucose intolerance by activation of peripheral AMPK is of assistance for the suppression of cerebral ischemic neuronal damage. 2010 Elsevier B.V. All rights reserved.

  11. Site-specific and synergistic stimulation of methylation on the bacterial chemotaxis receptor Tsr by serine and CheW

    Directory of Open Access Journals (Sweden)

    Weis Robert M

    2005-03-01

    Full Text Available Abstract Background Specific glutamates in the methyl-accepting chemotaxis proteins (MCPs of Escherichia coli are modified during sensory adaptation. Attractants that bind to MCPs are known to increase the rate of receptor modification, as with serine and the serine receptor (Tsr, which contributes to an increase in the steady-state (adapted methylation level. However, MCPs form ternary complexes with two cytoplasmic signaling proteins, the kinase (CheA and an adaptor protein (CheW, but their influences on receptor methylation are unknown. Here, the influence of CheW on the rate of Tsr methylation has been studied to identify contributions to the process of adaptation. Results Methyl group incorporation was measured in a series of membrane samples in which the Tsr molecules were engineered to have one available methyl-accepting glutamate residue (297, 304, 311 or 493. The relative rates at these sites (0.14, 0.05, 0.05 and 1, respectively differed from those found previously for the aspartate receptor (Tar, which was in part due to sequence differences between Tar and Tsr near site four. The addition of CheW generated unexpectedly large and site-specific rate increases, equal to or larger than the increases produced by serine. The increases produced by serine and CheW (added separately were the largest at site one, ~3 and 6-fold, respectively, and the least at site four, no change and ~2-fold, respectively. The rate increases were even larger when serine and CheW were added together, larger than the sums of the increases produced by serine and CheW added separately (except site four. This resulted in substantially larger serine-stimulated increases when CheW was present. Also, CheW enhanced methylation rates when either two or all four sites were available. Conclusion The increase in the rate of receptor methylation upon CheW binding contributes significantly to the ligand specificity and kinetics of sensory adaptation. The synergistic effect of

  12. A cytotoxic serine proteinase isolated from mouse submandibular gland.

    Science.gov (United States)

    Shimamura, T; Nagumo, N; Ikigai, H; Murakami, K; Okubo, S; Toda, M; Ohnishi, R; Tomita, M

    1989-08-01

    We have isolated a novel cytotoxic factor from the submandibular glands of male BALB/c mice by Sephadex G-50 gel filtration chromatography and reverse-phase HPLC. The cytotoxic factor is a serine proteinase, which belongs to the mouse glandular kallikrein (mGK) family, with an Mr of approximately 27,000. The purified serine proteinase showed cytotoxic activity against mouse thymocytes in a dose-dependent manner, and a serine proteinase inhibitor, diisopropyl fluorophosphate, blocked its cytotoxic activity.

  13. Squash inhibitor family of serine proteinases

    International Nuclear Information System (INIS)

    Otlewski, J.; Krowarsch, D.

    1996-01-01

    Squash inhibitors of serine proteinases form an uniform family of small proteins. They are built of 27-33 amino-acid residues and cross-linked with three disulfide bridges. The reactive site peptide bond (P1-P1') is between residue 5 (Lys, Arg or Leu) and 6 (always Ile). High resolution X-ray structures are available for two squash inhibitors complexed with trypsin. NMR solution structures have also been determined for free inhibitors. The major structural motif is a distorted, triple-stranded antiparallel beta-sheet. A similar folding motif has been recently found in a number of proteins, including: conotoxins from fish-hunting snails, carboxypeptidase inhibitor from potato, kalata B1 polypeptide, and in some growth factors (e.g. nerve growth factor, transforming growth factor β2, platelet-derived growth factor). Squash inhibitors are highly stable and rigid proteins. They inhibit a number of serine proteinases: trypsin, plasmin, kallikrein, blood clotting factors: X a and XII a , cathepsin G. The inhibition spectrum can be much broadened if specific amino-acid substitutions are introduced, especially at residues which contact proteinase. Squash inhibitors inhibit proteinases via the standard mechanism. According to the mechanism, inhibitors are substrates which exhibit at neutral pH a high k cat /K m index for hydrolysis and resynthesis of the reactive site, and a low value of the hydrolysis constant. (author)

  14. Conserved water molecules in bacterial serine hydroxymethyltransferases.

    Science.gov (United States)

    Milano, Teresa; Di Salvo, Martino Luigi; Angelaccio, Sebastiana; Pascarella, Stefano

    2015-10-01

    Water molecules occurring in the interior of protein structures often are endowed with key structural and functional roles. We report the results of a systematic analysis of conserved water molecules in bacterial serine hydroxymethyltransferases (SHMTs). SHMTs are an important group of pyridoxal-5'-phosphate-dependent enzymes that catalyze the reversible conversion of l-serine and tetrahydropteroylglutamate to glycine and 5,10-methylenetetrahydropteroylglutamate. The approach utilized in this study relies on two programs, ProACT2 and WatCH. The first software is able to categorize water molecules in a protein crystallographic structure as buried, positioned in clefts or at the surface. The other program finds, in a set of superposed homologous proteins, water molecules that occur approximately in equivalent position in each of the considered structures. These groups of molecules are referred to as 'clusters' and represent structurally conserved water molecules. Several conserved clusters of buried or cleft water molecules were found in the set of 11 bacterial SHMTs we took into account for this work. The majority of these clusters were not described previously. Possible structural and functional roles for the conserved water molecules are envisaged. This work provides a map of the conserved water molecules helpful for deciphering SHMT mechanism and for rational design of molecular engineering experiments. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  15. Understanding serine proteases implications on Leishmania spp lifecycle.

    Science.gov (United States)

    Alves, Carlos Roberto; Souza, Raquel Santos de; Charret, Karen Dos Santos; Côrtes, Luzia Monteiro de Castro; Sá-Silva, Matheus Pereira de; Barral-Veloso, Laura; Oliveira, Luiz Filipe Gonçalves; da Silva, Franklin Souza

    2018-01-01

    Serine proteases have significant functions over a broad range of relevant biological processes to the Leishmania spp lifecycle. Data gathered here present an update on the Leishmania spp serine proteases and the status of these enzymes as part of the parasite degradome. The serine protease genes (n = 26 to 28) in Leishmania spp, which encode proteins with a wide range of molecular masses (35 kDa-115 kDa), are described along with their degrees of chromosomal and allelic synteny. Amid 17 putative Leishmania spp serine proteases, only ∼18% were experimentally demonstrated, as: signal peptidases that remove the signal peptide from secretory pre-proteins, maturases of other proteins and with metacaspase-like activity. These enzymes include those of clans SB, SC and SF. Classical inhibitors of serine proteases are used as tools for the characterization and investigation of Leishmania spp. Endogenous serine protease inhibitors, which are ecotin-like, can act modulating host actions. However, crude or synthetic based-natural serine protease inhibitors, such as potato tuber extract, Stichodactyla helianthus protease inhibitor I, fukugetin and epoxy-α-lapachone act on parasitic serine proteases and are promising leishmanicidal agents. The functional interrelationship between serine proteases and other Leishmania spp proteins demonstrate essential functions of these enzymes in parasite physiology and therefore their value as targets for leishmaniasis treatment. Copyright © 2017 Elsevier Inc. All rights reserved.

  16. Neonatal disruption of serine racemase causes schizophrenia-like behavioral abnormalities in adulthood: clinical rescue by d-serine.

    Directory of Open Access Journals (Sweden)

    Hiroko Hagiwara

    Full Text Available D-Serine, an endogenous co-agonist of the N-methyl-D-aspartate (NMDA receptor, is synthesized from L-serine by serine racemase (SRR. Given the role of D-serine in both neurodevelopment and the pathophysiology of schizophrenia, we examined whether neonatal disruption of D-serine synthesis by SRR inhibition could induce behavioral abnormalities relevant to schizophrenia, in later life.Neonatal mice (7-9 days were injected with vehicle or phenazine methosulfate (Met-Phen: 3 mg/kg/day, an SRR inhibitor. Behavioral evaluations, such as spontaneous locomotion, novel object recognition test (NORT, and prepulse inhibition (PPI were performed at juvenile (5-6 weeks old and adult (10-12 weeks old stages. In addition, we tested the effects of D-serine on PPI deficits in adult mice after neonatal Met-Phen exposure. Finally, we assessed whether D-serine could prevent the onset of schizophrenia-like behavior in these mice. Neonatal Met-Phen treatment reduced D-serine levels in the brain, 24 hours after the final dose. Additionally, this treatment caused behavioral abnormalities relevant to prodromal symptoms in juveniles and to schizophrenia in adults. A single dose of D-serine improved PPI deficits in adult mice. Interestingly, chronic administration of D-serine (900 mg/kg/day from P35 to P70 significantly prevented the onset of PPI deficits after neonatal Met-Phen exposure.This study shows that disruption of D-serine synthesis during developmental stages leads to behavioral abnormalities relevant to prodromal symptoms and schizophrenia, in later life. Furthermore, early pharmacological intervention with D-serine may prevent the onset of psychosis in adult.

  17. Digestible threonine to lysine ratio in diets for laying hens aged 24-40 weeks

    Directory of Open Access Journals (Sweden)

    Tatiana Cristina da Rocha

    2013-12-01

    Full Text Available Two-hundred sixteen white laying hens were used to assess the ideal ratio of digestible threonine:lysine in diets for laying hens at 24 to 40 weeks of age. Birds were assigned to a randomized block design, with six treatments, six replicates per treatment and six birds per experimental unit. The cage was used as the blocking criterion. Experimental diets contained different digestible threonine:digestible lysine ratios (65, 70, 75, 80, 85 and 90% with 142 g/kg of crude protein. Experimental diets were formulated to be isonitrogenous and isocaloric with different contents of L-glutamic acid. Feed intake (g/hen/d, egg production (%, egg weight (g, egg mass (g/hen/d, feed conversion ratio (kg/dozen and kg/kg egg, eggshell weight (g, albumen weight (g, yolk weight (g and body weight gain (g were assessed. The maximum egg production was observed at 78% digestible threonine:digestible lysine ratio, while the best values of feed conversion ratio (kg/dozen egg and feed conversion ratio (kg/kg of egg were observed at 77.6% and 75%, respectively. Feed intake, egg mass and egg contents (yolk, albumen and eggshell were not affected by treatments. The estimated digestible threonine:digestible lysine ratio of Hy-Line W36 laying hens at 24 to 40 weeks of age is 78%, corresponding to 5.70 g/kg of dietary digestible threonine.

  18. Opioid withdrawal increases transient receptor potential vanilloid 1 activity in a protein kinase A-dependent manner.

    Science.gov (United States)

    Spahn, Viola; Fischer, Oliver; Endres-Becker, Jeannette; Schäfer, Michael; Stein, Christoph; Zöllner, Christian

    2013-04-01

    Hyperalgesia is a cardinal symptom of opioid withdrawal. The transient receptor potential vanilloid 1 (TRPV1) is a ligand-gated ion channel expressed on sensory neurons responding to noxious heat, protons, and chemical stimuli such as capsaicin. TRPV1 can be inhibited via μ-opioid receptor (MOR)-mediated reduced activity of adenylyl cyclases (ACs) and decreased cyclic adenosine monophosphate (cAMP) levels. In contrast, opioid withdrawal following chronic activation of MOR uncovers AC superactivation and subsequent increases in cAMP and protein kinase A (PKA) activity. Here we investigated (1) whether an increase in cAMP during opioid withdrawal increases the activity of TRPV1 and (2) how opioid withdrawal modulates capsaicin-induced nocifensive behavior in rats. We applied whole-cell patch clamp, microfluorimetry, cAMP assays, radioligand binding, site-directed mutagenesis, and behavioral experiments. Opioid withdrawal significantly increased cAMP levels and capsaicin-induced TRPV1 activity in both transfected human embryonic kidney 293 cells and dissociated dorsal root ganglion (DRG) neurons. Inhibition of AC and PKA, as well as mutations of the PKA phosphorylation sites threonine 144 and serine 774, prevented the enhanced TRPV1 activity. Finally, capsaicin-induced nocifensive behavior was increased during opioid withdrawal in vivo. In summary, our results demonstrate an increased activity of TRPV1 in DRG neurons as a new mechanism contributing to opioid withdrawal-induced hyperalgesia. Copyright © 2013 International Association for the Study of Pain. Published by Elsevier B.V. All rights reserved.

  19. High abundance of Serine/Threonine-rich regions predicted to be hyper-O-glycosylated in the secretory proteins coded by eight fungal genomes

    Directory of Open Access Journals (Sweden)

    González Mario

    2012-09-01

    Full Text Available Abstract Background O-glycosylation of secretory proteins has been found to be an important factor in fungal biology and virulence. It consists in the addition of short glycosidic chains to Ser or Thr residues in the protein backbone via O-glycosidic bonds. Secretory proteins in fungi frequently display Ser/Thr rich regions that could be sites of extensive O-glycosylation. We have analyzed in silico the complete sets of putatively secretory proteins coded by eight fungal genomes (Botrytis cinerea, Magnaporthe grisea, Sclerotinia sclerotiorum, Ustilago maydis, Aspergillus nidulans, Neurospora crassa, Trichoderma reesei, and Saccharomyces cerevisiae in search of Ser/Thr-rich regions as well as regions predicted to be highly O-glycosylated by NetOGlyc (http://www.cbs.dtu.dk. Results By comparison with experimental data, NetOGlyc was found to overestimate the number of O-glycosylation sites in fungi by a factor of 1.5, but to be quite reliable in the prediction of highly O-glycosylated regions. About half of secretory proteins have at least one Ser/Thr-rich region, with a Ser/Thr content of at least 40% over an average length of 40 amino acids. Most secretory proteins in filamentous fungi were predicted to be O-glycosylated, sometimes in dozens or even hundreds of sites. Residues predicted to be O-glycosylated have a tendency to be grouped together forming hyper-O-glycosylated regions of varying length. Conclusions About one fourth of secretory fungal proteins were predicted to have at least one hyper-O-glycosylated region, which consists of 45 amino acids on average and displays at least one O-glycosylated Ser or Thr every four residues. These putative highly O-glycosylated regions can be found anywhere along the proteins but have a slight tendency to be at either one of the two ends.

