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

Two peaks of mitogen-activated protein (MAP) kinase activator activity are resolved upon ion exchange chromatography of cytosolic extracts from epidermal growth factor-stimulated A431 cells. Two forms of the activator (1 and 2) have been purified from these peaks, using chromatography on Q-Sepharose, heparin-agarose, hydroxylapatite, ATP-agarose, Sephacryl S-300, Mono S, and Mono Q. The two preparations each contained one major protein band with an apparent molecular mass of 46 or 45 kDa, respectively, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Evidence identifying the MAP kinase activators as the 46- and 45-kDa proteins is presented. Using inactive mutants of MAP kinase as potential substrates, it was found that each preparation of MAP kinase activator catalyzes phosphorylation of the regulatory residues, threonine 188 and tyrosine 190, of Xenopus MAP kinase. These results support the concept that the MAP kinase activators are protein kinases. These MAP kinase kinases demonstrate an apparent high degree of specificity toward the native conformation of MAP kinase, although slow autophosphorylation on serine, threonine, and tyrosine residues and phosphorylation of myelin basic protein on serine and threonine residues is detected as well.
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PMID:Purification and characterization of mitogen-activated protein kinase activator(s) from epidermal growth factor-stimulated A431 cells. 132 Nov 46

Nerve growth factor (NGF) binds to two structurally unrelated transmembrane proteins on the surface of PC-12 cells, a 75-kDa glycoprotein with a short cytoplasmic sequence, and the trk protooncogene (pp140c-trk), a protein tyrosine kinase activated by NGF. Immediately after binding to cells, NGF induces changes in serine/threonine phosphorylation of several proteins. We have explored the relative roles of these two NGF binding proteins in mediating the activation of two intracellular kinases that may be responsible for some of these phosphorylations. The raf-1 protooncogene is a serine/threonine kinase activated by several growth factors and oncogenic proteins. Treatment of PC-12 cells with NGF increases the serine and threonine phosphorylation of raf-1 in an anti-raf-1 immunoprecipitate kinase assay. This increased phosphorylation observed in vitro is dose-dependent and transient and is accompanied by the NGF-dependent shift in the mobility of immunoblotted raf-1 on SDS sodium dodecyl sulfate-polyacrylamide gel electrophoresis, an effect thought to reflect phosphorylation. NGF-dependent activation of raf-1 is not dependent on protein kinase C, since prolonged exposure to phorbol esters under conditions that cause down-regulation of cellular protein kinase C activity has no effect on the NGF response. Expression of pp140c-trk in 3T3 fibroblasts (3T3-c-trk), as evidenced by cross-linking of 125I-NGF to the 140-kDa protein, permits the NGF-dependent activation of raf-1 kinase, detected in the immunoprecipitate kinase assay, anti-raf immunoblot shift on gel electrophoresis, and incorporation of [32P]orthophosphate into the raf-1 protein. The concentration dependence of raf-1 activation is identical in 3T3-c-trk and PC-12 cells, despite the absence of the 75-kDa NGF binding protein in 3T3-c-trk cells. NGF is without effect in untransfected 3T3 cells or in Chinese hamster ovary cells overexpressing p75, although raf-1 is present in these cells. Similarly, the NGF-dependent activation of mitogen-activated protein (MAP) kinase is detected in 3T3-c-trk cells, but not in untransfected 3T3 or Chinese hamster ovary cells overexpressing p75. As described for raf-1 activation, the NGF dose responses for MAP kinase activation in 3T3-c-trk and PC-12 cells are virtually superimposable. These data indicate that the activation of these two serine/threonine kinases by NGF is mediated solely by binding to and activating the pp140c-trk receptor.
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PMID:Nerve growth factor stimulates the activities of the raf-1 and the mitogen-activated protein kinases via the trk protooncogene. 132 11

