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)

We have studied the role of Raf-1 in mitogenesis and cellular transformation induced by G protein-coupled receptors in NIH 3T3 cells transfected with the human m1 muscarinic receptor. We have observed that in m1-expressing NIH 3T3 cells, the cholinergic agonist carbachol induces a dose- and time-dependent shift in the electrophoretic mobility of p72Raf-1, equivalent to that observed when using phorbol esters or platelet-derived growth factor as stimulants. Phosphoamino acid analysis of slower mobility forms of p72Raf-1 revealed both phosphoserine and phosphothreonine. Carbachol potently induced c-Raf activity as judged by its in vitro phosphorylating activity using MEK as a substrate. However, induction of Raf-1 kinase activity by carbachol occurred much earlier than changes in its electrophoretic mobility. Raf-1 kinase activation followed a kinetic similar to that exhibited by an epitope-tagged ERK2 protein when coexpressed in the same cells. Conventional protein kinase C (PKC) inactivation by means of sustained phorbol ester treatment or by a new nontoxic PKC-specific inhibitor, GF 109203X, abolished p72Raf-1 mobility shift induced by carbachol or by phorbol esters. However, c-Raf and ERK2 enzymatic activity in response to carbachol was at least 50-80% PKC-independent. Furthermore, inhibition of PKC failed to affect DNA synthesis or focus formation induced by carbachol in cells expressing m1 receptors. In contrast, cotransfection of NIH 3T3 cells with the Raf-1 dominant negative mutant Raf-301 (K375W) drastically decreased the transforming ability of m1 receptors. Thus, our findings implicate Raf-1 activation in transformation by G protein-coupled receptors. In addition, our data suggest that activation of p72Raf-1 and ERK2 by G protein-coupled receptors involves PKC-independent pathways.
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PMID:Signaling through transforming G protein-coupled receptors in NIH 3T3 cells involves c-Raf activation. Evidence for a protein kinase C-independent pathway. 806 29

To evaluate a possible mechanism for the chronic regulation of MAPK/ERK kinase-1 (MEK-1) and p42 mitogen-activated protein kinase (MAPK) we studied the long-term effects of the G-protein-coupled receptor agonist endothelin-1 (ET-1) and the protein tyrosine kinase-coupled receptor agonist platelet-derived growth factor BB (PDGF BB) on MEK-1 and p42 MAPK in glomerular mesangial cells (GMCs). ET-1 and PDGF BB led to a time-dependent increase in MEK-1 mRNA expression without altering p42 MAPK mRNA levels. The effect of ET-1 and PDGF BB on MEK-1 mRNA expression was maximal after 24 h (3.3-fold) or 6 h (2.9-fold). Furthermore, the effect of ET-1 and PDGF BB on MEK-1 mRNA expression was additive (4.2-fold after 6 h) and was inhibited by actinomycin D (5 micrograms/ml). Cycloheximide (10 micrograms/ml) inhibited MEK-1 mRNA induction but stimulated p42 MAPK mRNA expression in both the absence and the presence of ET-1 and/or PDGF BB. The ET-1 and PDGF BB-induced increase in MEK-1 mRNA was accompanied by sustained enhancement of both p45 MEK protein expression after 12 h and by elevation of p42 MAPK activity for up to 24 h. We conclude that, in GMCs, MEK-1 acts like a delayed-early gene, whereas p42 MAPK resembles an immediate-early gene. MEK-1 mRNA and protein levels, as well as p42 MAPK activity, can be chronically regulated by both a seven-transmembrane domain receptor-coupled peptide such as ET-1 and by an agonist binding to a receptor with intrinsic protein tyrosine kinase activity, such as PDGF BB.
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PMID:ET-1 and PDGF BB induce MEK mRNA and protein expression in mesangial cells. 858 80

