Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.12.2 (MEK)
18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

In addition to their role in bacterial killing, reactive oxygen intermediates (ROI) produced by the NADPH oxidase may participate in the regulation of intracellular pathways. We have recently demonstrated that ROI produced by the oxidase regulate tyrosine phosphorylation in neutrophils, possibly by alterations in the cellular redox state. The purpose of the present study was to characterize the identities of certain of the redox-sensitive tyrosine-phosphorylated substrates and the significance of the increased phosphorylation. As a prominent 42-44-kDa phosphorylated band was noted in oxidant-treated cells, we investigated the possible phosphorylation and activation of mitogen-activated protein (MAP) kinase under these conditions. Immunoprecipitation of MAP kinase followed by immunoblotting with anti-phosphotyrosine antibodies indicated that a 42-44-kDa polypeptide was tyrosine-phosphorylated in response to treatment of cells, either with the oxidizing agent diamide or with H2O2 in cells where catalase was inhibited. Using an in vitro renaturation assay with myelin basic protein as the substrate, oxidant-induced stimulation of kinase activity of a 42-44-kDa band was observed in both whole cell extracts and in MAP kinase immunoprecipitates. The mechanism of redox-sensitive activation of MAP kinase was examined. First, exposure of cells to oxidants caused a significant increase in the activity of MEK (the putative activator of MAP kinase), as determined by an in vitro kinase assay using recombinant catalytically inactive glutathione S-transferase-MAP kinase as the substrate. Additionally, oxidant treatment of cells resulted in inhibition of the activity of CD45, a protein tyrosine phosphatase known to dephosphorylate and inactivate MAP kinase. We conclude that oxidant treatment of neutrophils can activate MAP kinase by stimulating its tyrosine and (presumably) threonine phosphorylation via MEK activation, a response that may be potentiated by inhibition of MAP kinase dephosphorylation by phosphatases such as CD45.
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PMID:Activation of the mitogen-activated protein kinase signaling pathway in neutrophils. Role of oxidants. 798 67

The mitogen-activated protein kinase (MAPK) family is comprised of key regulatory proteins that control the cellular response to both proliferation and stress signals. In this study we investigated the factors controlling MAPK activation by H2O2 and explored the impact of altering the pathways to kinase activation on cell survival following H2O2 exposure. Potent activation (10-20-fold) of extracellular signal-regulated protein kinase (ERK2) occurred within 10 min of H2O2 treatment, whereupon rapid inactivation ensued. H2O2 activated ERK2 in several cell types and also moderately activated (3-5-fold) both c-Jun N-terminal kinase and p38/RK/CSBP. Additionally, H2O2 increased the mRNA expression of MAPK-dependent genes c-jun, c-fos, and MAPK phosphatase-1. Suramin pretreatment completely inhibited H2O2 stimulation of ERK2, highlighting a role for growth factor receptors in this activation. Further, ERK2 activation by H2O2 was blocked by pretreatment with either N-acetyl-cysteine, o-phenanthroline, or mannitol, indicating that metal-catalyzed free radical formation mediates the initiation of signal transduction by H2O2. H2O2-stimulated activation of ERK2 was abolished in PC12 cells by inducible or constitutive expression of the dominant negative Ras-N-17 allele. Interestingly, PC12/Ras-N-17 cells were more sensitive than wild-type PC12 cells to H2O2 toxicity. Moreover, NIH 3T3 cells expressing constitutively active MAPK kinase (MEK, the immediate upstream regulator of ERK) were more resistant to H2O2 toxicity, while those expressing kinase-defective MEK were more sensitive, than cells expressing wild-type MEK. Taken together, these studies provide insight into mechanisms of MAPK regulation by H2O2 and suggest that ERK plays a critical role in cell survival following oxidant injury.
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PMID:Activation of mitogen-activated protein kinase by H2O2. Role in cell survival following oxidant injury. 862 53