An insulin-stimulated phosphorylation cascade was examined in rat liver after insulin injection via a portal vein by the use of immune complex kinase assays specific to the mitogen-activated protein (MAP) kinase and S6 kinase II homologue (rsk) kinase. We have prepared an antibody against the peptide consisting of a carboxyl-terminal portion of the extracellular signal-regulated kinase 1 (alpha C92), one of the MAP kinases, and an antibody against the peptide consisting of the carboxyl terminus of the mouse S6 kinase II homologue (alpha rsk(m)C). In alpha C92 immune complex assay, maximal activation of rat liver MAP kinases (approximately 4.3-fold) were observed 4.5 min after insulin injection. We also observed an insulin-stimulated MAP kinase activity (approximately 3-fold) in liver extracts from insulin-treated rat in fractions eluted from phenyl-Sepharose with 30-50% ethylene glycol. Kinase assay in myelin basic protein (MBP)-containing gel after sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by denaturation with 6 M guanidine HCl, and renaturation revealed that insulin injection stimulated the kinase activity of the 42- and 44-kDa proteins, which corresponded to the two distinct MAP kinases. In alpha rsk(m)C immune complex assay, maximal stimulation (approximately 5-fold) of the S6 peptide (Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala) kinase activity was observed 7.5 min after insulin injection. In addition, MAP kinases purified from insulin-treated rat liver were able to activate S6 peptide kinase activity in vitro in alpha rsk(m)C immunoprecipitates from untreated rat liver, accompanied by the appearance of several phosphorylated bands including a major band at 88 kDa. We also examined whether insulin injection stimulates the MAP kinase activator (Ahn, N. G., Seger, R., Bratlien, R. L., Diltz, C. D., Tonks, N. K., and Krebs, E. G. (1991) J. Biol. Chem. 266, 4220-4227) in rat liver. Using recombinant Xenopus MAP kinase, fractions of Q-Sepharose eluted early in the NaCl gradient were found to have MAP kinase activator activity accompanied by the phosphorylation of 42-kDa recombinant Xenopus MAP kinase. From these data, we demonstrate three tiers of a cascade composed of the MAP kinase activator, MAP kinases, and an S6 peptide kinase activity in rat liver under physiological conditions in the intact animal.
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PMID:Sequential activation of MAP kinase activator, MAP kinases, and S6 peptide kinase in intact rat liver following insulin injection. 132 22

Two site-specific antibodies have been prepared by immunizing rabbits with chemically synthesized peptides derived from the partial cDNA-predicted amino acid sequence of extracellular signal-regulated kinase 1 (ERK1), which has been proposed to encode the microtubule-associated protein 2 (MAP2) kinase (Boulton, T. G., Yancopoulos, G. D., Gregory, J. S., Slauer, C., Moomaw, C., Hsu, J., and Cobb, M. H. (1990) Science 249, 64-67). With immunoprecipitation in the presence of sodium dodecyl sulfate (SDS) and Western blotting, an antibody to the peptide containing triple tyrosine residues (alpha Y91) resembling one of the insulin receptor autophosphorylation sites specifically recognized 42- and 44-kDa proteins. On the other hand, an antibody to the peptide corresponding to the COOH terminus portions (alpha C92) of the ERK1 cDNA gene product recognized the 44-kDa protein much more efficiently than the 42-kDa protein. With immunoprecipitation in the absence of SDS, alpha Y91 could barely recognize these two proteins and alpha C92 recognized the 44-kDa protein but failed to recognize the 42-kDa protein. Kinase assays in myelin basic protein (MBP)-containing gel, after SDS-polyacrylamide gel electrophoresis, revealed that insulin or 12-O-tetradecanoylphorbol-13-acetate (TPA)-stimulated MBP kinase activity in alpha Y91 immunoprecipitates comigrated at molecular mass 42 and 44 kDa. On the other hand, the stimulated MBP kinase activity in alpha C92 immunoprecipitates comigrated only at molecular mass 44 kDa. Insulin stimulated the MBP kinase activity in gels and phosphorylation of these two proteins by greater than 10-fold with a maximal level at 5 min. Insulin and TPA rapidly stimulate the phosphorylation of the 42- and 44-kDa proteins via de novo threonine and tyrosine phosphorylation. Tryptic phosphopeptide mapping analysis of the 42- and 44-kDa proteins, respectively, revealed a single major phosphopeptide containing phosphothreonine and phosphotyrosine, which was common to both insulin- and TPA-stimulated phosphoproteins. Protein phosphatase 2A treatment of these two phosphoproteins caused a complete loss of kinase activity with selective dephosphorylation of phosphothreonine. These data strongly suggest that these two proteins are highly related to the mitogen-activated protein (MAP) kinase with an apparent molecular mass of 42 kDa (Ray, L. B., and Sturgill, T. W. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3753-3757) and that these two immunologically similar but distinct MBP/MAP2 kinases may represent isozymic forms of MBP/MAP2 kinases. These data also demonstrate that insulin and TPA activate MBP/MAP2 kinase activity by de novo phosphorylation of threonine and tyrosine residues via a very similar pathway.
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PMID:Insulin and 12-O-tetradecanoylphorbol-13-acetate activation of two immunologically distinct myelin basic protein/microtubule-associated protein 2 (MBP/MAP2) kinases via de novo phosphorylation of threonine and tyrosine residues. 166 17

