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)

WAVE is a Wiskott-Aldrich syndrome protein (WASP)-family protein that functions in membrane-ruffling formation induced by Rac, a Rho family small GTPase. Here we report that WAVE is a phosphoprotein whose phosphorylation increases in response to various external stimuli that activate mitogen-activated protein (MAP) kinase signaling. When Swiss 3T3 cells are stimulated with platelet-derived growth factor, electrophoretic mobility shift occurs to WAVE, which reflects hyperphosphorylation. This is perfectly inhibited by the addition of PD98059, a specific inhibitor of MAP kinase kinase. Indeed, the ectopic expression of an activated mutant of MAP kinase kinase induces WAVE mobility shift. When MAP kinase activation is suppressed by PD98059, the intensity of platelet-derived growth factor-induced membrane ruffling is greatly reduced. In various cancer cell lines, the amount of WAVE mobility shift was found to increase significantly, suggesting the importance of WAVE hyperphosphorylation in the formation of membrane ruffles and oncogenic transformation.
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PMID:Phosphorylation of WAVE downstream of mitogen-activated protein kinase signaling. 1048 99

p21(Waf1/Cip1) (hereafter referred to as p21) is up-regulated in differentiating and DNA-damaged cells, but it is also up-regulated by serum and growth factors. We show here that fibroblast growth factor-2 (FGF-2), platelet-derived growth factor (PDGF), and transforming growth factor-beta1 (TGF-beta1) all induce p21 expression in mouse fibroblasts, but with markedly different kinetics. We link their effect on p21 to Ras and mitogen-activated protein kinase kinase-1(/2) [MEK1(/2)]-regulated pathways using either a specific MEK1(/2) inhibitor (PD 098059) or cells expressing conditionally activated Ras or dominant negative Ras. We demonstrate that p21 induction by PDGF and TGF-beta1 requires MEK1(/2) and, additionally, that the TGF-beta1 effect on p21 depends on Ras, whereas the PDGF effect does not. In contrast, FGF-2 regulation of p21 is largely independent of MEK and Ras. However, PD 098059 efficiently inhibited S-phase entry of quiescent cells induced by either FGF-2 or PDGF, suggesting separate signaling pathways for FGF-2 in induction of p21 and in S-phase entry. The results suggest different but partly overlapping signaling pathways in growth factor regulation of p21.
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PMID:Ras- and mitogen-activated protein kinase kinase-dependent and -independent pathways in p21Cip1/Waf1 induction by fibroblast growth factor-2, platelet-derived growth factor, and transforming growth factor-beta1. 1051 12

Vascular smooth muscle cells (VSMC) exist in either a contractile or a synthetic phenotype in vitro and in vivo. The molecular mechanisms regulating phenotypic modulation are unknown. Previous studies have suggested that the serine/threonine protein kinase mediator of nitric oxide (NO) and cyclic GMP (cGMP) signaling, the cGMP-dependent protein kinase (PKG) promotes modulation to the contractile phenotype in cultured rat aortic smooth muscle cells (RASMC). Because of the potential importance of the mitogen-activated protein kinase (MAP kinase) pathways in VSMC proliferation and phenotypic modulation, the effects of PKG expression in PKG-deficient and PKG-expressing adult RASMC on MAP kinases were examined. In PKG-expressing adult RASMC, 8-para-chlorophenylthio-cGMP activated extracellular signal- regulated kinases (ERK1/2) and c-Jun N-terminal kinase (JNK). The major effect of PKG activation was increased activation by MAP kinase kinase (MEK). The cAMP analog, 8-Br-cAMP inhibited ERK1/2 activation in PKG-deficient and PKG-expressing RASMC but had no effect on JNK activity. The effects of PKG on ERK and JNK activity were additive with those of platelet-derived growth factor (PDGF), suggesting that PKG activates MEK through a pathway not used by PDGF. The stimulatory effects of cGMP on ERK and JNK activation were also observed in low-passaged, contractile RASMC still expressing endogenous PKG, suggesting that the effects of PKG expression were not artifacts of cell transfections. These results suggest that in contractile adult RASMC, NO-cGMP signaling increases MAP kinase activity. Increased activation of these MAP kinase pathways may be one mechanism by which cGMP and PKG activation mediate c-fos induction and increased proliferation of contractile adult RASMC.
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PMID:Activation of mitogen-activated protein kinase pathways by cyclic GMP and cyclic GMP-dependent protein kinase in contractile vascular smooth muscle cells. 1056 6