The Raf-1 serine/threonine protein kinase plays a central role in many of the mitogenic signaling pathways regulating cell growth and differentiation. The regulation of Raf-1 is complex, and involves protein-protein interactions as well as changes in the phosphorylation state of Raf-1 that are accompanied by alterations in its electrophoretic mobility. We have previously shown that a 33-kDa COOH-terminal, kinase-inactive fragment of Raf-1 underwent a mobility shift in response to the stimulation of cells with serum or phorbol esters. Here we demonstrate that treatment of NIH 3T3 cells or Sf9 cells with hydrogen peroxide (H2O2) also induces the mobility shift of the kinase-inactive Raf-1 fragment. A series of deletion mutants of the Raf-1 COOH terminus were analyzed, and the region required for the mobility shift was localized to a 78-amino acid fragment (residues 566-643). Metabolic labeling revealed that the slower migrating forms of the 33-kDa and of the smaller fragment contained phosphorus. Mutation of a previously characterized phosphorylation site, serine 621, to alanine prevented the mobility shift as well as phosphate incorporation or Src and Ras-dependent kinase activation in Sf9 cells when this mutation was engineered into the full-length Raf-1. Mutation of 621 to aspartate yielded a protein that existed in both the shifted and unshifted forms, demonstrating that a negative charge at 621 was necessary, but not sufficient, for the mobility shift to occur; however, its full-length form was still resistant to activation in the Sf9 system. Additional mutation of nearby serine 624 to alanine blocked the shift, implicating this residue as the site of the second of a two-step modification process leading to the slower migrating form. Co-expression of the 33-kDa fragment with an activated form of mitogen-activated protein kinase kinase in NIH 3T3 led to the appearance of the shifted form in a serum-independent manner. These results demonstrate that a mitogen-activated protein kinase kinase-induced event involving modification of serines 621 and 624 leads to the mobility shift of Raf-1.
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PMID:Sequential modification of serines 621 and 624 in the Raf-1 carboxyl terminus produces alterations in its electrophoretic mobility. 899 14

The mechanism of mitogen-activated protein kinase (MAPK, ERK) stimulation by the GnRH analog [D-Trp6]GnRH (GnRH-a) was investigated in the gonadotroph-derived alphaT3-1 cell line. GnRH-a as well as the protein kinase C (PKC) activator 12-O-tetradecanoyl phorbol-13-acetate (TPA) stimulated a sustained response of MAPK activity, whereas epidermal growth factor (EGF) stimulated a transient response. MAPK kinase (MEK) is also activated by GnRH-a, but in a transient manner. GnRH-a and TPA apparently activated mainly the MAPK isoform ERK1, as revealed by Mono-Q fast protein liquid chromatography followed by Western blotting as well as by gel kinase assay. GnRH-a and TPA stimulated the tyrosine phosphorylation of several proteins, and this effect as well as the stimulation of MAPK activity were inhibited by the PKC inhibitor GF 109203X. Similarly, down-regulation of TPA-sensitive PKC subspecies nearly abolished the effect of GnRH-a and TPA on MAPK activity. Furthermore, the protein tyrosine kinase (PTK) inhibitor genistein inhibited protein tyrosine phosphorylation and reduced GnRH-a-stimulated MAPK activity by 50%, suggesting the participation of genistein-sensitive and insensitive pathways in GnRH-a action. Although Ca2+ ionophores have only a marginal stimulatory effect, the removal of Ca2+ markedly reduced MAPK activation by GnRH-a and TPA, but had no effect on GnRH-a and TPA stimulation of protein tyrosine phosphorylation. Interestingly, the removal of Ca2+ also partly inhibited the activation of MAPK by EGF and vanadate/H2O2. Thus, a calcium-dependent component(s) downstream of PKC and PTK might also participate in MAPK activation. Elevation of cAMP by forskolin exerted partial inhibition on EGF, but not on TPA or GnRH-a action, suggesting that MEK activators other than Raf-1 might be involved in GnRH action. We conclude that Ca2+, PTK, and PKC participate in the activation of MAPK by GnRH-a, with Ca2+ being necessary downstream to PKC and PTK.
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PMID:Mechanism of mitogen-activated protein kinase activation by gonadotropin-releasing hormone in the pituitary of alphaT3-1 cell line: differential roles of calcium and protein kinase C. 907 30