Treatment of BC3H1 myocytes or 3T3-L1 fibroblasts with fluoroaluminate (AlF4-), a direct activator of G proteins, increased the tyrosine phosphorylation of a 42-kDa cytosolic protein. AlF4- induced a parallel increase in protein kinase activity toward myelin basic protein (MBP) in partially purified cell extracts. To test whether AlF4- was activating the 42-kDa MAP (mitogen-activated protein) kinase, extracts from AlF4--treated cells were taken through the chromatographic steps routinely used to purify MAP kinase from growth factor-stimulated cells. Following phenyl-Superose chromatography, a peak of MBP kinase activity eluted at a position characteristic of MAP kinase. Immunoblotting of the active fractions with anti-phosphotyrosine antibodies revealed a single reactive protein band of Mr 42,000. Stimulation of MAP kinase by AlF4- was rapid, peaking within 15 min and persisting for at least 1 h. In contrast, the activation of MAP kinase by insulin was transient, characteristic of its activation by growth factors in other cell types. Although concentrations of sodium fluoride greater than 1 mM also activated MAP kinase, this effect was shown to be dependent upon the simultaneous presence of aluminum ions in the medium. Activation of MAP kinase by AlF4- was not affected by either cellular depletion of protein kinase C or pretreatment of cells with pertussis toxin. Potential sites of action of AlF4- are discussed. These findings suggest that activation of a G protein(s) in intact cells can initiate events that result in tyrosine phosphorylation and activation of MAP kinase.
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PMID:Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate. 170 25

The mechanism by which cAMP inhibits growth factor-induced DNA synthesis in fibroblasts is not understood. Here we show that in Rat-1 fibroblasts, cAMP-raising agents inhibit p21ras-mediated mitogen-activated protein (MAP) kinase activation induced by either epidermal growth factor or lysophosphatidic acid. Under the same conditions, however, epidermal growth factor- or lysophosphatidic acid-induced protein tyrosine phosphorylation, Ca2+ mobilization, and activation of Na+/H+ exchange are not attenuated. In ras-transformed Rat-1 cells, 8-bromo-cAMP rapidly deactivates constitutively active MAP kinase without reducing p21ras.GTP levels; long term 8-bromo-cAMP treatment of these cells leads to growth arrest and reversion of the transformed phenotype. These results show that elevation of intracellular cAMP levels abrogates the p21ras MAP kinase pathway at a step downstream of p21ras activation. This finding provides a molecular basis for the growth-inhibitory action of cAMP in normal and transformed fibroblasts.
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PMID:cAMP abrogates the p21ras-mitogen-activated protein kinase pathway in fibroblasts. 750 16

