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Cawley, K. C., C. G. Akita, and D. A. Walsh. "Expression of a cDNA for the catalytic subunit of skeletal-muscle phosphorylase kinase in transfected 3T3 cells." Biochemical Journal 263, no. 1 (October 1, 1989): 223–29. http://dx.doi.org/10.1042/bj2630223.
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Phosphorylase kinase is a multimeric enzyme of composition (alpha, beta, gamma, delta)4 whose catalytic activity resides in the gamma-subunit. As an approach to understand further its regulation, a cDNA for the gamma-subunit of phosphorylase kinase (gamma PhK) has been cloned into a mammalian expression vector behind the mouse metallothionein-1 promoter. NIH 3T3 cells were co-transfected with this construct (pEV gamma PhK) and pSV2neo, G418-resistant clones were selected, and several were found to have stably incorporated the gamma-subunit cDNA into their genomic DNA. Phosphorylase kinase activity was clearly present in extracts from cultures of pEV gamma PhK-transformed cells and increased several-fold after 24 h of incubation with Zn2+, whereas it was undetectable in the parent 3T3 cells. A significant, but variable, proportion (15-70%) of the activity was Ca2+-dependent. We conclude that the phosphorylase kinase activity expressed by the cells transformed with pEV gamma PhK is due to free gamma-subunit and gamma-subunit associated with cellular calmodulin, which replaces the delta-subunit normally associated with the gamma-subunit in the holoenzyme.
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Lai, Guangrui, Jingjing Wu, Xiaoliang Liu, and Yanyan Zhao. "20-HETE Induces Hyperglycemia through the cAMP/PKA-PhK-GP Pathway." Molecular Endocrinology 26, no. 11 (November 1, 2012): 1907–16. http://dx.doi.org/10.1210/me.2012-1139.
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Abstract We previously generated cytochrome P450 4F2 (CYP4F2) transgenic mice and showed high 20-hydroxyeicosatetraenoic acid (20-HETE) production, which resulted in an elevation of blood pressure. However, it was unclear whether 20-HETE affected glucose metabolism. We measured fasting plasma glucose, insulin, hepatic CYP4F2 expression, and 20-HETE production by hepatic microsomes, and hepatic 20-HETE levels in transgenic mice. We also assessed glycogen phosphorylase (GP) activity and the cAMP/protein kinase A (PKA)-phosphorylase kinase (PhK)-GP pathway, as well as expressions of insulin receptor substrate 1 and glucose transporters in vivo and in vitro. The transgenic mice had overexpressed hepatic CYP4F2, high hepatic 20-HETE and fasting plasma glucose levels but normal insulin level. The GP activity was increased and the cAMP/PKA-PhK-GP pathway was activated in the transgenic mice compared with wild-type mice. Moreover, these alterations were eliminated with the addition of N-hydroxy-N′-(4-butyl-2 methylphenyl) formamidine, which is a selective 20-HETE inhibitor. The results were further validated in Bel7402 cells. In addition, the transgenic mice had functional insulin signaling, and 20-HETE had no effect on insulin signaling in Bel7402 cells, excluding that the observed hyperglycemia in CYP4F2 transgenic mice resulted from insulin dysfunction, because the target tissues were sensitive to insulin. Our study suggested that 20-HETE can induce hyperglycemia, at least in part, through the cAMP/PKA-PhK-GP pathway but not through the insulin-signaling pathway.
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AYERS, Nancy A., Owen W. NADEAU, Mark W. READ, Partha RAY, and Gerald M. CARLSON. "Effector-sensitive cross-linking of phosphorylase b kinase by the novel cross-linker 4-phenyl-1,2,4-triazoline-3,5-dione." Biochemical Journal 331, no. 1 (April 1, 1998): 137–41. http://dx.doi.org/10.1042/bj3310137.
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The dienophile 4-phenyl-1,2,4-triazoline-3,5-dione (PTD) was identified as a novel protein cross-linker, and utilized as a conformational probe of phosphorylase b kinase (PhK), a hexadecameric enzyme with the subunit composition (αβγδ)4. In its reaction with this enzyme, PTD produced five major cross-linked conjugates as resolved by denaturing gel electrophoresis: αβ, βγγ, αγ and a doublet of differently migrating homodimers, ββ1 and ββ2. Cross-linking in the presence of six different activators of the kinase targeted to its various subunits caused substantial changes in the amounts of three of the conjugates. The formation of αγ was increased by all of the activators but the largest enhancement was caused by exogenous Ca2+/calmodulin. All except one of the activators decreased the amount of βγγ formed, with Mg2+ having the greatest effect, and all except two increased the amount of ββ1, with Mg2+ again having the largest influence. From the overall similarity of the changes in cross-linking by PTD induced by the various activators, we conclude that, even though they are targeted to different sites and subunits, they induce activated conformations of PhK that have certain structural features in common. Regarding the mechanism of cross-linking by PTD, its reaction with a model nucleophile suggests that its initial reaction with a side chain nucleophile of PhK involves a 1,4-conjugate addition to form a urazole adduct, with the secondary cross-linking reaction occurring through an as yet unknown pathway.
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Schepetkin, Igor A., Mark B. Plotnikov, Andrei I. Khlebnikov, Tatiana M. Plotnikova, and Mark T. Quinn. "Oximes: Novel Therapeutics with Anticancer and Anti-Inflammatory Potential." Biomolecules 11, no. 6 (May 22, 2021): 777. http://dx.doi.org/10.3390/biom11060777.
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Oximes have been studied for decades because of their significant roles as acetylcholinesterase reactivators. Over the last twenty years, a large number of oximes have been reported with useful pharmaceutical properties, including compounds with antibacterial, anticancer, anti-arthritis, and anti-stroke activities. Many oximes are kinase inhibitors and have been shown to inhibit over 40 different kinases, including AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (PI3K), cyclin-dependent kinase (CDK), serine/threonine kinases glycogen synthase kinase 3 α/β (GSK-3α/β), Aurora A, B-Raf, Chk1, death-associated protein-kinase-related 2 (DRAK2), phosphorylase kinase (PhK), serum and glucocorticoid-regulated kinase (SGK), Janus tyrosine kinase (JAK), and multiple receptor and non-receptor tyrosine kinases. Some oximes are inhibitors of lipoxygenase 5, human neutrophil elastase, and proteinase 3. The oxime group contains two H-bond acceptors (nitrogen and oxygen atoms) and one H-bond donor (OH group), versus only one H-bond acceptor present in carbonyl groups. This feature, together with the high polarity of oxime groups, may lead to a significantly different mode of interaction with receptor binding sites compared to corresponding carbonyl compounds, despite small changes in the total size and shape of the compound. In addition, oximes can generate nitric oxide. This review is focused on oximes as kinase inhibitors with anticancer and anti-inflammatory activities. Oximes with non-kinase targets or mechanisms of anti-inflammatory activity are also discussed.
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Bisciglia, Michela, Roseline Froissart, Anne Laure Bedat-Millet, Norma Beatriz Romero, Magali Pettazzoni, Jean-Yves Hogrel, François M. Petit, and Tanya Stojkovic. "A novel PHKA1 mutation associating myopathy and cognitive impairment: Expanding the spectrum of phosphorylase kinase b (PhK) deficiency." Journal of the Neurological Sciences 424 (May 2021): 117391. http://dx.doi.org/10.1016/j.jns.2021.117391.
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Li, Chunyun, Lihua Huang, Lang Tian, Jia Chen, Shentang Li, and Zuocheng Yang. "PHKG2 mutation spectrum in glycogen storage disease type IXc: a case report and review of the literature." Journal of Pediatric Endocrinology and Metabolism 31, no. 3 (March 28, 2018): 331–38. http://dx.doi.org/10.1515/jpem-2017-0170.
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AbstractBackground:PHKG2gene mutation can lead to liver phosphorylase kinase (PhK) deficiency, which is related to glycogen storage disease type IX (GSD IX). GSD IXc due toPHKG2mutation is the second most common GSD IX.Methods:We identified a novel mutation (c.553C>T, p.Arg185X) inPHKG2in a Chinese family and verified it by next-generation and Sanger sequencing. The mutation spectrum of thePHKG2gene was summarized based on 25 GSD IXc patients withPHKG2mutations.Results:We found that missense mutation (39%) was the most common type of mutation, followed by nonsense mutation (23%). Mutations were more prevalent in Asian (12/25) and European (9/25) populations than in populations from elsewhere. The exons had more sites of mutation than the introns, and exons 3 and 6 were the most frequent sites of mutations.Conclusions:This study expands our knowledge of thePHKG2gene mutation spectrum, providing a molecular basis for GSD IXc.
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Calalb, M. B., D. T. Fox, and S. K. Hanks. "Molecular cloning and enzymatic analysis of the rat homolog of “PhK-gamma T,” an isoform of phosphorylase kinase catalytic subunit." Journal of Biological Chemistry 267, no. 3 (January 1992): 1455–63. http://dx.doi.org/10.1016/s0021-9258(18)45967-4.
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Liu, Li, Stephen R. Rannels, Mary Falconieri, Karen S. Phillips, Ellen B. Wolpert, and Timothy E. Weaver. "The Testis Isoform of the Phosphorylase Kinase Catalytic Subunit (PhK-T) Plays a Critical Role in Regulation of Glycogen Mobilization in Developing Lung." Journal of Biological Chemistry 271, no. 20 (May 17, 1996): 11761–66. http://dx.doi.org/10.1074/jbc.271.20.11761.
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Chamberlain, J. S., P. VanTuinen, A. A. Reeves, B. A. Philip, and C. T. Caskey. "Isolation of cDNA clones for the catalytic gamma subunit of mouse muscle phosphorylase kinase: expression of mRNA in normal and mutant Phk mice." Proceedings of the National Academy of Sciences 84, no. 9 (May 1, 1987): 2886–90. http://dx.doi.org/10.1073/pnas.84.9.2886.
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Anderton, Brian H., Joanna Betts, Walter P. Blackstock, Jean-Pierre Brion, Sara Chapman, James Connell, Rejith Dayanandan, et al. "Sites of phosphorylation in tau and factors affecting their regulation." Biochemical Society Symposia 67 (February 1, 2001): 73–80. http://dx.doi.org/10.1042/bss0670073.
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The microtubule-associated protein, tau, is the principal component of paired helical filaments (PHFs) in Alzheimer's disease. PHF-tau is highly phosphorylated and a total of 25 sites of phosphorylation have so far been identified. Many of these sites are serine or threonine residues that are immediately followed in the sequence by proline residues, and hence are candidate phosphorylation sites for proline-directed kinases. In vitro, glycogen synthase kinase-3 (GSK-3), extracellular signal-related kinase-1 and -2, and mitogen-activated protein kinases, p38 kinase and c-jun N-terminal kinase, all phosphorylate many of these sites, although with different efficiencies for particular sites. Phosphorylation studies in transfected cells and neurons show that GSK-3 phosphorylates tau more extensively than do these other proline-directed kinases. Mutations in tau have been shown to affect in vitro phosphorylation of tau by GSK-3. The Arg406-->Trp (R406W) tau mutation also affects tau phosphorylation in cells.
