Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:2.7.12.2 (MEK)
 18,161 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Osmotic shock induces a variety of biochemical and physiological responses in vertebrate cells. By analyzing extracts obtained from rat 3Y1 fibroblastic cells exposed to hyper-osmolar media, we have found that mitogen-activated protein kinases (MAPKs) and stress-activated protein kinases (SAPKs, also known as JNKs) are both activated in response to osmotic shock. MAPKK1 (MEK1) was also activated markedly. Furthermore, Raf-1 and MEKK were activated strikingly by the osmotic shock. Activation of Raf-1 and MEKK in response to osmotic shock was detected also in PC12 cells, in which MEKK activation by the osmotic shock was much stronger than that by epidermal growth factor. Activation of SAPKs in PC12 cells by the osmotic shock was also more marked than that by epidermal growth factor. The activated MEKK phosphorylated not only MAPKKs but also XMEK2, which is distantly related to MAPKK. Recombinant wild-type XMEK2, but not kinase-negative XMEK2, was able to phosphorylate and activate recombinant SAPK alpha in vitro. In addition, this activity of XMEK2 was activated by the activated MEKK. These results suggest that the MAPK cascade consisting of Raf-1, MAPKK, and MAPK and the SAPK cascade consisting of MEKK, XMEK2, and SAPK are both activated in response to osmotic shock. Finally, it was found that XMEK2 is a good substrate for SAPK.
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PMID:Activation of protein kinase cascades by osmotic shock. 775 32

It is known that mechanical stress directly changes the conformation of the functional proteins, or directly activates enzymes such as phospholipase in the plasma membrane. The integrin-cytoskeleton complex may be an alternative candidate structure for a mechanoreceptor and a transducer. The cytoskeleton has been also shown to play an important role in secretion. Mechanical stress may stimulate the secretion of some cytokines or angiotensin II, which may generate multiple intracellular signals as a secondary event. External stimuli are generally transduced into the nucleus through the activation of protein kinase cascade. Stretching of cardiac myocytes stimulates the activity of PKC, Raf-1 kinase, MAP kinase kinase. MAP kinase and S6 kinase. In cardiac myocytes, mechanical stress directly induces gene expression as well as protein synthesis. Immediate early genes are first induced, and then fetal-type genes are reinduced. Both in hypertrophied hearts and in the experimental model of cardiac hypertrophy induced by pressure overload. Ca(2+)-ATPase content of cardiac myocytes is depressed. Reduced function of sarcoplasmic reticulum causes insufficient decrease of intracellular calcium in diastole and induces slowing of ventricular relaxation. In the interstitium of pressure overloaded hearts, the accumulation of collagen fiber is increased. The abnormal deposit leads to increased chamber stiffness and diastolic dysfunction. Furthermore, TGF-beta and tissue renin-angiotensin system are up-regulated in pressure overloaded hearts, both of which accelerate the interstitial fibrosis.
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PMID:Interaction of cardiac myocytes and non-myocytes in mechanical stress-induced hypertrophy. 777 62

Mitogen-activated protein kinases (MAPKs) are activated upon a variety of extracellular stimuli in different cells. In macrophages, colony-stimulating factor 1 (CSF-1) stimulates proliferation, while bacterial lipopolysaccharide (LPS) inhibits cell growth and causes differentiation and activation. Both CSF-1 and LPS rapidly activate the MAPK network and induce the phosphorylation of two distinct ternary complex factors (TCFs), TCF/Elk and TCF/SAP. CSF-1, but not LPS, stimulated the formation of p21ras. GTP complexes. Expression of a dominant negative ras mutant reduced, but did not abolish, CSF-1-mediated stimulation of MEK and MAPK. In contrast, activation of the MEK kinase Raf-1 was Ras independent. Treatment with the phosphatidylcholine-specific phospholipase C inhibitor D609 suppressed LPS-mediated, but not CSF-1-mediated, activation of Raf-1, MEK, and MAPK. Similarly, down-regulation or inhibition of protein kinase C blocked MEK and MAPK induction by LPS but not that by CSF-1. Phorbol 12-myristate 13-acetate pretreatment led to the sustained activation of the Raf-1 kinase but not that of MEK and MAPK. Thus, activated Raf-1 alone does not support MEK/MAPK activation in macrophages. Phosphorylation of TCF/Elk but not that of TCF/SAP was blocked by all treatments that interfered with MAPK activation, implying that TCF/SAP was targeted by a MAPK-independent pathway. Therefore, CSF-1 and LPS target the MAPK network by two alternative pathways, both of which induce Raf-1 activation. The mitogenic pathway depends on Ras activity, while the differentiation signal relies on protein kinase C and phosphatidylcholine-specific phospholipase C activation.
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PMID:Ras-dependent and -independent pathways target the mitogen-activated protein kinase network in macrophages. 779 56

