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)

Activation of MAP kinase/Erk Kinase (MEK) via direct phosphorylation by Mos may be crucial for cellular transformation by the activated c-mos or v-mos gene. Recent studies on a number of different protein kinases showed that phosphorylation within a subdomain of the catalytic domain may represent a common mode of activation. In this regard, activation of MEK1 by Raf involves phosphorylation of serine residues 218 and 222. Here we show that recombinant kinase-inactive MEK1 is phosphorylated by v-Mos with equal efficiency at both Ser 218 and Ser 222 in vitro. Tryptic phosphopeptide analysis of glutathione-S-transferase (GST)-MEK1 K97R and its alanine-for-serine mutants indicated that Ser 222 is the preferred phosphorylation site. Wild-type GST-MEK1 was phosphorylated at the same sites but contained a significantly lower amount of doubly phosphorylated species then its K97R kinase-inactive mutant. The ratio of GST-MEK1 species phosphorylated at two serines to those phosphorylated at one serine was similar in auto-phosphorylated and v-Mos-phosphorylated GST-MEK1. Consistent with the in vitro data, phosphopeptide mapping of MEK1 immunoprecipitated from mos transformed cells showed an increased amount of singly phosphorylated phosphopeptide compared to nontransformed cels. MEK1 was found to be more highly activated in NIH3T3 cells transformed by an activated c-mos or v-mos gene than in cells growing normally in medium containing serum. Our data indicate that Mos activated MEK1 in vitro as well as in vivo by phosphorylating Ser 222.
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PMID:Characterization of MEK1 phosphorylation by the v-Mos protein. 773 26

The product of the c-mos proto-oncogene functions not only as an initiator of oocyte maturation but also as a component of cytostatic factor that causes the natural arrest of the unfertilized egg at the second meiotic metaphase. It has been shown that Mos can phosphorylate and activate mitogen-activated protein (MAP) kinase kinase (MAPKK) in vitro, leading to activation of MAP kinase. In this study, by using an anti-MAPKK antibody that can specifically inhibit Xenopus MAPKK activity, we have shown that MAPKK mediates the cytostatic factor activity of Mos. Coinjection of this anti-MAPKK antibody with the bacterially expressed Mos protein into a two-cell embryo prevented the Mos-induced cleavage arrest as well as the Mos-induced MAP kinase activation. The analysis of individual embryos indicated that the degree of the cleavage arrest was correlated with the extent of the MAP kinase activation in the Mos- and the Mos/antibody-injected embryos. These observations suggest the involvement of a signal transmission pathway consisting of Mos, MAPKK, and MAP kinase in the metaphase arrest.
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PMID:Mitogen-activated protein kinase kinase is required for the mos-induced metaphase arrest. 796 74

The natural arrest of vertebrate unfertilized eggs in second meiotic metaphase results from the activity of cytostatic factor (CSF). The product of the c-mos(xe) proto-oncogene is thought to be a component of CSF and can induce metaphase arrest when injected into blastomeres of two-cell embryos. The c-Mos(xe) protein can directly activate the mitogen-activated protein kinase kinase (MAP kinase kinase) in vitro, leading to activation of MAP kinase. MAP kinase and c-Mos(xe) are active in unfertilized eggs and are rapidly inactivated after fertilization. Microinjection of thiophosphorylated MAP kinase into one blastomere of a two-cell embryo induced metaphase arrest similar to that induced by c-Mos(xe). However, only arrest with c-Mos(xe) was associated with activation of endogenous MAP kinase. These results indicate that active MAP kinase is a component of CSF in Xenopus and suggest that the CSF activity of c-Mos(xe) is mediated by MAP kinase.
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PMID:Induction of metaphase arrest in cleaving Xenopus embryos by MAP kinase. 823 56

We undertook a study to determine if the serine-threonine kinase-encoding v-mos oncogene regulated the expression of the urokinase-type plasminogen activator. An expression vector encoding v-mos, but not a kinase-inactive mutant, stimulated urokinase promoter activity in CAT assays employing a squamous cell carcinoma cell line. The induction of urokinase promoter activity by v-mos was mediated, in part, via an increased AP-1 activity since (a) mutation of 2 AP-1 binding sites (at -1967 and -1885), or the co-expression of a transactivation domain-lacking c-jun mutant reduced the induction of the urokinase promoter by v-mos and (b) expression of v-mos increased the activity of a CAT reporter driven by three AP-1 tandem repeats. The stimulation of the urokinase promoter by v-mos was partially countered by co-expression of an ERK1/ERK2-inactivating phosphatase. Western blotting and zymographic analysis indicated that v-mos-transformed NIH3T3 cells (MSV NIH-3T3) secreted more urokinase compared with NIH3T3 cells and this was associated with a higher level of activated ERK1 and ERK2. Expression of a catalytically-inactive MAPKK mutant reduced the activity of a urokinase promoter-driven CAT reporter in the MSV NIH-3T3 cells. In conclusion, the data herein indicate that urokinase expression is regulated by v-mos through a MAPKK-dependent signaling pathway.
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PMID:Regulation of urokinase-type plasminogen activator expression by the v-mos oncogene. 854 21