The modulation of the activity of mitogen-activated protein kinase (MAPK) by endogenous growth factors or growth inhibitors provides a potential means of regulating cell proliferation. We determined the effect of the endogenous anti-proliferative peptide, atrial natriuretic peptide (ANP), on the ability of MAPK to phosphorylate myelin basic protein. In astrocytes, MAPK activity was significantly stimulated (up to 3-fold) by three known glial mitogens, endothelin-3, platelet-derived growth factor, or phorbol 12-myristate 13-acetate. ANP inhibited by 55-70% the ability of each of these mitogens to activate MAPK. The effects of ANP were equipotent to those caused by C-ANP 4-23, a peptide that specifically binds to the natriuretic peptide clearance receptor. Additionally, both natriuretic peptides caused a 70-80% inhibition of the sodium vanadate-stimulated MAPK activity, complete inhibition of the okadaic acid-stimulated activity, and inhibition of the mitogen-stimulated phosphorylation of MAPK. To understand the potential mechanism by which the natriuretic peptides act, we found that both ANP and C-ANP inhibited the mitogen-stimulated activity of the immediate upstream kinase in the cascade, MAPK kinase (MEK). C-ANP also strongly inhibited the endothelin-3-, platelet-derived growth factor-, and phorbol 12-myristate 13-acetate-induced stimulation of DNA synthesis in the astrocytes, while both okadaic acid and sodium vanadate significantly reversed these anti-proliferative actions. Our results identify ANP as a peptide hormone that inhibits growth factor-stimulated MAPK. These data suggest that the ability of the natriuretic peptides to inhibit MAPK may be important for their anti-growth actions. This effect likely occurs via the inhibition of upstream kinase(s), including MEK, uniquely resulting from ligand binding to the natriuretic peptide clearance receptor.
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PMID:Atrial natriuretic peptide inhibits mitogen-activated protein kinase through the clearance receptor. Potential role in the inhibition of astrocyte proliferation. 866 98

Desensitization of p21(ras) after stimulation of cells by growth factors and phorbol 12-myristate 13-acetate (PMA) correlates with hyperphosphorylation of the guanine nucleotide exchange factor Son-of-sevenless (Sos) and its dissociation from the adaptor protein Grb2 (Cherniack, A., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488). To test the role of the Raf/mitogen-activated protein (MAP) kinase pathway, we utilized cells expressing a chimera composed of the catalytic domain of p74Raf-1 and the hormone binding domain of the estradiol receptor (DeltaRaf-1:ER). Estradiol markedly stimulated DeltaRaf-1:ER and the downstream MEK and MAP kinases in these cells as well as Sos phosphorylation. However, the dissociation of Grb2 from Sos observed in response to PMA was not apparent upon DeltaRaf-1:ER activation. Furthermore, stimulation of DeltaRaf-1:ER did not impair GTP loading of p21(ras) in response to platelet-derived growth factor or epidermal growth factor. We conclude that activation of the Raf/MAP kinase pathway alone in these cells is insufficient to cause disassembly of Sos from Grb2 or to interrupt the ability of Sos to catalyze activation of p21(ras).
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PMID:Role of the Raf/mitogen-activated protein kinase pathway in p21ras desensitization. 866 95

The duration of extracellular signal-regulated protein kinase (ERK) activation is critical for cell signaling decisions and probably determines whether a stimulus elicits proliferation or differentiation. We studied the intracellular signals regulating sustained ERK-2 activity in glomerular mesangial cells (GMC), utilizing combination of GMC mitogens of different potency. Incubation of GMC with both endothelin-1 (ET-1) and platelet-derived growth factor BB (PDGF-BB) led to a long-lasting, monophasic increase in ERK-2 activity. In contrast, when ET-1 was administered together with epidermal growth factor (EGF), a less pronounced and shorter activation occurred. Long-term stimulation of ERK-2 was accompanied by an increase in p45 MEK activity, which again was more pronounced when ET-1 was administered together with PDGF-BB compared with EGF. In the presence of actinomycin D (Act D), an inhibitor of RNA synthesis, ERK-2 activity induced by ET-1 and PDGF-BB but not by ET-1 and EGF remained elevated more than sixfold throughout the whole incubation period of 6 h. The effect of Act D on ET-1- and PDGF-BB-induced ERK-2 activation was mimicked by the protein phosphatase inhibitor sodium orthovanadate. In addition, vanadate also unmarked an ET-1- and EGF-induced ERK-2 activity after 6 h. The serine/threonine phosphatase inhibitor okadaic acid (OA) did neither alter agonist-induced ERK-2 activity after 6 h (0.5 nM OA) nor after 10 min or 1 h (250 nM). Together these results suggest that, in GMC, long-term activation of the mitogen-activated protein kinase ERK-2 is differentially regulated, depending on the combination of agonists administered. ET-1- and PDGF-BB-induced long-term activation of ERK-2 is regulated by a vanadate-sensitive protein phosphatase(s) and by a transcriptionally regulated protein(s). In contrast, ET-1- and EGF-induced sustained ERK-2 stimulation is regulated by a vanadate-sensitive protein phosphatase(s) but not by a transcriptionally regulated protein. Agonist-specific and time-dependent stimulation of ERK-2-regulating protein phosphatases may be critical for the length of ERK-2 activation in GMC and could thus be of pathophysiological significance in glomerular diseases associated with alterations in cell proliferation or cell differentiation.
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PMID:Sustained ERK-2 activation in rat glomerular mesangial cells: differential regulation by protein phosphatases. 877 Jan 75