Ras p21 in the GTP-bound form was shown to act as an upstream activator for mitogen-activated protein (MAP) kinase kinase (MAPKK) and MAP kinase, and Raf-1 was reported to act as a MAPKK kinase. Further, physical association between Ras and Raf-1 was demonstrated. Here we have shown that incubation of Xenopus immature oocyte extracts with Ras enhances the ability of endogenous Raf-1 to activate MAPKK. Moreover, a dominant negative form of Raf-1 blocked the Ras-induced activation of MAPKK and MAP kinase in the extracts, but not the cyclin A-dependent activation of MAP kinase. When the extracts were depleted of 45-kDa MAPKK with polyclonal anti-MAPKK antibody, no activation of MAP kinase occurred even after incubation with Ras. These results suggest that Ras can activate the MAPKK kinase activity of Raf-1 in the extracts and that MAPKK is indispensable for the Ras-induced MAP kinase activation. It is well known that Ras can induce oocyte maturation when injected into immature Xenopus oocytes. Co-injection of Ras with an anti-MAPKK antibody that inhibits the MAPKK activity prevented the Ras-induced germinal vesicle breakdown, suggesting that MAPKK mediates, at least, one of cellular functions of Ras.
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PMID:Analysis of the Ras p21/mitogen-activated protein kinase signaling in vitro and in Xenopus oocytes. 780 37

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.
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PMID:Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade. 786 10

MAP kinase kinase (MAPKK), a key component of the MAP kinase cascade, is activated through phosphorylation by several protein kinases, including the oncogene v-Mos and its cellular counterpart, c-Mos. The v-Mos-catalyzed phosphorylation sites on recombinant MAPKK1 were identified by electrospray ionization mass spectrometry as S218 and S222, located within a sequence that aligns with the T loop structure of cAMP-dependent protein kinase; these are the same as the Raf-1 phosphorylation site identified previously [Alessi, D. R., et al. (1994) EMBO J. 13, 1610-1619]. Phosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of these sites was kinetically ordered, with S222 preferred over S218. Intramolecular autophosphorylation of MAPKK occurred at several residues and was increased upon the stimulation of MAPKK activity by v-Mos. Major autophosphorylation sites were residues S298 and Y300. Minor autophosphorylation sites included T23, S299, S218, and either S24 or S25. Sequence similarities were noted between MAPKK autophosphorylation sites and exogenous phosphorylation sites on MAP kinase. Phosphorylation of either S218 or S222 was sufficient for partial MAPKK activation by Mos, and phosphorylation of S222 alone was sufficient for autophosphorylation at S298 and Y300. Mass spectral analysis was also performed on MAPKK1 purified from rabbit skeletal muscle. The peptide containing S218 and S222 was observed in only a singly phosphorylated form, and the peptide containing S298, S299, and Y300 was observed in multiply phosphorylated forms, suggesting that MAPKK is only partially phosphorylated within the T loop but significantly modified in the autophosphorylation loop under physiological conditions.
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PMID:Determination of v-Mos-catalyzed phosphorylation sites and autophosphorylation sites on MAP kinase kinase by ESI/MS. 787 42

