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)

The use of bisindolylmaleimide derivatives of staurosporine as selective inhibitors of protein kinase C (PKC) is in doubt following the report by Alessi [FEBS Lett. 402 (1997) 121-123] that Ro31-8220 and GF109203X are potent in vitro inhibitors of p70 S6 kinase and mitogen-activated protein kinase-activated protein kinase-1beta, as well as of PKC. Here we show that the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells due to phospholipid hydrolysis by phospholipase D (PLD) is not inhibited by rapamycin or PD98059, specific inhibitors respectively of p70 S6 kinase and MAPKK (MEK) and thus of MAPKAP kinase-1beta but is still completely blocked by Ro31-8220. We conclude therefore that p70S6k and MAPKAP kinase-1beta as well as MAPK are not involved in signalling pathways downstream of PKC that regulate phorbol ester-stimulated phospholipid turnover and that the inhibitory action of Ro31-8220 occurs by blocking PKC which regulates at least one pathway to PLD activation. The PI-3 kinase inhibitor, wortmannin, inhibits the phorbol ester-stimulated release of ethanolamine- but not choline-metabolites from C6 cells suggesting that different PLD isoforms regulate the turnover of PtdEth and PtdCho in C6 cells. Both PLD isoforms are activated via PKC but the PtdEth-PLD is also regulated via a wortmannin-sensitive pathway.
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PMID:Ro31-8220 inhibits protein kinase C to block the phorbol ester-stimulated release of choline- and ethanolamine-metabolites from C6 glioma cells: p70 S6 kinase and MAPKAP kinase-1beta do not function downstream of PKC in activating PLD. 939 70

This study revealed an important and unexpected finding: namely, that inhibitory melatonin receptors can inhibit a phorbol 12,13 myristate acetate (PMA)-induced, protein kinase C (PKC)-dependent increase in c-fos messenger RNA expression in ovine pars tuberalis (PT) cells. PMA induces dose-dependent stimulation of c-fos expression that is attenuated by melatonin in a dose-dependent and pertussis toxin-sensitive manner. The effect of 100 nM PMA is blocked by Ro31-8220 (1 microM), yet is not mimicked by 4alpha-PMA (100 nM). PMA (100 nM) induces PKC activity in PT cells (P < 0.05) within 5 min, but melatonin has no effect on this response. PMA (100 nM) stimulates both phospholipase D and mitogen-activated protein kinase (MAPK) (p42/44) activities in PT cells, but melatonin has no effect on these responses. The results indicate that neither of these second-messenger activities contribute to the melatonin-sensitive pathway of c-fos activation. The MEK (MAPK kinase) inhibitor, PD98059 (50 microM), does not block the induction of c-fos by PMA, although at the same dose it inhibits PMA-mediated activation of p42/44 MAPK by 50-70%, and activation by forskolin or insulin-like growth factor-I by 100%. These data suggest that p42/44 MAPK may not be the primary mediator of PKC-dependent c-fos induction. In contrast to the effect of melatonin on PMA-mediated c-fos induction in PT cells, in L cells stably transfected with the sheep Mel1 alphabeta receptor, melatonin potentiates the c-fos response in a pertussis toxin-sensitive manner. These data indicate the tissue-specific nature of melatonin receptor signaling, and reveal that a pertussis toxin-sensitive pathway can block PKC-mediated c-fos induction in PT cells.
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PMID:A novel interaction between inhibitory melatonin receptors and protein kinase C-dependent signal transduction in ovine pars tuberalis cells. 952 55

1. Extracellular adenosine triphosphate (ATP) is mitogenic for vascular smooth muscle cells (VSMC) and stimulates several events that are important for cell proliferation: DNA synthesis, protein synthesis, increase of cell number, immediate early genes, cell-cycle progression, and tyrosine phosphorylation. 2. Receptor characterization indicates mitogenic effects of both P2U and P2Y receptors. The P2X receptor is lost in cultured VSMC and is not involved. Several related biological substances such as UTP, ITP, GTP, AP4A, ADP, and UDP are also mitogenic. 3. Signal transduction is mediated via Gq-proteins, phospholipase C beta, phospholipase D, diacyl glycerol, protein kinase C alpha, delta, Raf-1, MEK, and MAPK. 4. ATP acts synergistically with polypeptide growth factors (PDGF, bFGF, IGF-1, EGF, insulin) and growth factors acting via G-protein-coupled receptors (noradrenaline, neuropeptide Y, 5-hydroxytryptamine, angiotensin II, endothelin-1). 5. The mitogenic effects have been demonstrated in rat, porcine, and bovine VSMC and cells from human coronary arteries, aorta, and subcutaneous arteries and veins. 6. The trophic effects on VSMC and the abundant sources for extracellular ATP in the vessel wall make a pathophysiological role probable in the development of atherosclerosis, neointima-formation after angioplasty, and possibly hypertension.
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PMID:Extracellular ATP: a growth factor for vascular smooth muscle cells. 959 70