Vascular smooth muscle cell (SMC) apoptosis contributes to physiological and pathological vascular remodeling. Autocrine fibroblast growth factor (FGF) signaling promotes survival in SMC in vitro. Interruption of autocrine FGF signaling results in apoptosis that can be rescued by other growth factors such as PDGF (platelet-derived growth factor) or EGF (epidermal growth factor). Such heterologous growth factor rescue is prevented by pharmacological inhibition of MAPK, implicating signaling through Ras in mediating survival. This study was designed to test the hypothesis that signaling through Ras is both necessary and sufficient to mediate SMC survival in vitro. Recombinant adenoviruses encoding dominant-negative (Ras(N17)) and constitutively active (Ras(L61)) mutants of Ras were used. Ras(N17) blocks growth factor-mediated MAPK activation and can itself induce SMC apoptosis. Ras(N17) is synergistic with inhibition of autocrine FGF signaling in triggering apoptosis and prevents heterologous growth factor rescue. Conversely, Ras(L61) prevents apoptosis resulting from inhibition of autocrine FGF signaling. Rescue by Ras(L61) can be partially prevented by pharmacological inhibition of MEK or phosphatidylinositol 3-kinase, two downstream effectors of Ras. These results suggest that Ras signaling is both necessary and sufficient to mediate survival in SMC in vitro. Further work is required to determine how these signaling events are regulated in the context of vascular remodeling in vivo.
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PMID:Autocrine signaling through Ras prevents apoptosis in vascular smooth muscle cells in vitro. 1064 48

Overgrowth of rheumatoid synoviocytes, which results in joint destruction, is due to impaired balance between cell proliferation and cell death (apoptosis). Ceramide is an important lipid messenger involved in mediating a variety of cell functions including apoptosis. We investigated the effects of ceramide on growth-promoting anti-apoptotic signals in rheumatoid synovial cells. Human synovial cells isolated from patients with rheumatoid arthritis (RA) were stimulated with platelet-derived growth factor (PDGF) in the presence or absence of C2-ceramide. The kinase activity of Akt, MEK, and ERK1/2 was analyzed in PDGF-stimulated synovial cells by Western blot analysis. Pretreatment with C2-ceramide completely inhibited PDGF-induced cell cycle progression of rheumatoid synovial cells. PDGF stimulation induced phosphorylation and activation of Akt, MEK, and ERK1/2 in rheumatoid synovial cells. C2-ceramide inhibited the activation of Akt, MEK and ERK1/2 in PDGF-stimulated synovial cells. Our data demonstrated that inhibition of anti-apoptotic kinases, such as Akt and ERK1/2, may play an important role in ceramide-mediated apoptosis of rheumatoid synovial cells.
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PMID:Regulation of rheumatoid synovial cell growth by ceramide. 1069 79