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LITERSKY, Joel M., Gail V. W. JOHNSON, Ross JAKES, Michel GOEDERT, Michael LEE, and Peter SEUBERT. "Tau protein is phosphorylated by cyclic AMP-dependent protein kinase and calcium/calmodulin-dependent protein kinase II within its microtubule-binding domains at Ser-262 and Ser-356." Biochemical Journal 316, no. 2 (June 1, 1996): 655–60. http://dx.doi.org/10.1042/bj3160655.
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Phosphorylation of tau protein at Ser-262 has been shown to diminish its ability to bind to taxol-stabilized microtubules. The paired helical filaments (PHFs) found in Alzheimer's disease brain are composed of PHF-tau, which is hyperphosphorylated at multiple sites including Ser-262. However, protein kinase(s) able to phosphorylate this site are still under investigation. In this study, the ability of cyclic AMP-dependent protein kinase (cAMP-PK) and calcium/calmodulin-dependent protein kinase II (CaMKII) to phosphorylate tau at Ser-262, as well as Ser-356, is demonstrated by use of a monoclonal antibody (12E8) which has been shown to recognize tau when these sites are phosphorylated. Cleavage of cAMP-PK-phosphorylated tau at cysteine residues by 2-nitro-5-thiocyanobenzoic acid, which cuts the protein into essentially two fragments and separates Ser-262 from Ser-356, revealed that cAMP-PK phosphorylates both Ser-262 and Ser-356. In addition, phosphorylation with cAMP-PK or CaMKII of recombinant tau in which Ser-262, Ser-356 or both had been mutated to alanines, clearly demonstrated that cAMP-PK and CaMKII were able to phosphorylate both sites. Mitogen-activated protein kinase or protein kinase C did not phosphorylate tau at Ser-262 and/or Ser-356. Finally, evidence is presented that phosphorylation of both these sites occurs in cultured nerve cells under certain conditions, indicating their potential physiological relevance.
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Pang, Huan, Zhenheng Guo, Zhongwen Xie, Wen Su, and Ming C. Gong. "Divergent kinase signaling mediates agonist-induced phosphorylation of phosphatase inhibitory proteins PHI-1 and CPI-17 in vascular smooth muscle cells." American Journal of Physiology-Cell Physiology 290, no. 3 (March 2006): C892—C899. http://dx.doi.org/10.1152/ajpcell.00378.2005.
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Phosphatase holoenzyme inhibitor (PHI)-1 is one of the newest members of the family of protein phosphatase inhibitor proteins. In isolated enzyme systems, several kinases, including PKC and rho kinase (ROCK), have been shown to phosphorylate PHI-1. However, it is largely unknown whether PHI-1 is phosphorylated in response to agonist stimulation in intact cells. We investigated this question in primary cultured rat aortic vascular smooth muscle cells (VSMCs). Using two-dimensional polyacrylamide gel electrophoresis and immunoblot, we found that there are two major PHI-1 spots under resting conditions: a minor spot with an acidic isoelectric point (pI) and a major spot with a more alkaline pI. Interestingly, U-46619, a G protein-coupled receptor agonist, caused a significant increase in the acidic spot, suggesting that it may represent a phosphorylated form of PHI-1. This was confirmed by phosphatase treatment and by a specific phospho-PHI-1 antibody. Furthermore, we found that angiotensin II, thrombin, and U-46619 increased phosphorylated PHI-1 from 9% of total PHI-1 in resting cells to 18%, 18%, and 30%, respectively. We also found that inhibition of ROCK by Y-27632 or H-1152 selectively diminished U-46619-induced CPI-17 phosphorylation, whereas it did not affect PHI-1 phosphorylation. Activation of ROCK by expressing V14RhoA selectively induced CPI-17 phosphorylation without affecting PHI-1 phosphorylation. In contrast, inhibition of PKC by GF-109203X or by PKC downregulation selectively diminished U-46619-induced PHI-1 phosphorylation without significantly affecting U-46619-induced CPI-17 phosphorylation. Activating PKC by PMA induced PHI-1 phosphorylation. Together, our results show for the first time that agonist induces PHI-1 phosphorylation in VSMCs and divergent kinase signaling couples agonist stimulation to PHI-1 and CPI-17 phosphorylation.
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DENG, Jing Ti, Cindy SUTHERLAND, David L. BRAUTIGAN, Masumi ETO, and Michael P. WALSH. "Phosphorylation of the myosin phosphatase inhibitors, CPI-17 and PHI-1, by integrin-linked kinase." Biochemical Journal 367, no. 2 (October 15, 2002): 517–24. http://dx.doi.org/10.1042/bj20020522.
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Integrin-linked kinase (ILK) has been implicated in Ca2+- independent contraction of smooth muscle via its ability to phosphorylate myosin. We investigated the possibility that this kinase might also phosphorylate and regulate the myosin light-chain phosphatase inhibitor proteins CPI-17 [protein kinase C (PKC)-dependent phosphatase inhibitor of 17kDa] and PHI-1 (phosphatase holoenzyme inhibitor-1), known substrates of PKC. Both phosphatase inhibitors were phosphorylated by ILK in an in-gel kinase assay and in solution. A Thr→Ala mutation at Thr38 of CPI-17 and Thr57 of PHI-1 eliminated phosphorylation by ILK. Phosphopeptide mapping, phospho amino acid analysis and immunoblotting using phospho-specific antibodies indicated that ILK predominantly phosphorylated the site critical for potent inhibition, i.e. Thr38 of CPI-17 or Thr57 of PHI-1. CPI-17 and PHI-1 thiophosphorylated by ILK at Thr38 or Thr57 respectively inhibited myosin light-chain phosphatase (MLCP) activity bound to myosin, whereas the site-specific mutants CPI-17-Thr38Ala and PHI-1-Thr57Ala, treated with ILK under identical conditions, like the untreated wild-type proteins had no effect on the phosphatase. Consistent with these effects, both thiophospho-CPI-17 and -PHI-1 induced Ca2+ sensitization of contraction of Triton X-100-demembranated rat-tail arterial smooth muscle, whereas CPI-17-Thr38Ala and PHI-1-Thr57Ala treated with ILK in the presence of adenosine 5′-[γ-thio]triphosphate failed to evoke a contractile response. We conclude that ILK may activate smooth-muscle contraction both directly, via phosphorylation of myosin, and indirectly, via phosphorylation and activation of CPI-17 and PHI-1, leading to inhibition of MLCP.
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THELEN, Jay J., Jan A. MIERNYK, and Douglas D. RANDALL. "Pyruvate dehydrogenase kinase from Arabidopsis thaliana: a protein histidine kinase that phosphorylates serine residues." Biochemical Journal 349, no. 1 (June 26, 2000): 195–201. http://dx.doi.org/10.1042/bj3490195.
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Pyruvate dehydrogenase kinase (PDK) is the primary regulator of flux through the mitochondrial pyruvate dehydrogenase complex (PDC). Although PDKs inactivate mitochondrial PDC by phosphorylating specific Ser residues, the primary amino acid sequence indicates that they are more closely related to prokaryotic His kinases than to eukaryotic Ser/Thr kinases. Unlike Ser/Thr kinases, His kinases use a conserved His residue for phosphotransfer to Asp residues. To understand these unique kinases better, a presumptive PDK from Arabidopsis thaliana was heterologously expressed and purified for this investigation. Purified, recombinant A. thaliana PDK could inactivate kinase-depleted maize mitochondrial PDC by phosphorylating Ser residues. Additionally, A. thaliana PDK was capable of autophosphorylating Ser residues near its N-terminus, although this reaction is not part of the phosphotransfer pathway. To elucidate the mechanism involved, we performed site-directed mutagenesis of the canonical His residue likely to be involved in phosphotransfer. When His-121 was mutated to Ala or Gln, Ser-autophosphorylation was decreased by 50% and transphosphorylation of PDC was decreased concomitantly. We postulate that either (1) His-121 is not the sole phosphotransfer His residue or (2) mutagenesis of His-121 exposes an additional otherwise cryptic phosphotransfer His residue. Thus His-121 is one residue involved in kinase function.
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Couture, C., G. Baier, C. Oetken, S. Williams, D. Telford, A. Marie-Cardine, G. Baier-Bitterlich, S. Fischer, P. Burn, and A. Altman. "Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation." Molecular and Cellular Biology 14, no. 8 (August 1994): 5249–58. http://dx.doi.org/10.1128/mcb.14.8.5249.
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The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.
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Couture, C., G. Baier, C. Oetken, S. Williams, D. Telford, A. Marie-Cardine, G. Baier-Bitterlich, S. Fischer, P. Burn, and A. Altman. "Activation of p56lck by p72syk through physical association and N-terminal tyrosine phosphorylation." Molecular and Cellular Biology 14, no. 8 (August 1994): 5249–58. http://dx.doi.org/10.1128/mcb.14.8.5249-5258.1994.
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The p56lck and p59fyn protein tyrosine kinases are important signal transmission elements in the activation of mature T lymphocytes by ligands to the T-cell antigen receptor (TCR)/CD3 complex. The lack of either kinase results in deficient early signaling events, and pharmacological agents that block tyrosine phosphorylation prevent T-cell activation altogether. After triggering of the TCR/CD3 complex, both kinases are moderately activated and begin to phosphorylate cellular substrates, but the molecular mechanisms responsible for these changes have remained unclear. We recently found that the p72syk protein tyrosine kinase is physically associated with the TCR/CD3 complex and is rapidly tyrosine phosphorylated and activated by receptor triggering also in T cells lacking p56lck. Here we examine the regulation of p72syk and its interaction with p56lck in transfected COS-1 cells. p72syk was catalytically active and heavily phosphorylated on its putative autophosphorylation site, Tyr-518/519. Mutation of these residues to phenylalanines abolished its activity in vitro and toward cellular substrates in vivo and reduced its tyrosine phosphorylation in intact cells by approximately 90%. Coexpression of lck did not alter the catalytic activity of p72syk, but the expressed p56lck was much more active in the presence of p72syk than when expressed alone. This activation was also seen as increased phosphorylation of cellular proteins. Concomitantly, p56lck was phosphorylated at Tyr-192 in its SH2 domain, and a Phe-192 mutant p56lck was no longer phosphorylated by p72syk. Phosphate was also detected in p56lck at Tyr-192 in lymphoid cells. These findings suggest that p56lck is positively regulated by the p72syk kinase.
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BelAiba, Rachida, John Hess, Thomas Kietzmann, and Agnes Görlach. "Thrombin activates the p21-activated kinase in pulmonary artery smooth muscle cells." Thrombosis and Haemostasis 93, no. 06 (2005): 1168–75. http://dx.doi.org/10.1160/th05-01-0006.
