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)

Vascular endothelial growth factor (VEGF) stimulated the tyrosine phosphorylation of multiple components in confluent human umbilical vein endothelial cells (HUVECs) including bands of Mr 205,000, corresponding to the VEGF receptors Flt-1 and KDR, and Mr 145,000, 120,000, 97,000, and 65,000-70,000. VEGF caused a striking and transient increase in mitogen-activated protein (MAP) kinase activity and stimulated phospholipase C-gamma tyrosine phosphorylation, but it had no effect on phosphatidylinositol 3'-kinase activity. VEGF caused a marked increase in tyrosine phosphorylation of p125 focal adhesion kinase (p125(FAK)), which was both rapid and concentration-dependent. VEGF produced similar effects on p125(FAK) in the endothelial cell line ECV.304. VEGF stimulated tyrosine phosphorylation of the 68-kDa focal adhesion-associated component, paxillin, with similar kinetics and concentration dependence to that for p125(FAK). Thrombin and the phorbol ester, phorbol 12-myristate 13-acetate, also increased p125(FAK) tyrosine phosphorylation in HUVECs. The effect of VEGF on p125(FAK) tyrosine phosphorylation was completely inhibited by the actin filament-disrupting agent cytochalasin D and was partially inhibited by the protein kinase C inhibitor GF109203X. Inhibition of the MAP kinase pathway using a specific inhibitor of MAP kinase kinase had no effect on p125(FAK) tyrosine phosphorylation. VEGF stimulated migration and actin stress fiber formation in confluent HUVEC, and VEGF-induced p125(FAK)/paxillin tyrosine phosphorylation was accompanied by increased immunofluorescent staining of p125(FAK), paxillin, and phosphotyrosine in focal adhesions in confluent cultures of HUVECs. These findings identify p125(FAK) and paxillin as components in a VEGF-stimulated signaling pathway and suggest a novel mechanism for VEGF regulation of endothelial cell functions.
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PMID:Vascular endothelial growth factor stimulates tyrosine phosphorylation and recruitment to new focal adhesions of focal adhesion kinase and paxillin in endothelial cells. 918 76

The role of mitogen-activated protein (MAP) kinase cascades in platelet function remains to be determined. Several studies have suggested a role in the activation of phospholipase A2; however, other functions seem likely. The object of the present study was to determine the role of the MAP kinase cascade in platelet function. An inhibitor of the mitogen-activated protein kinase kinase MEK1, 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059), was used, at concentrations consistent with those reported to inhibit MEK1, to examine the role that this enzyme plays in platelet function. PD98059 inhibited aggregation in response to low-dose collagen and arachidonic acid, but not that in response to high-dose collagen, thrombin, thrombin receptor-activating peptide (TRAP), 9,11-dideoxy-11alpha, 9alpha-epoxymethano-prostaglandin F2alpha (U46619), or phorbol ester. Thrombin, thrombin receptor-activating peptide, U46619, collagen, and arachidonic acid each caused the release of [3H]serotonin from dense granules, but only that elicited by low-dose collagen and arachidonic acid was inhibited by PD98059. The release of [3H]arachidonic acid in response to thrombin or collagen was unaffected by PD98059 pretreatment. In contrast, collagen- and arachidonic acid-induced thromboxane formation was inhibited by PD98059. These data suggest that MEK1 is not involved in the platelet response to thrombin or U46619. Furthermore, the inhibitory effects of PD98059 on collagen- and arachidonic acid-induced responses suggest that PD98059 may inhibit the conversion of arachidonic acid to thromboxane, in addition to its reported effects on MEK1.
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PMID:Effects of the mitogen-activated protein (MAP) kinase kinase inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) on human platelet activation. 971 93

