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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)

We have used quail skeletal myotubes expressing a temperature-sensitive allele of the v-src oncogene to address the issue of the homeostasis of sarcomeric myofibrils in differentiated muscle cells. Reactivation of the v-Src tyrosine kinase by shifting the cultures to the permissive temperature leads within minutes to the formation of F-actin-containing bodies (ABs), that originate in the ventral region of the myotubes and increase in number concomitantly with the dismantling of the I-Z-I complex of the sarcomeres. This process is detailed by confocal and electron microscopy. Indirect immunofluorescence reveals that ABs contain muscle-specific protein isoforms associated with the I-Z-I complexes and vinculin, a component of the cytoskeletal network. Anti-phosphotyrosine antibodies label proteins in ABs and Z-discs. Evidence is presented indicating that this phenomenon specifically depends on the persistent activation of v-Src, rather than on a general increase in phosphotyrosine content such as that induced by vanadate. AB formation is prevented by activation of protein kinase C by phorbol ester or by treatment with the kinase inhibitor 2-aminopurine, without any detectable effect on tyrosine phosphorylation. Taken together these findings indicate that phosphorylation of specific target proteins by v-Src, although necessary, is not sufficient per se to induce AB formation. In addition, the signal transduction cascade that culminates in MAP kinase activation and its nuclear translocation is activated both by v-Src and phorbol ester, and is relatively unaffected by 2-aminopurine. These findings imply that both phorbol esters and 2-aminopurine operate, at least in part, at the level of alternative pathways that may diverge upstream of the MAP kinase and are presumably mediating the early effects of v-Src on the differentiated phenotype.
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PMID:Maintenance of the differentiated state in skeletal muscle: activation of v-Src disrupts sarcomeres in quail myotubes. 764 4

The beta-isoform of glycogen synthase kinase-3 (GSK3 beta) isolated from rabbit skeletal muscle was inactivated 90-95% following incubation with MgATP and either MAP kinase-activated protein kinase-1 (MAPKAP kinase-1, also termed RSK-2) or p70 S6 kinase (p70S6K), and re-activated with protein phosphatase 2A. MAPKAP kinase-1 and p70S6K phosphorylated the same tryptic peptide on GSK3 beta, and the site of phosphorylation was identified as the serine located nine residues from the N-terminus of the protein. The inhibitory effect of Ser-9 phosphorylation on GSK3 beta activity was observed with three substrates, (inhibitor-2, c-jun and a synthetic peptide), and also with glycogen synthase provided that 0.15 M KCl was added to the assays. The results suggest that Ser-9 phosphorylation underlies the reported inhibition of GSK3 beta by insulin and that GSK3 may represent a point of convergence of two major growth-factor-stimulated protein kinase cascades.
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PMID:Inactivation of glycogen synthase kinase-3 beta by phosphorylation: new kinase connections in insulin and growth-factor signalling. 825 Aug 35

The alpha-isoform of glycogen synthase kinase-3 (GSK3 alpha) was inactivated by 80% towards a synthetic peptide substrate upon incubation with Mg-ATP and either MAP kinase-activated protein (MAPKAP) kinase-1 or p70 S6 kinase. Inactivation by either kinase resulted from the phosphorylation of Ser-21 and was reversed by treatment with protein phosphatase 2A1. Phosphorylation also decreased GSK3 alpha activity towards glycogen synthase, inhibitor-2 and c-jun. The specificity of GSK3 alpha was similar to GSK3 beta, but with the synthetic peptide substrate heparin stimulated the dephosphorylated form of GSK3 alpha (6-fold) more than GSK3 beta (1.8-fold). After phosphorylation, both isoforms were stimulated 15-20-fold by heparin.
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PMID:The alpha-isoform of glycogen synthase kinase-3 from rabbit skeletal muscle is inactivated by p70 S6 kinase or MAP kinase-activated protein kinase-1 in vitro. 830 53

