Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.24 (mitogen-activated protein kinase)
 95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Maximally effective concentrations of endothelin-1 (ET-1), acidic FGF (aFGF), or 12-O-tetradecanoylphorbol-13-acetate (TPA) activated mitogen-activated protein kinase (MAPK) by 3-4-fold in crude extracts of myocytes cultured from neonatal rat heart ventricles. Maximal activation was achieved after 5 min. Thereafter, MAPK activity stimulated by ET-1 or aFGF declined to control values within 1-2 h, whereas activation by TPA was more sustained. Two peaks of MAPK activity (a 42- and a 44-kDa MAPK) were resolved in cells exposed to ET-1 or aFGF by fast protein liquid chromatography on a Mono Q column. One major and one minor peak of MAPK kinase (MAPKK) was stimulated by ET-1 or aFGF. Cardiac myocytes expressed protein kinase C (PKC)-alpha, -delta, -epsilon and -zeta as shown immunoblotting. Exposure to 1 microM TPA for 24 h down-regulated PKC-alpha, -delta, and -epsilon, but not PKC-zeta. This maneuver wholly abolished the activation of MAPK on re-exposure to TPA but did not affect the response to aFGF. The effect of ET-1 was partially down-regulated. ET-1 stimulated phospho[3H]inositide hydrolysis 18-fold, whereas aFGF stimulated by only 30%. Agonists which initially utilize dissimilar signaling pathways may therefore converge at the level of MAPKK/MAPK and this may be relevant to the hypertrophic response of the heart.
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PMID:Endothelin-1 and fibroblast growth factors stimulate the mitogen-activated protein kinase signaling cascade in cardiac myocytes. The potential role of the cascade in the integration of two signaling pathways leading to myocyte hypertrophy. 750 4

The translocation of protein kinase C (PKC) isoforms PKC-alpha, PKC-delta, PKC-epsilon, and PKC-zeta from soluble to particulate fractions was studied in ventricular cardiomyocytes cultured from neonatal rats. Endothelin-1 (ET-1) caused a rapid ETA receptor-mediated translocation of PKC-delta and PKC-epsilon (complete in 0.5-1 min). By 3-5 min, both isoforms were returning to the soluble fraction, but a greater proportion of PKC-epsilon remained associated with the particulate fraction. The EC50 of translocation for PKC-delta was 11-15 nM ET-1 whereas that for PKC-epsilon was 1.4-1.7 nM. Phenylephrine caused a rapid translocation of PKC-epsilon (EC50 = 0.9 microM) but the proportion lost from the soluble fraction was less than with ET-1. Translocation of PKC-delta was barely detectable with phenylephrine. Neither agonist caused any consistent translocation of PKC-alpha or PKC-zeta. Activation of p42 and p44 mitogen-activated protein kinase (MAPK) by ET-1 or phenylephrine followed more slowly (complete in 3-5 min). Phosphorylation of p42-MAPK occurred simultaneously with its activation. The proportion of the total p42-MAPK pool phosphorylated in response to ET-1 (50%) was greater than with phenylephrine (20%). In addition to activation of MAPK, an unidentified p85 protein kinase was activated by ET-1 in the soluble fraction whereas an unidentified p58 protein kinase was activated in the particulate fraction.
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PMID:Differential activation of protein kinase C isoforms by endothelin-1 and phenylephrine and subsequent stimulation of p42 and p44 mitogen-activated protein kinases in ventricular myocytes cultured from neonatal rat hearts. 780 10

p74raf-1, a serine/threonine kinase, is structurally related to the protein kinase C (PKC) family and contains a cysteine motif in its N-terminal domain, which is essential for its regulation. It has been shown that p74raf-1 functions upstream of mitogen-activated protein (MAP) kinase kinase. We have constructed a p74raf-1 mutant (N delta raf) that only contains the N-terminal regulatory domain. When transiently expressed in COS-M6 cells, N delta raf efficiently blocked the activation of the MAP extracellular signal regulated kinase (ERK2), induced by either epidermal growth factor, phorbol ester, serum, or oncogenic p21ras. Similar constructs with the cysteine motifs from either PKC-alpha or diacylglycerol kinase did not inhibit activation of ERK2. Overexpression of full-length p74raf-1 rescued the inhibition of ERK2 by N delta raf in a stimulus dependent manner, indicating that N delta raf acts as a competitive inhibitor of wild-type p74raf-1. In contrast, overexpression of either PKC-alpha, -epsilon, or -zeta in N delta raf-containing cells could not rescue the inhibition of ERK2. We conclude that p74raf-1 is an essential mediator of epidermal growth factor- and phorbol ester-induced ERK2 activation and that the MAP kinase kinase activity of p74raf-1 cannot be substituted with either PKC-alpha, -epsilon or -zeta.
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PMID:A dominant-negative mutant of raf blocks mitogen-activated protein kinase activation by growth factors and oncogenic p21ras. 839 1