We have previously reported that hydrogen peroxide (H2O2) induced a considerable increase of phospholipase D (PLD) activity and phosphorylation of mitogen-activated protein (MAP) kinase in PC12 cells. H2O2-induced PLD activation and MAP kinase phosphorylation were dose-dependently inhibited by a specific MAP kinase kinase inhibitor, PD 098059. In contrast, carbachol-mediated PLD activation was not inhibited by the PD 098059 pretreatment whereas MAP kinase phosphorylation was prevented. These findings indicated that MAP kinase is implicated in the PLD activation induced by H2O2, but not by carbachol. In the present study, H2O2 also caused a marked release of oleic acid (OA) from membrane phospholipids in PC12 cells. As we have previously shown that OA stimulates PLD activity in PC12 cells, the mechanism of H2O2-induced fatty acid liberation and its relation to PLD activation were investigated. Pretreatment of the cells with methylarachidonyl fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor, almost completely prevented the release of [3H]OA by H2O2 treatment. From the preferential release of OA and sensitivity to other PLA2 inhibitors, the involvement of a Ca2+-independent cytosolic PLA2-type enzyme was suggested. In contrast to OA release, MAFP did not inhibit PLD activation by H2O2. The inhibitory profile of the OA release by PD 098059 did not show any correlation with that of MAP kinase. These results lead us to suggest that H2O2-induced PLD activation may be mediated by MAP kinase and also that H2O2-mediated OA release, which would be catalyzed by a Ca2+-independent cytosolic PLA2-like enzyme, is not linked to the PLD activation in PC12 cells.
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PMID:Possible involvement of mitogen-activated protein kinase in phospholipase D activation induced by H2O2, but not by carbachol, in rat pheochromocytoma PC12 cells. 983 25

The serine/threonine kinase Raf-1 is an essential component of the MAPK cascade. Activation of Raf-1 by extracellular signals is initiated by association with intracellular membranes. Recruitment of Raf-1 to membranes has been reported to be mediated by direct association with Ras and by the phospholipase D product phosphatidic acid (PA). Here we report that insulin stimulation of HIRcB fibroblasts leads to accumulation of Ras, Raf-1, phosphorylated MEK, phosphorylated MAPK, and PA on endosomal membranes. Mutations that disrupt Raf-PA interactions prevented recruitment of Raf-1 to membranes, whereas disruption of Ras-Raf interactions did not affect agonist-dependent translocation. Expression of a dominant-negative Ras mutant did not prevent insulin-dependent Raf-1 translocation, but inhibited phosphorylation of MAPK. Finally, the PA-binding region of Raf-1 was sufficient to target green fluorescent protein to membranes, and its overexpression blocked recruitment of Raf-1 to membranes and disrupted insulin-dependent MAPK phosphorylation. These results indicate that agonist-dependent Raf-1 translocation is primarily mediated by a direct interaction with PA and is independent of association with Ras.
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PMID:The recruitment of Raf-1 to membranes is mediated by direct interaction with phosphatidic acid and is independent of association with Ras. 1080 16

In skeletal myoblasts, Ras has been considered to be a strong inhibitor of myogenesis. Here, we demonstrate that Ras is involved also in the chemotactic response of skeletal myoblasts. Expression of a dominant-negative mutant of Ras inhibited chemotaxis of C2C12 myoblasts in response to basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), and insulin-like growth factor 1 (IGF-1), key regulators of limb muscle development and skeletal muscle regeneration. A dominant-negative Ral also decreased chemotactic migration by these growth factors, while inhibitors for phosphatidylinositol 3-kinase and mitogen-activated protein kinase kinase (MEK) showed no effect. Activation of the Ras-Ral pathway by expression of an activated mutant of either Ras, the guanine-nucleotide dissociation stimulator for Ral, or Ral resulted in increased motility of myoblasts. The ability of Ral to stimulate motility was reduced by introduction of a mutation which prevents binding to Ral-binding protein 1 or phospholipase D. These results suggest that the Ras-Ral pathway is essential for the migration of myoblasts. Furthermore, we found that Ras and Ral are activated in C2C12 cells by bFGF, HGF and IGF-1 and that the Ral activation is regulated by the Ras- and the intracellular Ca(2+)-mediated pathways. Taken together, our data indicate that Ras and Ral regulate the chemotactic migration of skeletal muscle progenitors.
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PMID:Involvement of Ras and Ral in chemotactic migration of skeletal myoblasts. 1084 92