A novel protein kinase activity present in nuclear and cytosolic extracts has been identified and partially purified as a consequence of its tight binding to and phosphorylation of the extracellular signal-regulated protein kinase (ERK) 3. This novel protein kinase is inactivated by treatment with phosphoprotein phosphatase 2A. The ERK3 protein kinase was immunologically distinct from mitogen-activated protein (MAP) kinase/ERK kinases (MEK) 1 and 2 which phosphorylate the ERK3-related MAP kinases ERK1 and ERK2. This ERK3 kinase phosphorylated a single site on ERK3, Ser189, comparable to Thr183, one of the two activating phosphorylation sites of ERK2. To test the specificity of the ERK3 kinase, mutants of ERK3 and ERK2 were made in which the phosphorylated residues were exchanged. The double mutant S189T,G191Y ERK3, in which the phosphorylated residues from ERK2 replaced the comparable residues in ERK3, was phosphorylated by the ERK3 kinase but only on threonine. The ERK3 kinase did not phosphorylate ERK2 or ERK2 mutants. These findings indicate that although the ERK3 kinase is highly specific for ERK3, it does not recognize tyrosine, a feature that distinguishes it from MEKs that phosphorylate other ERK/MAP kinase family members.
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PMID:Characterization of a protein kinase that phosphorylates serine 189 of the mitogen-activated protein kinase homolog ERK3. 866 49

Tumor necrosis factor-alpha (TNF-alpha) is a proposed mediator of insulin resistance in obese/diabetic animals through its effects on tyrosine phosphorylation of the insulin receptor and its substrate, insulin receptor substrate-1. In this study, the acute effects of TNF-alpha on the mitogen-activated protein kinase (MAPK) signalling cascade were examined in cultured rat skeletal muscle cell line, L6. Insulin treatment of L6 cells resulted in a rapid increase in MAPK activity (> twofold in 5 min with 10 nM insulin). Prior treatment with TNF-alpha for 60 min blocked subsequent insulin-induced activation of MAPK in a dose- and time-dependent manner. Metabolic labelling studies with inorganic [32P]phosphate followed by immuno-precipitation of MAPK and its upstream activator, mitogen-activated protein kinase kinase, indicated decreased phosphorylation of MAPK and its kinase in response to insulin in cells exposed to TNF-alpha. This effect of TNF-alpha was not due to inhibition of insulin-stimulated p21ras-GTP loading or Raf-1 phosphorylation. Low concentrations (2 nM) of okadaic acid, a serine/threonine phosphatase inhibitor, prevented TNF-alpha-induced inhibition of MAPK and restored insulin's effect on MAPK activity, while orthovanadate (a tyrosine phosphatase inhibitor), inhibitor 2 (phosphatase-1 inhibitor) and FK506 (phosphatase-2B inhibitor) were ineffective. These results suggested an involvement of an okadaic-acid-sensitive serine/threonine phosphatase in TNF-alpha-induced blockade of insulin's effect on MAPK and/or its kinase. Therefore, we examined the effect of TNF-alpha on protein phosphatase-1 (PP-1) and protein phosphatase-2A (PP-2A) activities. As reported by us earlier, insulin rapidly stimulated PP-1 and concomitantly inhibited PP-2A activities in control cells. TNF-alpha treatment blocked insulin-induced activation of PP-1. In contrast to PP-1, TNF-alpha caused a 60% increase in PP-2A activity and insulin failed to prevent this TNF-alpha effect. The time course of PP-2A activation by TNF-alpha preceded the kinetics of inhibition of MAPK. Cell-permeable ceramide analogs mimicked the TNF-alpha effect on MAPK inhibition and PP-2A activation. We conclude that TNF-alpha abrogates the insulin effect on MAPK activation by increasing dephosphorylation of MAPK kinase via an activated phosphatase.
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PMID:Effect of tumor necrosis factor-alpha on insulin-stimulated mitogen-activated protein kinase cascade in cultured rat skeletal muscle cells. 866 40

Results of this study document a biphasic activation of protein kinases of the MAP kinase cascade-MEK and MAP kinases-upon interleukin-1 stimulation in human HeLa cells. The specific activities of both MEK and MAP kinases were increased within 1 min, declined rapidly to control levels and increased again after 15 min of interleukin-1 stimulation. Inhibition by okadaic acid of serine/threonine specific phosphatases resulted in a marked increase in interleukin-1 stimulated MEK and MAP kinase activities. Elevation by interleukin-1 of the specific activities of MEK and MAP kinases correlated with suppression of serine/threonine phosphatases in the late phase of stimulation. The data indicate, that enhanced phosphorylation of cellular proteins by enzymes of the MAP kinase cascade might represent a fine balance between activated protein kinases and repressed phosphoprotein phosphatase 2A in interleukin-1 stimulated HeLa cells.
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PMID:Interleukin-1 induced signalling: biphasic activation of mitogen-activated protein kinase kinase and mitogen-activated protein kinases in HeLa cells. Involvement of phosphoprotein phosphatases. 901 Jun 81

