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Query: UNIPROT:P51812 (mitogen-activated protein)
 10,636 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We report a strategy for regulating the activity of a cytoplasmic signaling molecule, the protein kinase encoded by raf-1. Retroviruses encoding a gene fusion between an oncogenic form of human p74raf-1 and the hormone-binding domain of the human estrogen receptor (hrafER) were constructed. The fusion protein was nontransforming in the absence of estradiol but could be reversibly activated by the addition or removal of estradiol from the growth media. Activation of hrafER was accompanied in C7 3T3 cells by the rapid, protein synthesis-independent activation of both mitogen-activated protein (MAP) kinase kinase and p42/p44 MAP kinase and by phosphorylation of the resident p74raf-1 protein as demonstrated by decreased electrophoretic mobility. The phosphorylation of p74raf-1 had no effect on the kinase activity of the protein, indicating that mobility shift is an unreliable indicator of p74raf-1 enzymatic activity. Removal of estradiol from the growth media led to a rapid inactivation of the MAP kinase cascade. These results demonstrate that Raf-1 can activate the MAP kinase cascade in vivo, independent of other "upstream" signaling components. Parallel experiments performed with rat1a cells conditionally transformed by hrafER demonstrated activation of MAP kinase kinase in response to estradiol but no subsequent activation of p42/p44 MAP kinases or phosphorylation of p74raf-1. This result suggests that in rat1a cells, p42/p44 MAP kinase activation is not required for Raf-1-mediated oncogenic transformation. Estradiol-dependent activation of p42/p44 MAP kinases and phosphorylation of p74raf-1 was, however, observed in rat1a cells expressing hrafER when the cells were pretreated with okadaic acid. This result suggests that the level of protein phosphatase activity may play a crucial role in the regulation of the MAP kinase cascade. Our results provide the first example of a cytosolic signal transducer being harnessed by fusion to the hormone-binding domain of the estrogen receptor. This conditional system not only will aid the elucidation of the function of Raf-1 but also may be more broadly useful for the construction of conditional forms of other kinases and signaling molecules.
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PMID:Conditional transformation of cells and rapid activation of the mitogen-activated protein kinase cascade by an estradiol-dependent human raf-1 protein kinase. 841 24

Several serine/threonine kinase inhibitors have been described recently that are sufficiently selective, and therefore useful as biochemical probes, for studying the role of kinases in signaling pathways. In addition, these newer classes of kinase inhibitor may well provide an impetus for the development of drugs to attenuate certain cellular responses in the treatment of diseases. Importantly, within the past year, specific and potent inhibitiors have been reported for both the new mitogen-activated protein (MAP) kinase homolog CSBP and MAP kinase kinase-1.
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PMID:Inhibitors of serine/threonine kinases. 852 36

A paradigm has been established whereby mutant tyrosine kinase receptors such as the v-erbB and v-fms gene products function as oncoproteins in the absence of ligand. A spontaneously occurring deletional mutant of the human epidermal growth factor receptor (EGFR-vIII) has been isolated from astrocytic neoplasms and transforms NIH3T3 cells in the absence of ligand. The EGFRvIII is constitutively complexed with the majority of cellular GRB2, suggesting a link to the Ras-Mitogen-activated protein (MAP) kinase pathway (D. Moscatello, R. B. Montgomery, P. Sundareshan, H. McDanel, M. Y. Wong, and A. J. Wong, submitted for publication). In this report, we document that expression of EGFRvIII in fibroblasts is associated with downstream activation of mitogen-activated protein (MAP) kinase/extracellular signal-regulated kinase (MEK) and modest activation of p42 and p44 MAP kinases. The presence of EGFRvIII suppresses activation of p42 and p44 MAP kinases by phorbol 12-myristate 13-acetate (PMA) and serum; however, MEK activation by PMA is not suppressed by EGFRvIII. Basal and PMA-stimulated MAP kinase activity in EGFRvIII-transfected cells is augmented by the tyrosine phosphatase inhibitor sodium vanadate. EGFR-vIII is capable of transducing downstream signals through MAP kinase as evidenced by activation of cytoplasmic phospholipase A2 at levels similar to that induced by intact EGFR. Our results suggest that EGFR-vIII constitutively activates downstream signal transduction through MAP kinase, and this chronic stimulation of the MAP kinase pathway may represent one means by which mutant EGFR transduces an oncogenic signal.
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PMID:Differential modulation of mitogen-activated protein (MAP) kinase/extracellular signal-related kinase kinase and MAP kinase activities by a mutant epidermal growth factor receptor. 853 Apr 89