  20. The PTI1-like kinase ZmPti1a from maize (Zea mays L.) co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte.

    Science.gov (United States)

    Herrmann, Markus M; Pinto, Sheena; Kluth, Jantjeline; Wienand, Udo; Lorbiecke, René

    2006-10-06

    The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR) reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied. Here we report the identification and molecular analysis of four Pti1-like kinases from maize (ZmPti1a, -b, -c, -d). These kinase genes showed tissue-specific expression and their corresponding proteins were targeted to different cellular compartments. Sequence similarity, expression pattern and cellular localization of ZmPti1b suggested that this gene is a putative orthologue of Pti1 from tomato. In contrast, ZmPti1a was specifically expressed in pollen and sequestered to the plasma membrane, evidently owing to N-terminal modification by myristoylation and/or S-acylation. The ZmPti1a:GFP fusion protein was not evenly distributed at the pollen plasma membrane but accumulated as an annulus-like structure which co-localized with callose (1,3-beta-glucan) deposition. In addition, co-localization of ZmPti1a and callose was observed during stages of pollen mitosis I and pollen tube germination. Maize plants in which ZmPti1a expression was silenced by RNA interference (RNAi) produced pollen with decreased competitive ability. Hence, our data provide evidence that ZmPti1a plays an important part in a signalling pathway that accelerates pollen performance and male fitness. ZmPti1a from maize is involved in pollen-specific processes during the progamic phase of reproduction, probably in crucial signalling processes associated with regions of callose deposition. Pollen

  1. The PTI1-like kinase ZmPti1a from maize (Zea mays L. co-localizes with callose at the plasma membrane of pollen and facilitates a competitive advantage to the male gametophyte

    Directory of Open Access Journals (Sweden)

    Wienand Udo

    2006-10-01

    Full Text Available Abstract Background The tomato kinase Pto confers resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato in a gene for gene manner. Upon recognition of specific avirulence factors the Pto kinase activates multiple signal transduction pathways culminating in induction of pathogen defense. The soluble cytoplasmic serine/threonine kinase Pti1 is one target of Pto phosphorylation and is involved in the hypersensitive response (HR reaction. However, a clear role of Pti1 in plant pathogen resistance is uncertain. So far, no Pti1 homologues from monocotyledonous species have been studied. Results Here we report the identification and molecular analysis of four Pti1-like kinases from maize (ZmPti1a, -b, -c, -d. These kinase genes showed tissue-specific expression and their corresponding proteins were targeted to different cellular compartments. Sequence similarity, expression pattern and cellular localization of ZmPti1b suggested that this gene is a putative orthologue of Pti1 from tomato. In contrast, ZmPti1a was specifically expressed in pollen and sequestered to the plasma membrane, evidently owing to N-terminal modification by myristoylation and/or S-acylation. The ZmPti1a:GFP fusion protein was not evenly distributed at the pollen plasma membrane but accumulated as an annulus-like structure which co-localized with callose (1,3-β-glucan deposition. In addition, co-localization of ZmPti1a and callose was observed during stages of pollen mitosis I and pollen tube germination. Maize plants in which ZmPti1a expression was silenced by RNA interference (RNAi produced pollen with decreased competitive ability. Hence, our data provide evidence that ZmPti1a plays an important part in a signalling pathway that accelerates pollen performance and male fitness. Conclusion ZmPti1a from maize is involved in pollen-specific processes during the progamic phase of reproduction, probably in crucial signalling processes associated with regions

  2. A cyclic peptidic serine protease inhibitor

    DEFF Research Database (Denmark)

    Zhao, Baoyu; Xu, Peng; Jiang, Longguang

    2014-01-01

    Peptides are attracting increasing interest as protease inhibitors. Here, we demonstrate a new inhibitory mechanism and a new type of exosite interactions for a phage-displayed peptide library-derived competitive inhibitor, mupain-1 (CPAYSRYLDC), of the serine protease murine urokinase...... pocket, its carbonyl group aligning improperly relative to Ser195 and the oxyanion hole, explaining why the peptide is an inhibitor rather than a substrate. Substitution of the P1 Arg with novel unnatural Arg analogues with aliphatic or aromatic ring structures led to an increased affinity, depending......, in spite of a less favorable binding entropy and loss of a polar interaction. We conclude that increased flexibility of the peptide allows more favorable exosite interactions, which, in combination with the use of novel Arg analogues as P1 residues, can be used to manipulate the affinity and specificity...

  3. Highly potent fibrinolytic serine protease from Streptomyces.

    Science.gov (United States)

    Uesugi, Yoshiko; Usuki, Hirokazu; Iwabuchi, Masaki; Hatanaka, Tadashi

    2011-01-05

    We introduce a highly potent fibrinolytic serine protease from Streptomyces omiyaensis (SOT), which belongs to the trypsin family. The fibrinolytic activity of SOT was examined using in vitro assays and was compared with those of known fibrinolytic enzymes such as plasmin, tissue-type plasminogen activator (t-PA), urokinase, and nattokinase. Compared to other enzymes, SOT showed remarkably higher hydrolytic activity toward mimic peptides of fibrin and plasminogen. The fibrinolytic activity of SOT is about 18-fold higher than that of plasmin, and is comparable to that of t-PA by fibrin plate assays. Furthermore, SOT had some plasminogen activator-like activity. Results show that SOT and nattokinase have very different fibrinolytic and fibrinogenolytic modes, engendering significant synergetic effects of SOT and nattokinase on fibrinolysis. These results suggest that SOT presents important possibilities for application in the therapy of thrombosis. Copyright © 2010 Elsevier Inc. All rights reserved.

  4. Enhancement of L-Serine Production by Corynebacterium ...

    African Journals Online (AJOL)

    glutamicum SYPS-062 cultivation process for efficient production of L-serine on a large scale. ... central intermediate for a number of cellular .... impeller, oxygen and pH electrodes, under the ... equation. The yield of L-serine was regressed with respect to the medium ..... is not essential for activity but is required for inhibition.

  5. D-serine : The right or wrong isoform?

    NARCIS (Netherlands)

    Fuchs, Sabine A; Berger, Ruud; de Koning, Tom J

    2011-01-01

    Only recently, d-amino acids have been identified in mammals. Of these, d-serine has been most extensively studied. d-Serine was found to play an important role as a neurotransmitter in the human central nervous system (CNS) by binding to the N-methyl-d-aspartate receptor (NMDAr), similar to

  6. Conditional ablation of glycogen synthase kinase 3β in postnatal mouse kidney.

    Science.gov (United States)

    Ge, Yan; Si, Jin; Tian, Li; Zhuang, Shougang; Dworkin, Lance D; Gong, Rujun

    2011-01-01

    Glycogen synthase kinase (GSK)3 is a ubiquitously expressed serine/threonine kinase existing in two isoforms, namely GSK3α and GSK3β. Aside from the long-recognized role in insulin signal transduction and glycogen biosynthesis, GSK3β has been recently coined as a master control molecule in nuclear factor-κB activation and inflammatory kidney injury. Nevertheless, previous studies are less conclusive because they relied greatly on small molecule inhibitors, which lack selectivity and barely distinguish between the GSK3 isoforms. In addition, early embryonic lethality after global knockout of GSK3β precludes interrogation of the biological role of GSK3β in the adult kidney. To circumvent these issues, the Cre/loxP system was used to generate a conditional knockout mouse model in which the GSK3β gene was specifically deleted in kidney cortical tubules at postnatal mature stage. Kidney-specific ablation of GSK3β resulted in a phenotype no different from control littermates. Knockout mice (KO) were viable and exhibited normal development and normal kidney physiology in terms of kidney function, urine albumin excretion, and urine-concentrating ability. It is noteworthy that apart from normal glomerular and tubulointerstitial morphology, the kidneys from KO demonstrated more glycogen accumulation in the renal cortical tubules as assessed by both periodic acid-Schiff staining for light microscopy and direct biochemical assay, consistent with an elevated glycogen synthetic activity as evidenced by diminished inhibitory phosphorylation of glycogen synthase that occurred subsequent to GSK3β ablation. This finding was further validated by electron microscopic observations of increased deposition of glycogen particles in the renal tubules of KO, suggesting that GSK3α could not fully compensate for the loss of GSK3β in regulating glycogen metabolism in the kidney. Collectively, our study suggests that kidney-specific ablation of GSK3β barely affects kidney function

  7. Pim-1 Kinase Phosphorylates Cardiac Troponin I and Regulates Cardiac Myofilament Function

    Directory of Open Access Journals (Sweden)

    Ni Zhu

    2018-03-01

    Full Text Available Background/Aims: Pim-1 is a serine/threonine kinase that is highly expressed in the heart, and exerts potent cardiac protective effects through enhancing survival, proliferation, and regeneration of cardiomyocytes. Its myocardial specific substrates, however, remain unknown. In the present study, we aim to investigate whether Pim-1 modulates myofilament activity through phosphorylation of cardiac troponin I (cTnI, a key component in regulating myofilament function in the heart. Methods: Coimmunoprecipitation and immunofluorescent assays were employed to investigate the interaction of Pim-1 with cTnI in cardiomyocytes. Biochemical, site directed mutagenesis, and mass spectrometric analyses were utilized to identify the phosphorylation sites of Pim1 in cTnI. Myofilament functional assay using skinned cardiac fiber was used to assess the effect of Pim1-mediated phosphorylation on cardiac myofilament activity. Lastly, the functional significance of Pim1-mediated cTnI in heart disease was determined in diabetic mice. Results: We found that Pim-1 specifically interacts with cTnI in cardiomyocytes and this interaction leads to Pim1-mediated cTnI phosphorylation, predominantly at Ser23/24 and Ser150. Furthermore, our functional assay demonstrated that Pim-1 induces a robust phosphorylation of cTnI within the troponin complex, thus leading to a decreased Ca2+ sensitivity. Insulin-like growth factor 1 (IGF-1, a peptide growth factor that has been shown to stimulate myocardial contractility, markedly induces cTnI phosphorylation at Ser23/24 and Ser150 through increasing Pim-1 expression in cardiomyocytes. In a high-fat diabetic mice model, the expression of Pim1 in the heart is significantly decreased, which is accompanied by a decreased phosphorylation of cTnI at Ser23/24 and Ser150, further implicating the pathological significance of the Pim1/cTnI axis in the development of diabetic cardiomyopathy. Conclusion: Our results demonstrate that Pim-1 is a

  8. Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.

    Science.gov (United States)

    Mian, Elisabeth; Wiesmüller, Lisa

    2017-01-01

    Ataxia telangiectasia mutated (ATM) encodes a serine/threonine protein kinase, which is involved in various regulatory processes in mammalian cells. Its best-known role is apical activation of the DNA damage response following generation of DNA double-strand breaks (DSBs). When DSBs appear, sensor and mediator proteins are recruited, activating transducers such as ATM, which in turn relay a widespread signal to a multitude of downstream effectors. ATM mutation causes Ataxia telangiectasia (AT), whereby the disease phenotype shows differing characteristics depending on the underlying ATM mutation. However, all phenotypes share progressive neurodegeneration and marked predisposition to malignancies at the organismal level and sensitivity to ionizing radiation and chromosome aberrations at the cellular level. Expression and localization of the ATM protein can be determined via western blotting and immunofluorescence microscopy; however, detection of subtle alterations such as resulting from amino acid exchanges rather than truncating mutations requires functional testing. Previous studies on the role of ATM in DSB repair, which connects with radiosensitivity and chromosomal stability, gave at first sight contradictory results. To systematically explore the effects of clinically relevant ATM mutations on DSB repair, we engaged a series of lymphoblastoid cell lines (LCLs) derived from AT patients and controls. To examine DSB repair both in a quantitative and qualitative manners, we used an EGFP-based assay comprising different substrates for distinct DSB repair mechanisms. In this way, we demonstrated that particular signaling defects caused by individual ATM mutations led to specific DSB repair phenotypes. To explore the impact of ATM on carcinogenic chromosomal aberrations, we monitored chromosomal breakage at a breakpoint cluster region hotspot within the MLL gene that has been associated with therapy-related leukemia. PCR-based MLL-breakage analysis of HeLa cells

  9. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    Energy Technology Data Exchange (ETDEWEB)

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria); DeVaney, Trevor [Institute of Biophysics, Medical University of Graz (Austria); Zimmer, Andreas [Institute of Pharmaceutical Sciences, Department of Pharmaceutical Technology, Karl-Franzens University, Graz (Austria); Raynham, Tony; Ireson, Christopher [Cancer Research Technology Ltd, London (United Kingdom); Sattler, Wolfgang, E-mail: wolfgang.sattler@medunigraz.at [Institute of Molecular Biology and Biochemistry, Medical University of Graz, Graz (Austria)

