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)

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

Mitogen-activated protein kinase (MAPK) signaling cascades include MAPK or extracellular signal-regulated kinase (ERK), MAPK kinase (MKK or MEK), and MAPK kinase kinase (MAPKKK or MEKK). MAPKK kinase/MEKK phosphorylates and activates its downstream protein kinase, MAPK kinase/MEK, which in turn activates MAPK. We report herein the isolation of a cDNA encoding a novel protein kinase designated MAPKKK5 from a human macrophage library. The nucleotide sequence predicts that MAPKKK5 encodes an open reading frame of 1374 amino acids with all 11 kinase subdomains. The putative catalytic domain of MAPKKK5 shows significant sequence homology to the kinase domains of the MAPKKK/MEKK level protein kinases from mouse MEKK2 and -3, Drosophila melanogaster PK92B, Saccharomyces cerevisiae STE11, and Schizosaccharomyces pombe BYR2. Northern blot analysis showed that MAPKKK5 transcript is abundantly expressed in human heart and pancreas. When transiently expressed in COS and 293 cells, MAPKKK5 markedly activated c-Jun N-terminal kinase or stress-activated protein kinase, but not MAPK/ERK. Furthermore, MAPKKK5 that was immunoprecipitated from transfected 293 cells was able to phosphorylate and activate MKK4 in vitro, suggesting that MAPKKK5 may be an upstream activator of MKK4 in the c-Jun N-terminal kinase pathway.
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PMID:Molecular cloning and characterization of a novel protein kinase with a catalytic domain homologous to mitogen-activated protein kinase kinase kinase. 894 Jan 79

Pituitary adenylate cyclase-activating polypeptides (PACAP-27 and PACAP-38) are neuropeptides of the vasoactive intestinal polypeptide (VIP)/secretin/glucagon family. PACAP receptors are expressed in different brain regions, including cerebellum. We used primary culture of rat cerebellar granule neurons to study the effect of PACAP-38 on apoptosis induced by potassium deprivation. We demonstrated that PACAP-38 increased survival of cerebellar neurons in a dose-dependent manner by decreasing the extent of apoptosis estimated by DNA fragmentation. PACAP-38 induced activation of the extracellular signal-regulated kinase (ERK)-type of mitogen-activated protein (MAP) kinase through a cAMP-dependent pathway. PD98059, an inhibitor of MEK (MAP kinase kinase), completely abolished the antiapoptotic effect of PACAP-38, suggesting that MAP kinase pathway activation is necessary for PACAP-38 action.
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PMID:Pituitary adenylate cyclase-activating polypeptide (PACAP-38) protects cerebellar granule neurons from apoptosis by activating the mitogen-activated protein kinase (MAP kinase) pathway. 898 38

The extracellular signal-regulated kinase (ERK) pathway, the stress-activated protein kinase (SAPK) pathway, and the p38 pathway are three major mitogen-activated protein kinase (MAPK) cascades known to participate in the regulation of cellular responses to a variety of extracellular signals. Upstream regulatory components of these kinase cascades, the MAPK/ERK kinase kinases (MEKK), have been described in several systems. We have isolated a cDNA encoding human MEKK3. Transfected MEKK3 has the ability to activate both SAPK and ERK pathways, but does not induce p38 activity, in agreement with a previous report on murine MEKK3 (Blank, J. L., Gerwins, P., Elliott, E. M., Sather, S., and Johnson, G. L. (1996) J. Biol. Chem. 271, 5361-5368). We now demonstrate that MEKK3 activates SEK and MEK, the known kinases targeting SAPK and ERK, respectively. Utilizing an estrogen ligand-activated MEKK3 derivative, we furthermore demonstrate that MEKK3 regulates the SAPK and the ERK pathway directly. Consistent with the fact that several SAPK-inducing agents activate the transcription factor NFkappaB, we now show that MEKK3 also enhances transcription from an NFkappaB-dependent reporter gene in cotransfection assays. The ability of MEKK3 to simultaneously activate the SAPK and ERK pathways is remarkable, given that they have divergent roles in cellular homeostasis.
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PMID:Direct activation of the stress-activated protein kinase (SAPK) and extracellular signal-regulated protein kinase (ERK) pathways by an inducible mitogen-activated protein Kinase/ERK kinase kinase 3 (MEKK) derivative. 900 2