The expression of mitogen-activated protein kinases (MAPKs) and MAPK kinases (MEKs) in rat islets of Langerhans and the involvement of MAPKs in regulated insulin secretion were examined. Two major isoforms of both MEK (45 and 46 kDa) and MAPK (42 and 44 kDa) were detected in rat islets and shown to be localized to insulin-secreting beta cells by detection of their expression in the beta cell line MIN6. The tyrosine phosphatase inhibitor sodium pervanadate, and, to a lesser extent, the serine/threonine phosphatase inhibitor okadaic acid, stimulated MAPK phosphorylation, as assessed by a shift in its electrophoretic mobility and by increased phosphotyrosine immunoreactivity of immunoprecipitated MAPK. The increase in MAPK phosphorylation stimulated by sodium pervanadate was not coupled to an increase in MAPK activity, but okadaic acid, either alone or in the presence of sodium pervanadate, caused an increase in myelin basic protein phosphorylation by MAPK. Neither okadaic acid nor sodium pervanadate, either individually or combined, stimulated insulin secretion. 4 beta-phorbol myristate acetate stimulated an increase in phosphorylation of the 42 kDa isoform of MAPK (erk2) in human umbilical vein endothelial cells, but neither it nor glucose affected either the phosphorylation state of islet erk2 or the activities of immunoprecipitated islet MAPKs. These results provide evidence for the presence of a regulated MAPK pathway in adult rat islets, but our data suggest that MAPK activation alone is not a sufficient stimulus for insulin secretion.
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PMID:The mitogen-activated protein kinase pathway in rat islets of Langerhans: studies on the regulation of insulin secretion. 854 72

Thrombopoietin (Tpo) is a cytokine regulating megakaryocyte maturation and platelet formation. We studied Tpo-induced signal transduction, and found that Tpo induces phosphorylation of adapter molecules. Shc and Vav, and of serine/threonine kinases Raf-1 and mitogen-activated protein (MAP) kinases. Further, Tpo induced activation of Ras, MAP kinase kinase, MAP kinase and Pim-1. Taken together with other observations, we concluded that Tpo induces the activation of at least two distinct signaling pathways, a specific Tyk2-JAK2/STAT1-STAT3-STAT5 signaling cascade and a common Shc/Vav/Ras/Raf-1/MAP kinase kinase/MAP kinase signaling cascade.
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PMID:Thrombopoietin induces activation of at least two distinct signaling pathways. 854 84

Expression of phosphoenolpyruvate carboxykinase (PEPCK), the rate-limiting step in hepatic gluconeogenesis, is primarily regulated at the level of gene transcription. Insulin and phorbol esters inhibit basal PEPCK transcription and antagonize the induction of PEPCK gene expression by glucocorticoids and glucagon (or its second messenger cAMP). Insulin activates a signaling cascade involving Ras --> Raf --> p42/p44 mitogen-activated protein (MAP) kinase kinase (MEK) --> p42/p44 MAP kinase (ERK 1 and 2). Recent reports suggest that activation of this Ras/MAP kinase pathway is critical for the effects of insulin on mitogenesis and c-fos transcription but is not required for insulin action on metabolic processes such as glycogen synthesis, lipogenesis, and Glut-4-mediated glucose transport. We have used three distinct approaches to examine the role of the Ras/MAP kinase pathway in the regulation of PEPCK transcription by insulin in H4IIE-derived liver cells: (i) chemical inhibition of Ras farnesylation, (ii) infection of cells with an adenovirus vector encoding a dominant-negative mutant of Ras, and (iii) use of a chemical inhibitor of MEK. Although each of these methods blocks insulin activation of MAP kinase, none alters insulin antagonism of cAMP- and glucocorticoid-stimulated PEPCK transcription. Although phorbol esters activate MAP kinase and mimic the effects of insulin on PEPCK gene transcription, inhibition of MEK has no effect on phorbol ester inhibition of PEPCK gene transcription. Using the structurally and mechanistically distinct phosphatidylinositol 3-kinase (PI 3-kinase) inhibitors, wortmannin and LY 294002, we provide further evidence supporting a role for PI 3-kinase activation in the regulation of PEPCK gene transcription by insulin. We conclude that neither insulin nor phorbol ester regulation of PEPCK gene transcription requires activation of the Ras/MAP kinase pathway and that insulin signaling to the PEPCK promoter is dependent on PI 3-kinase activation.
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PMID:Insulin regulation of phosphoenolpyruvate carboxykinase gene expression does not require activation of the Ras/mitogen-activated protein kinase signaling pathway. 856 35