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPARgamma) regulates transcription in response to prostanoid and thiazolidinedione ligands and promotes adipocyte differentiation. The amino-terminal A/B domain of this receptor contains a consensus mitogen-activated protein kinase site in a region common to PPARgamma1 and -gamma2 isoforms. The A/B domain of human PPARgamma1 was phosphorylated in vivo, and this was abolished either by mutation of serine 84 to alanine (S84A) or coexpression of a phosphoprotein phosphatase. In vitro, this domain was phosphorylated by ERK2 and JNK, and this was markedly reduced in the S84A mutant. A wild type Gal4-PPARgamma(A/B) chimera exhibited weak constitutive transcriptional activity. Remarkably, this was significantly enhanced in the S84A mutant fusion. Ligand-dependent activation by full-length mouse PPARgamma2 was also augmented by mutation of the homologous serine in the A/B domain to alanine. The nonphosphorylatable form of PPARgamma was also more adipogenic. Thus, phosphorylation of a mitogen-activated protein kinase site in the A/B region of PPARgamma inhibits both ligand-independent and ligand-dependent transactivation functions. This observation provides a potential mechanism whereby transcriptional activation by PPARgamma may be modulated by growth factor or cytokine-stimulated signal transduction pathways involved in adipogenesis.
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PMID:Transcriptional activation by peroxisome proliferator-activated receptor gamma is inhibited by phosphorylation at a consensus mitogen-activated protein kinase site. 903 May 79

Mitogen-activated protein (MAP) kinase pathways include a three-kinase cascade terminating in a MAP kinase family member. The middle kinase in the cascade is a MAP/extracellular signal-regulated kinase (ERK) kinase or MEK family member and is highly specific for its MAP kinase target. The first kinase in the cascade, a MEK kinase (MEKK), is characterized by its ability to activate one or more MEK family members. A two-plasmid bacterial expression system was employed to express active forms of the following MEK and MAP kinase family members: ERK1, ERK2, alpha-SAPK, and p38 and their upstream activators, MEK1, -2, -3, and -4. In each kinase module, the upstream activator, a constitutively active mutant of MEK1 or MEKK1, was expressed from a low copy plasmid, while one or two downstream effector kinases were expressed from a high copy plasmid with different antibiotic resistance genes and origins of replication. Consistent with their high activity, ERK1 and ERK2 were doubly phosphorylated on Tyr and Thr, were recognized by an antibody specific to the doubly phosphorylated forms, and were inactivated by either phosphoprotein phosphatase 2A or phosphotyrosine phosphatase type 1. Likewise, activated p38 and alpha-stress-activated protein kinase could also be inactivated by either phosphatase, and alpha-stress-activated protein kinase was recognized by an antibody specific to the doubly phosphorylated forms. These three purified, active MAP kinases have specific activities in the range of 0.6-2.3 micromol/min/mg. Coexpression of protein kinases with their substrates in bacteria is of great value in the preparation of numerous phosphoproteins, heretofore not possible in procaryotic expression systems.
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PMID:Reconstitution of mitogen-activated protein kinase phosphorylation cascades in bacteria. Efficient synthesis of active protein kinases. 911 Sep 99