Tyrphostins are synthetic compounds that have been described as in vitro inhibitors of epidermal growth factor receptor (EGF-R) tyrosine kinase activity. The inhibitory effect of tyrphostins in intact cells has been shown only after prolonged treatment. However, these compounds appear to be readily incorporated, which suggests that tyrphostin acts indirectly on EGF-R. We studied the effects of a tyrphostin derivative, RG 50864, without preincubation in intact epithelial cells. We selected two human cell lines differing in degree of expression of the p185erbB2 protein, which is closely related to EGF-R. We showed that tyrphostin (RG 50864) had no effect on EGF-dependent EGF-R tyrosine phosphorylation in the parental cell line. On the contrary, it prolonged the EGF-dependent EGF-R and p185erbB2(V-E) tyrosine phosphorylation in p185erbB2(V-E)-expressing cells. Because tyrphostin has been shown to be an inhibitor of p185erbB2 and EGF-R in vitro, this finding indicates that the tyrphostin effect on p185erbB2(V-E) and EGF-R was the result of an indirect mechanism in transfected cells. Tyrphostin treatment alone led to the activation of mitogen-activated protein (MAP) kinase kinase or MAP kinase or extracellular signal-regulated kinase kinase (MEK), suggesting that one of the tyrphostin targets was upstream of MEK1. MAP kinase, however, was not activated after tyrphostin treatment. This finding indicates that tyrphostin had another target in intact cells because MEK1 activation by tyrphostin alone did not correlate with MAP kinase activation. In the two cell lines, tyrphostin modified the time course of EGF-dependent MEK and MAP kinase activation. We conclude that whereas tyrphostins were designed to inhibit EGF-R tyrosine kinase activity, under our conditions EGF-R is not a physiological target for tyrphostin, nor is one of its related protein tyrosine kinases, p185erbB2(V-E). On the contrary, our results show that tyrphostin targets are multiple, leading to complex effects on receptor signaling in these epithelial cells.
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PMID:Epidermal growth factor receptor signaling cascade as target for tyrphostin (RG 50864) in epithelial cells. Paradoxical effects on mitogen-activated protein kinase kinase and mitogen-activated protein kinase activities. 906 32

Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.
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PMID:Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases. 911 Sep 99

Overexpression of a constitutively active mitogen-activated protein kinase kinase (MAPKK or MEK) induces neuronal differentiation in adrenal pheochromocytoma 12 cells but transformation in fibroblasts. In the present study, we used a constitutively active MAPK/extracellular signal-regulated kinase (ERK) kinase 1 (MEK1) mutant to investigate the function of the highly conserved MEK1-ERK2 signaling module in renal epithelial cell differentiation and proliferation. Stable expression of constitutively active MEK1 (CA-MEK1) in epithelial MDCK-C7 cells led to an increased basal and serum-stimulated ERK1 and ERK2 phosphorylation as well as ERK2 activation when compared with mock-transfected cells. In both mock-transfected and CA-MEK1-transfected MDCK-C7 cells, basal and serum-stimulated ERK1 and ERK2 phosphorylation was almost abolished by the synthetic MEK inhibitor PD098059. Increased ERK2 activation due to stable expression of CA-MEK1 in MDCK-C7 cells was associated with epithelial dedifferentiation as shown by both a dramatic alteration in cell morphology and an abolished cytokeratin expression but increased vimentin expression. In addition, we obtained a delayed and reduced serum-stimulated cell proliferation in CA-MEK1-transfected cells (4.6-fold increase in cell number/cm2 after 5 days of serum stimulation) as compared with mock-transfected controls (12.9-fold increase in cell number/cm2 after 5 days). This result was confirmed by flow cytometric DNA analysis showing that stable expression of CA-MEK1 decreased the proportion of MDCK-C7 cells moving from G0/G1 to G2/M as compared with both untransfected and mock-transfected cells. Taken together, our data demonstrate an association of increased basal and serum-stimulated activity of the MEK1-ERK2 signaling module with epithelial dedifferentiation and growth inhibition in MDCK-C7 cells. Thus, the MEK1-ERK2 signaling pathway could act as a negative regulator of epithelial differentiation thereby leading to an attenuation of MDCK-C7 cell proliferation.
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PMID:Constitutively active mutant of the mitogen-activated protein kinase kinase MEK1 induces epithelial dedifferentiation and growth inhibition in madin-darby canine kidney-C7 cells. 911 Oct 53