Rap1 small GTP-binding protein has the same amino acid sequence at its effector domain as that of Ras. Rap1 has been shown to antagonize the Ras functions, such as the Ras-induced transformation of NIH 3T3 cells and the Ras-induced activation of the c-Raf-1 protein kinase-dependent mitogen-activated protein (MAP) kinase cascade in Rat-1 cells, whereas we have shown that Rap1 as well as Ras stimulates DNA synthesis in Swiss 3T3 cells. We have established a cell-free assay system in which Ras activates bovine brain B-Raf protein kinase. Here we have used this assay system and examined the effect of Rap1 on the B-Raf activity to phosphorylate recombinant MAP kinase kinase (MEK). Recombinant Rap1B stimulated the activity of B-Raf, which was partially purified from bovine brain and immunoprecipitated by an anti-B-Raf antibody. The GTP-bound form was active, but the GDP-bound form was inactive. The fully post-translationally lipid-modified form was active, but the unmodified form was nearly inactive. The maximum B-Raf activity stimulated by Rap1B was nearly the same as that stimulated by Ki-Ras. Rap1B enhanced the Ki-Ras-stimulated B-Raf activity in an additive manner. These results indicate that not only Ras but also Rap1 is involved in the activation of the B-Raf-dependent MAP kinase cascade.
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PMID:Activation of brain B-Raf protein kinase by Rap1B small GTP-binding protein. 857 7

Incubating rat diaphragm muscles with insulin increased the glycogen synthase activity ratio (minus glucose 6-phosphate/plus glucose 6-phosphate) by approximately 2-fold. Insulin increased the activities of mitogen-activated protein (MAP) kinase and the Mr = 90,000 isoform of ribosomal protein S6 kinase (Rsk) by approximately 1.5-2.0-fold. Epidermal growth factor (EGF) was more effective than insulin in increasing MAP kinase and Rsk activity, but in contrast to insulin, EGF did not affect glycogen synthase activity. The activation of both MAP kinase and Rsk by insulin was abolished by incubating muscles with the MAP kinase kinase (MEK) inhibitor, PD 098059; however, the MEK inhibitor did not significantly reduce the effect of insulin on activating glycogen synthase. Incubating muscles with concentrations of rapamycin that inhibited activation of p70S6K abolished the activation of glycogen synthase. Insulin also increased the phosphorylation of PHAS-I (phosphorylated heat- and acid-stable protein) and promoted the dissociation of the PHAS-I*eIF-4E complex. Increasing MAP kinase activity with EGF did not mimic the effect of insulin on PHAS-I phosphorylation, and the effect of insulin on increasing MAP kinase could be abolished with the MEK inhibitor without decreasing the effect of insulin on PHAS-I. The effects of insulin on PHAS-I were attenuated by rapamycin. Thus, activation of the MAP kinase/Rsk signaling pathway appears to be neither necessary nor sufficient for insulin action on glycogen synthase and PHAS-I in rat skeletal muscle. The results indicate that the effects of insulin on increasing the synthesis of glycogen and protein in skeletal muscle, two of the most important actions of the hormone, involve a rapamycin-sensitive mechanism that may include elements of the p70S6K signaling pathway.
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PMID:Regulation of both glycogen synthase and PHAS-I by insulin in rat skeletal muscle involves mitogen-activated protein kinase-independent and rapamycin-sensitive pathways. 861 80