Hydrogen peroxide stimulated tyrosine phosphorylation of several proteins in growth-arrested vascular smooth muscle cells (VSMC). One of these proteins was identified as fibroblast growth factor receptor type I (FGFR1). In addition, induced tyrosine phosphorylation of FGFR1 by hydrogen peroxide resulted in complex formation with Grb2. Hydrogen peroxide also caused a time-dependent activation of extracellular signal-regulated protein kinases (ERKs; p42&p44) group of mitogen-activated protein kinases (MAPKs) in VSMC. The time courses of the hydrogen peroxide-stimulated FGFR1 tyrosine phosphorylation and ERKs activation were followed by induced expression of c-fos and c-jun. Genistein, a potent inhibitor of protein tyrosine kinases, significantly blunted the hydrogen peroxide-induced FGFR1 tyrosine phosphorylation, ERKs activation and c-fos and c-jun expression. PD98059, a specific inhibitor of MEK1, attenuated the hydrogen peroxide-induced ERKs activation and c-fos and c-jun expression. Together, these results suggest that oxidants such as hydrogen peroxide stimulate tyrosine phosphorylation of receptor tyrosine kinases and these, in turn, mediate the down-stream signalling events including the recruitment of Grb2 by the receptor, activation of ERKs and induction of c-fos and c-jun expression.
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PMID:Protein tyrosine kinase activity is required for oxidant-induced extracellular signal-regulated protein kinase activation and c-fos and c-jun expression. 911 18

Phosphorylation of alphaB-crystallin, a member of the hsp27 family, in human glioma (U373 MG) cells was stimulated by exposure of the cells to various stimuli, which included heat, arsenite, phorbol 12-myristate 13-acetate (PMA), okadaic acid, H2O2, anisomycin, and high concentrations of NaCl or sorbitol, but not in response to agents that elevated intracellular levels of cyclic AMP. Cells exposed to PMA together with okadaic acid yielded three bands of 32P-labeled alphaB-crystallin when immunoprecipitated samples were subjected to electrophoresis on an isoelectric focusing gel. All of the phosphorylated residues were identified as serine, an indication that three different serine residues can act as sites of phosphorylation in alphaB-crystallin. Structural analysis by mass spectrometry revealed that phosphorylation of alphaB-crystallin occurred at serines 19, 45, and 59. Dithiothreitol and staurosporine selectively inhibited the phosphorylation induced by arsenite and the phorbol ester, respectively. SB202190, an inhibitor of p38 mitogen-activated protein (MAP) kinase, suppressed the phosphorylation induced by arsenite, anisomycin, H2O2, sorbitol, NaCl, and heat shock, but not that induced by PMA and okadaic acid. The PMA-induced phosphorylation was selectively suppressed by an inhibitor of p44 MAP kinase kinase, PD98059. Although PMA and arsenite preferentially stimulated the phosphorylation of Ser-45 and Ser-59, respectively, as determined with antibodies that recognized the respective phosphorylated forms of alphaB-crystallin, all three sites were phosphorylated in response to each stimulus. These results suggest that p38 MAP kinase or p44 MAP kinase might be involved in the signal transduction cascade that leads to the phosphorylation of alphaB-crystallin. The phosphorylation of alphaB-crystallin was also enhanced in the heart and diaphragm when rats were exposed to heat stress (42 degrees C for 20 min).
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PMID:Phosphorylation of alphaB-crystallin in response to various types of stress. 936 70

Stimulation of cell signaling cascades by oxidants may be important in the pathogenesis of pulmonary and pleural diseases. Here, we demonstrate in rat pleural mesothelial cells that apoptotic concentrations of crocidolite asbestos and H2O2 induce phosphorylation and activation of extracellular signal-regulated protein kinases (ERK). Activation of c-jun-NH2-terminal protein kinases (JNK)/stress-activated protein kinases was also observed in response to H2O2. In contrast, asbestos caused more protracted activation of ERK without JNK activation. Both H2O2- and asbestos-induced activation of ERK was abolished by catalase. Moreover, chelation of surface iron from crocidolite fibers or addition of N-acetyl-L-cysteine prevented ERK activation and apoptosis by crocidolite, indicating an oxidative mechanism of cell signaling. The MEK1 inhibitor PD-98059 abrogated asbestos-induced apoptosis, confirming a causal relationship between ERK activation and apoptosis. These results suggest that distinct cell-signaling cascades may be important in phenotypic responses elicited by oxidant stresses.
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PMID:Role of extracellular signal-regulated protein kinases in apoptosis by asbestos and H2O2. 937 31

