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 previously shown that stimulation of proliferation of avian embryonic muscle cells (myoblasts) by 1alpha,25(OH)(2)-vitamin D(3) (1alpha,25(OH)(2)D(3)) is mediated by activation of the mitogen-activated protein kinase (MAPK; ERK1/2). To understand how 1alpha,25(OH)(2)D(3) up-regulates the MAPK cascade, we have investigated whether the hormone acts upstream through stimulation of Raf-1 and the signaling mechanism by which this effect might take place. Treatment of chick myoblasts with 1alpha,25(OH)(2)D(3) (1 nm) caused a fast increase of Raf-1 serine phosphorylation (1- and 3-fold over basal at 1 and 2 min, respectively), indicating activation of Raf-1 by the hormone. These effects were abolished by preincubation of cells with a specific Ras inhibitor peptide that involves Ras in 1alpha,25(OH)(2)D(3) stimulation of Raf-1. 1alpha,25(OH)(2)D(3) rapidly induced tyrosine de-phosphorylation of Ras-GTPase-activating protein, suggesting that inhibition of Ras-GTP hydrolysis is part of the mechanism by which 1alpha,25(OH)(2)D(3) activates Ras in myoblasts. The protein kinase C (PKC) inhibitors calphostin C, bisindolylmaleimide I, and Ro 318220 blocked 1alpha,25(OH)(2)D(3)-induced Raf-1 serine phosphorylation, revealing that hormone stimulation of Raf-1 also involves PKC. In addition, transfection of muscle cells with an antisense oligodeoxynucleotide against PKCalpha mRNA suppressed serine phosphorylation by 1alpha,25(OH)(2)D(3). The increase in MAPK activity and tyrosine phosphorylation caused by 1alpha,25(OH)(2)D(3) could be abolished by Ras inhibitor peptide, compound PD 98059, which prevents the activation of MEK by Raf-1, or incubation of cell lysates before 1alpha,25(OH)(2)D(3) exposure with an anti-Raf-1 antibody. In conclusion, these results demonstrate for the first time in a 1alpha,25(OH)(2)D(3) target cell that activation of Raf-1 via Ras and PKCalpha-dependent serine phosphorylation plays a central role in hormone stimulation of the MAPK-signaling pathway leading to muscle cell proliferation.
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PMID:Activation of RAF-1 through Ras and protein kinase Calpha mediates 1alpha,25(OH)2-vitamin D3 regulation of the mitogen-activated protein kinase pathway in muscle cells. 1241 93

Transformation by oncogenic Ras requires signaling through Rho family proteins including RhoA, but the mechanism(s) whereby oncogenic Ras regulates the activity of RhoA is (are) unknown. We examined the effect of Ras on RhoA activity in NIH 3T3 cells either stably transfected with H-Ras(V12) under control of an inducible promoter or transiently expressing the activated H-Ras. Using a novel method to quantitate enzymatically the GTP bound to Rho, we found that expression of the oncogenic Ras increased Rho activity approximately 2-fold. Increased Rho activity was associated with increased plasma membrane binding of RhoA and decreased activity of the Rho/Ras-regulated p21(WAF1/CIP1) promoter. RhoA activation by oncogenic Ras could be explained by a decrease in cytosolic p190 Rho-GAP activity and translocation of p190 Rho-GAP from the cytosol to a detergent-insoluble cytoskeletal fraction. Pharmacologic inhibition of the Ras/Raf/MEK/ERK pathway prevented Ras-induced activation of RhoA and translocation of p190 Rho-GAP; expression of constitutively active Raf-1 kinase or MEK was sufficient to induce p190 Rho-GAP translocation. We conclude that in NIH 3T3 cells oncogenic Ras activates RhoA through the Raf/MEK/ERK pathway by decreasing the cytosolic activity and changing the subcellular localization of p190 Rho-GAP.
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PMID:Oncogenic Ras leads to Rho activation by activating the mitogen-activated protein kinase pathway and decreasing Rho-GTPase-activating protein activity. 1242 40