In rat aortic smooth muscle cells, platelet-derived growth factor (PDGF) stimulated a sustained activation of mitogen-activated protein kinase (MAP kinase) while the response to angiotensin II (AII) was transient. This was due to a relatively greater initial activation of MAP kinase kinase (MEK) and a correspondingly greater residual MEK activity at later time points. Pretreatment of cells with the novel MEK inhibitor PD 098059 reduced MEK activation at 5 min in response to each agonist by a similar proportion (70%); however, at this time point MAP kinase activation in response to PDGF was only marginally affected while the response to AII was substantially reduced. PD 098059 did, however, reduce PDGF-stimulated MEK activity after 30 min and this correlated with a loss in MAP kinase activity and DNA synthesis. Pretreatment with forskolin also caused a similar pattern of inhibition of agonist-stimulated MEK and MAP kinase activity. Only following protein kinase C down-regulation were both AII- and PDGF-stimulated MAP kinase activation substantially reduced and this correlated with the virtual loss of both MEK and c-Raf-1 activity in response to both agents. The differential inhibition of MAP kinase activation by forskolin was not due to specific activation of A-Raf by PDGF; both PDGF and AII stimulated A-Raf kinase and this activity was strongly inhibited by forskolin. These results suggest that the efficacy of MEK activation determines the duration of MAP kinase activation and the susceptibility of MAP kinase activation to inhibition by different agents. The results also argue against the selective activation of A-Raf by PDGF as a mechanism to explain the differences in the kinetics of MAP kinase activity stimulated by AII and PDGF.
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PMID:Efficacy of agonist-stimulated MEK activation determines the susceptibility of mitogen-activated protein (MAP) kinase to inhibition in rat aortic smooth muscle cells. 880 60

Vascular smooth muscle cell (VSMC) proliferation and migration are responses to arterial injury that are highly important to the processes of restenosis and atherosclerosis. In the arterial balloon injury model in the rat, platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are induced in the vessel wall and regulate these VSMC activities. Novel insulin sensitizing agents, thiazolidinediones, have been demonstrated to inhibit insulin and epidermal growth factor-induced growth of VSMCs. We hypothesized that these agents might also inhibit the effect of PDGF and bFGF on cultured VSMCs and intimal hyperplasia in vivo. Troglitazone (1 microM), a member of the thiazolidinedione class, produced a near complete inhibition of both bFGF-induced DNA synthesis as measured by bromodeoxyuridine incorporation (6.5+/-3.9 vs. 17.6+/-4.3% cells labeled, P < 0.05) and c-fos induction. This effect was associated with an inhibition (by 73+/-4%, P < 0.01) by troglitazone of the transactivation of the serum response element, which regulates c-fos expression. Inhibition of c-fos induction by troglitazone appeared to occur via a blockade of the MAP kinase pathway at a point downstream of MAP kinase activation by MAP kinase kinase. At this dose, troglitazone also inhibited PDGF-BB-directed migration of VSMC (by 70+/-6%, P < 0.01). These in vitro effects were operative in vivo. Quantitative image analysis revealed that troglitazone-treated rats had 62% (P < 0.001) less neointima/media area ratio 14 d after balloon injury of the aorta compared with injured rats that received no troglitazone. These results suggest troglitazone is a potent inhibitor of VSMC proliferation and migration and, thus, may be a useful agent to prevent restenosis and possibly atherosclerosis.
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PMID:Troglitazone inhibits vascular smooth muscle cell growth and intimal hyperplasia. 887 42