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SummaryThe p21-activated serine/threonine kinases (PAK) play an important role in a variety of cellular functions. However, their role in the smooth muscle response to thrombin, which is activated upon vascular injury and promotes vascular remodelling processes, is not resolved. Here we investigated the role of PAK in thrombin signalling and regulation of tissue factor (TF), the activator of the extrinsic coagulation cascade, in pulmonary artery smooth muscle cells (PASMC), the main cell type responsible for vascular remodelling in pulmonary hypertension. PAK was rapidly phosphorylated in response to thrombin. Thrombin and active PAKT423E phosphorylated p38 MAP kinase (p38MAPK), ERK1/2, phosphatidylinositol-dependent kinase-1 (PDK1) and protein kinase B/Akt (PKB) whereas kinase-deficient PAK1 prevented activation of these kinases by thrombin. In addition, kinase-deficient MKK3 inhibited activation of PDK1 and PKB by thrombin. Further, thrombin and active PAK1 induced TF expression and promoter activity while kinase-deficient PAK1 diminished thrombin-induced TF upregulation. Moreover, kinase-deficient MKK3, PDK1 and PKB inhibited thrombin- and PAK-dependent TF expression and promoter activity. Together these findings show that PAK is a critical element of thrombin signalling in PASMC which is involved in the regulation of TF expression by sequentially activating MKK3/p38MAPK, PDK1 and PKB. Thus, PAK may play an important role in promoting vascular remodelling processes in pulmonary hypertension.
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DEPREZ, Johan, Luc BERTRAND, Dario R. ALESSI, Ulrike KRAUSE, Louis HUE, and Mark H. RIDER. "Partial purification and characterization of a wortmannin-sensitive and insulin-stimulated protein kinase that activates heart 6-phosphofructo-2-kinase." Biochemical Journal 347, no. 1 (March 27, 2000): 305–12. http://dx.doi.org/10.1042/bj3470305.
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A wortmannin-sensitive and insulin-stimulated protein kinase (WISK), which phosphorylates and activates cardiac 6-phosphofructo-2-kinase (PFK-2), was partially purified from perfused rat hearts. Immunoblotting showed that WISK was devoid of protein kinase B (PKB), serum- and glucocorticoid-regulated protein kinase and protein kinase Cζ (PKCζ). Comparison of the inhibition of WISK, PKCα and PKCζ by different protein kinase inhibitors suggested that WISK was not a member of the PKC family. In addition, WISK contained no detectable phosphoinositide-dependent protein kinase-1 (PDK1) activity. WISK phosphorylated recombinant heart PFK-2 in a time-dependent manner to the extent of 0.4 mol of phosphate incorporated/mol of enzyme subunit, and increased the Vmax of PFK-2 twofold, without affecting the Km for fructose 6-phosphate. WISK phosphorylated Ser-466 to a greater extent than Ser-483 in recombinant heart PFK-2, and both sites were demonstrated to be phosphorylated to the same extent by PKB. Gel filtration and in-gel kinase analysis indicated that WISK was a monomer with a Mr of 56500. Treatment of WISK with protein phosphatase 2A (PP2A) catalytic subunits reversed the effect of insulin, suggesting the involvement of an upstream activating kinase. Indeed, PDK1 was able to partially reactivate the PP2A-treated WISK and this reactivation was not enhanced by PtdIns(3,4,5)P3-containing vesicles. Moreover, a single 57000-Mr band was labelled on incubation of the dephosphorylated WISK preparation with PDK1 and [γ-32P]ATP. These findings provide evidence for the existence of a new protein kinase in the insulin signalling pathway, probably downstream of PDK1.
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Stokoe, D., B. Caudwell, P. T. W. Cohen, and P. Cohen. "The substrate specificity and structure of mitogen-activated protein (MAP) kinase-activated protein kinase-2." Biochemical Journal 296, no. 3 (December 15, 1993): 843–49. http://dx.doi.org/10.1042/bj2960843.
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The substrate specificity of mitogen-activated protein (MAP) kinase-activated protein kinase-2 (MAPKAP kinase-2) was investigated by using synthetic peptides related to the N-terminus of glycogen synthase. The minimum sequence required for efficient phosphorylation was found to be Xaa-Xaa-Hyd-Xaa-Arg-Xaa-Xaa-Ser-Xaa-Xaa, where Hyd is a bulky hydrophobic residue (Phe > Leu > Val >> Ala), and the peptide Lys-Lys-Phe-Asn-Arg-Thr-Leu-Ser-Val-Ala was phosphorylated with a Km of 9.3 microM and Vmax. of 10 mumol/min per mg. MAPKAP kinase-1 (a homologue of ribosomal protein S6 kinase) also requires an arginine three residues N-terminal to the serine (position n-3), but not a hydrophobic residue at position n-5. Neither MAPKAP kinase-1 nor MAPKAP kinase-2 could tolerate a proline residue at position n + 1, indicating that their specificities do not overlap with that of MAP kinase. The specificity of calmodulin-dependent protein kinase-II resembled that of MAPKAP kinase-2, except that it could tolerate replacement of the arginine by a lysine and the phosphorylation-site serine by a threonine residue. Partial cDNAs encoding MAPKAP kinase-2 were isolated from rabbit and human skeletal muscle and human teratocarcinoma libraries, and Northern-blotting experiments revealed a single 3.3 kb mRNA transcript present at similar levels in six human tissues examined. The catalytic domain was most similar (35-40% identity) to calmodulin-dependent protein kinases II and IV, phosphorylase kinase, putative serine kinase H1 and the C-terminal domain of MAPKAP kinase-1, which form one branch of the protein kinase phylogenetic tree. The sequence N-terminal to the catalytic domain is proline-rich and contains two putative SH3-binding sites. The threonine residue phosphorylated by MAP kinase lies immediately C-terminal to the catalytic domain and is followed by a nuclear localization signal, Lys-Lys-(Xaa)10-Lys-Arg-Arg-Lys-Lys, near the C-terminus.
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Eblen, Scott T., Jill K. Slack-Davis, Adel Tarcsafalvi, J. Thomas Parsons, Michael J. Weber, and Andrew D. Catling. "Mitogen-Activated Protein Kinase Feedback Phosphorylation Regulates MEK1 Complex Formation and Activation during Cellular Adhesion." Molecular and Cellular Biology 24, no. 6 (March 15, 2004): 2308–17. http://dx.doi.org/10.1128/mcb.24.6.2308-2317.2004.
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ABSTRACT Cell adhesion and spreading depend on activation of mitogen-activated kinase, which in turn is regulated both by growth factor and integrin signaling. Growth factors, such as epidermal growth factor, are capable of activating Ras and Raf, but integrin signaling is required to couple Raf to MEK and MEK to extracellular signal-regulated protein kinase (ERK). It was previously shown that Rac-p21-activated kinase (PAK) signaling regulated the physical association of MEK1 with ERK2 through phosphorylation sites in the proline-rich sequence (PRS) of MEK1. It was also shown that activation of MEK1 and ERK by integrins depends on PAK phosphorylation of S298 in the PRS. Here we report a novel MEK1-specific regulatory feedback mechanism that provides a means by which activated ERK can terminate continued PAK phosphorylation of MEK1. Activated ERK can phosphorylate T292 in the PRS, and this blocks the ability of PAK to phosphorylate S298 and of Rac-PAK signaling to enhance MEK1-ERK complex formation. Preventing ERK feedback phosphorylation on T292 during cellular adhesion prolonged phosphorylation of S298 by PAK and phosphorylation of S218 and S222, the MEK1 activating sites. We propose that activation of ERK during adhesion creates a feedback system in which ERK phosphorylates MEK1 on T292, and this in turn blocks additional S298 phosphorylation in response to integrin signaling.
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Shi, Yuguang, Krishna M. Vattem, Ruchira Sood, Jie An, Jingdong Liang, Lawrence Stramm, and Ronald C. Wek. "Identification and Characterization of Pancreatic Eukaryotic Initiation Factor 2 α-Subunit Kinase, PEK, Involved in Translational Control." Molecular and Cellular Biology 18, no. 12 (December 1, 1998): 7499–509. http://dx.doi.org/10.1128/mcb.18.12.7499.
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ABSTRACT In response to various environmental stresses, eukaryotic cells down-regulate protein synthesis by phosphorylation of the α subunit of eukaryotic translation initiation factor 2 (eIF-2α). In mammals, the phosphorylation was shown to be carried out by eIF-2α kinases PKR and HRI. We report the identification and characterization of a cDNA from rat pancreatic islet cells that encodes a new related kinase, which we term pancreatic eIF-2α kinase, or PEK. In addition to a catalytic domain with sequence and structural features conserved among eIF-2α kinases, PEK contains a distinctive amino-terminal region 550 residues in length. Using recombinant PEK produced inEscherichia coli or Sf-9 insect cells, we demonstrate that PEK is autophosphorylated on both serine and threonine residues and that the recombinant enzyme can specifically phosphorylate eIF-2α on serine-51. Northern blot analyses indicate that PEK mRNA is expressed in all tissues examined, with highest levels in pancreas cells. Consistent with our mRNA assays, PEK activity was predominantly detected in pancreas and pancreatic islet cells. The regulatory role of PEK in protein synthesis was demonstrated both in vitro and in vivo. The addition of recombinant PEK to reticulocyte lysates caused a dose-dependent inhibition of translation. In theSaccharomyces model system, PEK functionally substituted for the endogenous yeast eIF-2α kinase, GCN2, by a process requiring the serine-51 phosphorylation site in eIF-2α. We also identified PEK homologs from both Caenorhabditis elegans and the puffer fish Fugu rubripes, suggesting that this eIF-2α kinase plays an important role in translational control from nematodes to mammals.
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Carnevalli, Larissa S., Catia M. Pereira, Carolina B. Jaqueta, Viviane S. Alves, Vanessa N. Paiva, Krishna M. Vattem, Ronald C. Wek, Luiz Eugênio A. M. Mello, and Beatriz A. Castilho. "Phosphorylation of the α subunit of translation initiation factor-2 by PKR mediates protein synthesis inhibition in the mouse brain during status epilepticus." Biochemical Journal 397, no. 1 (June 14, 2006): 187–94. http://dx.doi.org/10.1042/bj20051643.
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In response to different cellular stresses, a family of protein kinases phosphorylates eIF2α (α subunit of eukaryotic initiation factor-2), contributing to regulation of both general and genespecific translation proposed to alleviate cellular injury or alternatively induce apoptosis. Recently, we reported eIF2α(P) (phosphorylated eIF2α) in the brain during SE (status epilepticus) induced by pilocarpine in mice, an animal model of TLE (temporal lobe epilepsy) [Carnevalli, Pereira, Longo, Jaqueta, Avedissian, Mello and Castilho (2004) Neurosci. Lett. 357, 191–194]. We show in the present study that one eIF2α kinase family member, PKR (double-stranded-RNA-dependent protein kinase), is activated in the cortex and hippocampus at 30 min of SE, reflecting the levels of eIF2α(P) in these areas. In PKR-deficient animals subjected to SE, eIF2α phosphorylation was clearly evident coincident with activation of a secondary eIF2α kinase, PEK/PERK (pancreatic eIF2α kinase/RNA-dependent-protein-kinase-like endoplasmic reticulum kinase), denoting a compensatory mechanism between the two kinases. The extent of eIF2α phosphorylation correlated with the inhibition of protein synthesis in the brain, as determined from polysome profiles. We also found that C57BL/6 mice, which enter SE upon pilocarpine administration but are more resistant to seizure-induced neuronal degeneration, showed very low levels of eIF2α(P) and no inhibition of protein synthesis during SE. These results taken together suggest that PKR-mediated phosphorylation of eIF2α contributes to inhibition of protein synthesis in the brain during SE and that sustained high levels of eIF2α phosphorylation may facilitate ensuing cell death in the most affected areas of the brain in TLE.