Protein kinase C (PKC) and mitogen-activated protein (MAP) kinase are protein-serine/threonine kinases which are important regulators of diverse cellular processes including metabolism, proliferation and differentiation. This study shows that both hypoxia and X irradiation of serum-deprived Chinese hamster V79 cells cause the induction and phosphorylation of the PKC-alpha isoform. The increased induction and phosphorylation of PKC occur mainly in the nuclear fraction. Unlike the PKC activator TPA, neither hypoxic nor radiation stress causes translocation of PKC-alpha from the cytosol to the membrane. The induction of PKC-alpha by hypoxia is accompanied by an increased expression of MAP kinase but, in contrast, this does not occur when PKC-alpha is induced by radiation. Radiation, like TPA, causes a complete redistribution of MAP kinase from the cytosol to the nucleus.
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PMID:Induction and phosphorylation of protein kinase C-alpha and mitogen-activated protein kinase by hypoxia and by radiation in Chinese hamster V79 cells. 860 21

When HD3 colon carcinoma cells differentiate to fluid-transporting, enterocytic-like cells, they down-regulate their protein kinase C (PKC) beta levels 5-10-fold and lose two responses to basic fibroblast growth factor (FGF): proliferation and the ability to activate p57 mitogen-activated protein (MAP) kinase. HD3 cells were transfected with expression plasmids for the splice variants PKC-beta 1 and PKC-beta 2 and the empty vector for a control. Each of two PKC-beta 1 and each of two PKC-beta 2 transfectant clones exhibited elevated levels of Ca(2+)-and phosphatidylserine-dependent PKC activity. Both PKC-beta 1 transfectant clones had elevated levels of PKC-beta 1 protein compared with the PKC-beta 2 transfectants or controls, whereas both PKC-beta 2 transfectant clones had elevated levels of PKC-beta 2 protein compared with PKC-beta 1 transfectants. Control transfectants had no detectable PKC-beta 2 protein. Similar levels of PKC-alpha were found in all lines. Each PKC-beta transfectant was less differentiated than the parental line and had regained proliferative response to basic FGF. Increased growth rates in athymic mice were seen for PKC-beta 2 and PKC-beta 1 transfectant cells. Immunocytochemistry of the sectioned tumors showed enhanced protein levels of PKC-beta 2 and PKC-beta 1, correlating increased levels of these isonzymes with increased growth. Increased myelin-basic protein (MBP) kinase activities of M(r) 44,000, 57,000, 63,000, 110,000, and 130,000 by in-gel kinase assay characterized each PKC-beta transfectant. Both Western blotting and immunoprecipitation studies from 35S-prelabeled cells with a pan-erk antibody showed no increase in protein abundance of MAP kinases of M(r) 44,000, 57,000, and 63,000, suggesting that elevated PKC-beta levels led to activation of the smaller three MAP and MBP kinases. Activation of p57 MAP kinase in each PKC-beta transfectant was demonstrated by immunoprecipitation with an antiphosphotyrosine monoclonal antibody and then by assay of the immunoprecipitates by in-gel kinase assay on MBP. p57 MAP kinase was distinguished from the M(r) 54,000 stress-activated protein kinases, which migrated more rapidly on SDS gels and could be detected by in-gel kinase assay on MBP only after cellular stress. Thus, expression of elevated levels of PKC-beta 1 and PKC-beta 2 in differentiated HD3 colon carcinoma cells blocked their differentiation, enabled them to proliferate in response to basic FGF like undifferentiated cells, increased their growth rate in athymic mice, and activated several MBP kinases, among them, p57 MAP kinase.
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PMID:Protein kinase C beta 1 and protein kinase C beta 2 activate p57 mitogen-activated protein kinase and block differentiation in colon carcinoma cells. 873 68