In CCL39 cells thrombin is a potent growth factor which requires sustained activation of mitogen activated protein kinases (MAPKs) to promote DNA synthesis. Some of the effects of thrombin can be mimicked by peptides based on the new amino terminus of the cleaved receptor; however, these thrombin receptor peptides (TRPs) fail to induce sustained activation of MAPK or DNA synthesis. We have used thrombin, TRP-7 and other agonists which elicit sustained or transient MAPK activation to identify immediate-early and delayed-early genes which are only expressed under conditions of sustained MAPK activation focusing on cyclin D1, p21CiP1 and the AP-1 transcription factor. Of the stimuli tested only FBS and thrombin were able to stimulate a sustained activation of MAPK, expression of cyclin D1, p21Cip1 and cell cycle re-entry. The expression of cyclin D1 was strongly, though not completely, inhibited by the MEK1 inhibitor PD098059. Thrombin stimulated a rapid but transient accumulation of c-Fos whereas the expression of Fra-1, Fra-2, c-Jun and JunB was sustained throughout the G1 phase of the cell cycle. We focussed our analysis on c-Fos (typical of AP-1 genes which are expressed rapidly and transiently) and Fra-1 and JunB (typical of AP-1 genes expressed after a delay but in a sustained manner). The expression of c-Fos, Fra-1 and JunB was dependent upon the activation of MAPK since these responses were inhibited by PD098059. However, a comparison of responses to FBS, thrombin, TRPs, LPA and EGF revealed that Fra-1 and JunB expression required sustained activation of MAPK whereas c-Fos expression was strongly induced even by non-mitogenic stimuli which elicited only transient MAPK activation. The expression of c-Fos (in response to thrombin, TRP or LPA) or Fra-1, JunB and cyclin D1 (thrombin only) was also inhibited by pertussis toxin. This suggests that both early and late AP-1 gene expression is regulated by the same Gi-mediated, MEK-dependent MAPK signalling pathway but that expression of late AP-1 genes and cyclin D1 requires that this pathway be persistently activated. The results suggest that the duration of receptor signalling and therefore MAPK activation is a key determinant of qualitative changes in gene expression during cell cycle re-entry.
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PMID:Sustained MAP kinase activation is required for the expression of cyclin D1, p21Cip1 and a subset of AP-1 proteins in CCL39 cells. 1034 Mar 80

We have previously shown that glucocorticoids inhibit mitogen-stimulated proliferation of human cultured airway smooth muscle (ASM) cells. The present study analyzed the effect of glucocorticoids on key regulatory pathways leading to passage of cells through the restriction point of the cell cycle, including those mediated by extracellular-regulated kinases (ERK) 1 and 2; the ERK upstream regulator MAPK kinase (MEK1); cyclin D1 levels; and levels and phosphorylation of retinoblastoma protein (pRb). Fluticasone propionate, a new inhaled glucocorticoid, was at least 10-fold more potent than dexamethasone in inhibiting thrombin-stimulated DNA synthesis and increases in cell number. Thrombin-stimulated increases in the levels and hyperphosphorylation of pRb were inhibited by glucocorticoids, which also reduced thrombin-stimulated cyclin D1 protein and messenger RNA (mRNA) levels. PD98059 (10 microM), an inhibitor of MEK1 activation, markedly attenuated thrombin stimulation of ERK activity and phosphorylation, DNA synthesis, and cyclin D1 levels. However, glucocorticoids had no effect on ERK activity or phosphorylation at 5 min, 2 h, or 12 h after addition of thrombin. In conclusion, glucocorticoid-induced reduction of cyclin D1 mRNA and protein levels, and of pRb phosphorylation, is sufficient to account for inhibition of ASM proliferation. Furthermore, these inhibitory effects of glucocorticoids on cyclin D1 and pRb occur on a component of the mitogen signaling cascade that is either downstream of or parallel to the ERK pathway.
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PMID:Glucocorticoids inhibit proliferation, cyclin D1 expression, and retinoblastoma protein phosphorylation, but not activity of the extracellular-regulated kinases in human cultured airway smooth muscle. 1038 95

Thrombin has been shown to stimulate endothelin release by endothelial cells, but the ability of thrombin to induce endothelin in nonendothelial cells is less well-known. Incubation of rat aortic smooth muscle cells with thrombin resulted in a stimulation of preproendothelin-1 (preproET-1) mRNA expression. This induction of preproET-1 mRNA expression by thrombin was accompanied by the release of immunoreactive peptide ET-1 into the extracellular medium. The synthetic thrombin receptor activator peptide (TRAP) confirmed ligand-specific receptor action to induce preproET-1 mRNA. Nuclear run-on analysis revealed that the transcriptional rate of preproET-1 mRNA increases twofold after 1 h of incubation with thrombin. In cells treated with thrombin, the half-life of preproET-1 mRNA was identical to that in untreated control cells. These results demonstrated that thrombin regulates endothelin synthesis at a transcriptional level but does not influence mRNA stability. Inhibition of protein kinase C (PKC) with selective inhibitors (chelerythrine and bisindolylmaleimide I) before thrombin stimulation failed to significantly inhibit preproET-1 gene expression. Inhibition of mitogen-activated protein (MAP) kinase kinase and protein tyrosine kinase decreased preproET-1 mRNA expression in thrombin-stimulated smooth muscle cells. Furthermore, addition of an activator of peroxisome proliferator-activated receptors-alpha (PPARalpha), fenofibrate, prevented the preproET-1 gene induction in response to thrombin. These results demonstrated that thrombin-induced endothelin gene transcription involved MAP kinase kinase rather than the PKC cascade in smooth muscle cells, which was repressed by PPARalpha stimulation.
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PMID:Thrombin induces endothelin expression in arterial smooth muscle cells. 1077 40