Caveolin-1 is the major coat protein of caveolae and has been reported to interact with various intracellular signaling molecules including the epidermal growth factor (EGF) receptor. To investigate the involvement of caveolin-1 in EGF receptor action, we used mouse B82L fibroblasts transfected with (a) wild type EGF receptor, (b) a C-terminally truncated EGF receptor at residue 1022, (c) a C-terminally truncated EGF receptor at residue 973, or (d) a kinase-inactive EGF receptor (K721M). Following EGF treatment, there was a distinct electrophoretic mobility shift of the caveolin-1 present in cells expressing the truncated forms of the EGF receptor, but this shift was not detectable in cells bearing either normal levels of the wild type EGF receptor or a kinase-inactive receptor. This mobility shift was also not observed following the addition of other cell stimuli, such as platelet-derived growth factor, insulin, basic fibroblast growth factor, or phorbol 12-myristate 13-acetate. Analysis of caveolin-1 immunoprecipitates from EGF-stimulated or nonstimulated cells demonstrated that the EGF-induced mobility shift of caveolin-1 was associated with its tyrosine phosphorylation in cells expressing truncated EGF receptors. Maximal caveolin-1 phosphorylation was achieved within 5 min after exposure to 10 nM EGF and remained elevated for at least 2 h. Additionally, several distinct phosphotyrosine-containing proteins (60, 45, 29, 24, and 20 kDa) were co-immunoprecipitated with caveolin-1 in an EGF-dependent manner. Furthermore, the Src family kinase inhibitor, PP1, does not affect autophosphorylation of the receptor, but it does inhibit the EGF-induced mobility shift and phosphorylation of caveolin-1. Conversely, the MEK inhibitors PD98059 and UO126 could attenuate EGF-induced mitogen-activated protein kinase activation, they do not affect the EGF-induced mobility shift of caveolin-1. Because truncation and overexpression of the EGF receptor have been linked to cell transformation, these results provide the first evidence that the tyrosine phosphorylation of caveolin-1 occurs via an EGF-sensitive signaling pathway that can be potentiated by an aberrant activity or expression of various forms of the EGF receptor.
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PMID:Epidermal growth factor-stimulated tyrosine phosphorylation of caveolin-1. Enhanced caveolin-1 tyrosine phosphorylation following aberrant epidermal growth factor receptor status. 1071 51

Platelet-derived growth factor (PDGF), which consists of an A and/or B chain, stimulates migration and proliferation in vascular smooth muscle cells as well as a large number of other cell types. Investigations over recent years have defined roles for several positive regulatory transcription factors in the PDGF-B promoter. However, little is known about the transcriptional mechanisms that negatively regulate this gene. Here, we used transient transfection and 5' deletion analysis to define a specific region in the PDGF-B promoter-mediating repression in vascular smooth muscle cells. Gel retardation assays revealed this region is bound by nuclear protein(s) in a specific manner. Supershift assays excluded the direct association of Sp1, Sp3, and Egr-1. Mutation of the negative regulatory element no longer supported nucleoprotein complex formation and, when introduced into the PDGF-B promoter, rescued the promoter from repression. Promoter activity was also restored by transfection of oligonucleotide decoys bearing the repressor binding site. The MEK1/2 inhibitor, PD98059, and a dominant negative construct generating inactive ERK1 increased reporter expression driven by the PDGF-B promoter. In contrast, the MEK inhibitor had no effect on the activity of the mutant PDGF-B promoter. These effects were cell type-specific, since neither suppression of the PDGF-B promoter nor nucleoprotein complex formation was observed in vascular endothelial cells. These findings define a distinct negative regulatory element in the PDGF-B promoter that interacts with nuclear protein(s) and inhibits PDGF-B promoter-dependent gene expression in an ERK-dependent manner.
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PMID:Novel negative regulatory element in the platelet-derived growth factor B chain promoter that mediates ERK-dependent transcriptional repression. 1075 66

