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)

Intermediate filament proteins have been isolated from ME-180, cells of a human cervical carcinoma. Eight of these proteins have been identified as keratins by immunologic cross-reactivity to antibodies raised against authentic human epidermal keratins. The ME-180 keratin proteins consist of two major subunits designated MEK-1 and MEK-2 with approximate molecular weights of 58,000 and 53,000, respectively, and six minor subunits of 59, 57, 52.5, 50.5, 45, and 40 kilodaltons. When ME-180 cells were incubated for 2-24 h in the presence of [32P]orthophosphate, MEK-1 and MEK-2 as well as the 52.5- and 40-kilodalton keratins were phosphorylated at their serine residues. V8 protease digests revealed that phosphorylation of MEK-2 is restricted to one peptide representing approximately half the molecule. Regulation of MEK-1 and MEK-2 phosphorylation has been studied by prelabeling the cells for 2 h in 32P-labeled medium. This was followed by up to 2 h of continued incubation in the same medium after the addition of a variety of perturbing agents. The phosphorylation of MEK-2 increased in the presence of 10(-4) M dibutyryl cyclic AMP (twofold), 1 mM methylisobutylxanthine (2.5-fold), 10(-5) M isoproterenol (fivefold), and 10(-9) M cholera toxin (sevenfold). In contrast, MEK-1 phosphorylation was unaffected by these agents. Neither cyclic GMP, Ca++, hydrocortisone, nor epidermal growth factor had any effect on the phosphorylation of MEK-1 or MEK-2. The results indicate that the phosphorylation of these two keratins is independently controlled by cyclic AMP-dependent kinase for MEK-2 and by cyclic nucleotide-independent kinase for MEK-1. The observed differences in control suggest distinct functions for MEK-1 and MEK-2 within the cytoskeletal network.
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PMID:Dual regulation of intermediate filament phosphorylation. 619 63

Activation of mitogen-activated protein kinase (MAP kinase) plays an important role in the cellular effects of nerve growth factor (NGF). Although the precise pathway by which NGF activates MAP kinase is not clear, several enzymes have been identified that may form a linear phosphorylation cascade, in which MAP kinase is activated by MAP kinase kinase (MEK). A key enzyme that links the ras-GTP complex to MEK is widely believed to be the raf kinase. However, immunoprecipitation experiments in PC-12 cells revealed that raf is not the major NGF-dependent MEK kinase [Zheng, Ohmichi, Saltiel and Guan (1994) Biochemistry 33, 5595-5599]. We have identified a protein kinase from PC-12 cells that catalyses both the phosphorylation and activation of MEK. This activity is stimulated 3-fold in cells treated with NGF. The partial purification on FPLC and characterization of this MEK kinase indicate that it is distinct from raf, MEK, MAP kinase and other previously described NGF-stimulated protein kinases. The activity of this enzyme is unaffected by direct addition to the assay of heparin, staurosporine, K252A and the heat-stable cyclic AMP-dependent kinase peptide inhibitor, but is slightly inhibited by NaF and calcium ions. Comparison of its behaviour on gel permeation and sucrose-density gradients indicates a molecular mass in the region of 50,000 Da. Moreover, isoelectric focusing of the enzyme revealed a pI of approx. 7.3. The kinase activity is specific for ATP as substrate with a Km of 11 microM, and requires Mg2+ as a cofactor. Analysis of the activation of this enzyme in PC-12 cells transfected with a dominant inhibitory mutant of p21ras suggests that this MEK kinase resides downstream of ras in the MAP kinase activation pathway. Moreover, site-directed mutation of the residues on MEK that are phosphorylated by raf does not completely abrogate phosphorylation by the MEK kinase, suggesting that this enzyme may share some phosphorylation sites with raf, but also phosphorylates MEK on other sites.
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PMID:Nerve growth factor stimulates a novel protein kinase in PC-12 cells that phosphorylates and activates mitogen-activated protein kinase kinase (MEK). 773 91