The interaction of platelets with subendothelial von Willebrand factor (VWF), especially under high shear stress, is considered to be the first activation step which primes platelets for subsequent haemostatic events. The signalling cascade which results from the interaction of VWF and its receptor GPIbIX has only been partially defined. Mitogen-activated protein kinases (MAPKs) are a family of downstream transmembrane signalling serine-threonine kinases and have been demonstrated to be present and functional in platelets; these include the extracellular signal-related kinases (ERKs), c-Jun amino-terminal kinases (JNKs) and p38 MAPK. Previously, we showed that p38 MAPK was not required in VWF-induced human platelet activation. It is not known whether VWF-dependent platelet activation involves the activation of the JNK and ERK family of signalling molecules. This report demonstrates that porcine von Willebrand factor (pVWF) induced a sustained and stable JNK activation measurable by 1 min after activation. Thrombin also induced JNK activation assessed at 1 min after activation. In contrast to thrombin, pVWF did not induce ERK2 activation at any time point tested. To ensure that ERK activation was unnecessary for pVWF-dependent platelet activation, we functionally inhibited ERK-dependent signalling with PD98059, a potent and selective inhibitor of the MAP kinase kinase (MEK-1), which is the upstream kinase of ERK1 and ERK2. Although PD98059 inhibited ERK2 activation in platelets, it had no effect on pVWF- or thrombin-induced platelet alpha or lysozomal granule release, modulation of membrane glycoprotein CD41, microparticle formation, platelet shape change or platelet agglutination. It is concluded that pVWF and thrombin induced JNK activation, but whereas thrombin induced ERK2 activation VWF did not; functional ERK2 activity was also not required for pVWF- or thrombin-dependent platelet activation.
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PMID:Porcine von Willebrand factor and thrombin induce the activation of c-Jun amino-terminal kinase (JNK/SAPK) whereas only thrombin induces activation of extracellular signal-related kinase 2 (ERK2) in human platelets. 1092 41

The relationship between persistent ERK (extracellular signal-regulated kinase) activity, cyclin D1 protein and mRNA levels and cell cycle progression in human cultured airway smooth muscle was examined in response to stimulation by ET-1 (endothelin-1), thrombin and bFGF (basic fibroblast growth factor). Thrombin (0.3 and 3 u ml(-1)) and bFGF (0.3 and 3 nM) increased ERK activity for more than 2 h and increased cell number, whereas ET-1 (100 nM) transiently stimulated ERK activity and was non-mitogenic. The MEK1 (mitogen-activated ERK kinase) inhibitor, PD 98059 (30 microM), inhibited both ERK phosphorylation and activity, and either prevented (thrombin 0.3 and 3 u ml(-1), bFGF 300 pM) or attenuated (bFGF 3 nM) DNA synthesis. Thrombin and bFGF increased both cyclin D1 mRNA and protein levels. PD 98059 decreased cyclin D1 protein levels stimulated by the lower but not higher thrombin concentrations. Moreover, increases in cyclin D1 mRNA levels were unaffected by PD 98059 pretreatment, irrespective of the mitogen or its concentration, suggesting that inhibition of cyclin D1 protein levels occurred by a post-transcriptional mechanism. These findings indicate that the control of cyclin D1 protein levels may occur independently of the MEK1/ERK signalling pathways. The inhibition of S phase entry by PD 98059 at higher thrombin concentrations appears to result from effects on pathways downstream or parallel to those regulating cyclin D1 protein levels. These findings suggest heterogeneity in the signalling of DNA synthesis in human cultured airway smooth muscle.
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PMID:The importance of ERK activity in the regulation of cyclin D1 levels and DNA synthesis in human cultured airway smooth muscle. 1096 64