The phospholipase A2 (PLA2) enzymes play a central role in diverse cellular processes including phospholipid digestion and metabolism, host defense, and cell signaling. We investigated the ability of CD16 clustering to trigger PLA2 and extracellular signal-regulated kinase (ERK) activation in human NK cells, as well as their possible involvement in CD16-stimulated degranulation. Both secretory (sPLA2) and cytosolic (cPLA2) PLA2 were rapidly activated upon CD16 cross-linking; sPLA2 was found in the supernatant and also in a cell-associated form. cPLA2 activation was controlled by the ERK pathway as indicated by the close correlation between their kinetics of activation and by the ability of the specific MEK inhibitor, PD 098059, to abolish cPLA2 activation. CD16 stimulation also resulted in the generation of platelet-activating factor (PAF) and leukotrienes; both phospholipases contributed to their biosynthesis. Using the pharmacologic inhibitors AACOCF3, p-bromophenacyl bromide (pBPB), and PD 098059, which specifically inhibit cPLA2, sPLA2, and MEK, respectively, we demonstrated that the ERK signaling pathway, but not cytosolic or secretory PLA2, is required for CD16-triggered granule release.
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PMID:CD16 cross-linking induces both secretory and extracellular signal-regulated kinase (ERK)-dependent cytosolic phospholipase A2 (PLA2) activity in human natural killer cells: involvement of ERK, but not PLA2, in CD16-triggered granule exocytosis. 912 Feb 68

We tested whether activation of mitogen-activated protein kinase/ extracellular signal-regulated kinase kinase-1 (MEK1) is required and sufficient for extracellular signal-regulated kinase (ERK) activation in airway smooth muscle cells. First, we transiently cotransfected bovine tracheal myocytes with an epitope-tagged ERK2 and a dominant-negative or a constitutively active form of the gene encoding MEK1 and assessed ERK2 activation by in vitro phosphorylation assay. Expression of the dominant-negative MEK1 inhibited platelet-derived growth factor (PDGF)-induced ERK2 activation, whereas expression of the constitutively active MEK1 induced ERK2 activation, suggesting that MEK1 is required and sufficient for ERK activation in these cells. Next, we assessed the effect of PD-98059, a synthetic MEK inhibitor, on PDGF-induced MEK1 and ERK activation. PD-98059 (10 microM) inhibited MEK1 and ERK activation, confirming that MEK1 is required for ERK activation in bovine tracheal myocytes. PD-98059 had no effect on Src or Raf-1 activity, evidence that PD-98059 is a specific inhibitor of MEK in this system. Finally, PD-98059 reduced PDGF-induced [(3)H]thymidine incorporation in a concentration-dependent manner, suggesting that catalytic activation of MEK1 and ERKs is required for DNA synthesis. We conclude that MEK1 is required for PDGF-induced ERK activation in bovine tracheal myocytes and that MEK1 and ERKs are required for PDGF-induced DNA synthesis in these cells.
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PMID:MEK1 is required for PDGF-induced ERK activation and DNA synthesis in tracheal myocytes. 912 14

The activation of protein kinase C (PKC) found in diabetic glomeruli and glomerular mesangial cells cultured under high glucose conditions has been proposed to contribute to the development of diabetic nephropathy. However, the abnormalities distal to PKC have not been fully elucidated yet. Herein, we provide the evidence that mitogen-activated protein kinase (MAPK) cascade, an important kinase cascade downstream to PKC and an activator of cytosolic phospholipase A2 (cPLA2) by direct phosphorylation, is activated in glomeruli isolated from streptozotocin-induced diabetic rats. MAPK cascade was also activated in glomerular mesangial cells cultured under high glucose (27.8 mmol/l) conditions for 5 days, and the activation of MAPK cascade was inhibited by treating the cells with calphostin C, an inhibitor of PKC. Furthermore, the activities of cPLA2 also increased in cells cultured under the same conditions and this activation was inhibited by both calphostin C and PD 098059, an inhibitor of MEK (MAPK or extracellular signal-regulated kinase [ERK] kinase). These results indicate that MAPK cascade is activated in glomeruli and mesangial cells under the diabetic state possibly through the activation of PKC. Activated MAPK, in turn, may induce various functional changes of mesangial cells at least through the activation of cPLA2 and contribute to the development of diabetic nephropathy.
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PMID:Mitogen-activated protein kinase cascade is activated in glomeruli of diabetic rats and glomerular mesangial cells cultured under high glucose conditions. 913 54


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