Ustilago maydis, a pathogen of maize, is a useful model for the analysis of mating, pathogenicity, and the morphological transition between budding and filamentous growth in fungi. As in other fungi, these processes are regulated by conserved signaling mechanisms, including the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and at least one mitogen-activated protein kinase (MAP kinase) pathway. A current challenge is to identify additional factors that lie downstream of the cAMP pathway and that influence morphogenesis in U. maydis. In this study, we identified suppressor mutations that restored budding growth to a constitutively filamentous mutant with a defect in the gene encoding a catalytic subunit of PKA. Complementation of one suppressor mutation unexpectedly identified the ras2 gene, which is predicted to encode a member of the well-conserved ras family of small GTP-binding proteins. Deletion of the ras2 gene in haploid cells altered cell morphology, eliminated pathogenicity on maize seedlings, and revealed a role in the production of aerial hyphae during mating. We also used an activated ras2 allele to demonstrate that Ras2 promotes pseudohyphal growth via a MAP kinase cascade involving the MAP kinase kinase Fuz7 and the MAP kinase Ubc3. Overall, our results reveal an additional level of crosstalk between the cAMP signaling pathway and a MAP kinase pathway influenced by Ras2.
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PMID:ras2 Controls morphogenesis, pheromone response, and pathogenicity in the fungal pathogen Ustilago maydis. 1247 96

We report that the multidomain protein POSH (plenty of SH3s) acts as a scaffold for the JNK pathway of neuronal death. This pathway consists of a sequential cascade involving activated Rac1/Cdc42, mixed-lineage kinases (MLKs), MAP kinase kinases (MKKs) 4 and 7, c-Jun N-terminal kinases (JNKs) and c-Jun, and is required for neuronal death induced by various means including nerve growth factor (NGF) deprivation. In addition to binding GTP-Rac1 as described previously, we find that POSH binds MLKs both in vivo and in vitro, and complexes with MKKs 4 and 7 and with JNKs. POSH overexpression promotes apoptotic neuronal death and this is suppressed by dominant-negative forms of MLKs, MKK4/7 and c-Jun, and by an MLK inhibitor. Moreover, a POSH antisense oligonucleotide and a POSH small interfering RNA (siRNA) suppress c-Jun phosphorylation and neuronal apoptosis induced by NGF withdrawal. Thus, POSH appears to function as a scaffold in a multiprotein complex that links activated Rac1 and downstream elements of the JNK apoptotic cascade.
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PMID:POSH acts as a scaffold for a multiprotein complex that mediates JNK activation in apoptosis. 1251 31

In cardiac myocytes, stimulation of alpha(1)-adrenoceptor (AR) leads to a hypertrophic phenotype. The G(h) protein (transglutaminase II, TGII) is tissue type transglutaminase and transmits the alpha(1B)-adrenoceptor signal with GTPase activity. Recently, it has been shown that the calreticulin (CRT) down-regulates both GTP binding and transglutaminase activities of TGII. To elucidate whether G(h) mediates norepinephrine-stimulated intracellular signal transductions leading to activation of extracellular signal-regulated kinases (ERKs) and neonatal rat cardiomyocyte hypertrophy, we examined the effects of G(h) on the activation of ERKs and inhibitory effects of CRT on alpha(1)-adrenoceptor/G(h) signaling. In neonatal rat cardiomyocytes, norepinephrine-induced ERKs activation was inhibited by an alpha(1)-adrenoceptor blocker (prazosin), but not by an beta-adrenoceptor blocker (propranolol). Overexpression of the G(h) protein stimulated norepinephrine-induced ERKs activation, which was inhibited by alpha-adrenoceptor blocker (prazosin). Co-overexpression of G(h) and CRT abolished norepinephrine-induced ERKs activation. Taken together, norepinephrine induces hypertrophy in neonatal rat cardiomyocytes through alpha(1)-AR stimulation and G(h) is partly involved in norepinephrine-induced MEK1,2/ERKs activation. Activation of G(h)-mediated MEK1,2/ERKs was completely inhibited by CRT.
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PMID:Calreticulin inhibits the MEK1,2-ERK1,2 pathway in alpha 1-adrenergic receptor/Gh-stimulated hypertrophy of neonatal rat cardiomyocytes. 1264 29