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Vitari, Alberto C., Jacob Thastrup, Fatema H. Rafiqi, Maria Deak, Nick A. Morrice, Håkan K. R. Karlsson, and Dario R. Alessi. "Functional interactions of the SPAK/OSR1 kinases with their upstream activator WNK1 and downstream substrate NKCC1." Biochemical Journal 397, no. 1 (June 14, 2006): 223–31. http://dx.doi.org/10.1042/bj20060220.
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The SPAK (STE20/SPS1-related proline/alanine-rich kinase) and OSR1 (oxidative stress-responsive kinase-1) kinases interact and phosphorylate NKCC1 (Na+–K+–2Cl− co-transporter-1), leading to its activation. Recent studies indicated that SPAK and OSR1 are phosphorylated and activated by the WNK1 [with no K (lysine) protein kinase-1] and WNK4, genes mutated in humans affected by Gordon's hypertension syndrome. In the present study, we have identified three residues in NKCC1 (Thr175/Thr179/Thr184 in shark or Thr203/Thr207/Thr212 in human) that are phosphorylated by SPAK and OSR1, and have developed a peptide substrate, CATCHtide (cation chloride co-transporter peptide substrate), to assess SPAK and OSR1 activity. Exposure of HEK-293 (human embryonic kidney) cells to osmotic stress, which leads to phosphorylation and activation of NKCC1, increased phosphorylation of NKCC1 at the sites targeted by SPAK/OSR1. The residues on NKCC1, phosphorylated by SPAK/OSR1, are conserved in other cation co-transporters, such as the Na+–Cl− co-transporter, the target of thiazide drugs that lower blood pressure in humans with Gordon's syndrome. Furthermore, we characterize the properties of a 92-residue CCT (conserved C-terminal) domain on SPAK and OSR1 that interacts with an RFXV (Arg-Phe-Xaa-Val) motif present in the substrate NKCC1 and its activators WNK1/WNK4. A peptide containing the RFXV motif interacts with nanomolar affinity with the CCT domains of SPAK/OSR1 and can be utilized to affinity-purify SPAK and OSR1 from cell extracts. Mutation of the arginine, phenylalanine or valine residue within this peptide abolishes binding to SPAK/OSR1. We have identified specific residues within the CCT domain that are required for interaction with the RFXV motif and have demonstrated that mutation of these in OSR1 inhibited phosphorylation of NKCC1, but not of CATCHtide which does not possess an RFXV motif. We establish that an intact CCT domain is required for WNK1 to efficiently phosphorylate and activate OSR1. These data establish that the CCT domain functions as a multipurpose docking site, enabling SPAK/OSR1 to interact with substrates (NKCC1) and activators (WNK1/WNK4).
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Loo, Tsui-Han, and Mohan Balasubramanian. "Schizosaccharomyces pombe Pak-related protein, Pak1p/Orb2p, phosphorylates myosin regulatory light chain to inhibit cytokinesis." Journal of Cell Biology 183, no. 5 (November 24, 2008): 785–93. http://dx.doi.org/10.1083/jcb.200806127.
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p21-activated kinases (Paks) have been identified in a variety of eukaryotic cells as key effectors of the Cdc42 family of guanosine triphosphatases. Pak kinases play important roles in regulating the filamentous actin cytoskeleton. In this study, we describe a function for the Schizosaccharomyces pombe Pak-related protein Pak1p/Orb2p in cytokinesis. Pak1p localizes to the actomyosin ring during mitosis and cytokinesis. Loss of Pak1p function leads to accelerated cytokinesis. Pak1p mediates phosphorylation of myosin II regulatory light chain Rlc1p at serine residues 35 and 36 in vivo. Interestingly, loss of Pak1p function or substitution of serine 35 and serine 36 of Rlc1p with alanines, thereby mimicking a dephosphorylated state of Rlc1p, leads to defective coordination of mitosis and cytokinesis. This study reveals a new mechanism involving Pak1p kinase that helps ensure the fidelity of cytokinesis.
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Fiory, Francesca, Anna Teresa Alberobello, Claudia Miele, Francesco Oriente, Iolanda Esposito, Vincenzo Corbo, Menotti Ruvo, et al. "Tyrosine Phosphorylation of Phosphoinositide-Dependent Kinase 1 by the Insulin Receptor IsNecessary for Insulin Metabolic Signaling." Molecular and Cellular Biology 25, no. 24 (December 15, 2005): 10803–14. http://dx.doi.org/10.1128/mcb.25.24.10803-10814.2005.
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ABSTRACT In L6 myoblasts, insulin receptors with deletion of the C-terminal 43 amino acids (IRΔ43) exhibited normal autophosphorylation and IRS-1/2 tyrosine phosphorylation. The L6 cells expressing IRΔ43 (L6IRΔ43) also showed no insulin effect on glucose uptake and glycogen synthase, accompanied by a >80% decrease in insulin induction of 3-phosphoinositide-dependent protein kinase 1 (PDK-1) activity and tyrosine phosphorylation and of protein kinase B (PKB) phosphorylation at Thr308. Insulin induced the phosphatidylinositol 3 kinase-dependent coprecipitation of PDK-1 with wild-type IR (IRWT), but not IRΔ43. Based on overlay blotting, PDK-1 directly bound IRWT, but not IRΔ43. Insulin-activated IRWT, and not IRΔ43, phosphorylated PDK-1 at tyrosines 9, 373, and 376. The IR C-terminal 43-amino-acid peptide (C-terminal peptide) inhibited in vitro PDK-1 tyrosine phosphorylation by the IR. Tyr→Phe substitution prevented this inhibitory action. In the L6hIR cells, the C-terminal peptide coprecipitated with PDK-1 in an insulin-stimulated fashion. This peptide simultaneously impaired the insulin effect on PDK-1 coprecipitation with IRWT, on PDK-1 tyrosine phosphorylation, on PKB phosphorylation at Thr308, and on glucose uptake. Upon insulin exposure, PDK-1 membrane persistence was significantly reduced in L6IRΔ43 compared to control cells. In L6 cells expressing IRWT, the C-terminal peptide also impaired insulin-dependent PDK-1 membrane persistence. Thus, PDK-1 directly binds to the insulin receptor, followed by PDK-1 activation and insulin metabolic effects.
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Brown, Michael C., Leslie A. Cary, Jennifer S. Jamieson, Jonathan A. Cooper, and Christopher E. Turner. "Src and FAK Kinases Cooperate to Phosphorylate Paxillin Kinase Linker, Stimulate Its Focal Adhesion Localization, and Regulate Cell Spreading and Protrusiveness." Molecular Biology of the Cell 16, no. 9 (September 2005): 4316–28. http://dx.doi.org/10.1091/mbc.e05-02-0131.
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The ArfGAP paxillin kinase linker (PKL)/G protein-coupled receptor kinase-interacting protein (GIT)2 has been implicated in regulating cell spreading and motility through its transient recruitment of the p21-activated kinase (PAK) to focal adhesions. The Nck-PAK-PIX-PKL protein complex is recruited to focal adhesions by paxillin upon integrin engagement and Rac activation. In this report, we identify tyrosine-phosphorylated PKL as a protein that associates with the SH3-SH2 adaptor Nck, in a Src-dependent manner, after cell adhesion to fibronectin. Both cell adhesion and Rac activation stimulated PKL tyrosine phosphorylation. PKL is phosphorylated on tyrosine residues 286/392/592 by Src and/or FAK and these sites are required for PKL localization to focal adhesions and for paxillin binding. The absence of either FAK or Src-family kinases prevents PKL phosphorylation and suppresses localization of PKL but not GIT1 to focal adhesions after Rac activation. Expression of an activated FAK mutant in the absence of Src-family kinases partially restores PKL localization, suggesting that Src activation of FAK is required for PKL phosphorylation and localization. Overexpression of the nonphosphorylated GFP-PKL Triple YF mutant stimulates cell spreading and protrusiveness, similar to overexpression of a paxillin mutant that does not bind PKL, suggesting that failure to recruit PKL to focal adhesions interferes with normal cell spreading and motility.
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Koukourakis, M. I., A. Giatromanolaki, E. Sivridis, K. C. Gatter, A. L. Harris, T. Trarbach, G. Folprecht, M. M. Shi, and G. Meinhardt. "Intratumoral lactate dehydrogenase 5 (LDH5) protein expression is associated with expression of angiogenesis markers and hypoxia in patients with colorectal cancer (CRC)." Journal of Clinical Oncology 25, no. 18_suppl (June 20, 2007): 4107. http://dx.doi.org/10.1200/jco.2007.25.18_suppl.4107.
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4107 Background: Recent clinical trials (CONFIRM 1 and CONFIRM 2) have shown that metastatic CRC patients (pts) with high serum lactate dehydrogenase (LDH) derive the greatest therapeutic benefit from PTK787/ZK 222584 (PTK/ZK). PTK/ZK is a novel, oral tyrosine kinase inhibitor (TKI), which blocks all known VEGF receptors (VEGFR). From previous studies, total LDH and isoenzyme LDH5 have been associated with tumor aggressiveness and hypoxia. In the present study, we tested whether CRC pts with high levels of tumor LDH5 have increased expression of proteins involved with hypoxia (hypoxia inducible factors [HIF1a and 2a], pyruvate dehydrokinase [PDHK]), increased vessel density (VD), angiogenesis (VEGFA; phosphorylated VEGFR2 [pKDR]), acidity (carbonic anhydrase 9 [CA9]), and others. Methods: Baseline sections from either primary or metastatic tumor sites from 42 pts of the CONFIRM 1 and 2 trials were analysed with immunohistochemistry utilizing an established nominal scoring system. The degree of association between the scores of protein expressed was estimated by the phi-coefficient (correlation coefficient) and assessed by means of p-values from pairwise Fisher’s exact test (two- sided). Results: Associations were observed between LDH5 and the following: pKDR (Phi=.53; p<.001), VEGF (Phi=.41; p=.006), HIF-1a (Phi=.56; p<.001), VD (Phi=.34; p=.052), and PDHK (Phi=.58; p=.014), respectively. HIF-1a associated with pKDR (Phi=.38; p=.027), VD (Phi=.34; p=.045), and VEGFA (Phi=.33; p=.067). VEGFA associated with PDHK (Phi=.52; p=.035). Conclusions: Our results demonstrate that mCRC patients with high level of intratumoral protein expression of LDH5 have elevated HIF-1a, pKDR, VEGFA, PDHK expression and VD. These results support the concept that tumor hypoxia and angiogenesis are associated and that elevated LDH protein expression may serve as a surrogate marker for activated HIF-1a pathway. No significant financial relationships to disclose.