Exposure of NIH3T3 cells to elevated temperatures induces the phosphorylation and activation of mitogen-activated protein (MAP) kinases [or extracellular signal-regulated kinases (ERKs)]. To investigate the significance of MAP kinase activation by heat shock, we examined the effect of inhibiting the activity of MAP kinase on heat shock protein 70 (hsp 70) expression. Overexpression of a dominant inhibitory mutant of ERK1, but not ERK2, in heat-shocked cells increased hsp70 reporter gene activity, suggesting that ERK1 acts as a repressor of hsp70 gene expression. Increases in ERK1 activity through treatment of cells with sodium vanadate (SV), an inhibitor of the dual-specificity MAP kinase phosphatase 1 (PAC1), resulted in increased phosphorylation of the heat shock transcription factor-1 (HSF-1) in unheated cells, delayed the activation of HSF-1 by heat shock, and inhibited the induction of hsp 70 by heat shock. Furthermore, the induction of thermotolerance was reduced significantly in cells that increased ERK1 activity by SV pretreatment. Immune complex kinase assays of heat shocked or SV-pretreated cells indicated that HSF-1 is a potential in vivo substrate for ERK1 phosphorylation. Taken together, these results suggest that agents that modulate MAP kinase act as negative regulators of the heat shock response in mammalian cells by modulating HSF-1 activity and hsp 70 expression.
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PMID:Mitogen-activated protein kinase acts as a negative regulator of the heat shock response in NIH3T3 cells. 758 24

To investigate the efficacy and mechanism of action of sodium metavanadate as an oral hypoglycemic agent, five insulin-dependent diabetes mellitus (IDDM) and five noninsulin-dependent diabetes mellitus (NIDDM) patients were studied before and after 2 weeks of oral sodium metavanadate (NaVO3; 125 mg/day). Glucose metabolism measured during a two-step euglycemic insulin clamp was not significantly increased by vanadate therapy in patients with IDDM, but was improved by 29% during the low dose (0.5 mU/kg.min) insulin infusion and 39% during the high dose (1.0 mU/kg.min) in patients with NIDDM. The changes in glucose metabolism were largely accounted for by an increase in nonoxidative glucose disposal, as measured by indirect calorimetry. Basal hepatic glucose production and suppression of hepatic glucose production by insulin were unchanged by vanadate therapy. There was a significant decrease in insulin requirements in the patients with IDDM (39.1 +/- 6.6 to 33.8 +/- 4.7 U/day; P < 0.05). Cholesterol levels significantly decreased in both IDDM (4.53 +/- 0.16 vs. 4.27 +/- 0.22 mmol/L; P = 0.06) and NIDDM (6.92 +/- 0.75 vs. 5.28 +/- 0.46 mmol/L; P < 0.05). After NaVO3 therapy, there was a 1.7- to 3.9-fold increase in basal mitogen-activated protein and S6 kinase activities in mononuclear cells from patients with IDDM and NIDDM that mimicked the effect of insulin stimulation in controls. The most common adverse effect of oral NaVO3 was mild gastrointestinal intolerance. These data suggest that vanadate or related agents may have a potential role as adjunctive therapy in patients with diabetes mellitus.
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PMID:Metabolic effects of sodium metavanadate in humans with insulin-dependent and noninsulin-dependent diabetes mellitus in vivo and in vitro studies. 759 44