The Ras-GTPase-activating protein (RasGAP) is an important modulator of p21ras - dependent signal transduction in Xenopus oocytes and in mammalian cells. We investigated the role of the RasGAP SH3 domain in signal transduction with a monoclonal antibody against the SH3 domain of RasGaP. This antibody prevented the activation of the maturation-promoting factor complex (cyclin B-p34cdc2) by oncogenic Ras. The antibody appears to be specific because as little as 5 ng injected per oocyte reduced the level of Cdc2 activation by 50% whereas 100 ng of nonspecific immunoglobulin G did not affect Cdc2 activation. The antibody blocked the Cdc2 activation induced by oncogenic Ras but not that induced by progesterone, which acts independently of Ras. A peptide corresponding to positions 317 to 326 of a sequence in the SH3 domain of human RasGAP blocked Cdc2 activation, whereas a peptide corresponding to positions 273 to 305 of a sequence in the N-terminal moiety of the SH3 domain of RasGAP had no effect. The antibody did not block the mitogen-activated protein (MAP) kinase cascade (activation of MAPK/ERK kinase [MEK], MAP kinase, and S6 kinase p90rsk). Surprisingly, injection of the negative MAP kinase mutant protein ERK2 K52R (containing a K-to-R mutation at position 52) blocked the Cdc2 activation induced by oncogenic Ras as well as blocking the activation of MAP kinase. Thus, MAP kinase is also implicated in the regulation of Cdc2 activity. In this study, we further investigated the regulation of the synthesis of the c-mos oncogene product, which is necessary for the activation of Cdc2. We report that the synthesis of the c-mos oncogene product, which is necessary for the activation antibody to the SH3 domain of RasGAP and by injecting the negative MAP kinase mutant protein ERK2 K52R. These results suggest that oncogenic Ras activates two signaling mechanisms: the MAP kinase cascade and a signaling pathway implicating the SH3 domain of RasGAP. These mechanisms might control Mos protein expression implicated in Cdc2 activation.
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PMID:The Ras-GTPase-activating protein SH3 domain is required for Cdc2 activation and mos induction by oncogenic Ras in Xenopus oocytes independently of mitogen-activated protein kinase activation. 864 28

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 kinases (MKKs or MEKs) are dual specificity tyrosine/threonine protein kinases that are activated by phosphorylation at two closely spaced serine residues (serines-218 and -222) by the c-mos and raf proto-oncogenes. This double phosphorylation is both necessary and sufficient for MEKs to activate the MAP kinase enzymes in vitro. The specificity or regulation of in vivo signaling to the mammalian MEKs (MEK1 and MEK2) was recently reported also to involve the differential phosphorylation of a proline-rich peptide located between the MEK kinase-subdomains IX and X. Here we report the purification and characterization of an auto-activating protein kinase from bovine brain that phosphorylates serine-298 of the MEK1 and MEK2 proline-rich insert peptides. The auto-activation of the MEK-S298 peptide kinase is the result of an intermolecular phosphorylation event that can be prevented by the peptide substrates. The inactive kinase migrates on gel filtration as a 90 kDa protein, and after activation as a 43 kDa phosphoprotein. Incorporation of 32P[phosphate] into 40-42 kDa proteins on SDS-PAGE parallels the activation of the enzyme, and dephosphorylation by protein phosphatase 2Ac reverses the activation. SDS-PAGE renaturation assays show that the 40 kDa protein has the capacity to autophosphorylate, and exhibits kinase activity towards myelin basic protein after activation. Phosphorylation of purified bovine brain MEK or recombinant MEK1 by the auto-activated kinase does not activate the enzyme, and does not interfere with the in vitro raf-mediated MEK activation. We conclude that still unknown kinases may control the MAP kinase pathway by targeting MEK.
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PMID:Identification and characterization of an auto-activating MEK kinase from bovine brain: phosphorylation of serine-298 in the proline-rich domain of the mammalian MEKs. 941 3