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Leung, Thomas, Xiang-Qun Chen, Ivan Tan, Edward Manser, and Louis Lim. "Myotonic Dystrophy Kinase-Related Cdc42-Binding Kinase Acts as a Cdc42 Effector in Promoting Cytoskeletal Reorganization." Molecular and Cellular Biology 18, no. 1 (January 1, 1998): 130–40. http://dx.doi.org/10.1128/mcb.18.1.130.
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ABSTRACT The Rho GTPases play distinctive roles in cytoskeletal reorganization associated with growth and differentiation. The Cdc42/Rac-binding p21-activated kinase (PAK) and Rho-binding kinase (ROK) act as morphological effectors for these GTPases. We have isolated two related novel brain kinases whose p21-binding domains resemble that of PAK whereas the kinase domains resemble that of myotonic dystrophy kinase-related ROK. These ∼190-kDa myotonic dystrophy kinase-related Cdc42-binding kinases (MRCKs) preferentially phosphorylate nonmuscle myosin light chain at serine 19, which is known to be crucial for activating actin-myosin contractility. The p21-binding domain binds GTP-Cdc42 but not GDP-Cdc42. The multidomain structure includes a cysteine-rich motif resembling those of protein kinase C andn-chimaerin and a putative pleckstrin homology domain. MRCKα and Cdc42V12 colocalize, particularly at the cell periphery in transfected HeLa cells. Microinjection of plasmid encoding MRCKα resulted in actin and myosin reorganization. Expression of kinase-dead MRCKα blocked Cdc42V12-dependent formation of focal complexes and peripheral microspikes. This was not due to possible sequestration of the p21, as a kinase-dead MRCKα mutant defective in Cdc42 binding was an equally effective blocker. Coinjection of MRCKα plasmid with Cdc42 plasmid, at concentrations where Cdc42 plasmid by itself elicited no effect, led to the formation of the peripheral structures associated with a Cdc42-induced morphological phenotype. These Cdc42-type effects were not promoted upon coinjection with plasmids of kinase-dead or Cdc42-binding-deficient MRCKα mutants. These results suggest that MRCKα may act as a downstream effector of Cdc42 in cytoskeletal reorganization.
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BROOKS, STEPHEN P., and KENNETH B. STOREY. "Glycolytic Enzyme Binding and Metabolic Control in Estivation and Anoxia in the Land Snail Otala Lactea." Journal of Experimental Biology 151, no. 1 (July 1, 1990): 193–204. http://dx.doi.org/10.1242/jeb.151.1.193.
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The mechanisms controlling glycolytic rate were examined in foot muscle of the terrestrial snail Otala lactea (Miiller) (Pulmonata, Helicidae), during short and long periods of estivation and anoxia. Binding associations between glycolytic enzymes and the particulate fraction of the cell were assessed in both states. The percentage of enzyme activity bound to particulate matter decreased significantly over the short term (4 days estivation and 14.5 h anoxia); significant changes were seen for hexokinase (HK), phosphofructokinase (PFK), aldolase and lactate dehydrogenase (LDH) in estivation and, for these enzymes plus triosephosphate isomerase and pyruvate kinase (PK), in anoxia. Over the longer term in estivation (22 days) and anoxia (45 h), enzyme binding returned to control values. Tissue content of fructose-2,6-bisphosphate, a potent phosphofructokinase activator, decreased under all experimental conditions. Total glycogen phosphorylase activity decreased during short-term anoxia (14.5 h) and during long-term estivation (22 days), but the percentage of the active a form decreased significantly during anoxia only. Significant changes in the maximal activities of several enzymes were observed during both estivation and anoxia. Decreases inthe maximal activity of HK, PFK, glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase (PGK) and LDH were observed during long-term estivation. Increases in PGK and PK maximal activity in short-term anoxia and aldolase and PGK in long-term anoxia were also observed. These results suggest that changes in glycolytic enzyme binding may be part of an immediate mechanism used to cause a rapid decrease in glycolytic flux and initiate glycolytic rate depression, which also includes a reduction of fructose-2,6-bisphosphate content and decreased glycogen phosphorylase activity. In the long term, however, control of snail glycolytic rate is reorganized, so that enzyme binding associations revert to the control values. In the long term, then, control is mediated by lower fructose- 2,6-bisphosphate concentrations and, during estivation, also by a decrease in maximal enzyme activities.
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Schlaepfer, David D., K. C. Jones, and Tony Hunter. "Multiple Grb2-Mediated Integrin-Stimulated Signaling Pathways to ERK2/Mitogen-Activated Protein Kinase: Summation of Both c-Src- and Focal Adhesion Kinase-Initiated Tyrosine Phosphorylation Events." Molecular and Cellular Biology 18, no. 5 (May 1, 1998): 2571–85. http://dx.doi.org/10.1128/mcb.18.5.2571.
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ABSTRACT Fibronectin receptor integrin-mediated cell adhesion triggers intracellular signaling events such as the activation of the Ras/mitogen-activated protein (MAP) kinase cascade. In this study, we show that the nonreceptor protein-tyrosine kinases (PTKs) c-Src and focal adhesion kinase (FAK) can be independently activated after fibronectin (FN) stimulation and that their combined activity promotes signaling to extracellular signal-regulated kinase 2 (ERK2)/MAP kinase through multiple pathways upstream of Ras. FN stimulation of NIH 3T3 fibroblasts promotes c-Src and FAK association in the Triton-insoluble cell fraction, and the time course of FN-stimulated ERK2 activation paralleled that of Grb2 binding to FAK at Tyr-925 and Grb2 binding to Shc. Cytochalasin D treatment of fibroblasts inhibited FN-induced FAK in vitro kinase activity and signaling to ERK2, but it only partially inhibited c-Src activation. Treatment of fibroblasts with protein kinase C inhibitors or with the PTK inhibitor herbimycin A or PP1 resulted in reduced Src PTK activity, no Grb2 binding to FAK, and lowered levels of ERK2 activation. FN-stimulated FAK PTK activity was not significantly affected by herbimycin A treatment and, under these conditions, FAK autophosphorylation promoted Shc binding to FAK. In vitro, FAK directly phosphorylated Shc Tyr-317 to promote Grb2 binding, and in vivo Grb2 binding to Shc was observed in herbimycin A-treated fibroblasts after FN stimulation. Interestingly, c-Src in vitro phosphorylation of Shc promoted Grb2 binding to both wild-type and Phe-317 Shc. In vivo, Phe-317 Shc was tyrosine phosphorylated after FN stimulation of human 293T cells and its expression did not inhibit signaling to ERK2. Surprisingly, expression of Phe-925 FAK with Phe-317 Shc also did not block signaling to ERK2, whereas FN-stimulated signaling to ERK2 was inhibited by coexpression of an SH3 domain-inactivated mutant of Grb2. Our studies show that FN receptor integrin signaling upstream of Ras and ERK2 does not follow a linear pathway but that, instead, multiple Grb2-mediated interactions with Shc, FAK, and perhaps other yet-to-be-determined phosphorylated targets represent parallel signaling pathways that cooperate to promote maximal ERK2 activation.
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RIDER, Mark H., Luc BERTRAND, Didier VERTOMMEN, Paul A. MICHELS, Guy G. ROUSSEAU, and Louis HUE. "6-Phosphofructo-2-kinase/fructose-2,6-bisphosphatase: head-to-head with a bifunctional enzyme that controls glycolysis." Biochemical Journal 381, no. 3 (July 27, 2004): 561–79. http://dx.doi.org/10.1042/bj20040752.
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Fru-2,6-P2 (fructose 2,6-bisphosphate) is a signal molecule that controls glycolysis. Since its discovery more than 20 years ago, inroads have been made towards the understanding of the structure–function relationships in PFK-2 (6-phosphofructo-2-kinase)/FBPase-2 (fructose-2,6-bisphosphatase), the homodimeric bifunctional enzyme that catalyses the synthesis and degradation of Fru-2,6-P2. The FBPase-2 domain of the enzyme subunit bears sequence, mechanistic and structural similarity to the histidine phosphatase family of enzymes. The PFK-2 domain was originally thought to resemble bacterial PFK-1 (6-phosphofructo-1-kinase), but this proved not to be correct. Molecular modelling of the PFK-2 domain revealed that, instead, it has the same fold as adenylate kinase. This was confirmed by X-ray crystallography. A PFK-2/FBPase-2 sequence in the genome of one prokaryote, the proteobacterium Desulfovibrio desulfuricans, could be the result of horizontal gene transfer from a eukaryote distantly related to all other organisms, possibly a protist. This, together with the presence of PFK-2/FBPase-2 genes in trypanosomatids (albeit with possibly only one of the domains active), indicates that fusion of genes initially coding for separate PFK-2 and FBPase-2 domains might have occurred early in evolution. In the enzyme homodimer, the PFK-2 domains come together in a head-to-head like fashion, whereas the FBPase-2 domains can function as monomers. There are four PFK-2/FBPase-2 isoenzymes in mammals, each coded by a different gene that expresses several isoforms of each isoenzyme. In these genes, regulatory sequences have been identified which account for their long-term control by hormones and tissue-specific transcription factors. One of these, HNF-6 (hepatocyte nuclear factor-6), was discovered in this way. As to short-term control, the liver isoenzyme is phosphorylated at the N-terminus, adjacent to the PFK-2 domain, by PKA (cAMP-dependent protein kinase), leading to PFK-2 inactivation and FBPase-2 activation. In contrast, the heart isoenzyme is phosphorylated at the C-terminus by several protein kinases in different signalling pathways, resulting in PFK-2 activation.
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Badolia, Rachit. "PAK and Akt Interactions Regulate PAR-Mediated Akt Translocation to Membrane in Platelets: A Novel PIP3-Independent Mechanism." Blood 120, no. 21 (November 16, 2012): 2166. http://dx.doi.org/10.1182/blood.v120.21.2166.2166.