In normal development, embryonic astrocytes progress through their cell lineage by acquiring differentiation, by apoptosis, and by proliferation. In this study, we show that embryonic astrocytes may maintain and make gains in differentiation as they simultaneously progress through one cell cycle when induced by prolactin (PRL). Prolactin induced the majority of astrocytes to incorporate bromodeoxyuridine (BrdU) with a four-fold increase over controls after 18 h of exposure. Investigating possible mitogenic signaling pathways we show for the first time that prolactin is coupled to a sustained phospholipase D (PLD) activation, with an efficacy similar to the phorbol ester and astrocytic mitogen 12-tetradecanoylphorbol-13-acetate (TPA). Both cyclosporine and suramin abolished this activation. Staurosporine and calphostin C also inhibited the PRL effect by 50%, consistent with involvement of protein kinase C-(PKC)-alpha, the major PKC isoform in astrocytes. Genistein and PP1 blocked the activation indicating additional regulation by cytosolic tyrosine kinases. This profile of PLD activation was suggestive of a PLD I isoform and a mitogenic response. Upon completion of the cell cycle, analysis of glia fibrillary acidic protein (GFAP) and vimentin abundance, and glutamine synthetase (GS) activity showed that astrocytes had gained in expression of differentiation markers. Moreover, the intensity of GFAP immunofluorescence was greater per cell, as was the length of the cell processes. In exploring the signaling for prolactin-induced differentiation we found that prolactin activated the tyrosine kinase Janus kinase (JAK) 2 and significantly stimulated tyrosine, phosphorylation of the prolactin receptor. Stat 1 and 3 were also activated presumably downstream to JAK2 activation. A rapid translocation of the cytosolic Stats over the nucleus was seen in nearly every astrocyte corresponding well with the gains in GFAP per cell. The Stats translocation did not depend on MEK-ERK inhibition by PD98059, inhibition of p38 by 1 microm SB203580, or Src kinase family inhibition by PP1. Our results demonstrate the ability of PRL to concurrently induce activation of PLD, a mitogenic signaling pathway in astrocytes, and prolonged stimulation of Stat1, compatible with the increased GFAP upregulation and cell differentiation. Considered together this data may provide an explanation on the fast gain in both numbers and differentiation in the astrocytic population during development (HD 09402, CRF).
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PMID:Prolactin concurrently activates src-PLD and JAK/Stat signaling pathways to induce proliferation while promoting differentiation in embryonic astrocytes. 1097 48

Angiotensin II (Ang II) activates cytosolic phospholipase A(2) (cPLA(2)) and phospholipase D (PLD) in rabbit vascular smooth muscle cells (VSMCs). Ang II also activates ras/mitogen-activated protein (MAP) kinase in VSMCs; this activation is mediated by 20-hydroxyeicosatetraenoic acid (HETE) and 12(S)-HETE, which are metabolites of arachidonic acid generated by cytochrome P450 4A and lipoxygenase, respectively, produced on activation of cPLA(2). The purpose of this study was to determine if Ang II-induced PLD activation in VSMCs is mediated through the ras/extracellular signal-regulating kinase (ERK) pathway by arachidonic acid metabolites that are generated consequent to cPLA(2) stimulation. Inhibitors of PLD (C(2) ceramide), phosphatidate phosphohydrolase (propranolol), and diacylglycerol lipase (RHC 80267) attenuated Ang II-induced arachidonic acid release. Ang II-induced PLD activation, as measured by [(3)H]phosphatidylethanol production, was inhibited by C(2) ceramide but not by propranolol or RHC 80267. Ang II-induced PLD activation was decreased by the inhibitor methyl arachidonylfluorophosphate (MAFP) and the antisense oligonucleotide of cPLA(2). Inhibitors of lipoxygenases (baicalein) and cytochrome P450 4A (ODYA) attenuated Ang II-induced PLD activation. 20-HETE and 12(S)-HETE increased PLD activity. Inhibitors of ras farnesyltransferase (FPT III and BMS-191563) and MAP kinase kinase (UO126) attenuated the increase in PLD activity elicited by 20-HETE and Ang II. PLD2 was the main isoform activated by Ang II in VSMCs. These data suggest that the CYP4A metabolite 20-HETE, which is generated from arachidonic acid after cPLA(2) activation by Ang II, stimulates the ras/MAP kinase pathway, which in turn activates PLD2 and releases further arachidonic acid for prostaglandin synthesis through the phosphatidate phosphohydrolase/diacylglycerol lipase pathway.
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PMID:20-Hydroxyeicosatetraenoic acid mediates angiotensin ii-induced phospholipase d activation in vascular smooth muscle cells. 1123 Mar 46