Hepatocyte growth factor (HGF) stimulated mitogen-activated protein (MAP) kinases and MAP kinase kinase in primary cultured rat hepatocytes. Inhibitors for protein kinase C (PKC), Ro31-8425, H-7, and calphostin C, reduced HGF-induced MAP kinase activity. A PKC activator, phorbol myristate acetate (PMA), induced MAP kinase activation in a concentration-dependent manner. Protein tyrosine kinase (PTK) inhibitors, genistein, and ST638 also inhibited HGF-induced MAP kinase activation. Furthermore, HGF increased formation of Ras guanosine triphosphate (GTP) complex, indicating Ras activation. Genistein inhibited HGF-induced Ras activation, but Ro31-8425 was without effect. On the other hand, Ro31-8425 decreased HGF-induced [3H]arachidonic acid (AA) release and [3H]thymidine incorporation. Genistein also prevented [3H]AA release and [3H]-thymidine incorporation. Moreover, a commonly used phospholipase A2 (PLA2) inhibitor, quinacrine, decreased HGF-induced [3H]AA release and [3H]thymidine incorporation. The inhibitory profile of [3H]AA release was well correlated with that of [3H]thymidine incorporation in Ro31-8425-, genistein-, and quinacrine-treated cells. A cyclooxygenase inhibitor, indomethacin, which suppressed HGF-induced DNA synthesis, had minimal effect on MAP kinase activation. In contrast, prostaglandin (PG) E1, E2, or F2 alpha, which stimulate [3H]thymidine incorporation to the same level as that caused by HGF in hepatocytes, caused very weak activation of MAP kinases. These results suggest that PTK, Ras, and PKC play roles in MAP kinase activation induced by HGF and that MAP kinase activation resulting in AA release is involved in DNA synthesis in rat hepatocytes.
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PMID:Mitogen-activated protein kinase activation in hepatocyte growth factor-stimulated rat hepatocytes: involvement of protein tyrosine kinase and protein kinase C. 862 Nov 60

Insulin activation of Ras is mediated by the plasma membrane targeting of the guanylnucleotide exchange factor SOS associated with the small adapter protein Grb2. SOS also lies in an insulin-stimulated feedback pathway in which the serine/threonine phosphorylation of SOS results in disassociation of the Grb2-SOS complex thereby limiting the extent of Ras activation. To examine the relative role of the mitogen-activated protein kinases in the feedback phosphorylation of SOS we determined the signaling specificity of insulin, osmotic shock, and anisomycin to activate the ERK (extracellular-signal regulated kinase) and JNK (c-Jun kinase) pathways. In Chinese hamster ovary cells expressing the human insulin receptor and murine 3T3L1 adipocytes, insulin specifically activated ERK with no significant effect on JNK, whereas anisomycin specifically activated JNK but was unable to activate ERK. In contrast, osmotic shock was equally effective in the activation of both kinase pathways. Insulin and osmotic shock, but not anisomycin, resulted in SOS phosphorylation and disassociation of the Grb2-SOS complex, demonstrating that the JNK pathway was not involved in the insulin-stimulated feedback uncoupling of the Grb2- SOS complex. Both the insulin and osmotic shock-induced activation of ERK was prevented by treatment of cells with the specific MEK inhibitor (PD98059). However, expression of dominant-interfering Ras (N17Ras) inhibited the insulin- but not osmotic shock-stimulated phosphorylation of ERK and SOS. These data demonstrate that activation of the ERK pathway, but not JNK, is responsible for the feedback phosphorylation and disassociation of the Grb2-SOS complex.
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PMID:SOS phosphorylation and disassociation of the Grb2-SOS complex by the ERK and JNK signaling pathways. 862 28


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