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Abstract Abstract 2166 Akt has been shown to be an important signaling molecule regulating platelet aggregation. It was proposed that Akt translocates to the membrane for its activation where it is phosphorylated on Thr308 by PDK1 and Ser473 by mTORC2. However, the specific signaling events that regulate the Akt phosphorylation and its mechanism of translocation to the membrane are not clearly defined. It has been shown that Akt phosphorylation by many agonists requires signaling events downstream of Gi activation, involving PI3-kinaseb. In addition, it was proposed that Akt translocates to the plasma membrane by binding to PIP3 (Phosphatidylinositol-3,4,5-trisphosphate) generated by PI3-kinase. Our previous studies have revealed that Gq and G12/13 pathways enhance Akt phosphorylation in platelets, suggesting a role for these pathways independently of PIP3. Hence we investigated the mechanisms of Akt translocation in platelets. Stimulation of platelets with the PAR4 activation peptide (AYPGKF) caused translocation of Akt to the membrane rapidly (as early as 20 seconds) whereas, the phosphorylation of Akt occurred at the later time points (Figure 1). Pan-phoshatidylinositide 3-kinase (PI3-Kinase) inhibitors, LY-294002 (Figure 2) and Wortmannin inhibited Ser473 phosphorylation but failed to affect translocation of Akt to the membrane indicating that Akt translocation can occur independent of PI3-kinase activity. However, translocation of Akt was dramatically inhibited in the presence of a Gq-selective inhibitor, YM254890, indicating that Akt translocation is regulated downstream of Gqsignaling pathways (Figure 3). The p21-activated kinases (PAKs) are a family of serine/threonine kinases and are key regulators of actin polymerization and cell migration. PAK functions as a scaffolding protein and is reported to interact with numerous proteins including Akt. Here, we evaluated whether PAK activated by the Gqpathways plays a significant role in the translocation of Akt to the membrane. The co-immunoprecipitation studies revealed that Akt associates with PAK upon stimulation with AYPGKF (Figure 4). This association was abolished when platelets were pre-treated with YM254890 (Figure 5). These results suggest that PAK1/2 activation downstream of Gq pathway mediates Akt translocation to the membrane when stimulated by PAR agonist in platelets and that this translocation is PIP3- independent. Disclosures: No relevant conflicts of interest to declare.
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Cano, E., C. A. Hazzalin, and L. C. Mahadevan. "Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun." Molecular and Cellular Biology 14, no. 11 (November 1994): 7352–62. http://dx.doi.org/10.1128/mcb.14.11.7352.
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Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.
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Cano, E., C. A. Hazzalin, and L. C. Mahadevan. "Anisomycin-activated protein kinases p45 and p55 but not mitogen-activated protein kinases ERK-1 and -2 are implicated in the induction of c-fos and c-jun." Molecular and Cellular Biology 14, no. 11 (November 1994): 7352–62. http://dx.doi.org/10.1128/mcb.14.11.7352-7362.1994.
Full textAbstract:
Independent of its ability to block translation, anisomycin intrinsically initiates intracellular signals and immediate-early gene induction [L. C. Mahadevan and D. R. Edwards, Nature (London) 349:747-749, 1991]. Here, we characterize further its action as a potent, selective signalling agonist. In-gel kinase assays show that epidermal growth factor (EGF) transiently activates five kinases: the mitogen-activated protein (MAP) kinases ERK-1 and -2, and three others, p45, p55, and p80. Anisomycin, at inhibitory and subinhibitory concentrations, does not activate ERK-1 and -2 but elicits strong sustained activation of p45 and p55, which are unique in being serine kinases whose detection is enhanced with poly-Glu/Tyr or poly-Glu/Phe copolymerized in these gels. Translational arrest using emetine or puromycin does not activate p45 and p55 but does prolong EGF-stimulated ERK-1 and -2 activation. Rapamycin, which blocks anisomycin-stimulated p70/85S6k activation without affecting nuclear responses, has no effect on p45 or p55 kinase. p45 and p55 are activable by okadaic acid or UV irradiation, and both kinases phosphorylate the c-Jun NH2-terminal peptide 1-79, putatively placing them within c-Jun NH2-terminal kinase/stress-activated protein kinase (JNK/SAPK) subfamily of MAP kinases. Thus, the EGF- and anisomycin-activated kinases p45 and p55 are strongly implicated in signalling to c-fos and c-jun, whereas the MAP kinases ERK-1 and -2 are not essential for this process.
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Zhu, Guozhi, Koichi Fujii, Yin Liu, Vlad Codrea, Juan Herrero, and Stephen Shaw. "A Single Pair of Acidic Residues in the Kinase Major Groove Mediates Strong Substrate Preference for P-2 or P-5 Arginine in the AGC, CAMK, and STE Kinase Families." Journal of Biological Chemistry 280, no. 43 (August 30, 2005): 36372–79. http://dx.doi.org/10.1074/jbc.m505031200.
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Most basophilic serine/threonine kinases preferentially phosphorylate substrates with Arg at P-3 but vary greatly in additional strong preference for Arg at P-2 or P-5. The structural basis for P-2 or P-5 preference is known for two AGC kinases (family of protein kinases A, G, and C) in which it is mediated by a single pair of acidic residues (PEN+1 and YEM+1). We sought a general understanding of P-2 and P-5 Arg preference. The strength of Arg preference at each position was assessed in 15 kinases using a new degenerate peptide library approach. Strong P-2 or P-5 Arg preference occurred not only in AGC kinases (7 of 8 studied) but also in calmodulin-dependent protein kinase (CAMK, 1 of 3) and Ste20 (STE) kinases (2 of 4). Analysis of sequence conservation demonstrated almost perfect correlation between (a) strong P-2 or P-5 Arg preference and (b) acidic residues at both PEN+1 and YEM+1. Mutation of two kinases (PKC-θ and p21-activated kinase 1 (PAK1)) confirmed critical roles of both PEN+1 and YEM+1 residues in determining strong R-2 Arg preference. PAK kinases were unique in having exceptionally strong Arg preference at P-2 but lacking strong Arg preference at P-3. Preference for Arg at P-2 was so critical to PAK recognition that PAK1 activity was virtually eliminated by mutating the PEN+1 or YEM+1 residues. The fact that this specific pair of acidic residues has been repeatedly and exclusively used by evolution for conferring strong Arg preference at two different substrate positions in three different kinase families implies it is uniquely well suited to mediate sufficiently good substrate binding without unduly restricting product release.
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Brown, Amanda, Xia Wang, Earl Sawai, and Cecilia Cheng-Mayer. "Activation of the PAK-Related Kinase by Human Immunodeficiency Virus Type 1 Nef in Primary Human Peripheral Blood Lymphocytes and Macrophages Leads to Phosphorylation of a PIX-p95 Complex." Journal of Virology 73, no. 12 (December 1, 1999): 9899–907. http://dx.doi.org/10.1128/jvi.73.12.9899-9907.1999.
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ABSTRACT Human immunodeficiency virus type 1 (HIV-1) Nef enhances virus replication in both primary T lymphocytes and monocyte-derived macrophages. This enhancement phenotype has been linked to the ability of Nef to modulate the activity of cellular kinases. We find that despite the reported high-affinity interaction between Nef and the Src kinase Hck in vitro, a Nef-Hck interaction in the context of HIV-1-infected primary macrophages is not detectable. However, Nef binding and activation of the PAK-related kinase and phosphorylation of its substrate could be readily detected in both infected primary T lymphocytes and macrophages. Furthermore, we show that this substrate is a complex composed of the recently characterized PAK interacting partner PIX (PAK-interacting guanine nucleotide exchange factor) and its tightly associated p95 protein. PAK and PIX-p95 appear to be differentially activated and phosphorylated depending on the intracellular environment in which nef is expressed. These results identify the PIX-p95 complex as a novel effector of Nef in primary cells and suggest that the regulation of the PAK signaling pathway may differ in T cells and macrophages.
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Zhu, Jianxin, Ortal Attias, Lamine Aoudjit, Ruihua Jiang, Hiroshi Kawachi, and Tomoko Takano. "p21-Activated kinases regulate actin remodeling in glomerular podocytes." American Journal of Physiology-Renal Physiology 298, no. 4 (April 2010): F951—F961. http://dx.doi.org/10.1152/ajprenal.00536.2009.
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The tyrosine phosphorylation of nephrin is reported to regulate podocyte morphology via the Nck adaptor proteins. The Pak family of kinases are regulators of the actin cytoskeleton and are recruited to the plasma membrane via Nck. Here, we investigated the role of Pak in podocyte morphology. Pak1/2 were expressed in cultured podocytes. In mouse podocytes, Pak2 was predominantly phosphorylated, concentrated at the tips of the cellular processes, and its expression and/or phosphorylation were further increased when differentiated. Overexpression of rat nephrin in podocytes increased Pak1/2 phosphorylation, which was abolished when the Nck binding sites were mutated. Furthermore, dominant-negative Nck constructs blocked the Pak1 phosphorylation induced by antibody-mediated cross linking of nephrin. Transient transfection of constitutively kinase-active Pak1 into differentiated mouse podocytes decreased stress fibers, increased cortical F-actin, and extended the cellular processes, whereas kinase-dead mutant, kinase inhibitory construct, and Pak2 knockdown by shRNA had the opposite effect. In a rat model of puromycin aminonucleoside nephrosis, Pak1/2 phosphorylation was decreased in glomeruli, concomitantly with a decrease of nephrin tyrosine phosphorylation. These results suggest that Pak contributes to remodeling of the actin cytoskeleton in podocytes. Disturbed nephrin-Nck-Pak interaction may contribute to abnormal morphology of podocytes and proteinuria.
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Merla, Ramanna, Yumei Ye, Yu Lin, Saraswathy Manickavasagam, Ming-He Huang, Regino J. Perez-Polo, Barry F. Uretsky, and Yochai Birnbaum. "The central role of adenosine in statin-induced ERK1/2, Akt, and eNOS phosphorylation." American Journal of Physiology-Heart and Circulatory Physiology 293, no. 3 (September 2007): H1918—H1928. http://dx.doi.org/10.1152/ajpheart.00416.2007.
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Statins activate phosphatidylinositol-3-kinase, which activates ecto-5′-nucleotidase and phosphorylates 3-phosphoinositide-dependent kinase-1 (PDK-1). Phosphorylated (P-)PDK-1 phosphorylates Akt, which phosphorylates endothelial nitric oxide synthase (eNOS). We asked if the blockade of adenosine receptors (A1, A2A, A2B, or A3 receptors) could attenuate the induction of Akt and eNOS by atorvastatin (ATV) and whether ERK1/2 is involved in the ATV regulation of Akt and eNOS. In protocol 1, mice received intraperitoneal ATV, theophylline (TH), ATV + TH, or vehicle. In protocol 2, mice received intraperitoneal injections of ATV, U0126 (an ERK1/2 inhibitor), ATV + U0126, or vehicle; 8 h later, hearts were assessed by immunoblot analysis. In protocol 3, mice received intraperitoneal ATV alone or with 8-sulfophenyltheophylline (SPT); 1, 3, and 6 h after injection, hearts were assessed by immunoblot analysis. In protocol 4, mice received intraperitoneal ATV alone or with SPT, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX), 1,3,7-trimethyl-8-(3-chlorostyryl)xanthine (CSC), alloxazine, or MRS-1523; 3 h after injection, hearts were assessed by immunoblot analysis. ATV increased P-ERK, P-PDK-1, Ser473 P-Akt, Thr308 P-Akt, and P-eNOS levels. TH blocked ATV-induced increases in P-ERK, Ser473 P-Akt, Thr308 P-Akt, and P-eNOS levels without affecting the induction of P-PDK-1 by ATV. U0126 blocked the ATV induction of Ser473 P-Akt and Thr308 P-Akt while attenuating the induction of P-eNOS. A detectable increase in P-ERK, Ser473 P-Akt and P-eNOS was seen 3 and 6 h after injection but not at 1 h. DPCPX, CSC, and alloxazine partially blocked the ATV induction of P-ERK, Ser473 P-Akt, and P-eNOS. In conclusion, blockade of adenosine A1, A2A, and A2B receptors but not A3 receptors inhibited the induction of Akt and eNOS by statins. Adenosine was required for ERK1/2 activation by statins, which resulted in Akt and eNOS phosphorylation.