    2013-08-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun{sup S73} phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

  10. Protein kinase D2 regulates migration and invasion of U87MG glioblastoma cells in vitro

    International Nuclear Information System (INIS)

    Bernhart, Eva; Damm, Sabine; Wintersperger, Andrea; DeVaney, Trevor; Zimmer, Andreas; Raynham, Tony; Ireson, Christopher; Sattler, Wolfgang

    2013-01-01

    Glioblastoma multiforme (GBM) is the most common malignant brain tumor, which, despite combined modality treatment, reoccurs and is invariably fatal for affected patients. Recently, a member of the serine/threonine protein kinase D (PRKD) family, PRKD2, was shown to be a potent mediator of glioblastoma growth. Here we studied the role of PRKD2 in U87MG glioblastoma cell migration and invasion in response to sphingosine-1-phosphate (S1P), an activator of PRKD2 and a GBM mitogen. Time-lapse microscopy demonstrated that random cell migration was significantly diminished in response to PRKD2 silencing. The pharmacological PRKD family inhibitor CRT0066101 decreased chemotactic migration and invasion across uncoated or matrigel-coated Transwell inserts. Silencing of PRKD2 attenuated migration and invasion of U87MG cells even more effectively. In terms of downstream signaling, CRT0066101 prevented PRKD2 autophosphorylation and inhibited p44/42 MAPK and to a smaller extent p54/46 JNK and p38 MAPK activation. PRKD2 silencing impaired activation of p44/42 MAPK and p54/46 JNK, downregulated nuclear c-Jun protein levels and decreased c-Jun S73 phosphorylation without affecting the NFκB pathway. Finally, qPCR array analyses revealed that silencing of PRKD2 downregulates mRNA levels of integrin alpha-2 and -4 (ITGA2 and -4), plasminogen activator urokinase (PLAU), plasminogen activator urokinase receptor (PLAUR), and matrix metallopeptidase 1 (MMP1). Findings of the present study identify PRKD2 as a potential target to interfere with glioblastoma cell migration and invasion, two major determinants contributing to recurrence of glioblastoma after multimodality treatment. Highlights: • Sphingosine-1-phosphate induces glioma cell migration and invasion. • Part of the effects is mediated by protein kinase D2 (PRKD2) activation. • Inactivation of PRKD2 attenuates glioblastoma cell migration and invasion. • Both, RNAi and pharmacological inhibition of PRKD2 inhibits MAPK

  11. RNAi screen reveals host cell kinases specifically involved in Listeria monocytogenes spread from cell to cell.

    Directory of Open Access Journals (Sweden)

    Ryan Chong

    Full Text Available Intracellular bacterial pathogens, such as Listeria monocytogenes and Rickettsia conorii display actin-based motility in the cytosol of infected cells and spread from cell to cell through the formation of membrane protrusions at the cell cortex. Whereas the mechanisms supporting cytosolic actin-based motility are fairly well understood, it is unclear whether specific host factors may be required for supporting the formation and resolution of membrane protrusions. To address this gap in knowledge, we have developed high-throughput fluorescence microscopy and computer-assisted image analysis procedures to quantify pathogen spread in human epithelial cells. We used the approach to screen a siRNA library covering the human kinome and identified 7 candidate kinases whose depletion led to severe spreading defects in cells infected with L. monocytogenes. We conducted systematic validation procedures with redundant silencing reagents and confirmed the involvement of the serine/threonine kinases, CSNK1A1 and CSNK2B. We conducted secondary assays showing that, in contrast with the situation observed in CSNK2B-depleted cells, L. monocytogenes formed wild-type cytosolic tails and displayed wild-type actin-based motility in the cytosol of CSNK1A1-depleted cells. Furthermore, we developed a protrusion formation assay and showed that the spreading defect observed in CSNK1A1-depleted cells correlated with the formation of protrusion that did not resolve into double-membrane vacuoles. Moreover, we developed sending and receiving cell-specific RNAi procedures and showed that CSNK1A was required in the sending cells, but was dispensable in the receiving cells, for protrusion resolution. Finally, we showed that the observed defects were specific to Listeria monocytogenes, as Rickettsia conorii displayed wild-type cell-to-cell spread in CSNK1A1- and CSNK2B-depleted cells. We conclude that, in addition to the specific host factors supporting cytosolic actin

  12. Purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae.

    Science.gov (United States)

    Elbing, Karin; McCartney, Rhonda R; Schmidt, Martin C

    2006-02-01

    Members of the Snf1/AMPK family of protein kinases are activated by distinct upstream kinases that phosphorylate a conserved threonine residue in the Snf1/AMPK activation loop. Recently, the identities of the Snf1- and AMPK-activating kinases have been determined. Here we describe the purification and characterization of the three Snf1-activating kinases of Saccharomyces cerevisiae. The identities of proteins associated with the Snf1-activating kinases were determined by peptide mass fingerprinting. These kinases, Sak1, Tos3 and Elm2 do not appear to require the presence of additional subunits for activity. Sak1 and Snf1 co-purify and co-elute in size exclusion chromatography, demonstrating that these two proteins form a stable complex. The Snf1-activating kinases phosphorylate the activation loop threonine of Snf1 in vitro with great specificity and are able to do so in the absence of beta and gamma subunits of the Snf1 heterotrimer. Finally, we showed that the Snf1 kinase domain isolated from bacteria as a GST fusion protein can be activated in vitro and shows substrate specificity in the absence of its beta and gamma subunits.

  13. Microbial Production of l-Serine from Renewable Feedstocks.

    Science.gov (United States)

    Zhang, Xiaomei; Xu, Guoqiang; Shi, Jinsong; Koffas, Mattheos A G; Xu, Zhenghong

    2018-07-01

    l-Serine is a non-essential amino acid that has wide and expanding applications in industry with a fast-growing market demand. Currently, extraction and enzymatic catalysis are the main processes for l-serine production. However, such approaches limit the industrial-scale applications of this important amino acid. Therefore, shifting to the direct fermentative production of l-serine from renewable feedstocks has attracted increasing attention. This review details the current status of microbial production of l-serine from renewable feedstocks. We also summarize the current trends in metabolic engineering strategies and techniques for the typical industrial organisms Corynebacterium glutamicum and Escherichia coli that have been developed to address and overcome major challenges in the l-serine production process. Copyright © 2018 Elsevier Ltd. All rights reserved.

  14. Down-Regulation of the Na+-Coupled Phosphate Transporter NaPi-IIa by AMP-Activated Protein Kinase

    Directory of Open Access Journals (Sweden)

    Miribane Dërmaku-Sopjani

    2013-11-01

    Full Text Available Background/Aims: The Na+-coupled phosphate transporter NaPi-IIa is the main carrier accomplishing renal tubular phosphate reabsorption. It is driven by the electrochemical Na+ gradient across the apical cell membrane, which is maintained by Na+ extrusion across the basolateral cell membrane through the Na+/K+ ATPase. The operation of NaPi-IIa thus requires energy in order to avoid cellular Na+ accumulation and K+ loss with eventual decrease of cell membrane potential, Cl- entry and cell swelling. Upon energy depletion, early inhibition of Na+-coupled transport processes may delay cell swelling and thus foster cell survival. Energy depletion is sensed by the AMP-activated protein kinase (AMPK, a serine/threonine kinase stimulating several cellular mechanisms increasing energy production and limiting energy utilization. The present study explored whether AMPK influences the activity of NAPi-IIa. Methods: cRNA encoding NAPi-IIa was injected into Xenopus oocytes with or without additional expression of wild-type AMPK (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1-HA, of inactive AMPKαK45R (AMPKα1K45R+AMPKβ1-Flag+AMPKγ1-HA or of constitutively active AMPKγR70Q (AMPKα1-HA+AMPKβ1-Flag+AMPKγ1R70Q. NaPi-IIa activity was estimated from phosphate-induced current in dual electrode voltage clamp experiments. Results: In NaPi-IIa-expressing, but not in water-injected Xenopus oocytes, the addition of phosphate (1 mM to the extracellular bath solution generated a current (Ip, which was significantly decreased by coexpression of wild-type AMPK and of AMPKγR70Q but not of AMPKαK45R. The phosphate-induced current in NaPi-IIa- and AMPK-expressing Xenopus ooocytes was significantly increased by AMPK inhibitor Compound C (20 µM. Kinetic analysis revealed that AMPK significantly decreased the maximal transport rate. Conclusion: The AMP-activated protein kinase AMPK is a powerful regulator of NaPi-IIa and thus of renal tubular phosphate transport.

  15. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2004-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  16. Activation of ATM by DNA Damaging Agents

    National Research Council Canada - National Science Library

    Kurz, Ebba U; Lees-Miller, Susan P

    2005-01-01

    Ataxia-telangiectasia mutated (ATM) is a serine/threonine protein kinase that acts as a master switch controlling the cell cycle in response to ionizing radiation-induced DNA double-strand breaks (DSBs...

  17. A New Paradigm for Ovarian Sex Cord-Stromal Tumor Development

    Science.gov (United States)

    2017-05-01

    Transforming growth factor beta (TGFB) family members regulate multiple cellular functions and key reproductive processes in a contextually dependent manner ...key reproductive processes in a contextually dependent manner via the interaction with membrane associated serine/threonine kinase receptor

  18. Membrane Heterogeneity in Akt Activation in Prostate Cancer

    National Research Council Canada - National Science Library

    Hager, Martin H

    2008-01-01

    This project focuses on the novel finding from our group that the serine-threonine kinase Akt1 partitions into specialized membrane microdomains, termed lipid rafts, and that this localization event...

  19. Influence of Threonine Metabolism on S-adenosyl-methionine and Histone Methylation

    Science.gov (United States)

    Shyh-Chang, Ng; Locasale, Jason W.; Lyssiotis, Costas A.; Zheng, Yuxiang; Teo, Ren Yi; Ratanasirintrawoot, Sutheera; Zhang, Jin; Onder, Tamer; Unternaehrer, Juli J.; Zhu, Hao; Asara, John M.; Daley, George Q.; Cantley, Lewis C.

    2013-01-01

    Threonine is the only amino acid critically required for the pluripotency of mouse embryonic stem cells (mESCs) but the detailed mechanism remains unclear. We found that threonine (Thr) and S-adenosyl-methionine (SAM) metabolism are coupled in pluripotent stem cells, resulting in regulation of histone methylation. Isotope labeling of mESCs revealed that Thr provides a substantial fraction of both the cellular glycine (Gly) and the acetyl-coenzyme A (CoA) needed for SAM synthesis. Depletion of Thr from the culture medium or threonine dehydrogenase (Tdh) from mESCs decreased accumulation of SAM and decreased tri-methylation of histone H3 lysine-4 (H3K4me3), leading to slowed growth, and increased differentiation. Thus abundance of SAM appears to influence H3K4me3, providing a possible mechanism by which modulation of a metabolic pathway might influence stem cell fate. PMID:23118012

  20. Enhancement of L-Threonine Production by Controlling Sequential Carbon-Nitrogen Ratios during Fermentation.

    Science.gov (United States)

    Lee, Hyeok-Won; Lee, Hee-Suk; Kim, Chun-Suk; Lee, Jin-Gyeom; Kim, Won-Kyo; Lee, Eun-Gyo; Lee, Hong-Weon

    2018-02-28

    Controlling the residual glucose concentration is important for improving productivity in L-threonine fermentation. In this study, we developed a procedure to automatically control the feeding quantity of glucose solution as a function of ammonia-water consumption rate. The feeding ratio (R C/N ) of glucose and ammonia water was predetermined via a stoichiometric approach, on the basis of glucose-ammonia water consumption rates. In a 5-L fermenter, 102 g/l L -threonine was obtained using our glucose-ammonia water combined feeding strategy, which was then successfully applied in a 500-L fermenter (89 g/l). Therefore, we conclude that an automatic combination feeding strategy is suitable for improving L-threonine production.

  1. Threonine deaminase from extremely halophilic bacteria - Cooperative substrate kinetics and salt dependence.

    Science.gov (United States)

    Lieberman, M. M.; Lanyi, J. K.

    1972-01-01

    The effect of salt on the activity, stability, and allosteric properties of catabolic threonine deaminase from Halobacterium cutirubrum was studied. The enzyme exhibits sigmoidal kinetics with the substrate, threonine. The Hill slope is 1.55 at pH 10. The enzyme is activated by ADP at low substrate concentrations. In the presence of this effector, sigmoidal kinetics are no longer observed. At pH 10, in the absence of ADP, enzyme activity increases with increasing NaCl concentration from 0 to 4 M.