In human osteoblast-like MG-63 cells, extracellular ATP increased [(3)H]thymidine incorporation and cell proliferation and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced [(3)H]thymidine incorporation. ATP-induced [(3)H]thymidine incorporation was mimicked by the nonhydrolyzable ATP analogs adenosine 5'-O-(3-thiotriphosphate) and adenosine 5'-adenylylimidodiphosphate and was inhibited by the P2 purinoceptor antagonist suramin, suggesting involvement of P2 purinoceptors. The P2Y receptor agonist UTP and UDP and a P2Y receptor antagonist reactive blue 2 did not affect [(3)H]thymidine incorporation, whereas the P2X receptor antagonist pyridoxal phosphate-6-azophenyl-2',4-disulfonic acid inhibited ATP-induced [(3)H]thymidine incorporation, suggesting that ATP-induced DNA synthesis was mediated by P2X receptors. RT-PCR analysis revealed that MG-63 cells expressed P2X(4), P2X(5), P2X(6), and P2X(7), but not P2X(1), P2X(2), and P2X(3), receptors. In fura 2-loaded cells, not only ATP, but also UTP, increased intracellular Ca(2+) concentration, and inhibitors for several Ca(2+)-activated protein kinases had no effect on ATP-induced DNA synthesis, suggesting that an increase in intracellular Ca(2+) concentration is not indispensable for ATP-induced DNA synthesis. ATP increased mitogen-activated protein kinase activity in a Ca(2+)-independent manner and synergistically enhanced platelet-derived growth factor- or insulin-like growth factor I-induced kinase activity. Furthermore, the mitogen-activated protein kinase kinase inhibitor PD-98059 totally abolished ATP-induced DNA synthesis. We conclude that ATP increases DNA synthesis and enhances the proliferative effects of growth factors through P2X receptors by activating a mitogen-activated protein kinase pathway.
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PMID:ATP activates DNA synthesis by acting on P2X receptors in human osteoblast-like MG-63 cells. 1091 18

CeReS-18, a cell regulatory sialoglycopeptide, has been shown to inhibit proliferation of a wide array of target cells. In the present study, the effect of CeReS-18 on vascular smooth muscle cell (SMC) proliferation was characterized in cultured rat aorta SMCs (A7r5). More extensively, the effect of CeReS-18 on platelet-derived growth factor (PDGF)-induced SMC migration was examined using a modified Boyden's chamber assay. CeReS-18 inhibits both SMC proliferation and migration in a concentration-dependent, calcium-sensitive, and reversible manner. Furthermore, cells preincubated with the inhibitor had an increased sensitivity to CeReS-18-mediated inhibition of SMC migration. Immunoprecipitation and in vitro phosphorylation assays demonstrated that MAP kinase activity was inhibited in the CeReS-18-treated cells and pretreatment with CeReS-18 suppressed the activation of MAP kinase stimulated by PDGF. However, it is not likely that the suppression of the MAP kinase pathway was directly responsible for the ability of CeReS-18 to inhibit migration of the rat aorta smooth muscle cells since a MEK-specific inhibitor, PD98059, did not influence A7r5 cell migration.
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PMID:CeReS-18, a cell regulatory sialoglycopeptide, inhibits proliferation and migration of rat vascular smooth muscle cells. 1103 12

Previous studies have demonstrated that a number of biochemical actions of ceramide are mediated through protein kinase signalling pathways, such as p42/p44 mitogen-activated protein kinase (p42/p44 MAPK) and c-Jun N-terminal directed protein kinase (JNK). Ceramide-activated protein kinases, such as the kinase suppressor of Ras (KSR) and protein kinase Czeta (PKCzeta), are involved in the regulation of c-Raf, which promotes sequential activation of MEK-1 and p42/p44 MAPK in mammalian cells. However, in cultured airway smooth muscle (ASM) cells, neither KSR nor PKCzeta are involved in the C2-ceramide (C2-Cer)-dependent activation of this kinase cascade. Instead, we found that C2-Cer utilises a novel pathway involving tyrosine kinases, phosphoinositide 3-kinase (PI3K) and conventional PKC isoform(s). We also found that despite its ability to stimulate p42/p44 MAPK, C2-Cer inhibited platelet-derived growth factor (PDGF)-stimulated DNA synthesis. The possibility that growth arrest could be mediated by JNK was discounted on the basis that PDGF, as well as ceramide, stimulated JNK in these cells. Therefore, growth arrest in response to ceramide is mediated by an alternative mechanism.
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PMID:Ceramide-dependent regulation of p42/p44 mitogen-activated protein kinase and c-Jun N-terminal-directed protein kinase in cultured airway smooth muscle cells. 1115 59


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