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Yamanashi, Yuji, Takahiro Fukuda, Hirofumi Nishizumi, Tetsuya Inazu, Ken-ichi Higashi, Daisuke Kitamura, Takaomi Ishida, Hirohei Yamamura, Takeshi Watanabe, and Tadashi Yamamoto. "Role of Tyrosine Phosphorylation of HS1 in B Cell Antigen Receptor-mediated Apoptosis." Journal of Experimental Medicine 185, no. 7 (April 7, 1997): 1387–92. http://dx.doi.org/10.1084/jem.185.7.1387.
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The 75-kD HS1 protein is highly tyrosine-phosphorylated during B cell antigen receptor (BCR)-mediated signaling. Owing to low expression of HS1, WEHI-231-derived M1 cells, unlike the parental cells, are insensitive to BCR-mediated apoptosis. Here, we show that BCR-associated tyrosine kinases Lyn and Syk synergistically phosphorylate HS1, and that Tyr378 and Tyr-397 of HS1 are the critical residues for its BCR-induced phosphorylation. In addition, unlike wild-type HS1, a mutant HS1 carrying the mutations Phe-378 and Phe-397 was unable to render M1 cells sensitive to apoptosis. Wild-type HS1, but not the mutant, localized to the nucleus under the synergy of Lyn and Syk. Thus, tyrosine phosphorylation of HS1 is required for BCR-induced apoptosis and nuclear translocation of HS1 may be a prerequisite for B cell apoptosis.
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Johnston, J. B., John W. Barrett, Wen Chang, Che-Sheng Chung, Wei Zeng, Jennefer Masters, Melissa Mann, Fuan Wang, Jingxin Cao, and Grant McFadden. "Role of the Serine-Threonine Kinase PAK-1 in Myxoma Virus Replication." Journal of Virology 77, no. 10 (May 15, 2003): 5877–88. http://dx.doi.org/10.1128/jvi.77.10.5877-5888.2003.
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ABSTRACT Subversion or appropriation of cellular signal transduction pathways is a common strategy employed by viruses to promote an environment within infected cells that supports the viral replicative cycle. Using subsets of 3T3 murine fibroblasts previously shown to differ in their ability to support myxoma virus (MV) replication, we investigated the role of host serine-threonine kinases (STKs) as potential mediators of the permissive phenotype. Both permissive and nonpermissive 3T3 cells supported equivalent levels of virion binding, entry, and early virus gene expression, indicating that MV tropism in 3T3 cells was not determined by receptor-mediated entry. In contrast, late virus gene expression and viral DNA replication were selectively compromised in restrictive 3T3 cells. Addition of specific protein kinase inhibitors, many of which shared the ability to influence the activity of the STKs p21-activated kinase 1 (PAK-1) and Raf-1 attenuated MV replication in permissive 3T3 cells. Western blot detection of the phosphorylated forms of PAK-1 (Thr423) and Raf-1 (Ser338) confirmed activation of these kinases in permissive cells after MV infection or gamma interferon treatment, but the activated forms of both kinases were greatly reduced or absent in restrictive 3T3 cells. The biological significance of these activations was demonstrated by using the autoinhibitory domain of PAK-1 (amino acids 83 to 149), expression of which reduced the efficiency of MV infection in permissive 3T3 cells concurrent with a decrease in PAK-1 activation. In comparison, overexpression of a constitutively active PAK-1 (T423E) mutant increased MV replication in restrictive 3T3 cells. These observations suggest that induced signaling via cellular STKs may play important roles in determining the permissiveness of host cells to poxvirus infection.
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Renkema, G. Herma, Kati Pulkkinen, and Kalle Saksela. "Cdc42/Rac1-Mediated Activation Primes PAK2 for Superactivation by Tyrosine Phosphorylation." Molecular and Cellular Biology 22, no. 19 (October 1, 2002): 6719–25. http://dx.doi.org/10.1128/mcb.22.19.6719-6725.2002.
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ABSTRACT The involvement of p21-activated kinases (PAKs) in important cellular processes such as regulation of the actin skeleton morphology, transduction of signals controlling gene expression, and execution of programmed cell death has directed attention to the regulation of the activity of these kinases. Here we report that activation of PAK2 by p21 GTPases can be strongly potentiated by cellular tyrosine kinases. PAK2 became tyrosine phosphorylated in its N-terminal regulatory domain, where Y130 was identified as the major phosphoacceptor site. Tyrosine phosphorylation-mediated superactivation of PAK2 could be induced by overexpression of different Src kinases or by inhibiting cellular tyrosine phosphatases with pervanadate and could be blocked by the Src kinase inhibitor PP1 or by mutating the Y130 residue. Analysis of PAK2 mutants activated by amino acid changes in the autoinhibitory domain or the catalytic domain indicated that GTPase-induced conformational changes, rather than catalytic activation per se, rendered PAK2 a target for tyrosine phosphorylation. Thus, PAK activation represents a potentially important point of convergence of tyrosine kinase- and p21 GTPase-dependent signaling pathways.
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Zhou, Guo-Lei, Ya Zhuo, Charles C. King, Benjamin H. Fryer, Gary M. Bokoch, and Jeffrey Field. "Akt Phosphorylation of Serine 21 on Pak1 Modulates Nck Binding and Cell Migration." Molecular and Cellular Biology 23, no. 22 (November 15, 2003): 8058–69. http://dx.doi.org/10.1128/mcb.23.22.8058-8069.2003.
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ABSTRACT The p21-activated protein kinases (Paks) regulate cellular proliferation, differentiation, transformation, and survival through multiple downstream signals. Paks are activated directly by the small GTPases Rac and Cdc42 and several protein kinases including Akt and PDK-1. We found that Akt phosphorylated and modestly activated Pak1 in vitro. The major site phosphorylated by Akt on Pak1 mapped to serine 21, a site originally shown to be weakly autophosphorylated on Pak1 when Cdc42 or Rac activates it. A peptide derived from the region surrounding serine 21 was a substrate for Akt but not Pak1 in vitro, and Akt stimulated serine 21 phosphorylation on the full-length Pak1 much better than Rac did. The adaptor protein Nck binds Pak near serine 21, and its association is regulated by phosphorylation of this site. We found that either treatment of Pak1 in vitro with Akt or coexpression of constitutively active Akt with Pak1 reduced Nck binding to Pak1. In HeLa cells, green fluorescent protein-tagged Pak1 was concentrated at focal adhesions and was released when Akt was cotransfected. A peptide containing the Nck binding site of Pak1 fused to a portion of human immunodeficiency virus Tat to allow it to enter cells was used to test the functional importance of Nck/Pak binding in Akt-stimulated cell migration. This Tat-Nck peptide reduced Akt-stimulated cell migration. Together, these data suggest that Akt modulates the association of Pak with Nck to regulate cell migration.
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Ido, Masaru, Tatsuya Hayashi, Esteban Gabazza, and Koji Suzuki. "Identification of a Novel 33-kDa Ser/Thr Kinase that Phosphorylates the Cytoplasmic Tail of Protease-activated Receptor 1 (Thrombin Receptor) in Human Platelets." Thrombosis and Haemostasis 83, no. 04 (2000): 617–21. http://dx.doi.org/10.1055/s-0037-1613873.
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SummaryStimulation of human platelets with thrombin or thrombin receptor agonist peptide (TRAP/ Ser-Phe-Leu-Leu-Arg-Asn) resulted in phosphorylation of the protease-activated receptor 1 (PAR1). However, protein kinase(s), capable of phosphorylating PAR1 upon activation of this receptor, has not been as yet identified in human platelets. The present study was undertaken to assess the presence of protein kinase(s) that may interact with PAR1 using a procedure based on the ability of protein kinase to undergo renaturation and phosphorylate a protein substrate fixed in a gel. We employed a fusion protein that was prepared using a glutathione S-transferase (GST) and the cytoplasmic tail of PAR1 (Pro368-Thr425)(GST-PAR1) or a reverse sequenced peptide of this domain (GST-rPAR1). The results showed that treatment of platelets with thrombin induced about 10-fold increase in the activity of the 33-kDa Ser/Thr protein kinase, which was also activated by TRAP, but not by hirudin-treated thrombin or diisopropylfluorophosphate-inactivated thrombin, suggesting that it is activated through PAR1. Furthermore, treatment of platelets with thromboxane A2 analog, STA2, led to an activation of this protein kinase and phosphorylation of PAR1. In conclusion, the present study provides evidence of homologous and heterologous activation of a novel 33-kDa Ser/Thr kinase that phosphorylates the cytoplasmic tail of PAR1.
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Naik, Ulhas P., and Meghna U. Naik. "Inhibition of Polo-Like Kinase 3 by CIB1 Facilitates Outside-In Signaling through αIIbβ3 in Platelets." Blood 108, no. 11 (November 16, 2006): 214. http://dx.doi.org/10.1182/blood.v108.11.214.214.
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Abstract Integrin αIIbβ3, the platelet fibrinogen receptor, is involved in bi-directional signaling during platelet activation. It has been shown that the β3 subunit of this receptor is phosphorylated on both threonine and tyrosine. Although, the role of tyrosine phosphorylation is well studied, the role of threonine phosphorylation is not well understood. Here we identify the kinase responsible for threonine phosphoruylation of β3 and provide a mechanistic significance of this phosphorylation in outside-in signaling. We found that calcium- and integrin-binding protein 1 (CIB1), which specifically interacts with the cytoplasmic domain of αIIb, is not involved in inside-out signaling, but regulates outside-in signaling through αIIbβ3. CIB1 is known to interact with and regulate several serine/threonine protein kinases. Among these, p21 activated kinase 1 (PAK-1) is highly expressed in platelets. We found that CIB1 does not colocalize with PAK-1 during outside in signaling induced by adhesion of platelets to immobilized fibrinogen. When searched for the expression of other kinases that are known to interact with CIB1, we found that polo-like kinase 3 (Plk3) is expressed in platelets and is responsible for the constitutive phosphorylation of β3. Plk3 is constitutively active in resting platelets but is rendered inactive during outside in signaling as a result of CIB1 binding in a calcium dependent manner. Plk3 becomes colocalized with CIB1 at the filopodia when platelets change shape in response to an agonist and remain associated with both CIB1 and αIIb as platelets spread on immobilized fibrinogen, as indicated by immunoprecipitation experiments. These results suggest that in resting platelets, constitutively active Plk3 maintains β3 in a threonine phosphorylated state which is nonpermissive for activation. Upon platelet activation by agonist CIB1 associates with Plk3 and renders it inactive in a calcium-dependent manner. This allows serine/threonine phosphatases, such as PP1-c to dephosphorylate β3 on threonine and facilitate outside-in signaling. Thus we have identified the kinase responsible for threonine phosphorylation of β3 and have provided a possible mechanism of the regulation of outside-in signaling.