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

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

The cDNA encoding the platelet-activating factor (PAF) receptor was cloned from a guinea pig lung cDNA library by using a Xenopus laevis oocyte expression system. In the CHO cells which expressed guinea-pig PAF receptor, PAF triggered production of inositol phosphates, the release of arachidonic acid, and inhibited cyclic AMP accumulation. PAF also activated mitogen-activated protein (MAP) kinase and MAP kinase kinase in the CHO cells. These effects were partially regulated by pertussis toxin-sensitive G proteins. The analysis of the human PAF receptor gene revealed that it contains no intron in its coding region, but introns are distributed in the 5'-untranslated region. Two 5'-noncoding exons were identified, which are alternatively spliced to a common splice acceptor site on the third exon to yield two different species of functional mRNA. Existence of distinct promoters may regulated the PAF receptor gene expression in different tissues and cells.
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PMID:[Biological regulation with platelet activating factor--molecular cloning and signal transduction of PAF]. 802 85

The platelet-activating factor (PAF) was seen to potently activate mitogen-activated protein (MAP) kinase and MAP kinase kinase through the cloned guinea pig PAF receptor stably expressed in Chinese hamster ovary (CHO) cells. Both 42- and 44-kDa MAP kinases were activated and tyrosine-phosphorylated in response to PAF. The PAF receptor also triggered the production of inositol phosphates and the release of arachidonic acid and inhibited cyclic AMP accumulation. Differential inhibitory effects of pertussis toxin (PTX) on these signals suggested that the PAF receptor couples to both PTX-sensitive and -insensitive G proteins in CHO cells. MAP kinase and MAP kinase activations were partially regulated by PTX-sensitive G proteins. The PAF receptor did not trigger any detectable increase in the GTP form of Ras under the conditions in which the human insulin receptor expressed in the same parent CHO cells potently increased the level. Since these agonists induced comparable MAP kinase activations through cognate receptors, Ras seems to play different roles in MAP kinase activation by the two different classes of receptors. The activation of MAP kinase by the cloned PAF receptor may explain part of the mechanisms underlying PAF-induced differentiation and proliferation in non-inflammatory cells.
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PMID:Transfected platelet-activating factor receptor activates mitogen-activated protein (MAP) kinase and MAP kinase kinase in Chinese hamster ovary cells. 829 89

We have investigated the mechanisms that bring about the termination of mitogen-activated protein kinase (MAP kinase) activation in response to UTP in EAhy 926 endothelial cells. UTP-stimulated MAP kinase activity was transient, returning to basal values by 60 min. At this time MAP kinase activation was desensitized; re-application of UTP did not further activate MAP kinase, full re-activation of MAP kinase being only apparent after a 1-2 h wash period. However, activation of MAP kinase by UTP could be sustained beyond 60 min by preincubation of the cells with the protein synthesis inhibitor cycloheximide. UTP also stimulated expression of MAP kinase phosphatase-1 and this was abolished after pretreatment with cycloheximide. Pretreatment of cells with forskolin abolished the initial activation of MAP kinase kinase or c-Raf-1 by UTP, but only affected MAP kinase activity during prolonged stimulation. The effect of forskolin on prolonged MAP kinase activation was also prevented by cycloheximide. These results suggest that the termination of MAP kinase activity in response to UTP involves a number of interacting mechanisms including receptor desensitization and the induction of a phosphatase. However, several pieces of evidence do not support a major role for MAP kinase phosphatase-1 in termination of the MAP kinase signal. Raising intracellular cyclic AMP may also be involved but only after an initial protein-synthesis step and by a mechanism that does not involve the inactivation of c-Raf-1 or MAP kinase kinase.
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PMID:Role of receptor desensitization, phosphatase induction and intracellular cyclic AMP in the termination of mitogen-activated protein kinase activity in UTP-stimulated EAhy 926 endothelial cells. 861 30