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.
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PMID:B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP. 793 74

We have previously reported that immobilized p21ras forms a GMPPNP-dependent complex with a MEK activity. Furthermore, the association of the MEK activity was found to be independent of the presence of Raf-1. We have extended those observations to show that MEK1 is the MEK activity previously described to associate with immobilized p21ras.GMPPNP. The association between MEK1 and immobilized p21ras.GMPPNP increased its specific activity towards p42MAPK. We detected the specific association of B-Raf with immobilized p21ras.GMPPNP. In contrast to Raf-1-immunodepleted lysates, preclearance of the cytosolic B-Raf significantly reduced, by 96%, the amount of MEK1 activity associated with immobilized p21ras.GMPPNP. The decrease in MEK1 activity correlated with complete loss in the binding of both B-Raf and MEK1 proteins with immobilized p21ras.GMPPNP. These data suggest that the p21ras.GMPPNP-dependent activation of MEK1 in brain extracts is dependent on the presence of the B-Raf protein kinase.
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PMID:Association of MEK1 with p21ras.GMPPNP is dependent on B-Raf. 793 30

Mitogenic signals initiated at the plasma membrane by extracellular factors acting on receptor tyrosine kinases or G protein-coupled receptors are transmitted to the nucleus through an intricate signaling network. Components of this network participate, upon stimulation, in a complex array of phosphorylation-dependent protein-protein interactions which leads to the formation of transient multimolecular complexes. Complexes containing products of the protooncogenes ras and raf-1 and the protein kinase MEK-1 activate the mitogen-activated protein kinases (MAPKs), which play a central role in the integration of different mitogenic signals by directly phosphorylating cytoplasmic and nuclear targets. In this report we present evidence that the kinase encoded by the tumor progression locus 2 gene (Tpl-2) contributes to the activation of the MAPK cascade. MAPK activation induced by the Tpl-2 protein is blocked by dominant negative mutants of Ras and Raf-1, whereas a kinase-deficient Tpl-2 mutant down-regulates mitogenic signals induced by v-Ha-Ras or v-Raf. These data suggest that Tpl-2 activates the MAPK cascade, perhaps through its participation in the assembly of Ras/Raf-1-containing multimolecular complexes.
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PMID:Tpl-2 acts in concert with Ras and Raf-1 to activate mitogen-activated protein kinase. 793 86

Mitogen-activated protein (MAP) kinase kinases (MKKs) are dual-specificity protein kinases which activate p42mapk and p44mapk by phosphorylation of regulatory tyrosine and threonine residues. cDNAs for two isotypes of MKK, MKK1 and MKK2, have been isolated from several species. Here we describe construction of recombinant baculoviruses for high-level expression of histidine-tagged rat MKK1 and MKK2, and procedures for production of nearly homogeneous MKK1 and MKK2 fusion proteins, in both inactive and active forms. Co-infection of Sf9 cells with either MKK1 or MKK2 virus together with recombinant viruses for Raf-1, pp60src (Y527F) and c-Ha-Ras resulted in activations of 250-fold and 150-fold for MKK1 and MKK2 respectively. Specific activities towards kinase-defective p42mapk were of the order of several hundred nanomoles of phosphate transferred/min per mg of MKK protein. The Michaelis constants for both enzymes were approx. 1 microM. Preparations of activated MKK were apparently free of Raf-1 as assessed by Western blotting. Raf-1 phosphorylated MKK1 on one major tryptic phosphopeptide, the phosphorylation of which increased with time. This phosphopeptide contained only phosphoserine and possessed neutral overall charge at pH 1.9 on two-dimensional peptide mapping. Phosphorylation of MKK1 by Raf-1 correlated with activation and reached a plateau of approximately 2 mol/mol.
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PMID:Expression, purification and characterization of recombinant mitogen-activated protein kinase kinases. 794 29


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