  2. Antitumor efficacy of PKI-587, a highly potent dual PI3K/mTOR kinase inhibitor.

    Science.gov (United States)

    Mallon, Robert; Feldberg, Larry R; Lucas, Judy; Chaudhary, Inder; Dehnhardt, Christoph; Santos, Efren Delos; Chen, Zecheng; dos Santos, Osvaldo; Ayral-Kaloustian, Semiramis; Venkatesan, Aranapakam; Hollander, Irwin

    2011-05-15

    The aim of this study was to show preclinical efficacy and clinical development potential of PKI-587, a dual phosphoinositide 3-kinase (PI3K)/mTOR inhibitor. In vitro class 1 PI3K enzyme and human tumor cell growth inhibition assays and in vivo five tumor xenograft models were used to show efficacy. In vitro, PKI-587 potently inhibited class I PI3Ks (IC(50) vs. PI3K-α = 0.4 nmol/L), PI3K-α mutants, and mTOR. PKI-587 inhibited growth of 50 diverse human tumor cell lines at IC(50) values of less than 100 nmol/L. PKI-587 suppressed phosphorylation of PI3K/mTOR effectors (e.g., Akt), and induced apoptosis in human tumor cell lines with elevated PI3K/mTOR signaling. MDA-MB-361 [breast; HER2(+), PIK3CA mutant (E545K)] was particularly sensitive to this effect, with cleaved PARP, an apoptosis marker, induced by 30 nmol/L PKI-587 at 4 hours. In vivo, PKI-587 inhibited tumor growth in breast (MDA-MB-361, BT474), colon (HCT116), lung (H1975), and glioma (U87MG) xenograft models. In MDA-MB-361 tumors, PKI-587 (25 mg/kg, single dose i.v.) suppressed Akt phosphorylation [at threonine(T)308 and serine(S)473] for up to 36 hours, with cleaved PARP (cPARP) evident up to 18 hours. PKI-587 at 25 mg/kg (once weekly) shrank large (∼1,000 mm(3)) MDA-MB-361 tumors and suppressed tumor regrowth. Tumor regression correlated with suppression of phosphorylated Akt in the MDA-MB-361 model. PKI-587 also caused regression in other tumor models, and efficacy was enhanced when given in combination with PD0325901 (MEK 1/2 inhibitor), irinotecan (topoisomerase I inhibitor), or HKI-272 (neratinib, HER2 inhibitor). Significant antitumor efficacy and a favorable pharmacokinetic/safety profile justified phase 1 clinical evaluation of PKI-587. ©2011 AACR.

  3. Protein kinase activity of phosphoinositide 3-kinase regulates cytokine-dependent cell survival.

    Directory of Open Access Journals (Sweden)

    Daniel Thomas

    Full Text Available The dual specificity protein/lipid kinase, phosphoinositide 3-kinase (PI3K, promotes growth factor-mediated cell survival and is frequently deregulated in cancer. However, in contrast to canonical lipid-kinase functions, the role of PI3K protein kinase activity in regulating cell survival is unknown. We have employed a novel approach to purify and pharmacologically profile protein kinases from primary human acute myeloid leukemia (AML cells that phosphorylate serine residues in the cytoplasmic portion of cytokine receptors to promote hemopoietic cell survival. We have isolated a kinase activity that is able to directly phosphorylate Ser585 in the cytoplasmic domain of the interleukin 3 (IL-3 and granulocyte macrophage colony stimulating factor (GM-CSF receptors and shown it to be PI3K. Physiological concentrations of cytokine in the picomolar range were sufficient for activating the protein kinase activity of PI3K leading to Ser585 phosphorylation and hemopoietic cell survival but did not activate PI3K lipid kinase signaling or promote proliferation. Blockade of PI3K lipid signaling by expression of the pleckstrin homology of Akt1 had no significant impact on the ability of picomolar concentrations of cytokine to promote hemopoietic cell survival. Furthermore, inducible expression of a mutant form of PI3K that is defective in lipid kinase activity but retains protein kinase activity was able to promote Ser585 phosphorylation and hemopoietic cell survival in the absence of cytokine. Blockade of p110α by RNA interference or multiple independent PI3K inhibitors not only blocked Ser585 phosphorylation in cytokine-dependent cells and primary human AML blasts, but also resulted in a block in survival signaling and cell death. Our findings demonstrate a new role for the protein kinase activity of PI3K in phosphorylating the cytoplasmic tail of the GM-CSF and IL-3 receptors to selectively regulate cell survival highlighting the importance of targeting

  4. Protein kinase D1 stimulates proliferation and enhances tumorigenesis of MCF-7 human breast cancer cells through a MEK/ERK-dependent signaling pathway

    International Nuclear Information System (INIS)

    Karam, Manale; Legay, Christine; Auclair, Christian; Ricort, Jean-Marc

    2012-01-01

    Protein kinase D1, PKD1, is a novel serine/threonine kinase whose altered expression and dysregulation in many tumors as well as its activation by several mitogens suggest that this protein could regulate proliferation and tumorigenesis. Nevertheless, the precise signaling pathways used are still unclear and the potential direct role of PKD1 in tumor development and progression has not been yet investigated. In order to clarify the role of PKD1 in cell proliferation and tumorigenesis, we studied the effects of PKD1 overexpression in a human adenocarcinoma breast cancer cell line, MCF-7 cells. We demonstrated that overexpression of PKD1 specifically promotes MCF-7 cell proliferation through accelerating G0/G1 to S phase transition of the cell cycle. Moreover, inhibition of endogenous PKD1 significantly reduced cell proliferation. Taken together, these results clearly strengthen the regulatory role of PKD1 in cell growth. We also demonstrated that overexpression of PKD1 specifically diminished serum- and anchorage-dependence for proliferation and survival in vitro and allowed MCF-7 cells to form tumors in vivo. Thus, all these data highlight the central role of PKD1 in biological processes which are hallmarks of malignant transformation. Analysis of two major signaling pathways implicated in MCF-7 cell proliferation showed that PKD1 overexpression significantly increased ERK1/2 phosphorylation state without affecting Akt phosphorylation. Moreover, PKD1 overexpression-stimulated cell proliferation and anchorage-independent growth were totally impaired by inhibition of the MEK/ERK kinase cascade. However, neither of these effects was affected by blocking the PI 3-kinase/Akt signaling pathway. Thus, the MEK/ERK signaling appears to be a determining pathway mediating the biological effects of PKD1 in MCF-7 cells. Taken together, all these data demonstrate that PKD1 overexpression increases the aggressiveness of MCF-7 breast cancer cells through enhancing their oncogenic

  5. Casein kinases

    DEFF Research Database (Denmark)

    Issinger, O G

    1993-01-01

    The present review on casein kinases focuses mainly on the possible metabolic role of CK-2, with special emphasis on its behavior in pathological tissues. From these data at least three ways to regulate CK-2 activity emerge: (i) CK-2 activity changes during embryogenesis, being high at certain...

  6. Fibrin(ogen)olytic activity of bumblebee venom serine protease

    International Nuclear Information System (INIS)

    Qiu Yuling; Choo, Young Moo; Yoon, Hyung Joo; Jia Jingming; Cui Zheng; Wang Dong; Kim, Doh Hoon; Sohn, Hung Dae; Jin, Byung Rae

    2011-01-01

    Bee venom is a rich source of pharmacologically active components; it has been used as an immunotherapy to treat bee venom hypersensitivity, and venom therapy has been applied as an alternative medicine. Here, we present evidence that the serine protease found in bumblebee venom exhibits fibrin(ogen)olytic activity. Compared to honeybee venom, bumblebee venom contains a higher content of serine protease, which is one of its major components. Venom serine proteases from bumblebees did not cross-react with antibodies against the honeybee venom serine protease. We provide functional evidence indicating that bumblebee (Bombus terrestris) venom serine protease (Bt-VSP) acts as a fibrin(ogen)olytic enzyme. Bt-VSP activates prothrombin and directly degrades fibrinogen into fibrin degradation products. However, Bt-VSP is not a plasminogen activator, and its fibrinolytic activity is less than that of plasmin. Taken together, our results define roles for Bt-VSP as a prothrombin activator, a thrombin-like protease, and a plasmin-like protease. These findings offer significant insight into the allergic reaction sequence that is initiated by bee venom serine protease and its potential usefulness as a clinical agent in the field of hemostasis and thrombosis. - Graphical abstract: Display Omitted Highlights: → Bumblebee venom serine protease (Bt-VSP) is a fibrin(ogen)olytic enzyme. → Bt-VSP activates prothrombin. → Bt-VSP directly degrades fibrinogen into fibrin degradation products. → Bt-VSP is a hemostatically active protein that is a potent clinical agent.

  7. Kinases and Cancer

    OpenAIRE

    Jonas Cicenas; Egle Zalyte; Amos Bairoch; Pascale Gaudet

    2018-01-01

    Protein kinases are a large family of enzymes catalyzing protein phosphorylation. The human genome contains 518 protein kinase genes, 478 of which belong to the classical protein kinase family and 40 are atypical protein kinases [...

  8. Identification of the protein kinase C phosphorylation site in neuromodulin

    International Nuclear Information System (INIS)

    Apel, E.D.; Byford, M.F.; Au, D.; Walsh, K.A.; Storm, D.R.

    1990-01-01

    Neuromodulin (P-57, GAP-43, B-50, F-1) is a neurospecific calmodulin binding protein that is phosphorylated by protein kinase C. Phosphorylation by protein kinase C has been shown to abolish the affinity of neuromodulin for calmodulin and the authors have proposed that the concentration of free CaM in neurons may be regulated by phosphorylation and dephosphorylation of neuromodulin. The purpose of this study was to identify the protein kinase C phosphorylation site(s) in neuromodulin using recombinant neuromodulin as a substrate. Toward this end, it was demonstrated that recombinant neuromodulin purified from Escherichia coli and bovine neuromodulin were phosphorylated with similar K m values and stoichiometries and that protein kinase C mediated phosphorylation of both proteins abolished binding to calmodulin-Sepharose. Recombinant neuromodulin was phosphorylated by using protein kinase C and [γ- 32 P]ATP and digested with trypsin, and the resulting peptides were separated by HPLC. Only one 32 P-labeled tryptic peptide was generated from phosphorylated neuromodulin. They conclude that serine-41 is the protein kinase C phosphorylation site of neuromodulin and that phosphorylation of this amino acid residue blocks binding of calmoculin to neuromodulin. The proximity of serine-41 to the calmodulin binding domain in neuromodulin very likely explains the effect of phosphorylation on the affinity of neuromodulin for calmodulin

  9. Evaluating acetaldehyde synthesis from L-/sup 14/C(U)) threonine by Streptococcus thermophilus and Lactobacillus bulgaricus

    Energy Technology Data Exchange (ETDEWEB)

    Wilkins, D.W.; Schmidt, R.H.; Shireman, R.B.; Smith, K.L.; Jezeski, J.J.

    1986-05-01

    To evaluate the synthesis of acetaldehyde from threonine during growth of yogurt cultures, Streptococcus thermophilus MS1 and Lactobacillus bulgaricus MR1 were grown in defined medium in which 10% of the total threonine was composed of L-(carbon-14(U))threonine. Acetaldehyde production was monitored by formation of 2,4-dinitrophenylhydrazone followed by separation and analysis using high performance liquid chromatography. After growth for 8 h at 42/sup 0/C, approximately 2.0% of the total acetaldehyde (780.4 nmol) produced was from L-(carbon-14)threonine. Threonine aldolase activity was determined in cell-free extracts from S. thermophilus and L. bulgaricus grown in Elliker broth. Increasing incubation temperature from 30 to 42/sup 0/C decreased threonine aldolase activity in cells of the streptococcus harvested after 8 h of incubation. Effect of incubation temperature was more dramatic in cells harvested after 18 h where the activity of cells grown at 48/sup 0/C was 89% lower than that of cells grown at 30/sup 0/C. Cell extracts from S. thermophilus MS1 possessed higher threonine aldolase activity than did those from L. bulgaricus MR1. Increased assay temperature from 30 to 42/sup 0/C increased threonine aldolase activity in S. thermophilus MS1.

  10. Identification of an evolutionarily conserved extracellular threonine residue critical for surface expression and its potential coupling of adjacent voltage-sensing and gating domains in voltage-gated potassium channels.

    Science.gov (United States)

    Mckeown, Lynn; Burnham, Matthew P; Hodson, Charlotte; Jones, Owen T

    2008-10-31

    The dynamic expression of voltage-gated potassium channels (Kvs) at the cell surface is a fundamental factor controlling membrane excitability. In exploring possible mechanisms controlling Kv surface expression, we identified a region in the extracellular linker between the first and second of the six (S1-S6) transmembrane-spanning domains of the Kv1.4 channel, which we hypothesized to be critical for its biogenesis. Using immunofluorescence microscopy, flow cytometry, patch clamp electrophysiology, and mutagenesis, we identified a single threonine residue at position 330 within the Kv1.4 S1-S2 linker that is absolutely required for cell surface expression. Mutation of Thr-330 to an alanine, aspartate, or lysine prevented surface expression. However, surface expression occurred upon co-expression of mutant and wild type Kv1.4 subunits or mutation of Thr-330 to a serine. Mutation of the corresponding residue (Thr-211) in Kv3.1 to alanine also caused intracellular retention, suggesting that the conserved threonine plays a generalized role in surface expression. In support of this idea, sequence comparisons showed conservation of the critical threonine in all Kv families and in organisms across the evolutionary spectrum. Based upon the Kv1.2 crystal structure, further mutagenesis, and the partial restoration of surface expression in an electrostatic T330K bridging mutant, we suggest that Thr-330 hydrogen bonds to equally conserved outer pore residues, which may include a glutamate at position 502 that is also critical for surface expression. We propose that Thr-330 serves to interlock the voltage-sensing and gating domains of adjacent monomers, thereby yielding a structure competent for the surface expression of functional tetramers.

  11. The fibronectin III-1 domain activates a PI3-Kinase/Akt signaling pathway leading to αvβ5 integrin activation and TRAIL resistance in human lung cancer cells

    International Nuclear Information System (INIS)

    Cho, Christina; Horzempa, Carol; Jones, David; McKeown-Longo, Paula J.