Phenylephrine and noradrenaline (alpha-adrenergic agonism) or isoprenaline (beta-adrenergic agonism) stimulated protein synthesis rates, increased the activity of the atrial natriuretic factor gene promoter and activated mitogen-activated protein kinase (MAPK). The EC50 for MAPK activation by noradrenaline was 2-4 microM and that for isoprenaline was 0.2-0.3 microM. Maximal activation of MAPK by isoprenaline was inhibited by the beta-adrenergic antagonist, propranolol, whereas the activation by noradrenaline was inhibited by the alpha1-adrenergic antagonist, prazosin. FPLC on a Mono-Q column separated two peaks of MAPK (p42MAPK and p44MAPK) and two peaks of MAPK-activating activity (MEK) activated by isoprenaline or noradrenaline. Prolonged phorbol ester exposure partially down-regulated the activation of MAPK by noradrenaline but not by isoprenaline. This implies a role for protein kinase C in MAPK activation by noradrenaline but not isoprenaline. A role for cyclic AMP in activation of the MAPK pathway was eliminated when other agonists that elevate cyclic AMP in the cardiac myocyte did not activate MAPK. In contrast, MAPK was activated by exposure to ionomycin, Bay K8644 or thapsigargin that elevate intracellular Ca2+. Furthermore, depletion of extracellular Ca2+ concentrations with bis-(o-aminophenoxy)ethane-NNN'N'-tetra-acetic acid (BAPTA) or blocking of the L-type Ca2+ channel with nifepidine or verapamil inhibited the response to isoprenaline without inhibiting the responses to noradrenaline. We conclude that alpha- and beta-adrenergic agonists can activate the MEK/MAPK pathway in the heart by different signalling pathways. Elevation of intracellular Ca2+ rather than cyclic AMP appears important in the activation of MAPK by isoprenaline in the cardiac myocyte.
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PMID:Adrenergic receptor stimulation of the mitogen-activated protein kinase cascade and cardiac hypertrophy. 866 Feb 71

In the course of screening for inhibitors of tumorigenic phenotype of K-ras-transformed NIH3T3 cells (DT cells), we found a novel compound, oxamflatin, an aromatic sulfonamide hydroxamate derivative, which induces flat phenotype in these cells and suppresses their anchorage-independent growth. In contrast to DT cells, in v-raf-transformed NIH3T3 cells, no change in their morphology and no specific inhibition of their anchorage-independent growth was observed. Interestingly, oxamflatin was effective to NIH3T3 cells transformed by constitutively activated mutant of MEK, indicating the possibility that oncogene-induced morphological change is not necessarily induced by common signaling pathway such as MAP kinase cascade. In oxamflatin-treated DT cells, the expression of transcription factor junD was highly augmented, resulting in trans-activation of fibronectin gene by junD via cyclic AMP responsive element in its promoter. This behavior of junD was confirmed to correlate well with partial blocking of malignant phenotype in DT cells. Thus, oxamflatin can be categorized as the first reagent which induces genes whose products can interfere with oncogene-dependent transformation.
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PMID:Oxamflatin: a novel compound which reverses malignant phenotype to normal one via induction of JunD. 870 May 40

The effects of cyclic AMP on mitogen-activated protein (MAP) kinase activation were investigated in 3T3-F442A preadipocytes. Several agents, which raise intracellular cyclic AMP levels by distinct mechanisms, induced a transient activation of the p42 and p44 isoforms of MAP kinase and of a MAP kinase kinase. Activation of MAP kinase by cyclic AMP was prevented by two distinct inhibitors of cyclic AMP-dependent protein kinase and by PD 098059, a specific inhibitor of the activation of the MAP kinase kinase MEK 1. Therefore, in contrast to most cell types studied, cyclic AMP exerts a positive influence on the MAP kinase pathway in 3T3-F442A preadipocytes at a level upstream of the activation of MAP kinase kinase.
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PMID:Cyclic AMP stimulates the phosphorylation and activation of p42 and p44 mitogen-activated protein kinases in 3T3-F442A preadipocytes. 871 15


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