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van der Geer, P., and T. Hunter. "Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription." Molecular and Cellular Biology 11, no. 9 (September 1991): 4698–709. http://dx.doi.org/10.1128/mcb.11.9.4698.
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The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.
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van der Geer, P., and T. Hunter. "Tyrosine 706 and 807 phosphorylation site mutants in the murine colony-stimulating factor-1 receptor are unaffected in their ability to bind or phosphorylate phosphatidylinositol-3 kinase but show differential defects in their ability to induce early response gene transcription." Molecular and Cellular Biology 11, no. 9 (September 1991): 4698–709. http://dx.doi.org/10.1128/mcb.11.9.4698-4709.1991.
Full textAbstract:
The receptor for colony-stimulating factor-1 (CSF-1) is a receptor protein-tyrosine kinase. To study the possible function of CSF-1 receptor autophosphorylation, two autophosphorylation sites, Tyr-706, located in the kinase insert, and Tyr-807, a residue conserved in all protein-tyrosine kinases, were changed independently to either phenylalanine or glycine. Wild-type and mutant receptors were stably expressed in Rat-2 cells. In response to CSF-1, cells expressing Phe- or Gly-706 mutant receptors showed increased growth rate and altered cell morphology. Both the Phe- and Gly-706 mutant receptors associated with and phosphorylated phosphatidylinositol-3 kinase at levels comparable with those of wild-type receptors. However, these mutant receptors differed subtly from each other and from the wild-type receptor in their ability to induce different aspects of the response to CSF-1. The Phe-706 mutant receptor was most strongly affected in its ability to increase growth rate or elevate the levels of c-fos and NGF1A mRNAs, whereas the Gly-706 mutant receptor was most markedly affected in its ability to induce a change in cell morphology or increase the levels of c-jun and NGF1A mRNAs. These findings indicate that Tyr-706 itself, or this region of the receptor, may be important for interaction of the CSF-1 receptor with different signalling pathways. Gly-807 mutant receptors lacked protein-tyrosine kinase activity, failed to respond to CSF-1, and were defective in biosynthetic processing. Phe-807 mutant receptors had 40 to 60% reduced protein-tyrosine kinase activity in vitro. Although cells expressing Phe-807 receptors were able to respond to CSF-1, the changes in growth rate and cell morphology were significantly less than seen with wild-type receptors, and the induction of early response genes was also slightly lower than for the wild-type receptor. In contrast, Phe-807 receptors were equivalent to wild-type receptors when tested for their ability to interact with phosphatidylinositol-3 kinase. These findings indicate that phosphorylation of Tyr-807 may be important for full activation of the receptor.
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Cui, Xiang, Da-Wei Qian, Shu Jiang, Er-Xin Shang, Zhen-Hua Zhu, and Jin-Ao Duan. "Scutellariae Radix and Coptidis Rhizoma Improve Glucose and Lipid Metabolism in T2DM Rats via Regulation of the Metabolic Profiling and MAPK/PI3K/Akt Signaling Pathway." International Journal of Molecular Sciences 19, no. 11 (November 18, 2018): 3634. http://dx.doi.org/10.3390/ijms19113634.
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Aim Scutellariae Radix (SR) and Coptidis Rhizoma (CR) have often been combined to cure type 2 diabetes mellitus (T2DM) in the clinical practice for over thousands of years, but their compatibility mechanism is not clear. Mitogen-activated protein kinase (MAPK) signaling pathway has been suggested to play a critical role during the process of inflammation, insulin resistance, and T2DM. This study was designed to investigate their compatibility effects on T2DM rats and explore the underlying mechanisms by analyzing the metabolic profiling and MAPK/PI3K/Akt signaling pathway. Methods The compatibility effects of SR and CR were evaluated with T2DM rats induced by a high-fat diet (HFD) along with a low dose of streptozocin (STZ). Ultra performance liquid chromatography-quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS) was performed to discover potential biomarkers. The levels of pro-inflammatory cytokines; biochemical indexes in serum, and the activities of key enzymes related to glycometabolism in liver were assessed by ELISA kits. qPCR was applied to examine mRNA levels of key targets in MAPK and insulin signaling pathways. Protein expressions of p65; p-p65; phosphatidylinositol-4,5-bisphosphate 3-kinase (PI3K); phosphorylated-PI3K (p-PI3K); protein kinase B (Akt); phosphorylated Akt (p-Akt) and glucose transporter 2 (Glut2) in liver were investigated by Western blot analysis. Results Remarkably, hyperglycaemia, dyslipidemia, inflammation, and insulin resistance in T2DM were ameliorated after oral administration of SR and CR, particularly their combined extracts. The effects of SR, CR, low dose of combined extracts (LSC) and high dose of combined extracts (HSC) on pro-inflammatory cytokine transcription in T2DM rats showed that the MAPK pathway might account for the phenomenon with down-regulation of MAPK (P38 mitogen-activated protein kinases (P38), extracellular regulated protein kinases (ERK), and c-Jun N-terminal kinase (JNK)) mRNA, and protein reduction in p-P65. While mRNA levels of key targets such as insulin receptor substrate 1 (IRS1), PI3K, Akt2, and Glut2 in the insulin signaling pathway were notably up-modulated, phosphorylations of PI3K, Akt, and expression of Glut2 were markedly enhanced. Moreover, the increased activities of phosphoenolpyruvate carboxykinase (PEPCK), fructose-1,6-bisphosphatase (FBPase), glucose 6-phosphatase (G6Pase), and glycogen phosphorylase (GP) were highly reduced and the decreased activities of glucokinase (GK), phosphofructokinase (PFK), pyruvate kinase (PK), and glycogen synthase (GS) in liver were notably increased after treatment. Further investigation indicated that the metabolic profiles of plasma and urine were clearly improved in T2DM rats. Fourteen potential biomarkers (nine in plasma and five in urine) were identified. After intervention, these biomarkers returned to normal level to some extent. Conclusion The results showed that SR, CR, and combined extract groups were normalized. The effects of combined extracts were more remarkable than single herb treatment. Additionally, this study also showed that the metabonomics method is a promising tool to unravel how traditional Chinese medicines work.
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Wek, R. C., H. Y. Jiang, and T. G. Anthony. "Coping with stress: eIF2 kinases and translational control." Biochemical Society Transactions 34, no. 1 (January 20, 2006): 7–11. http://dx.doi.org/10.1042/bst0340007.
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In response to environmental stresses, a family of protein kinases phosphorylate eIF2 (eukaryotic initiation factor 2) to alleviate cellular injury or alternatively induce apoptosis. Phosphorylation of eIF2 reduces global translation, allowing cells to conserve resources and to initiate a reconfiguration of gene expression to effectively manage stress conditions. Accompanying this general protein synthesis control, eIF2 phosphorylation induces translation of specific mRNAs, such as that encoding the bZIP (basic leucine zipper) transcriptional regulator ATF4 (activating transcription factor 4). ATF4 also enhances the expression of additional transcription factors, ATF3 and CHOP (CCAAT/enhancer-binding protein homologous protein)/GADD153 (growth arrest and DNA-damage-inducible protein), that assist in the regulation of genes involved in metabolism, the redox status of the cells and apoptosis. Reduced translation by eIF2 phosphorylation can also lead to activation of stress-related transcription factors, such as NF-κB (nuclear factor κB), by lowering the steady-state levels of short-lived regulatory proteins such as IκB (inhibitor of NF-κB). While many of the genes induced by eIF2 phosphorylation are shared between different environmental stresses, eIF2 kinases function in conjunction with other stress-response pathways, such as those regulated by mitogen-activated protein kinases, to elicit gene expression programmes that are tailored for the specific stress condition. Loss of eIF2 kinase pathways can have important health consequences. Mice devoid of the eIF2 kinase GCN2 [general control non-derepressible-2 or EIF2AK4 (eIF2α kinase 4)] show sensitivity to nutritional deficiencies and aberrant eating behaviours, and deletion of PEK [pancreatic eIF2α kinase or PERK (RNA-dependent protein kinase-like endoplasmic reticulum kinase) or EIF2AK3] leads to neonatal insulin-dependent diabetes, epiphyseal dysplasia and hepatic and renal complications.
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Fryer, Benjamin H., Changhui Wang, Srilakshmi Vedantam, Guo-Lei Zhou, Shenghao Jin, Linda Fletcher, M. Celeste Simon, and Jeffrey Field. "cGMP-dependent Protein Kinase Phosphorylates p21-activated Kinase (Pak) 1, Inhibiting Pak/Nck Binding and Stimulating Pak/Vasodilator-stimulated Phosphoprotein Association." Journal of Biological Chemistry 281, no. 17 (February 20, 2006): 11487–95. http://dx.doi.org/10.1074/jbc.m600279200.
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Jiang, Hao-Yuan, Sheree A. Wek, Barbara C. McGrath, Dan Lu, Tsonwin Hai, Heather P. Harding, Xiaozhong Wang, David Ron, Douglas R. Cavener, and Ronald C. Wek. "Activating Transcription Factor 3 Is Integral to the Eukaryotic Initiation Factor 2 Kinase Stress Response." Molecular and Cellular Biology 24, no. 3 (February 1, 2004): 1365–77. http://dx.doi.org/10.1128/mcb.24.3.1365-1377.2004.
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ABSTRACT In response to environmental stress, cells induce a program of gene expression designed to remedy cellular damage or, alternatively, induce apoptosis. In this report, we explore the role of a family of protein kinases that phosphorylate eukaryotic initiation factor 2 (eIF2) in coordinating stress gene responses. We find that expression of activating transcription factor 3 (ATF3), a member of the ATF/CREB subfamily of basic-region leucine zipper (bZIP) proteins, is induced in response to endoplasmic reticulum (ER) stress or amino acid starvation by a mechanism requiring eIF2 kinases PEK (Perk or EIF2AK3) and GCN2 (EIF2AK4), respectively. Increased expression of ATF3 protein occurs early in response to stress by a mechanism requiring the related bZIP transcriptional regulator ATF4. ATF3 contributes to induction of the CHOP transcriptional factor in response to amino acid starvation, and loss of ATF3 function significantly lowers stress-induced expression of GADD34, an eIF2 protein phosphatase regulatory subunit implicated in feedback control of the eIF2 kinase stress response. Overexpression of ATF3 in mouse embryo fibroblasts partially bypasses the requirement for PEK for induction of GADD34 in response to ER stress, further supporting the idea that ATF3 functions directly or indirectly as a transcriptional activator of genes targeted by the eIF2 kinase stress pathway. These results indicate that ATF3 has an integral role in the coordinate gene expression induced by eIF2 kinases. Given that ATF3 is induced by a very large number of environmental insults, this study supports involvement of eIF2 kinases in the coordination of gene expression in response to a more diverse set of stress conditions than previously proposed.