Norepinephrine (NE) stimulates phospholipase D (PLD) through a Ras/MAPK pathway in rabbit vascular smooth muscle cells (VSMC). NE also activates calcium influx and calmodulin (CaM)-dependent protein kinase II-dependent cytosolic phospholipase A(2) (cPLA(2)). Arachidonic acid (AA) released by cPLA(2)-catalyzed phospholipid hydrolysis is then metabolized into hydroxyeicosatetraenoic acids (HETEs) through lipoxygenase and cytochrome P450 4A (CYP4A) pathways. HETEs, in turn, have been shown to stimulate Ras translocation and to increase MAPK activity in VSMC. This study was conducted to determine the contribution of cPLA(2)-derived AA and its metabolites (HETEs) to the activation of PLD. NE-induced PLD activation was reduced by two structurally distinct CaM antagonists, W-7 and calmidazolium, and by CaM-dependent protein kinase II inhibition. Blockade of cPLA(2) activity or protein depletion with selective cPLA(2) antisense oligonucleotides abolished NE-induced PLD activation. The increase in PLD activity elicited by NE was also blocked by inhibitors of lipoxygenases (baicalein) and CYP4A (17-octadecynoic acid), but not of cyclooxygenase (indomethacin). AA and its metabolites (12(S)-, 15(S)-, and 20-HETEs) increased PLD activity. PLD activation by AA and HETEs was reduced by inhibitors of Ras farnesyltransferase (farnesyl protein transferase III and BMS-191563) and MEK (U0126 and PD98059). These data suggest that HETEs are the mediators of cPLA(2)-dependent PLD activation by NE in VSMC. In addition to cPLA(2), PLD was also found to contribute to AA release for prostacyclin production via the phosphatidate phosphohydrolase/diacylglycerol lipase pathway. Finally, a catalytically inactive PLD(2) (but not PLD(1)) mutant inhibited NE-induced PLD activity, and PLD(2) was tyrosine-phosphorylated in response to NE by a MAPK-dependent pathway. We conclude that NE stimulates cPLA(2)-dependent PLD(2) through lipoxygenase- and CYP4A-derived HETEs via the Ras/ERK pathway by a mechanism involving tyrosine phosphorylation of PLD(2) in rabbit VSMC.
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PMID:Phospholipase D activation by norepinephrine is mediated by 12(s)-, 15(s)-, and 20-hydroxyeicosatetraenoic acids generated by stimulation of cytosolic phospholipase a2. tyrosine phosphorylation of phospholipase d2 in response to norepinephrine. 1127 12

Exposure to hydrogen peroxide induced considerable activation of phospholipase D (PLD) in rat pheochromocytoma PC12 cells. This PLD activation was potentiated by orthovanadate and okadaic acid, suggesting that tyrosine kinase and serine/threonine kinase are involved. Furthermore, H2O2-induced PLD activation was partially inhibited by either MEK1 inhibitor (PD98059) or p38 MAP kinase inhibitor (SB203580), but a combination of both inhibitors resulted in nearly 80% suppression. The major isozyme was found to be PLD2 in PC12 cells by Western blotting analysis. When the PLD2-transfected COS-7 cells were exposed to H2O2, the PLD activation was markedly inhibited by the combined pretreatment with PD98059 and SB203580. To our knowledge, this study is the first demonstration that both ERK1/2 and p38 MAP kinase are involved in the PLD2 activation in PC12 cells exposed to H2O2.
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PMID:Involvement of ERK and p38 MAP kinase in oxidative stress-induced phospholipase D activation in PC12 cells. 1144 48


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