    2016-01-01

    Fibronectin is a mechanically sensitive protein which is organized in the extracellular matrix as a network of interacting fibrils. The lung tumor stroma is enriched for fibronectin which is thought to contribute to metastasis and drug resistance. Fibronectin is an elastic, multi-modular protein made up of individually folded domains, some of which can stretch in response to increased mechanical tension. Very little is known about the relationship of fibronectin’s unfolded domains to lung cancer resistance to chemotherapy. In the present study, we evaluated the impact of unfolding the first Type III domain of fibronectin (FnIII-1c) on TNF-related apoptosis inducing ligand (TRAIL) resistance. NCI-H460 non-small cell lung cancer cells were treated with FnIII-1c then assessed for TRAIL-induced apoptosis. Subsequent analysis of FnIII-1c-mediated signaling pathways was also completed. Human non-small cell lung cancer tissue sections were assessed for the expression of vitronectin by immunohistochemistry. FnIII-1c inhibited TRAIL-induced activation of caspase 8 and subsequent apoptosis in NCI-H460 lung cancer cells. FnIII-1c treatment was associated with the activation of the phosphatidylinositol-3-kinase/alpha serine/threonine kinase (PI3K/Akt) pathway and the αvβ5 integrin receptor for vitronectin, both of which were required for TRAIL resistance. Immunohistochemical staining of sections from non-small cell lung cancers showed that vitronectin was localized around blood vessels and in the tumor-stroma interface. Unfolding of Type III domains within the fibronectin matrix may promote TRAIL resistance through the activation of a PI3K/Akt/αvβ5 signaling axis and point to a novel mechanism by which changes in secondary structure of fibronectin contribute to cancer cell resistance to apoptosis

  12. Flow synthesis of phenylserine using threonine aldolase (TA) immobilized on Eupergit support

    NARCIS (Netherlands)

    Tibhe, J.; Fu, Hui; Noel, T.; Wang, Q.; Meuldijk, J.; Hessel, V.

    2013-01-01

    Threonine aldolase (TA) from Thermotoga maritima was immobilized on Eupergit support by both a direct and an indirect method. The incubation time for the direct immobilization method was optimized for the highest amount of enzyme on the support. By introducing the immobilized TA in a packed-bed

  13. Investigations on the optical, thermal and surface modifications of electron irradiated L-threonine single crystals

    Energy Technology Data Exchange (ETDEWEB)

    Ramesh Kumar, G.; Gokul Raj, S. [Department of Physics, Presidency College, Chepauk, Chennai 600005 (India); Bogle, K.A.; Dhole, S.D.; Bhoraskar, V.N. [Department of Physics, University of Pune, Pune 411007 (India); Mohan, R. [Department of Physics, Presidency College, Chepauk, Chennai 600005 (India)], E-mail: professormohan@yahoo.co.in

    2008-06-15

    L-Threonine single crystals have been irradiated by 6 MeV electrons. Irradiated crystals at various electron fluences were subjected to various techniques such as UV-vis-NIR, atomic force microscopy (AFM) and thermomechanical analyses. Thermal strength of the irradiated crystals has also been studied through differential scanning calorimetry (DSC) measurements. The results have been discussed in detail.

  14. Angiotensin II-induced hypertension blunts thick ascending limb NO production by reducing NO synthase 3 expression and enhancing threonine 495 phosphorylation.

    Science.gov (United States)

    Ramseyer, Vanesa D; Gonzalez-Vicente, Agustin; Carretero, Oscar A; Garvin, Jeffrey L

    2015-01-15

    Thick ascending limbs reabsorb 30% of the filtered NaCl load. Nitric oxide (NO) produced by NO synthase 3 (NOS3) inhibits NaCl transport by this segment. In contrast, chronic angiotensin II (ANG II) infusion increases net thick ascending limb transport. NOS3 activity is regulated by changes in expression and phosphorylation at threonine 495 (T495) and serine 1177 (S1177), inhibitory and stimulatory sites, respectively. We hypothesized that NO production by thick ascending limbs is impaired by chronic ANG II infusion, due to reduced NOS3 expression, increased phosphorylation of T495, and decreased phosphorylation of S1177. Rats were infused with 200 ng·kg(-1)·min(-1) ANG II or vehicle for 1 and 5 days. ANG II infusion for 5 days decreased NOS3 expression by 40 ± 12% (P thick ascending limbs from ANG II-infused animals [ANG II -0.01 ± 0.06 arbitrary fluorescence units (AFU)/min vs. 0.17 ± 0.02 AFU/min in controls; P thick ascending limbs is impaired due to decreased NOS3 expression and altered phosphorylation. Copyright © 2015 the American Physiological Society.

  15. Chromatin-Bound MDM2 Regulates Serine Metabolism and Redox Homeostasis Independently of p53.

    Science.gov (United States)

    Riscal, Romain; Schrepfer, Emilie; Arena, Giuseppe; Cissé, Madi Y; Bellvert, Floriant; Heuillet, Maud; Rambow, Florian; Bonneil, Eric; Sabourdy, Frédérique; Vincent, Charles; Ait-Arsa, Imade; Levade, Thierry; Thibaut, Pierre; Marine, Jean-Christophe; Portais, Jean-Charles; Sarry, Jean-Emmanuel; Le Cam, Laurent; Linares, Laetitia K

    2016-06-16

    The mouse double minute 2 (MDM2) oncoprotein is recognized as a major negative regulator of the p53 tumor suppressor, but growing evidence indicates that its oncogenic activities extend beyond p53. Here, we show that MDM2 is recruited to chromatin independently of p53 to regulate a transcriptional program implicated in amino acid metabolism and redox homeostasis. Identification of MDM2 target genes at the whole-genome level highlights an important role for ATF3/4 transcription factors in tethering MDM2 to chromatin. MDM2 recruitment to chromatin is a tightly regulated process that occurs during oxidative stress and serine/glycine deprivation and is modulated by the pyruvate kinase M2 (PKM2) metabolic enzyme. Depletion of endogenous MDM2 in p53-deficient cells impairs serine/glycine metabolism, the NAD(+)/NADH ratio, and glutathione (GSH) recycling, impacting their redox state and tumorigenic potential. Collectively, our data illustrate a previously unsuspected function of chromatin-bound MDM2 in cancer cell metabolism. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Identification of Ser/Thr kinase and Forkhead Associated Domains in Mycobacterium ulcerans: Characterization of Novel Association between Protein Kinase Q and MupFHA

    Science.gov (United States)

    Singhal, Anshika; Joshi, Jayadev; Virmani, Richa; Gupta, Meetu; Verma, Nupur; Maji, Abhijit; Misra, Richa; Baronian, Grégory; Pandey, Amit K.; Molle, Virginie; Singh, Yogendra

    2014-01-01

    Background Mycobacterium ulcerans, the causative agent of Buruli ulcer in humans, is unique among the members of Mycobacterium genus due to the presence of the virulence determinant megaplasmid pMUM001. This plasmid encodes multiple virulence-associated genes, including mup011, which is an uncharacterized Ser/Thr protein kinase (STPK) PknQ. Methodology/Principal Findings In this study, we have characterized PknQ and explored its interaction with MupFHA (Mup018c), a FHA domain containing protein also encoded by pMUM001. MupFHA was found to interact with PknQ and suppress its autophosphorylation. Subsequent protein-protein docking and molecular dynamic simulation analyses showed that this interaction involves the FHA domain of MupFHA and PknQ activation loop residues Ser170 and Thr174. FHA domains are known to recognize phosphothreonine residues, and therefore, MupFHA may be acting as one of the few unusual FHA-domain having overlapping specificity. Additionally, we elucidated the PknQ-dependent regulation of MupDivIVA (Mup012c), which is a DivIVA domain containing protein encoded by pMUM001. MupDivIVA interacts with MupFHA and this interaction may also involve phospho-threonine/serine residues of MupDivIVA. Conclusions/Significance Together, these results describe novel signaling mechanisms in M. ulcerans and show a three-way regulation of PknQ, MupFHA, and MupDivIVA. FHA domains have been considered to be only pThr specific and our results indicate a novel mechanism of pSer as well as pThr interaction exhibited by MupFHA. These results signify the need of further re-evaluating the FHA domain –pThr/pSer interaction model. MupFHA may serve as the ideal candidate for structural studies on this unique class of modular enzymes. PMID:25412098

  17. A central role for CK1 in catalysing phosphorylation of the P53 transactivation domain at serine 20 after HHV-6B viral infection

    DEFF Research Database (Denmark)

    Maclaine, NJ; Øster, Bodil; Bundgaard, Bettina

    2008-01-01

    The tumour suppressor protein p53 is activated by distinct cellular stresses including radiation, hypoxia, type-I interferon, and DNA/RNA virus infection. The transactivation domain of p53 contains a phosphorylation site at serine 20 (Ser20) whose modification stabilises the binding of the transc...... was not blocked by D4476. These data highlight a central role for CK1 as the Ser20-site kinase for p53 in DNA virus-infected cells, but also suggest that distinct stresses may selectively trigger different protein kinases to modify the transactivation domain of p53 at Ser20....

  18. Lauric acid and myristic acid from Allium sativum inhibit the growth of Mycobacterium tuberculosis H37Ra: in silico analysis reveals possible binding to protein kinase B.

    Science.gov (United States)

    Muniyan, Rajiniraja; Gurunathan, Jayaraman

    2016-12-01

    The bulb of Allium sativum Linn (Alliaceae) has numerous medicinal values. Though the petroleum ether extract of the bulb has shown to exhibit antimycobacterial activity, the phytochemical(s) responsible for this inhibitory activity is not known. To characterize the bioactive compounds in the petroleum ether extract of Allium sativum (garlic) that inhibit the growth of Mycobacterium tuberculosis H37Ra. Bioactivity-guided fractionation was employed to isolate the bioactive compounds. Antimycobacterial activity was evaluated by well-diffusion method and microplate alamar blue assay (MABA). Infrared spectroscopy, mass spectrometry and nuclear magnetic resonance spectroscopy were used to characterize the bioactive compounds. Autodock was used to obtain information on molecular recognition, and molecular dynamics simulation was performed using GROMACS. The bioactive compounds that inhibited the growth of M. tuberculosis H37Ra were found to be lauric acid (LA) and myristic acid (MA). The minimal inhibitory concentration of LA and MA was found to be 22.2 and 66.7 μg/mL, respectively. In silico analysis revealed that these fatty acids could bind at the cleft between the N-terminal and C-terminal lobes of the cytosolic domain of serine/threonine protein kinase B (PknB). The inhibition activity was dependent on the alkyl chain length of the fatty acid, and the amino acid residues involved in binding to fatty acid was found to be conserved across the Pkn family of proteins. The study indicates the possibility of using fatty acid derivatives, involving Pkn family of proteins, to inhibit the signal transduction processes in M. tuberculosis.

  19. Augmented O-GlcNAcylation of AMP-activated kinase promotes the proliferation of LoVo cells, a colon cancer cell line.

    Science.gov (United States)

    Ishimura, Emi; Nakagawa, Takatoshi; Moriwaki, Kazumasa; Hirano, Seiichi; Matsumori, Yoshinobu; Asahi, Michio

    2017-12-01

    Increasing incidence of various cancers has been reported in diabetic patients. O-linked N-acetylglucosamine (O-GlcNAc) modification of proteins at serine/threonine residues (O-GlcNAcylation) is an essential post-translational modification that is upregulated in diabetic patients and has been implicated in tumor growth. However, the mechanisms by which O-GlcNAcylation promotes tumor growth remain unclear. Given that AMP-activated kinase (AMPK) has been thought to play important roles in suppressing tumor growth, we evaluated the involvement of AMPK O-GlcNAcylation on the growth of LoVo cells, a human colon cancer cell line. Results revealed that treatment with Thiamet G (TMG), an inhibitor of O-GlcNAc hydrolase, increased both anchorage-dependent and -independent growth of the cells. O-GlcNAc transferase overexpression also increased the growth. These treatments increased AMPK O-GlcNAcylation in a dose-dependent manner, which led to reduced AMPK phosphorylation and mTOR activation. Chemical inhibition or activation of AMPK led to increased or decreased growth, respectively, which was consistent with the data with genetic inhibition of AMPK. In addition, TMG-mediated acceleration of tumor growth was abolished by both chemical and genetic inhibition of AMPK. To examine the effects of AMPK O-GlcNAcylation in vivo, the LoVo cells were s.c. transplanted onto the backs of BALB/c-nu/nu mice. Injection of TMG promoted the growth and enhanced O-GlcNAcylation of the tumors of the mice. Consistent with in vitro data, AMPK O-GlcNAcylation was increased, which reduced AMPK phosphorylation and resulted in activation of mTOR. Collectively, the higher colon cancer risk of diabetic patients could be due to O-GlcNAcylation-mediated AMPK inactivation and subsequent activation of mTOR. © 2017 The Authors. Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.