Fibroblasts synthesize, organize, and maintain connective tissues during development and in response to injury and fibrotic disease. Studies on cells in three-dimensional collagen matrices have shown that fibroblasts switch between proliferative and quiescence phenotypes, depending upon whether matrices are attached or floating during matrix remodeling. Previous work showed that cell signaling through the ERK pathway was decreased in fibroblasts in floating matrices. In the current research, we extend the previous findings to show that serum stimulation of fibroblasts in floating matrices does not result in ERK translocation to the nucleus. In addition, there was decreased serum activation of upstream members of the ERK signaling pathway, MEK and Raf, even though Ras became GTP loaded. The findings suggest that quiescence of fibroblasts in floating collagen matrices may result from a defect in Ras coupling to its downstream effectors.
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PMID:Fibroblast quiescence in floating collagen matrices: decrease in serum activation of MEK and Raf but not Ras. 1266 62

Azathioprine and its metabolite 6-mercaptopurine (6-MP) are immunosuppressive drugs that are used in organ transplantation and autoimmune and chronic inflammatory diseases such as Crohn disease. However, their molecular mechanism of action is unknown. In the present study, we have identified a unique and unexpected role for azathioprine and its metabolites in the control of T cell apoptosis by modulation of Rac1 activation upon CD28 costimulation. We found that azathioprine and its metabolites induced apoptosis of T cells from patients with Crohn disease and control patients. Apoptosis induction required costimulation with CD28 and was mediated by specific blockade of Rac1 activation through binding of azathioprine-generated 6-thioguanine triphosphate (6-Thio-GTP) to Rac1 instead of GTP. The activation of Rac1 target genes such as mitogen-activated protein kinase kinase (MEK), NF-kappaB, and bcl-x(L) was suppressed by azathioprine, leading to a mitochondrial pathway of apoptosis. Azathioprine thus converts a costimulatory signal into an apoptotic signal by modulating Rac1 activity. These findings explain the immunosuppressive effects of azathioprine and suggest that 6-Thio-GTP derivates may be useful as potent immunosuppressive agents in autoimmune diseases and organ transplantation.
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PMID:CD28-dependent Rac1 activation is the molecular target of azathioprine in primary human CD4+ T lymphocytes. 1269 31

We recently reported that Rho kinase is required for sustained ERK signaling and the consequent mid-G(1) phase induction of cyclin D1 in fibroblasts. The results presented here indicate that these Rho kinase effects are mediated by the formation of stress fibers and the consequent clustering of alpha5beta1 integrin. Mechanistically, alpha5beta1 signaling and stress fiber formation allowed for the sustained activation of MEK, and this effect was mediated upstream of Ras-GTP loading. Interestingly, disruption of stress fibers with ML-7 led to G(1) phase arrest while comparable disruption of stress fibers with Y27632 (an inhibitor of Rho kinase) or dominant-negative Rho kinase led to a more rapid progression through G(1) phase. Inhibition of either MLCK or Rho kinase blocked sustained ERK signaling, but only Rho kinase inhibition allowed for the induction of cyclin D1 and activation of cdk4 via Rac/Cdc42. The levels of cyclin E, cdk2, and their major inhibitors, p21(cip1) and p27(kip1), were not affected by inhibition of MLCK or Rho kinase. Overall, our results indicate that Rho kinase-dependent stress fiber formation is required for sustained activation of the MEK/ERK pathway and the mid-G(1) phase induction of cyclin D1, but not for other aspects of cdk4 or cdk2 activation. They also emphasize that G(1) phase cell cycle progression in fibroblasts does not require stress fibers if Rac/Cdc42 signaling is allowed to induce cyclin D1.
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PMID:Effects of rho kinase and actin stress fibers on sustained extracellular signal-regulated kinase activity and activation of G(1) phase cyclin-dependent kinases. 1764 1