We have previously demonstrated that hydrogen peroxide (H2O2) treatment of bovine tracheal myocytes increases the activity of extracellular signal-regulated kinases (ERK), serine/threonine kinases of the mitogen-activated protein (MAP) kinase superfamily thought to play a key role in the transduction of mitogenic signals to the cell nucleus. Moreover, H2O2-induced ERK activation was partially reduced by pretreatment with phorbol 12,13-dibutyrate, which depletes protein kinase C (PKC). In this study, we further examined the signaling intermediates responsible for ERK activation by H2O2 in airway smooth muscle, focusing on MAP kinase/ERK kinase (MEK), a dual-function kinase which is required and sufficient for ERK activation in bovine tracheal myocytes; Raf-1, a serine/threonine kinase known to activate MEK; and PKC. Pretreatment of cells with inhibitors of MEK (PD98059), Raf-1 (forskolin), and PKC (chelerythrine) each reduced H2O2-induced ERK activity. In addition, H2O2 treatment significantly increased both MEK1 and Raf-1 activity. No activation of MEK2 was detected. Together these data suggest that H2O2 may stimulate ERK via successive activation of PKC, Raf-1, and MEK1.
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PMID:Hydrogen peroxide activates extracellular signal-regulated kinase via protein kinase C, Raf-1, and MEK1. 953 45

Hydrogen peroxide (H2O2) is a potent stimulator of signal-responsive phospholipase A2 (PLA2) in vascular smooth muscle and cultured endothelial cells. We investigated whether H2O2 plays a similar regulatory role in neurons. H2O2 did not stimulate a release of arachidonic acid from cultured neurons when applied alone but strongly enhanced the liberation of arachidonic acid evoked by maximally effective concentrations of either glutamate, the glutamate receptor agonist N-methyl-D-aspartate (NMDA), the muscarinic receptor agonist carbachol, the Na+-channel opener veratridine, or the Ca2+-ionophore ionomycin. The potentiating effects of H2O2 were strongly inhibited in the presence of the PLA2 inhibitor mepacrine, suggesting that the site of action was within the signal responsive arachidonic acid cascade. The enhancing effect of H2O2 was not reversed by protein kinase C inhibitors (chelerythrine chloride or GF 109203X) nor was it mimicked by phorbol ester treatment. H2O2 alone strongly enhanced the levels of immunodetectable activated mitogen-activated protein kinase (activated MAP kinases ERK1 and ERK2) in a Ca2+-dependent manner and this effect was additive with increases in the levels of activated MAP kinase evoked by glutamate. The enhanced release of arachidonic acid, however, was not clearly reversed by the MAP kinase kinase (MEK) inhibitor PD 98059, although this treatment effectively abolished H2O2 activation of MAP kinase. Thus, MAP kinase activation and Ca2+-dependent arachidonic acid release are regulated by oxidative stress in cultured striatal neurons.
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PMID:Hydrogen peroxide enhances signal-responsive arachidonic acid release from neurons: role of mitogen-activated protein kinase. 957 94

Several recently identified intracellular proteins associate with the tumor necrosis factor (TNF) receptor and activate nuclear transcription factor (NF)-kappaB, c-Jun kinase, and apoptosis. However, the mechanism is not understood. In the present report, we investigated the role of reactive oxygen intermediates in TNF-induced signaling. Overexpression of manganese superoxide dismutase (Mn-SOD) in human breast cancer MCF-7 cells completely abolished TNF-mediated NF-kappaB activation, IkappaB alpha degradation, p65 nuclear translocation, and NF-kappaB-dependent reporter gene expression. Besides TNF, phorbol ester-, okadaic acid-, ceramide-, and lipopolysaccharide-induced activation of NF-kappaB was blocked by Mn-SOD, indicating a common pathway of activation. H2O2-induced NF-kappaB activation, however, was potentiated. In addition, Mn-SOD blocked the TNF-mediated activation of activated protein-1, stress-activated c-Jun protein kinase, and mitogen-activated protein kinase kinase. TNF-induced antiproliferative effects and caspase-3 activation, indicators of apoptosis, were also completely suppressed by transfection of cells with Mn-SOD. Suppression of apoptosis induced by okadaic acid, H2O2, and taxol was also inhibited by Mn-SOD but not that induced by vincristine, vinblastine, or daunomycin. Overall, these results demonstrate that, in addition to several recently identified signaling molecules, reactive oxygen intermediates play a critical role in activation of NF-kappaB, activated protein-1, c-Jun kinase, and apoptosis induced by TNF and other agents.
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PMID:Overexpression of manganese superoxide dismutase suppresses tumor necrosis factor-induced apoptosis and activation of nuclear transcription factor-kappaB and activated protein-1. 958 69


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