Xenopus laevis oocytes undergo an increase in intracellular pH (pHi) from 7.2 to 7.7 due to the up-regulation of Na+/H+ antiporters in their plasma membrane during oocyte meiotic maturation. Up-regulation of Na+/H+ exchangers (NHE) found in other cell systems appears to be controlled, in some cases, by direct phosphorylation of the exchanger. A number of active protein kinases can be found in maturing Xenopus oocytes. These include, c-mos kinase, Raf-1 kinase, mitogen-activated kinase kinase (MAPKK), MAPK, ribosomal S6 kinase (RSK), and histone H-1 kinase. Our previous study indicated that c-mos kinase, was involved in regulating the increase in oocyte pHi. In the current study, we show that when mRNA coding for a constitutively active form of Raf-1 kinase (delta N-Raf-1) was microinjected into oocytes, the protein product induced an increase in oocyte pHi. On the contrary, the injection of mRNA coding for wild-type Raf-1 (WT-Raf-1) or a kinase-deficient form of Raf-1 (KD-Raf-1) had no effect on the recipient oocyte pHi. 8-Br-cAMP and forskolin blocked the increase in pHi during oocyte meiotic maturation, but had no effect on the Raf-1-induced increase in oocyte pHi. Studies using antisense c-mos oligos demonstrated that Raf-1 was not working via a feedback loop to endogenous c-mos mRNA within the recipient oocytes. Experiments using the selective MAPKK inhibitor, PD 98059, indicated that the Raf-1 effect on oocyte pHi was not mediated by the downstream kinase, MAPKK. Therefore, Raf-1 appears to act independently of c-mos kinase in a pathway, not involving MAPKK, leading to the up-regulation of the Na+/H+ antiporters in Xenopus oocytes.
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PMID:Raf-1 kinase, a potential regulator of intracellular pH in Xenopus oocytes. 992 73

To investigate the role of c-mos in rat spermatogenesis, expression of c-mos, MAP kinase kinase (MAPKK), MAP kinase (MAPK), cdc2 and protein kinase A (PKA) by spermatogenic cell culture of 14 day-old rats was examined. MAPKK and PKA expressions were constitutive, whereas the expression of MAPK and cdc2 in spermatogonia initially decreased, but later increased on meiotic maturation of spermatocytes. c-mos expression was definitive of late meiotic prophase. c-mos immunoprecipitates prepared from the c-mos-enriched fraction (pI9.0-9.6) could form complex(es) in the cultured spermatogenic cell lysates. In vitro phosphorylation of the c-mos immune complexes revealed a 34 kDa protein that was phosphorylated at serine and threonine residues as a target of the c-mos signal. Its pI value was 4.4-4.5, and cdc2 was not detected, making it different from cdc2 (p34). These results suggest that the phosphorylation of the 34 kDa protein by the c-mos signal may play a crucial role in the meiotic division of rat spermatocytes.
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PMID:Definitive expression of c-mos in late meiotic prophase leads to phosphorylation of a 34 kda protein in cultured rat spermatocytes. 1184 49

The inhibition of mitogen activated protein kinase (MAPK) activation during porcine oocyte maturation leads to decreased maturation promoting factor (MPF) activity and to the induction of parthenogenetic activation. In the present study, in order to analyze the mechanism underlying the suppression of MPF activity in MAPK-inhibited porcine oocytes, we injected mRNA of SASA-MEK, a dominant negative MAPK kinase, or antisense RNA of c-mos, a MAPK kinase kinase, into immature porcine oocyte cytoplasm. The injection of SASA-MEK mRNA or c-mos antisense RNA inhibited the MAPK activity partially or completely, respectively, decreased the MPF activity slightly or significantly, respectively, and induced parthenogenetic activation in 17.1% or 96.6% of mature oocytes, respectively, although no parthenogenetic activation was observed in the control oocytes. Immunoblotting experiments revealed that cyclin B accumulation in these MAPK-suppressed porcine oocytes was increased significantly after 50 h of culture and that a considerable amount of MPF was converted into inactive pre-MPF by hyperphosphorylation. These results indicate that the inhibition of MAPK activity in porcine oocytes did not promote cyclin B degradation but rather suppressed it; also the decrease in MPF activity in MAPK-suppressed porcine oocytes correlated with the conversion of active MPF into inactive pre-MPF.
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PMID:Inhibition of mitogen activated protein kinase activity induces parthenogenetic activation and increases cyclin B accumulation during porcine oocyte maturation. 1603 93


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