Accumulating evidence suggests that p21(Cip1) located in the cytoplasm might play a role in promoting transformation and tumor progression. Here we show that oncogenic H-RasV12 contributes to the loss of actin stress fibers by inducing cytoplasmic localization of p21(Cip1), which uncouples Rho-GTP from stress fiber formation by inhibiting Rho kinase (ROCK). Concomitant with the loss of stress fibers in Ras-transformed cells, there is a decrease in the phosphorylation level of cofilin, which is indicative of a compromised ROCK/LIMK/cofilin pathway. Inhibition of MEK in Ras-transformed NIH3T3 results in restoration of actin stress fibers accompanied by a loss of cytoplasmic p21(Cip1), and increased phosphorylation of cofilin. Ectopic expression of cytoplasmic but not nuclear p21(Cip1) in Ras-transformed cells was effective in preventing stress fibers from being restored upon MEK inhibition and inhibited phosphorylation of cofilin. p21(Cip1) was also found to form a complex with ROCK in Ras-transformed cells in vivo. Furthermore, inhibition of the PI 3-kinase pathway resulted in loss of p21(Cip1) expression accompanied by restoration of phosphocofilin, which was not accompanied by stress fiber formation. These results suggest that restoration of cofilin phosphorylation in Ras-transformed cells is necessary but not sufficient for stress fiber formation. Our findings define a novel mechanism for coupling cytoplasmic p21(Cip1) to the control of actin polymerization by compromising the Rho/ROCK/LIMK/cofilin pathway by oncogenic Ras. These studies suggest that localization of p21(Cip1) to the cytoplasm in transformed cells contributes to pathways that favor not only cell proliferation, but also cell motility thereby contributing to invasion and metastasis.
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PMID:Cytoplasmic p21Cip1 is involved in Ras-induced inhibition of the ROCK/LIMK/cofilin pathway. 1455 14

We have reported previously that protein kinase C (PKC) signaling can mediate a program of cell cycle withdrawal in IEC-18 nontransformed intestinal crypt cells, involving rapid disappearance of cyclin D1, increased expression of Cip/Kip cyclin-dependent kinase inhibitors, and activation of the growth suppressor function of pocket proteins. In the current study, we present evidence to support a requisite role for PKC alpha in mediating these effects. Furthermore, analysis of the signaling events linking PKC/PKC alpha activation to changes in the cell cycle regulatory machinery implicate the Ras/Raf/MEK/ERK cascade. PKC/PKC alpha activity promoted GTP loading of Ras, activation of Raf-1, and phosphorylation/activation of ERK. ERK activation was found to be required for critical downstream effects of PKC/PKC alpha activation, including cyclin D1 down-regulation, p21(Waf1/Cip1) induction, and cell cycle arrest. PKC-induced ERK activation was strong and sustained relative to that produced by proliferative signals, and the growth inhibitory effects of PKC agonists were dominant over proliferative events when these opposing stimuli were administered simultaneously. PKC signaling promoted cytoplasmic and nuclear accumulation of ERK activity, whereas growth factor-induced phospho-ERK was localized only in the cytoplasm. Comparison of the effects of PKC agonists that differ in their ability to sustain PKC alpha activation and growth arrest in IEC-18 cells, together with the use of selective kinase inhibitors, indicated that the length of PKC-mediated cell cycle exit is dictated by the magnitude/duration of input signal (i.e. PKC alpha activity) and of activation of the ERK cascade. The extent/duration of phospho-ERK nuclear localization may also be important determinants of the duration of PKC agonist-induced growth arrest in this system. Taken together, the data point to PKC alpha and the Ras/Raf/MEK/ERK cascade as key regulators of cell cycle withdrawal in intestinal epithelial cells.
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PMID:Involvement of the ERK signaling cascade in protein kinase C-mediated cell cycle arrest in intestinal epithelial cells. 1467 Sep 56


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