The molecular mechanisms by which overloaded cardiac myocytes increase the cell size (hypertrophy) remain unknown. We have previously shown that mechanical loading increased the protein synthesis and the expression of proto-oncogene c-fos mRNA (Komuro, I., Kaida, T., Shibazaki, Y., Kurabayashi, M., Katoh, Y. Hoh, E., Takaku, F., and Yazaki, Y. (1990) J. Biol. Chem. 265, 3595-3598; Komuro, I., Katoh, Y., Kaida, T., Shibazaki, Y., Kurabayashi, M., Hoh, E., Takaku, F., and Yazaki, Y. (1991) J. Biol. Chem. 266, 1265-1268). It has been known that both mitogen-activated protein (MAP) kinase and S6 kinase can be activated by many kinds of growth factors. To clarify whether MAP kinase(s) and S6 kinase(s) are associated with the intracellular signaling of cardiac hypertrophy induced by mechanical loading, we cultured neonatal rat cardiac myocytes in deformable dishes and imposed an in vitro mechanical loading by stretching the adherent myocytes. In this study, we demonstrated that 1) myocyte stretching maximally activated a kinase activity toward myelin basic protein (MBP) at 10 min after stretching, and the kinase activity returned to the control level at 30 min after stretching; 2) kinase assays in MBP-containing gel, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that stretch-induced MBP kinase activity mainly migrated at 42 kDa in the immunoprecipitated fraction of anti-MAP kinase antibody, suggesting that the stretching mainly increased the 42-kDa MAP kinase activity in cardiac myocytes; 3) phosphorylation of MAP kinase was induced after stretching cardiac myocytes; 4) when protein kinase C was depleted by preincubating myocytes with 100 nM 12-O-tetradecanoyl-phorbol-13-acetate for 24 h or 2 nM staurosporine for 30 min, stretch-induced MBP kinase activity was decreased by approximately 60-70% as compared with the kinase activity in myocytes without protein kinase C depletion; 5) although the receptor tyrosine kinases were depleted by preincubating myocytes with 50 microM tyrphostin or 20 microM genistein for 30 min, there was no change in the stretch-induced MBP kinase activity; 6) stretch-induced MBP kinase activity was partially dependent on transsarcolemmal influx of Ca2+; 7) myocyte stretching also increased S6 peptide (RRLSSLRA) kinase activity in the anti-S6 kinase II antibody immunoprecipitates. From these results, we conclude that myocyte stretching increases the activities of MAP kinase and S6 peptide kinase, which may play an important role in the induction of the specific genes and the increase in the protein synthesis.
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PMID:Mechanical loading activates mitogen-activated protein kinase and S6 peptide kinase in cultured rat cardiac myocytes. 768 31

Using the human Intestine 407 cell line as a model, we investigated a possible role for tyrosine kinase(s) in regulating the ion efflux pathways induced by hyposmotic stimulation (regulatory volume decrease, RVD). Pretreatment of 125I(-)-and 86Rb(+)-loaded cells with the phosphotyrosine phosphatase inhibitor sodium orthovanadate (200 microM) potentiated isotope efflux triggered by mild hypotonicity (10-20%) but did not further increase the efflux in response to more vigorous osmotic stimulation (30% hypotonicity). The tyrosine kinase inhibitors herbimycin A and genistein largely reduced the osmoshock-induced efflux in both control and vanadate-pretreated cells, while not affecting calcium-activated 86Rb+ efflux. Potentiation of the RVD response by vanadate was confirmed by direct measurements of hypotonicity-induced changes in cell volume. Hypotonic shock alone triggered a rapid and transient increase in tyrosine phosphorylation of several proteins as well as phosphorylation of mitogen-activated protein kinase. Furthermore, the potentiating effects of vanadate on hypotonicity-induced ion efflux and mitogen-activated protein (MAP) kinase phosphorylation were mimicked by epidermal growth factor. Neither vanadate nor epidermal growth factor provoked a RVD-like ionic response under isotonic conditions. These results indicate that tyrosine phosphorylation is an essential step in the RVD response and suggest a novel role of growth factors in the cellular defense against osmotic stress.
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PMID:Protein tyrosine phosphorylation is involved in osmoregulation of ionic conductances. 769 Jul 49


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