The serine/threonine kinase Raf-1 functions downstream of Rats in a signal transduction cascade which transmits mitogenic stimuli from the plasma membrane to the nucleus. Raf-1 integrates signals coming from extracellular factors and, in turn, activates its substrate, MEK kinase. MEK activates mitogen-activated protein kinase (MAPK), which phosphorylates other kinases as well as transcription factors. Raf-1 exists in a complex with HSP90 and other proteins. The benzoquinone ansamycin geldanamycin (GA) binds to HSP90 and disrupts the Raf-1-HSP90 multimolecular complex, leading to destabilization of Raf-1. In this study, we examined whether Raf-1 destabilization is sufficient to block the Raf-1-MEK-MAPK signalling pathway and whether GA specifically inactivates the Raf-1 component of this pathway. Using the model system of NIH 3T3 cells stimulated with phorbol 12-myristate 13-acetate (PMA), we show that GA does not affect the ability of protein kinase C alpha to be activated by phorbol esters, but it does block activation of MEK and MAPK. Further, GA does not decrease the activity of constitutively active MEK in transiently transfected cells. Finally, disruption of the Raf-1-MEK-MAPK signalling pathway by GA prevents both the PMA-induced proliferative response and PMA-induced activation of a MAPK-sensitive nuclear transcription factor. Thus, we demonstrate that interaction between HSP90 and Raf-1 is a sine qua non for Raf stability and function as a signal transducer and that the effects observed cannot be attributed to a general impairment of protein kinase function.
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PMID:Destabilization of Raf-1 by geldanamycin leads to disruption of the Raf-1-MEK-mitogen-activated protein kinase signalling pathway. 881 98

Recent evidence has implicated caveolae/DIGs in various aspects of signal transduction, a process in which polyphosphoinositides play a central role. We therefore undertook a study to determine the distribution of phosphoinositides and the enzymes that utilize them in these detergent-insoluble domains. We report here that the polyphosphoinositide phosphatase, but not several other phosphoinositide-utilizing enzymes, is highly enriched in a low density, Triton-insoluble membrane fraction that contains caveolin. This fraction is also enriched in polyphosphoinositides, containing approximately one-fifth of the total cellular phosphatidylinositol (4,5)P2. Treatment of cells with the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate (PMA), did not alter the distribution of polyphosphoinositides or the polyphosphoinositide phosphatase. However, PMA treatment did lead to a decrease in the mitogen-activated protein kinase and actin present in these domains. PMA also induced the recruitment of protein kinase C alpha to the caveolae/DIGs fraction. These findings suggest that polyphosphoinositides, the polyphosphoinositide phosphatase and protein kinase C play an important role in the structure or function of detergent-insoluble membrane domains.
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PMID:Phosphoinositides and phosphoinositide-utilizing enzymes in detergent-insoluble lipid domains. 881 92

The mitogenic effect of extracellular ATP was examined in cultured rat aortic smooth muscle cells (VSMCs). ATP, 2-methylthio-ATP, and ADP stimulated [3H]thymidine and [3H]leucine incorporation and cell growth. AMP, adenosine, UTP, and P2x agonists showed little of these effects. Reactive blue 2, a P2Y purinoceptor antagonist, was effective in suppressing the mitogenic effect of ATP and 2-methylthio-ATP, indicating that extracellular ATP-induced VSMC proliferation is mediated by P2Y purinoceptors. The P2Y purinoceptor activation was coupled to a pertussis toxin (PTX)-insensitive G protein (Gq) and triggered phosphoinositide hydrolysis with subsequent activation of protein kinase C (PKC), Raf-1, and mitogen-activated protein kinase (MAPK) in VSMCs. In response to ATP, both 42-and 44-kDa MAPKs were activated, and tyrosine was phosphorylated. Western blot analysis using PKC isozyme-specific antibodies indicated that VSMCs express PKC-alpha, PKC-delta, and PKC-zeta. A complete down-regulation of PKC-alpha and PKC-delta was seen after 24-hr treatment with 12-O-tetradecanoylphorbol-13-acetate. When cells were pretreated with 12-O-tetradecanoyl-phorbol-13-acetate for 24 hr and subsequently challenged with ATP, Raf-1 activation and 42-kDa as well as 44-kDa MAPK tyrosine phosphorylation failed to be induced. These results demonstrate that ATP-induced Raf-1 and MAPK activations involve the activation of PKC-alpha and PKC-delta. P2Y purinoceptor stimulation with ATP also caused accumulation of c-fos and c-myc mRNAs. Both Reactive blue 2 and staurosporine significantly blocked this increase by ATP. In conclusion, the mitogenic effect of ATP seemed to be triggered by activation of the Gq protein-coupled P2Y purinoceptor that led to the formation of inositol trisphosphate and activation of PKC. PKC and, in turn, Raf-1 and MAPK were then activated, leading eventually to DNA synthesis and cell proliferation.
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PMID:Mechanism of extracellular ATP-induced proliferation of vascular smooth muscle cells. 949 67