Thrombin is primarily known for its role in homeostasis and thrombosis. However, this enzyme also plays important roles in wound healing and pathologic situations such as inflammation and tumorigenesis. Among the molecules stimulated by thrombin in these latter processes are the stress response proteins, chemokines. Chemokines are also known for their roles in inflammatory responses and tumor development. These correlative observations strongly suggest that chemokines may be mediators of some of thrombin's functions in these processes. Elucidation of the molecular mechanisms of stimulation of chemokines by thrombin may help to unravel the ways in which their expression can be modulated. Up-regulation of the chemokine 9E3/cCAF by thrombin occurs via its proteolytically activated receptor with subsequent transactivation of the epidermal growth factor receptor tyrosine kinase. This study shows that stimulation by thrombin very rapidly activates this chemokine at the transcriptional level, that 2 Elk1 binding elements located between -534 and -483 bp of the promoter are major thrombin response elements, that activation occurs via the Elk1 transcription factor, and that the latter is directly activated by MEK1/ERK2. The common occurrence of Elk1 binding domains in the promoters of immediate early response genes suggests that it may be characteristically involved in gene activation by stress-inducing agents. (Blood. 2000;96:3696-3706)
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PMID:Novel nuclear target for thrombin: activation of the Elk1 transcription factor leads to chemokine gene expression. 1109 49

The elevated level of thrombin has been detected in the airway fluids of asthmatic patients. However, the implication of thrombin in the pathogenesis of bronchial hyperreactivity was not completely understood. Therefore, in this study we investigated the effect of thrombin on cell proliferation and p42/p44 mitogen-activated protein kinase (MAPK) activation in human tracheal smooth muscle cells (TSMCs). Thrombin stimulated [3H]thymidine incorporation and p42/p44 MAPK phosphorylation in a time- and concentration-dependent manner in TSMCs. Pretreatment of TSMCs with pertussis toxin (PTX) significantly inhibited [3H]thymidine incorporation and phosphorylation of MAPK induced by thrombin. These responses were attenuated by tyrosine kinase inhibitors genistein and herbimycin A, phosphatidyl inositide (PI)-phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor GF109203X, removal of Ca(2+) by addition of BAPTA/AM plus EGTA, and PI 3-kinase inhibitors wortmannin and LY294002. In addition, thrombin-induced [3H]-thymidine incorporation and p42/p44 MAPK phosphorylation was completely inhibited by PD98059 (an inhibitor of MEK1/2), indicating that activation of MEK1/2 was required for these responses. Furthermore, overexpression of dominant negative mutants, RasN17 and Raf-301, significantly suppressed p42/p44 MAPK activation induced by thrombin and PDGF-BB, indicating that Ras and Raf may be required for activation of these kinases. These results conclude that the mitogenic effect of thrombin was mediated through the activation of Ras/Raf/MEK/MAPK pathway. Thrombin-mediated MAPK activation was modulated by PI-PLC, Ca(2+), PKC, tyrosine kinase, and PI 3-kinase associated with cell proliferation in cultured human TSMCs.
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PMID:Mechanisms of thrombin-induced MAPK activation associated with cell proliferation in human cultured tracheal smooth muscle cells. 1130 43

The stimulation of platelet-derived growth factor (PDGF) receptors shifts vascular smooth muscle (VSM) cells toward a more proliferative phenotype. Thrombin activates the same signaling cascades in VSM cells, namely the Ras/Raf/MEK/ERK and the phosphatidylinositol 3-kinase (PI 3-kinase)/Akt pathways. Nonetheless, thrombin was not mitogenic, but rather increased the expression of the smooth muscle-specific myosin heavy chain (SM-MHC) indicative of an in vitro re-differentiation of VSM cells. A more detailed analysis of the temporal pattern and relative signal intensities revealed marked differences. The strong and biphasic phosphorylation of ERK1/2 in response to thrombin correlated with its ability to increase the activity of the SM-MHC promoter whereas Akt was only partially and transiently phosphorylated. By contrast, PDGF, a potent mitogen in VSM cells, induced a short-lived ERK1/2 phosphorylation but a complete and sustained phosphorylation of Akt. The phosphorylated form of Akt physically interacted with Raf. Moreover, Akt phosphorylated Raf at Ser(259), resulting in a reduced Raf kinase activity and a termination of MEK and ERK1/2 phosphorylation. Disruption of the PI 3-kinase signaling prevented the PDGF-induced Akt and Raf-Ser(259) phosphorylation. Under these conditions, PDGF elicited a more sustained MEK and ERK phosphorylation and increased SM-MHC promoter activity. Consistently, in cells that express dominant negative Akt, PDGF increased SM-MHC promoter activity. Furthermore, expression of constitutively active Akt blocked the thrombin-stimulated SM-MHC promoter activity. Thus, we present evidence that the balance and cross-regulation between the PI 3-kinase/Akt and Ras/Raf/MEK signaling cascades determine the temporal pattern of ERK1/2 phosphorylation and may thereby guide the phenotypic modulation of vascular smooth muscle cells.
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PMID:Regulation of Raf by Akt controls growth and differentiation in vascular smooth muscle cells. 1144 34


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