  20. A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

    Science.gov (United States)

    Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

    2013-06-10

    We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

  1. Coumestrol Epigenetically Suppresses Cancer Cell Proliferation: Coumestrol Is a Natural Haspin Kinase Inhibitor

    Directory of Open Access Journals (Sweden)

    Jong-Eun Kim

    2017-10-01

    Full Text Available Targeting epigenetic changes in gene expression in cancer cells may offer new strategies for the development of selective cancer therapies. In the present study, we investigated coumestrol, a natural compound exhibiting broad anti-cancer effects against skin melanoma, lung cancer and colon cancer cell growth. Haspin kinase was identified as a direct target protein of coumestrol using kinase profiling analysis. Histone H3 is a direct substrate of haspin kinase. We observed haspin kinase overexpression as well as greater phosphorylation of histone H3 at threonine 3 (Thr-3 in the cancer cells compared to normal cells. Computer modeling using the Schrödinger Suite program identified the binding interface within the ATP binding site. These findings suggest that the anti-cancer effect of coumestrol is due to the direct targeting of haspin kinase. Coumestrol has considerable potential for further development as a novel anti-cancer agent.

  2. Cross genome comparisons of serine proteases in Arabidopsis and rice

    Directory of Open Access Journals (Sweden)

    Sowdhamini R

    2006-08-01

    Full Text Available Abstract Background Serine proteases are one of the largest groups of proteolytic enzymes found across all kingdoms of life and are associated with several essential physiological pathways. The availability of Arabidopsis thaliana and rice (Oryza sativa genome sequences has permitted the identification and comparison of the repertoire of serine protease-like proteins in the two plant species. Results Despite the differences in genome sizes between Arabidopsis and rice, we identified a very similar number of serine protease-like proteins in the two plant species (206 and 222, respectively. Nearly 40% of the above sequences were identified as potential orthologues. Atypical members could be identified in the plant genomes for Deg, Clp, Lon, rhomboid proteases and species-specific members were observed for the highly populated subtilisin and serine carboxypeptidase families suggesting multiple lateral gene transfers. DegP proteases, prolyl oligopeptidases, Clp proteases and rhomboids share a significantly higher percentage orthology between the two genomes indicating substantial evolutionary divergence was set prior to speciation. Single domain architectures and paralogues for several putative subtilisins, serine carboxypeptidases and rhomboids suggest they may have been recruited for additional roles in secondary metabolism with spatial and temporal regulation. The analysis reveals some domain architectures unique to either or both of the plant species and some inactive proteases, like in rhomboids and Clp proteases, which could be involved in chaperone function. Conclusion The systematic analysis of the serine protease-like proteins in the two plant species has provided some insight into the possible functional associations of previously uncharacterised serine protease-like proteins. Further investigation of these aspects may prove beneficial in our understanding of similar processes in commercially significant crop plant species.

  3. Microstructure and nanomechanical properties of enamel remineralized with asparagine-serine-serine peptide

    Energy Technology Data Exchange (ETDEWEB)

    Chung, Hsiu-Ying, E-mail: hychung@mail.fcu.edu.tw; Li, Cheng Che

    2013-03-01

    A highly biocompatible peptide, triplet repeats of asparagine-serine-serine (3NSS) was designed to regulate mineral deposition from aqueous ions in saliva for the reconstruction of enamel lesions. Healthy human enamel was sectioned and acid demineralized to create lesions, then exposed to the 3NSS peptide solution, and finally immersed in artificial saliva for 24 h. The surface morphology and roughness were examined using scanning electron microscopy (SEM) and atomic force microscopy (AFM), respectively. X-ray diffraction (XRD) was used to identify the phases and crystallinity of the deposited minerals observed on the enamel surface. Attenuated total reflection Fourier transform infrared spectroscopy (ATR-FTIR) was used to quantitatively analyze the mineral variation by calculating the relative integrated-area of characteristic bands. Nanohardness and elastic modulus measured by nanoindentation at various treatment stages were utilized to evaluate the degree of recovery. Biomimetic effects were accessed according to the degree of nanohardness recovery and the amount of hydroxyapatite deposition. The charged segments in the 3NSS peptide greatly attracted aqueous ions from artificial saliva to form hydroxyapatite crystals to fill enamel caries, in particular the interrod areas, resulting in a slight reduction in overall surface roughness. Additionally, the deposited hydroxyapatites were of a small crystalline size in the presence of the 3NSS peptide, which effectively restrained the plastic deformations and thus resulted in greater improvements in nanohardness and elastic modulus. The degree of nanohardness recovery was 5 times greater for remineralized enamel samples treated with the 3NSS peptide compared to samples without peptide treatment. - Highlights: Black-Right-Pointing-Pointer The degree of nanohardness recovery of enamel was 4 times greater with the aid of 3NSS peptide. Black-Right-Pointing-Pointer 3NSS peptide promoted the formation of hydroxyapatites with

  4. Refractometry of TGS crystals doped with L-threonine impurity under uniaxial pressure

    International Nuclear Information System (INIS)

    Stadnyk, V. I.; Kiryk, Yu. I.

    2013-01-01

    The temperature and spectral dependences of the refractive indices of triglycine sulphate (TGS) crystals doped with L-threonine impurity have been investigated. It is established that the introduction of an impurity weakens the temperature dependence of refractive indices. The electronic polarizability, refractions, and parameters of UV oscillators of mechanically deformed impurity crystals are calculated. The temperature coefficients of the phase transition shift are determined

  5. Refractometry of TGS crystals doped with L-threonine impurity under uniaxial pressure

    Energy Technology Data Exchange (ETDEWEB)

    Stadnyk, V. I., E-mail: vasylstadnyk@ukr.net; Kiryk, Yu. I. [Lviv National University (Ukraine)

    2013-07-15

    The temperature and spectral dependences of the refractive indices of triglycine sulphate (TGS) crystals doped with L-threonine impurity have been investigated. It is established that the introduction of an impurity weakens the temperature dependence of refractive indices. The electronic polarizability, refractions, and parameters of UV oscillators of mechanically deformed impurity crystals are calculated. The temperature coefficients of the phase transition shift are determined.

  6. LmxMPK4, an essential mitogen-activated protein kinase of Leishmania mexicana is phosphorylated and activated by the STE7-like protein kinase LmxMKK5

    DEFF Research Database (Denmark)

    John von Freyend, Simona; Rosenqvist, Heidi; Fink, Annette

    2010-01-01

    The essential mitogen-activated protein kinase (MAP kinase), LmxMPK4, of Leishmania mexicana is minimally active when purified following recombinant expression in Escherichia coli and was therefore unsuitable for drug screening until now. Using an E. coli protein co-expression system we identified...... LmxMKK5, a STE7-like protein kinase from L. mexicana, which phosphorylates and activates recombinant LmxMPK4 in vitro. LmxMKK5 is comprised of 525 amino acids and has a calculated molecular mass of 55.9kDa. The co-expressed, purified LmxMPK4 showed strong phosphotransferase activity in radiometric...... kinase assays and was confirmed by immunoblot and tandem mass spectrometry analyses to be phosphorylated on threonine 190 and tyrosine 192 of the typical TXY MAP kinase activation motif. The universal protein kinase inhibitor staurosporine reduced the phosphotransferase activity of co...

  7. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance {sup 13}C NMR spectroscopy

    Energy Technology Data Exchange (ETDEWEB)

    Daley, Margaret E.; Sykes, Brian D. [University of Alberta, Department of Biochemistry, CIHR Group in Protein Structure and Function and Protein Engineering Network of Centres of Excellence (Canada)

    2004-06-15

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance {sup 13}C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the {sup 1}H-{sup 13}C NOE were determined in this study. The C{alpha}H relaxation measurements were compared to the previously measured {sup 15}N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the {chi}{sub 1} dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than {+-}25 deg.

  8. Characterization of threonine side chain dynamics in an antifreeze protein using natural abundance 13C NMR spectroscopy

    International Nuclear Information System (INIS)

    Daley, Margaret E.; Sykes, Brian D.

    2004-01-01

    The dynamics of threonine side chains of the Tenebrio molitor antifreeze protein (TmAFP) were investigated using natural abundance 13 C NMR. In TmAFP, the array of threonine residues on one face of the protein is responsible for conferring its ability to bind crystalline ice and inhibit its growth. Heteronuclear longitudinal and transverse relaxation rates and the 1 H- 13 C NOE were determined in this study. The CαH relaxation measurements were compared to the previously measured 15 N backbone parameters and these are found to be in agreement. For the analysis of the threonine side chain motions, the model of restricted rotational diffusion about the χ 1 dihedral angle was employed [London and Avitabile (1978) J. Am. Chem. Soc., 100, 7159-7165]. We demonstrate that the motion experienced by the ice binding threonine side chains is highly restricted, with an approximate upper limit of less than ±25 deg

  9. The binding mechanism of a peptidic cyclic serine protease inhibitor

    DEFF Research Database (Denmark)

    Jiang, Longguang; Svane, Anna Sigrid P.; Sørensen, Hans Peter

    2011-01-01

    Serine proteases are classical objects for studies of catalytic and inhibitory mechanisms as well as interesting as therapeutic targets. Since small-molecule serine protease inhibitors generally suffer from specificity problems, peptidic inhibitors, isolated from phage-displayed peptide libraries......, have attracted considerable attention. Here, we have investigated the mechanism of binding of peptidic inhibitors to serine protease targets. Our model is upain-1 (CSWRGLENHRMC), a disulfide-bond-constrained competitive inhibitor of human urokinase-type plasminogen activator with a noncanonical...... inhibitory mechanism and an unusually high specificity. Using a number of modified variants of upain-1, we characterised the upain-1-urokinase-type plasminogen activator complex using X-ray crystal structure analysis, determined a model of the peptide in solution by NMR spectroscopy, and analysed binding...

  10. Mosaic serine proteases in the mammalian central nervous system.

    Science.gov (United States)

    Mitsui, Shinichi; Watanabe, Yoshihisa; Yamaguchi, Tatsuyuki; Yamaguchi, Nozomi

    2008-01-01

    We review the structure and function of three kinds of mosaic serine proteases expressed in the mammalian central nervous system (CNS). Mosaic serine proteases have several domains in the proenzyme fragment, which modulate proteolytic function, and a protease domain at the C-terminus. Spinesin/TMPRSS5 is a transmembrane serine protease whose presynaptic distribution on motor neurons in the spinal cord suggests that it is significant for neuronal plasticity. Cell type-specific alternative splicing gives this protease diverse functions by modulating its intracellular localization. Motopsin/PRSS12 is a mosaic protease, and loss of its function causes mental retardation. Recent reports indicate the significance of this protease for cognitive function. We mention the fibrinolytic protease, tissue plasminogen activator (tPA), which has physiological and pathological functions in the CNS.

  11. Dosage compensation of serine-4 transfer RNA in Drosophila melanogaster

    International Nuclear Information System (INIS)

    Birchler, J.A.; Owenby, R.K.; Jacobson, K.B.

    1982-01-01

    A dosage series of the X chromosome site for serine-4 transfer RNA consisting of one of three copies in females and one to two in males was constructed to test whether transfer RNA expression is governed by dosage compensation. A dosage effect on the level of the serine-4 isoacceptor was observed in both females and males when the structural locus was varied. However, in males, each dose had a relatively greater expression so the normal one dose was slightly greater than the total female value and the duplicated male had the highest relative expression of all the types examined. Serine-4 levels in males and females from an isogenic Oregon-R stock were similar. Thus the transfer RNA levels conform to the expectations of dosage compensation

  12. Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.

    Science.gov (United States)

    Tuteja, Narendra; Reddy, Malireddy Kodandarami; Mudgil, Yashwanti; Yadav, Badam Singh; Chandok, Meena Rani; Sopory, Sudhir Kumar

    2003-08-01

    DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.

  13. Convergent synthesis of a deuterium-labeled serine dipeptide lipid for analysis of biological samples.

    Science.gov (United States)

    Dietz, Christopher; Clark, Robert B; Nichols, Frank C; Smith, Michael B

    2017-05-30

    Bacterial serine dipeptide lipids are known to promote inflammatory processes and are detected in human tissues associated with periodontal disease or atherosclerosis. Accurate quantification of bacterial serine lipid, specifically lipid 654 [((S)-15-methyl-3-((13-methyltetradecanoyl)oxy)hexadecanoyl)glycyl-l-serine, (3S)-l-serine] isolated from Porphyromonas gingivalis, in biological samples requires the preparation of a stable isotope internal standard for sample supplementation and subsequent mass spectrometric analysis. This report describes the convergent synthesis of a deuterium-substituted serine dipeptide lipid, which is an isotopically labeled homologue that represents a dominant form of serine dipeptide lipid recovered in bacteria. Copyright © 2017 John Wiley & Sons, Ltd.

  14. The VA, VCD, Raman and ROA spectra of tri-L-serine in aqueous solution

    DEFF Research Database (Denmark)

    Jürgensen, Vibeke Würtz; Jalkanen, Karl J.

    2006-01-01

    The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory with the ......The structures of one conformer of the nonionic neutral and zwitterionic species of L-serinyl L-serinyl L-serine (SSS or tri-L-serine), together with its cationic and anionic species and the capped N-acetyl tri-L-serine N'-methylamide analog were optimized with density functional theory...

  15. Fatal cerebral edema associated with serine deficiency in CSF

    NARCIS (Netherlands)

    Keularts, Irene M. L. W.; Leroy, Piet L. J. M.; Rubio-Gozalbo, Estela M.; Spaapen, Leo J. M.; Weber, Biene; Dorland, Bert; de Koning, Tom J.; Verhoeven-Duif, Nanda M.