Both angiotensin II (Ang II) and platelet-derived growth factor (PDGF) rapidly increase intracellular Ca2+ and activate protein kinase C (PKC) and MAP kinase in vascular smooth muscle cells (VSMCs). However, Ang II causes cell hypertrophy, whereas PDGF causes hyperplasia. These findings indicate that VSMCs are a good model for studying the relationship between cell growth and the MAP kinase pathway. In this study, we investigated the role of Raf in activation of 42- and 44-kD MAP kinases. Western blot analysis showed that c-Raf-1 was the predominant Raf isozyme in cultured rat aortic VSMCs. In response to Ang II, there was translocation of Raf to the membrane, which occurred significantly earlier than MAP kinase activation, suggesting that Raf activation precedes MAP kinase activation. Translocation of Raf to the membrane resulted in association with H-Ras as shown by c-Raf-1 coprecipitation with anti-Ras anti-bodies. Western blot analysis of H-Ras immunoprecipitates revealed c-Raf-1, but c-mos, MEK (MAP kinase/extracellular signal-regulated kinase) kinase-1 (MEKK-1), and Raf-B were not present. MAP kinase kinase kinase (MAPKKK) activity was assayed in c-Raf-1 and H-Ras immunoprecipitates by MAP kinase kinase-dependent phosphorylation of catalytically inactive 42-kD MAP kinase. In Ras immunoprecipitates, MAPKKK activity was stimulated approximately threefold by both Ang II and PDGF, with a peak at 5 minutes. Downregulation of PKC by 24-hour exposure to phorbol ester significantly inhibited Ang II-stimulated and PDGF-stimulated MAPKKK activity (approximately 80% decrease) and Raf translocation (approximately 90% decrease), suggesting that a phorbol-responsive PKC is upstream from MAPKKK and Raf. In contrast, Ang II (but not PDGF) stimulation of MAP kinase was unaffected by PKC downregulation or pharmacological PKC inhibition. These findings demonstrate for the first time that Ang II stimulation of MAP kinase may occur via a pathway independent of c-Raf-1 and of the phorbol-responsive PKC isozymes. The differing effects of Ang II and PDGF on VSMC growth may be a consequence of specific signal transduction events, as demonstrated here for activation of MAP kinase.
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PMID:Angiotensin II stimulates MAP kinase kinase kinase activity in vascular smooth muscle cells, Role of Raf. 888 93

Prostaglandin synthase 2 (PGS2) is an immediate-early gene induced in a variety of cellular contexts. We investigate here the transcriptional activation of the murine PGS2 gene in NIH 3T3 cells, in response to the mitogens platelet-derived growth factor (PDGF) or serum. Site-directed mutagenesis experiments demonstrate that a consensus cyclic AMP response element (CRE) in the murine PGS2 promoter is essential for optimal PGS2 gene expression in response to PDGF or to serum. Overexpression of c-Jun potentiates PDGF- or serum-induced luciferase expression from a reporter construct containing the first 371 nucleotides of the PGS2 promoter. In contrast, overexpression of other transcription factors binding to the CRE element of the PGS2 gene inhibits induction by PDGF or serum. Moreover, positioning the c-Jun activation domain next to the minimal PGS2 promoter via a GAL4 DNA binding site rather than the CRE is sufficient to permit serum or PDGF stimulation of luciferase expression from this modified reporter construct. PDGF or serum treatment both activate c-Jun N-terminal kinase (JNK), the mitogen-activated protein kinase responsible for phosphorylation and activation of c-Jun. Cotransfection of plasmids expressing dominant-negative Ras, Rac1, MEKK-1, or JNK along with the [PGS2][luciferase] reporter prevents induction by PDGF or serum, demonstrating that serum and PDGF induction of the PGS2 gene in NIH 3T3 cells requires activation of a Ras/Rac1/MEKK-1/JNK kinase/JNK signal transduction leading to phosphorylation of c-Jun. Additional cotransfection experiments with plasmids expressing dominant-negative Raf1 and ERK demonstrate that induction of PGS2 gene expression by PDGF and serum also requires activation of a Ras/Raf1/mitogen-activated protein kinase kinase (MAPKK)/ERK signal transduction pathway.
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PMID:Transcriptional regulation of prostaglandin synthase 2 gene expression by platelet-derived growth factor and serum. 894 Jan 99

We have investigated the requirement for mitogen-activated protein (MAP) kinase in the stimulation of DNA synthesis by platelet-derived growth factor (PDGF) in rat aortic smooth muscle cells using a phosphorothioate-modified oligodeoxy-nucleotide (ODN) to deplete MAP kinase. Treatment for 72 h with MAP kinase antisense ODN directed against both the p42 and p44 isoforms of MAP kinase abolished the expression of MAP kinase and reduced agonist-stimulated MAP kinase activity by approx. 95%. The scrambled control ODN was without effect, but the sense control ODN slightly enhanced the expression of both isoforms. Abolition of MAP kinase activity by antisense ODN treatment prevented angiotensin II- and PDGF-stimulated activation of p90 ribosomal S6 kinase activity, but did not affect activation of MAP kinase kinase. In addition, antisense ODN pretreatment reduced PDGF-stimulated [3H]thymidine incorporation to < 5% of control, and decreased basal incorporation by approx. 90%. In contrast, basal [3H]thymidine incorporation was enhanced approx. 60% by control sense ODN treatment. These results indicate an obligatory role for MAP kinase in the activation of a number of early events in mitogenesis, including DNA synthesis, in vascular smooth muscle cells.
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PMID:Treatment of vascular smooth muscle cells with antisense phosphorothioate oligodeoxynucleotides directed against p42 and p44 mitogen-activated protein kinases abolishes DNA synthesis in response to platelet-derived growth factor. 894 76


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