Activation of 44 and 42 kDa extracellular signal-regulated kinases (ERK)1/2 by angiotensin II (angII) plays an important role in vascular smooth muscle cell (VSMC) function. The dual specificity mitogen-actived protein (MAP) kinase/ERK kinase (MEK) activates ERK1/2 in response to angII, but the MEK activating kinases remain undefined. Raf is a candidate MEK kinase. However, a kinase other than Raf appears responsible for angII-mediated signal transduction because we showed previously that treatment with 1 microM phorbol 12, 13-dibutyrate (PDBU) for 24 h completely blocked Raf-Ras association in VSMC but did not inhibit activation of MEK and ERK1/2 by angII. We hypothesized that an atypical protein kinase C (PKC) isoform, which lacks a phorbol ester binding domain, mediated ERK1/2 activation by angII. Western blot analysis of rat aortic VSMC with PKC isoform-specific antibodies showed PKC-alpha, -beta1, -delta, -epsilon, and -zeta in relative abundance. All isoforms except PKC-zeta were down-regulated by 1 microM PDBU for 24 h suggesting that PKC-zeta was responsible for angII-mediated ERK1/2 activation. In response to angII, PKC-zeta associated with Ras as shown by co-precipitation of PKC-zeta with anti-H-Ras antibody. To characterize further the role of PKC-zeta, PKC-zeta protein was depleted specifically by transfection with antisense PKC-zeta oligonucleotides. Antisense PKC-zeta oligonucleotide treatment significantly decreased PKC-zeta protein expression (without effect on other PKC isoforms) and angII-mediated ERK1/2 activation in a concentration-dependent manner. In contrast, ERK1/2 activation by platelet-derived growth factor and phorbol ester was not significantly inhibited. These results demonstrate an important difference in signal transduction by angII compared with PDGF and phorbol ester in VSMC, and suggest a critical role for PKC-zeta and Ras in angII stimulation of ERK1/2.
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PMID:Protein kinase C-zeta mediates angiotensin II activation of ERK1/2 in vascular smooth muscle cells. 904 26

1. The expression and functional roles of protein kinase C (PKC) isoenzymes in purinoceptor signalling have been examined in rat renal mesangial cells. 2. It is observed that rat mesangial cells express four PKC isoenzymes, PKC-alpha, -delta, -epsilon and zeta, as determined by Western blot analysis. No PKC-beta, -gamma, -eta, -theta, or -mu isoforms were detected. 3. By using specific PKC inhibitors and down-regulation experiments we provide evidence that PKC alpha acts as a negative feedback regulator of ATP- and UTP-stimulated phosphoinositide turnover, whereas PKC epsilon triggers arachidonic acid release and subsequent prostaglandin synthesis and stimulates a phosphatidylcholine-hydrolysing phospholipase D. Moreover, PKC delta may activate the mitogen-activated protein kinase cascade and thus promote mesangial cell proliferation in response to extracellular ATP and UTP. 4. In summary our data identify mesangial cells in culture as an excellently characterized cell culture system with well-defined functions of PKC isoenzymes. Functional identification of PKC isoenzymes involved in specific cell responses is one of the most promising steps towards understanding of molecular mechanisms of cell regulation and identifies new targets for drug development.
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PMID:Regulatory functions of protein kinase C isoenzymes in purinoceptor signalling in mesangial cells. 913 6


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