Vascular 5-Hydroxytryptamine2A (5-HT2A) receptor signaling and contraction has been associated with the activation of L-type calcium channels, phospholipase C (PLC) and, as we previously demonstrated, tyrosine kinase activation. We hypothesize the 5-HT2A receptor activates all three pathways independently to elicit contraction and that one of the tyrosine kinases activated by 5-HT is mitogen-activated protein kinase kinase (MEK). Endothelium-denuded rat thoracic aorta was mounted into isolated tissue baths for measurement of isometric contractile force. 5-HT, alpha-methyl-5-HT and 2,5-dimethoxy-4-iodoamphetamine all contracted the rat aorta, whereas the 5-HT2A receptor antagonist ketanserin (30 nM) blocked contraction to 5-HT. The tyrosine kinase inhibitor genistein (5 microM) shifted contraction to 5-HT, alpha-methyl-5-HT and DOI approximately 10-fold to the right, whereas daidzein (5 microM), the inactive isomer of genistein, was unable to shift 5-HT-induced contraction. PD098059 (10 microM), an inhibitor of MEK, shifted contraction to 5-HT approximately 7-fold to the right. We next examined the integration of tyrosine kinase activation in 5-HT2A receptor signaling. 5-HT-induced contraction was reduced individually by the PLC inhibitor 2-nitro-4-carboxyphenyl-N,N-diphenylcarbamate (NCDC; 100 microM) or the Ca++ channel inhibitor nifedipine (50 nM); the remaining response to 5-HT was reduced by further addition of either genistein or PD098059. When nifedipine and NCDC were used in combination, a part of the contraction to 5-HT remained: this contraction was further reduced by genistein or PD098059. In cultured aortic smooth muscle cells, 5-HT (0.01-100 microM) stimulated tyrosyl-phosphorylation of 42- and 44-kDa proteins identified as Erk MAPKs; this phosphorylation was reduced by PD098059 (10 microM). Neither nifedipine nor NCDC reduced 5-HT (1 microM)-stimulated Erk MAPK tyrosyl-phosphorylation, but the combination of nifedipine, NCDC and PD098059 abolished 5-HT (1 microM)-stimulated Erk MAPK tyrosyl-phosphorylation. Taken together, these studies indicate that stimulation of a vascular 5-HT2A receptor activates Ca++ channels and PLC as well as MEK to cause rat aortic contraction and that MEK activation is at least partially independent of the two pathways classically associated with 5-HT2A receptors.
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PMID:Integration of mitogen-activated protein kinase kinase activation in vascular 5-hydroxytryptamine2A receptor signal transduction. 943 97

Numerous studies with PC12 cells have suggested that the mitogen-activated protein (MAP) kinase pathway might play a major role in the neuronal differentiation that is induced by nerve growth factor (NGF). Cells of the PC12D subline extend neurites within several hours in response to NGF in the presence of inhibitors of the synthesis of RNA and protein. We examined the effects of a specific inhibitor 2-(2'-amino-3'-methoxyphenyl)-oxanaphthalen-4-one (PD98059) of the MAP kinase kinase (MEK)/MAP kinase pathway on the NGF-induced outgrowth of neurites in PC12D cells. The increase in MAP kinase activity in response to NGF was reduced by 80% upon treatment of PC12D cells with 50 microM PD98059, whereas the NGF-dependent formation of ruffles and the subsequent outgrowth of neurites were not blocked by PD98059 at this concentration. The outgrowth of neurites from conventional PC12 cells by NGF was suppressed by the addition of 50 microM PD98059 as reported by Pang et al. [L. Pang, T. Sawada, J. Stuart,S.J. Decker, A.R. Saltiel, Inhibition of MAP kinase kinase blocks the differentiation of PC12 cells induced by nerve growth factor, J. Biol. Chem. 270 (1995) 13585-13588]. In contrast, the rapid regeneration of neurites from PC12 cells primed with NGF, was not altered in the presence of the same dose of the inhibitor of MEK. It appeared, therefore, that the activation of the MAP kinase pathway was not necessarily required for the NGF-dependent extension of neurites. When PC12D cells were transfected with the dominant inhibitory Ha-ras Asn-17 gene, the induction of the mutant Ras protein led the suppression of the rapid outgrowth of neurites in response to NGF but not to dibutyryl cyclic AMP (dbcAMP). The result implies a direct involvement of Ras protein in the NGF-induced signal transduction that lead to the formation of neurites in PC12D cells. We can conclude that the activation of MAP kinase and selective gene expression are required for the differentiation of conventional PC12 cells to sympathetic neuron-like cells and that activation of Ras protein and, subsequently, of a MAP kinase-independent pathway might be involved in the extension of neurites from PC12D cells or in the regeneration of neurites from primed PC12 cells in response to NGF.
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PMID:Activation of mitogen-activated protein kinases is not required for the extension of neurites from PC12D cells triggered by nerve growth factor. 951 60


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