    2010-01-01

    Two young girls without a notable medical history except for asthma presented with an acute toxic encephalopathy with very low serine concentrations both in plasma and cerebrospinal fluid (CSF) comparable to patients with 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency. Clinical symptoms and

  16. The Hunger Games: p53 regulates metabolism upon serine starvation.

    Science.gov (United States)

    Tavana, Omid; Gu, Wei

    2013-02-05

    Cancer cells reprogram their metabolism to support a high proliferative rate. A new study shows that, upon serine starvation, the tumor suppressor p53 activates p21 to shift metabolic flux from purine biosynthesis to glutathione production, which enhances cellular proliferation and viability by combating ROS (Maddocks et al., 2013). Copyright © 2013 Elsevier Inc. All rights reserved.

  17. Serine protease from midgut of Bombus terrestris males

    Czech Academy of Sciences Publication Activity Database

    Brabcová, Jana; Kindl, Jiří; Valterová, Irena; Pichová, Iva; Zarevúcka, Marie; Brabcová, J.; Jágr, Michal; Mikšík, Ivan

    2013-01-01

    Roč. 82, č. 3 (2013), s. 117-128 ISSN 0739-4462 R&D Projects: GA ČR GA203/09/1446; GA TA ČR TA01020969 Institutional support: RVO:61388963 ; RVO:67985823 Keywords : Bombus terrestris * midgut * serine protease * bumblebee Subject RIV: CE - Biochemistry; CE - Biochemistry (FGU-C) Impact factor: 1.160, year: 2013

  18. LOK is a major ERM kinase in resting lymphocytes and regulates cytoskeletal rearrangement through ERM phosphorylation.

    Science.gov (United States)

    Belkina, Natalya V; Liu, Yin; Hao, Jian-Jiang; Karasuyama, Hajime; Shaw, Stephen

    2009-03-24

    ERM (ezrin-radixin-moesin) proteins mediate linkage of actin cytoskeleton to plasma membrane in many cells. ERM activity is regulated in part by phosphorylation at a C-terminal threonine, but the identity of ERM kinases is unknown in lymphocytes and incompletely defined in other mammalian cells. Our studies show that lymphocyte-oriented kinase (LOK) is an ERM kinase in vitro and in vivo. Mass spectrometric analysis indicates LOK is abundant at the lymphocyte plasma membrane and immunofluorescence studies show LOK enrichment at the plasma membrane near ERM. In vitro peptide specificity analyses characterize LOK as a basophilic kinase whose optimal substrate sequence resembles the ERM site, including unusual preference for tyrosine at P-2. LOK's activity on moesin peptide and protein was comparable to reported ERM kinases ROCK and PKC but unlike them LOK displayed preferential specificity for moesin compared to traditional basophilic kinase substrates. Two genetic approaches demonstrate a role for LOK in ERM phosphorylation: cell transfection with LOK kinase domain augments ERM phosphorylation and lymphocytes from LOK knockout mice have >50% reduction in ERM phosphorylation. The findings on localization and specificity argue that LOK is a direct ERM kinase. The knockout mice have normal hematopoietic cell development but notably lymphocyte migration and polarization in response to chemokine are enhanced. These functional alterations fit the current understanding of the role of ERM phosphorylation in regulating cortical reorganization. Thus, these studies identify a new ERM kinase of importance in lymphocytes and confirm the role of ERM phosphorylation in regulating cell shape and motility.

  19. Aurora B kinase inhibition in mitosis: strategies for optimising the use of aurora kinase inhibitors such as AT9283.

    Science.gov (United States)

    Curry, Jayne; Angove, Hayley; Fazal, Lynsey; Lyons, John; Reule, Matthias; Thompson, Neil; Wallis, Nicola

    2009-06-15

    Aurora kinases play a key role in regulating mitotic division and are attractive oncology targets. AT9283, a multi-targeted kinase inhibitor with potent activity against Aurora A and B kinases, inhibited growth and survival of multiple solid tumor cell lines and was efficacious in mouse xenograft models. AT9283-treatment resulted in endoreduplication and ablation of serine-10 histone H3 phosphorylation in both cells and tumor samples, confirming that in these models it acts as an Aurora B kinase inhibitor. In vitro studies demonstrated that exposure to AT9283 for one complete cell cycle committed an entire population of p53 checkpoint-compromised cells (HCT116) to multinucleation and death whereas treatment of p53 checkpoint-competent cells (HMEC, A549) for a similar length of time led to a reversible arrest of cells with 4N DNA. Further studies in synchronized cell populations suggested that exposure to AT9283 during mitosis was critical for optimal cytotoxicity. We therefore investigated ways in which these properties might be exploited to optimize the efficacy and therapeutic index of Aurora kinase inhibitors for p53 checkpoint compromised tumors in vivo. Combining Aurora B kinase inhibition with paclitaxel, which arrests cells in mitosis, in a xenograft model resulted in promising efficacy without additional toxicity. These findings have implications for optimizing the efficacy of Aurora kinase inhibitors in clinical practice.

  20. Engineering of High Yield Production of L-serine in Escherichia coli

    DEFF Research Database (Denmark)

    Mundhada, Hemanshu; Schneider, Konstantin; Christensen, Hanne Bjerre

    2016-01-01

    by deletion of three L-serine deaminases sdaA, sdaB, and tdcG, as well as serine hydroxyl methyl transferase (SHMT) encoded by glyA. Upon overexpression of the serine production pathway, consisting of a feedback resistant version of serA along with serB and serC, this quadruple deletion strain showed a very...

  1. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

    Energy Technology Data Exchange (ETDEWEB)

    Chen, Shang-Der, E-mail: chensd@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Lin, Tsu-Kung [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Yang, Ding-I. [Institute of Brain Science and Brain Research Center, National Yang-Ming University, Taipei, Taiwan (China); Lee, Su-Ying [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Shaw, Fu-Zen [Department of Psychology, National Cheng Kung University, Tainan, Taiwan (China); Liou, Chia-Wei [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Chuang, Yao-Chung, E-mail: ycchuang@adm.cgmh.org.tw [Department of Neurology, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China); Center for Translational Research in Biomedical Sciences, Kaohsiung Chang Gung Memorial Hospital, Chang Gung University College of Medicine, Taiwan (China)

    2015-05-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield.

  2. The Importance of G Protein-Coupled Receptor Kinase 4 (GRK4 in Pathogenesis of Salt Sensitivity, Salt Sensitive Hypertension and Response to Antihypertensive Treatment

    Directory of Open Access Journals (Sweden)

    Brian Rayner

    2015-03-01

    Full Text Available Salt sensitivity is probably caused by either a hereditary or acquired defect of salt excretion by the kidney, and it is reasonable to consider that this is the basis for differences in hypertension between black and white people. Dopamine acts in an autocrine/paracrine fashion to promote natriuresis in the proximal tubule and thick ascending loop of Henle. G-protein receptor kinases (or GRKs are serine and threonine kinases that phosphorylate G protein-coupled receptors in response to agonist stimulation and uncouple the dopamine receptor from its G protein. This results in a desensitisation process that protects the cell from repeated agonist exposure. GRK4 activity is increased in spontaneously hypertensive rats, and infusion of GRK4 antisense oligonucleotides attenuates the increase in blood pressure (BP. This functional defect is replicated in the proximal tubule by expression of GRK4 variants namely p.Arg65Leu, p.Ala142Val and p.Val486Ala, in cell lines, with the p.Ala142Val showing the most activity. In humans, GRK4 polymorphisms were shown to be associated with essential hypertension in Australia, BP regulation in young adults, low renin hypertension in Japan and impaired stress-induced Na excretion in normotensive black men. In South Africa, GRK4 polymorphisms are more common in people of African descent, associated with impaired Na excretion in normotensive African people, and predict blood pressure response to Na restriction in African patients with mild to moderate essential hypertension. The therapeutic importance of the GRK4 single nucleotide polymorphisms (SNPs was emphasised in the African American Study of Kidney Disease (AASK where African-Americans with hypertensive nephrosclerosis were randomised to receive amlodipine, ramipril or metoprolol. Men with the p.Ala142Val genotype were less likely to respond to metoprolol, especially if they also had the p.Arg65Leu variant. Furthermore, in the analysis of response to treatment in

  3. Roles of PTEN-induced putative kinase 1 and dynamin-related protein 1 in transient global ischemia-induced hippocampal neuronal injury

    International Nuclear Information System (INIS)

    Chen, Shang-Der; Lin, Tsu-Kung; Yang, Ding-I.; Lee, Su-Ying; Shaw, Fu-Zen; Liou, Chia-Wei; Chuang, Yao-Chung

    2015-01-01

    Recent studies showed that increased mitochondrial fission is an early event of cell death during cerebral ischemia and dynamin-related protein 1 (Drp1) plays an important role in mitochondrial fission, which may be regulated by PTEN-induced putative kinase 1 (PINK1), a mitochondrial serine/threonine-protein kinase thought to protect cells from stress-induced mitochondrial dysfunction and regulate mitochondrial fission. However, the roles of PINK1 and Drp1 in hippocampal injury caused by transient global ischemia (TGI) remain unknown. We therefore tested the hypothesis that TGI may induce PINK1 causing downregulation of Drp1 phosphorylation to enhance hippocampal neuronal survival, thus functioning as an endogenous neuroprotective mechanism. We found progressively increased PINK1 expression in the hippocampal CA1 subfield1-48 h following TGI, reaching the maximal level at 4 h. Despite lack of changes in the expression level of total Drp1 and phosphor-Drp1 at Ser637, TGI induced a time-dependent increase of Drp1 phosphorlation at Ser616 that peaked after 24 h. Notably, PINK1-siRNA increased p-Drp1(Ser616) protein level in hippocampal CA1 subfield 24 h after TGI. The PINK1 siRNA also aggravated the TGI-induced oxidative DNA damage with an increased 8-hydroxy-deoxyguanosine (8-OHdG) content in hippocampal CA1 subfield. Furthermore, PINK1 siRNA also augmented TGI-induced apoptosis as evidenced by the increased numbers of TUNEL-positive staining and enhanced DNA fragmentation. These findings indicated that PINK1 is an endogenous protective mediator vital for neuronal survival under ischemic insult through regulating Drp1 phosphorylation at Ser616. - Highlights: • Transient global ischemia increases expression of PINK1 and p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA decreases PINK1 expression but increases p-Drp1 at Ser616 in hippocampal CA1 subfield. • PINK1-siRNA augments oxidative stress and neuronal damage in hippocampal CA1 subfield

  4. Functional human sperm capacitation requires both bicarbonate-dependent PKA activation and down-regulation of Ser/Thr phosphatases by Src family kinases.

    Science.gov (United States)

    Battistone, M A; Da Ros, V G; Salicioni, A M; Navarrete, F A; Krapf, D; Visconti, P E; Cuasnicú, P S

    2013-09-01

    In all mammalian species studied so far, sperm capacitation correlates with an increase in protein tyrosine (Tyr) phosphorylation mediated by a bicarbonate-dependent cAMP/protein kinase A (PKA) pathway. Recent studies in mice revealed, however, that a Src family kinase (SFK)-induced inactivation of serine/threonine (Ser/Thr) phosphatases is also involved in the signaling pathways leading to Tyr phosphorylation. In view of these observations and with the aim of getting a better understanding of the signaling pathways involved in human sperm capacitation, in the present work we investigated the involvement of both the cAMP/PKA and SFK/phosphatase pathways in relation to the capacitation state of the cells. For this purpose, different signaling events and sperm functional parameters were analyzed as a function of capacitation time. Results revealed a very early bicarbonate-dependent activation of PKA indicated by the rapid (1 min) increase in both phospho-PKA substrates and cAMP levels (P < 0.05). However, a complete pattern of Tyr phosphorylation was detected only after 6-h incubation at which time sperm exhibited the ability to undergo the acrosome reaction (AR) and to penetrate zona-free hamster oocytes. Sperm capacitated in the presence of the SFK inhibitor SKI606 showed a decrease in both PKA substrate and Tyr phosphorylation levels, which was overcome by exposure of sperm to the Ser/Thr phosphatase inhibitor okadaic acid (OA). However, OA was unable to induce phosphorylation when sperm were incubated under PKA-inhibitory conditions (i.e. in the absence of bicarbonate or in the presence of PKA inhibitor). Moreover, the increase in PKA activity by exposure to a cAMP analog and a phosphodiesterase inhibitor did not overcome the inhibition produced by SKI606. Whereas the presence of SKI606 during capacitation produced a negative effect (P < 0.05) on sperm motility, progesterone-induced AR and fertilizing ability, none of these inhibitions were observed when sperm

  5. The General Amino Acid Permease FfGap1 of Fusarium fujikuroi Is Sorted to the Vacuole in a Nitrogen-Dependent, but Npr1 Kinase-Independent Manner.

    Directory of Open Access Journals (Sweden)

    Andreas Pfannmüller

    Full Text Available The rice pathogenic fungus Fusarium fujikuroi is well known for the production of a broad spectrum of secondary metabolites (SMs such as gibberellic acids (GAs, mycotoxins and pigments. The biosynthesis of most of these SMs strictly depends on nitrogen availability and of the activity of permeases of nitrogen sources, e.g. the ammonium and amino acid permeases. One of the three ammonium permeases, MepB, was recently shown to act not only as a transporter but also as a nitrogen sensor affecting the production of nitrogen-repressed SMs. Here we describe the identification of a general amino acid permease, FfGap1, among the 99 putative amino acid permeases (AAPs in the genome of F. fujikuroi. FfGap1 is able to fully restore growth of the yeast gap1∆ mutant on several amino acids including citrulline and tryptophane. In S. cerevisiae, Gap1 activity is regulated by shuttling between the plasma membrane (nitrogen limiting conditions and the vacuole (nitrogen sufficiency, which we also show for FfGap1. In yeast, the Npr1 serine/threonine kinase stabilizes the Gap1 position at the plasma membrane. Here, we identified and characterized three NPR1-homologous genes, encoding the putative protein kinases FfNpr1-1, FfNpr1-2 and FfNpr1-3 with significant similarity to yeast Npr1. Complementation of the yeast npr1Δ mutant with each of the three F. fujikuroi NPR1 homologues, resulted in partial restoration of ammonium, arginine and proline uptake by FfNPR1-1 while none of the three kinases affect growth on different nitrogen sources and nitrogen-dependent sorting of FfGap1 in F. fujikuroi. However, exchange of the putative ubiquitin-target lysine 9 (K9A and 15 (K15A residues of FfGap1 resulted in extended localization to the plasma membrane and increased protein stability independently of nitrogen availability. These data suggest a similar regulation of FfGap1 by nitrogen-dependent ubiquitination, but differences regarding the role of Fusarium Npr1

  6. Localized hydroxylamine mutagenesis, and cotransduction of threonine and lysine genes, in Streptomyces venezuelae.

    Science.gov (United States)

    Stuttard, C

    1983-01-01

    A lysate of the generalized transducing phage SV1, grown on the prototrophic type strain 10712 of Streptomyces venezuelae, was mutagenized with hydroxylamine and used to transduce a lysineless auxotroph to lysine independence on supplemented minimal agar. A complex threonine mutant, strain VS95, was isolated from among the transductants and was shown to be carrying at least two different thr mutations. These were about 50% cotransducible with alleles of four independently isolated lysA mutations, as were two other independently isolated threonine mutations, thr-1 and hom-5. The location of thr genes close to lysA occurs in at least three other streptomycetes, but apparently not in Streptomyces coelicolor A3(2), in which the lysA and thr loci are at diametrically opposite locations on the linkage map. This first observation of cotransduction between loci governing the biosynthesis of different amino acids in the genus Streptomyces demonstrates the feasibility of fine-structure genetic analysis by transduction in these antibiotic-producing bacteria. PMID:6411685

  7. Adaptive evolution of threonine deaminase in plant defense against insect herbivores

    Energy Technology Data Exchange (ETDEWEB)

    Gonzales-Vigil, Eliana; Bianchetti, Christopher M.; Phillips, Jr., George N.; Howe, Gregg A. (MSU); (UW)

    2011-11-07

    Gene duplication is a major source of plant chemical diversity that mediates plant-herbivore interactions. There is little direct evidence, however, that novel chemical traits arising from gene duplication reduce herbivory. Higher plants use threonine deaminase (TD) to catalyze the dehydration of threonine (Thr) to {alpha}-ketobutyrate and ammonia as the committed step in the biosynthesis of isoleucine (Ile). Cultivated tomato and related Solanum species contain a duplicated TD paralog (TD2) that is coexpressed with a suite of genes involved in herbivore resistance. Analysis of TD2-deficient tomato lines showed that TD2 has a defensive function related to Thr catabolism in the gut of lepidopteran herbivores. During herbivory, the regulatory domain of TD2 is removed by proteolysis to generate a truncated protein (pTD2) that efficiently degrades Thr without being inhibited by Ile. We show that this proteolytic activation step occurs in the gut of lepidopteran but not coleopteran herbivores, and is catalyzed by a chymotrypsin-like protease of insect origin. Analysis of purified recombinant enzymes showed that TD2 is remarkably more resistant to proteolysis and high temperature than the ancestral TD1 isoform. The crystal structure of pTD2 provided evidence that electrostatic interactions constitute a stabilizing feature associated with adaptation of TD2 to the extreme environment of the lepidopteran gut. These findings demonstrate a role for gene duplication in the evolution of a plant defense that targets and co-opts herbivore digestive physiology.

  8. Evolutionary analysis of a novel zinc ribbon in the N-terminal region of threonine synthase.

    Science.gov (United States)

    Kaur, Gurmeet; Subramanian, Srikrishna

    2017-10-18

    Threonine synthase (TS) catalyzes the terminal reaction in the biosynthetic pathway of threonine and requires pyridoxal phosphate as a cofactor. TSs share a common catalytic domain with other fold type II PALP dependent enzymes. TSs are broadly grouped into two classes based on their sequence, quaternary structure, and enzyme regulation. We report the presence of a novel zinc ribbon domain in the N-terminal region preceding the catalytic core in TS. The zinc ribbon domain is present in TSs belonging to both classes. Our sequence analysis reveals that archaeal TSs possess all zinc chelating residues to bind a metal ion that are lacking in the structurally characterized homologs. Phylogenetic analysis suggests that TSs with an N-terminal zinc ribbon likely represents the ancestral state of the enzyme while TSs without a zinc ribbon must have diverged later in specific lineages. The zinc ribbon and its N- and C-terminal extensions are important for enzyme stability, activity and regulation. It is likely that the zinc ribbon domain is involved in higher order oligomerization or mediating interactions with other biomolecules leading to formation of larger metabolic complexes.

  9. Antinociceptive Effect of Rat D-Serine Racemase Inhibitors, L-Serine-O-Sulfate, and L-Erythro-3-Hydroxyaspartate in an Arthritic Pain Model

    Directory of Open Access Journals (Sweden)

    Claudio Laurido

    2012-01-01

    Full Text Available N-methyl-D-aspartic acid receptor (NMDAr activation requires the presence of D-serine, synthesized from L-serine by a pyridoxal 5′-phosphate-dependent serine racemase (SR. D-serine levels can be lowered by inhibiting the racemization of L-serine. L-serine-O-sulfate (LSOS and L-erythro-3-hydroxyaspartate (LEHA, among others, have proven to be effective in reducing the D-serine levels in culture cells. It is tempting then to try these compounds in their effectiveness to decrease nociceptive levels in rat arthritic pain. We measured the C-reflex paradigm and wind-up potentiation in the presence of intrathecally injected LSOS (100 μg/10 μL and LEHA (100 μg/10 μL in normal and monoarthritic rats. Both compounds decreased the wind-up activity in normal and monoarthritic rats. Accordingly, all the antinociceptive effects were abolished when 300 μg/10 μL of D-serine were injected intrathecally. Since no in vivo results have been presented so far, this constitutes the first evidence that SR inhibitions lower the D-serine levels, thus decreasing the NMDAr activity and the consequent development and maintenance of chronic pain.

  10. Disruption of δ-opioid receptor phosphorylation at threonine 161 attenuates morphine tolerance in rats with CFA-induced inflammatory hypersensitivity.

    Science.gov (United States)

    Chen, Hai-Jing; Xie, Wei-Yan; Hu, Fang; Zhang, Ying; Wang, Jun; Wang, Yun

    2012-04-01

    Our previous study identified Threonine 161 (Thr-161), located in the second intracellular loop of the δ-opioid receptor (DOR), as the only consensus phosphorylation site for cyclin-dependent kinase 5 (Cdk5). The aim of this study was to assess the function of DOR phosphorylation by Cdk5 in complete Freund's adjuvant (CFA)-induced inflammatory pain and morphine tolerance. Dorsal root ganglion (DRG) neurons of rats with CFA-induced inflammatory pain were acutely dissociated and the biotinylation method was used to explore the membrane localization of phosphorylated DOR at Thr-161 (pThr-161-DOR), and paw withdrawal latency was measured after intrathecal delivery of drugs or Tat-peptide, using a radiant heat stimulator in rats with CFA-induced inflammatory pain. Both the total amount and the surface localization of pThr-161-DOR were significantly enhanced in the ipsilateral DRG following CFA injection. Intrathecal delivery of the engineered Tat fusion-interefering peptide corresponding to the second intracellular loop of DOR (Tat-DOR-2L) increased inflammatory hypersensitivity, and inhibited DOR- but not µ-opioid receptor-mediated spinal analgesia in CFA-treated rats. However, intrathecal delivery of Tat-DOR-2L postponed morphine antinociceptive tolerance in rats with CFA-induced inflammatory pain. Phosphorylation of DOR at Thr-161 by Cdk5 attenuates hypersensitivity and potentiates morphine tolerance in rats with CFA-induced inflammatory pain, while disruption of the phosphorylation of DOR at Thr-161 attenuates morphine tolerance.

  11. Protein kinase C-delta inactivation inhibits the proliferation and survival of cancer stem cells in culture and in vivo

    International Nuclear Information System (INIS)

    Chen, Zhihong; Forman, Lora W; Williams, Robert M; Faller, Douglas V

    2014-01-01

    A subpopulation of tumor cells with distinct stem-like properties (cancer stem-like cells, CSCs) may be responsible for tumor initiation, invasive growth, and possibly dissemination to distant organ sites. CSCs exhibit a spectrum of biological, biochemical, and molecular features that are consistent with a stem-like phenotype, including growth as non-adherent spheres (clonogenic potential), ability to form a new tumor in xenograft assays, unlimited self-renewal, and the capacity for multipotency and lineage-specific differentiation. PKCδ is a novel class serine/threonine kinase of the PKC family, and functions in a number of cellular activities including cell proliferation, survival or apoptosis. PKCδ has previously been validated as a synthetic lethal target in cancer cells of multiple types with aberrant activation of Ras signaling, using both genetic (shRNA and dominant-negative PKCδ mutants) and small molecule inhibitors. In contrast, PKCδ is not required for the proliferation or survival of normal cells, suggesting the potential tumor-specificity of a PKCδ-targeted approach. shRNA knockdown was used validate PKCδ as a target in primary cancer stem cell lines and stem-like cells derived from human tumor cell lines, including breast, pancreatic, prostate and melanoma tumor cells. Novel and potent small molecule PKCδ inhibitors were employed in assays monitoring apoptosis, proliferation and clonogenic capacity of these cancer stem-like populations. Significant differences among data sets were determined using two-tailed Student’s t tests or ANOVA. We demonstrate that CSC-like populations derived from multiple types of human primary tumors, from human cancer cell lines, and from transformed human cells, require PKCδ activity and are susceptible to agents which deplete PKCδ protein or activity. Inhibition of PKCδ by specific genetic strategies (shRNA) or by novel small molecule inhibitors is growth inhibitory and cytotoxic to multiple types of human

  12. Intervention with Serine Protease Activity with Small Peptides

    DEFF Research Database (Denmark)

    Xu, Peng

    2015-01-01

    Serine proteases perform proteolytic reactions in many physiological and metabolic processes and have been certified as targets for therapeutics. Small peptides can be used as potent antagonists to target serine proteases and intervene with their activities. Urokinase-type plasminogen activator (u......PA) plays an important role in plasminogen activation system, which has many physiological and pathological functions and is closely associated with the metastasis of tumor cells. Based on a mono-cyclic peptidic inhibitor of murine uPA (muPA), mupain-1, which was screened out from a phage-display library...... before, we elucidated the binding and inhibitory mechanism by using multiple techniques, like X-ray crystallography, site-directed mutagenesis, isothermal titration calorimetry and surface plasmon resonance analysis. By studying the peptide-enzyme interaction, we discovered an unusual inhibitor...

  13. Site-specific DNA Inversion by Serine Recombinases

    Science.gov (United States)

    2015-01-01

    Reversible site-specific DNA inversion reactions are widely distributed in bacteria and their viruses. They control a range of biological reactions that most often involve alterations of molecules on the surface of cells or phage. These programmed DNA rearrangements usually occur at a low frequency, thereby preadapting a small subset of the population to a change in environmental conditions, or in the case of phages, an expanded host range. A dedicated recombinase, sometimes with the aid of additional regulatory or DNA architectural proteins, catalyzes the inversion of DNA. RecA or other components of the general recombination-repair machinery are not involved. This chapter discusses site-specific DNA inversion reactions mediated by the serine recombinase family of enzymes and focuses on the extensively studied serine DNA invertases that are stringently controlled by the Fis-bound enhancer regulatory system. The first section summarizes biological features and general properties of inversion reactions by the Fis/enhancer-dependent serine invertases and the recently described serine DNA invertases in Bacteroides. Mechanistic studies of reactions catalyzed by the Hin and Gin invertases are then discussed in more depth, particularly with regards to recent advances in our understanding of the function of the Fis/enhancer regulatory system, the assembly of the active recombination complex (invertasome) containing the Fis/enhancer, and the process of DNA strand exchange by rotation of synapsed subunit pairs within the invertasome. The role of DNA topological forces that function in concert with the Fis/enhancer controlling element in specifying the overwhelming bias for DNA inversion over deletion and intermolecular recombination is emphasized. PMID:25844275

  14. Antibacterial activity of silver nanoparticles synthesized from serine

    Energy Technology Data Exchange (ETDEWEB)

    Jayaprakash, N. [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); SRM Valliammai Engineering College, Department of Chemistry, Chennai 603 203 (India); Judith Vijaya, J., E-mail: jjvijayaloyola@yahoo.co.in [Catalysis and Nanomaterials Research Laboratory, Department of Chemistry, Loyola College, Chennai 600 034 (India); John Kennedy, L. [Materials Division, School of Advanced Sciences, VIT University, Chennai Campus, Chennai 600 048 (India); Priadharsini, K.; Palani, P. [Department of Center for Advanced Study in Bot