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:2.7.11.24 (mitogen-activated protein kinase)
95,810 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The normal cellular homologue of the acutely transforming oncogene v-raf is c-raf-1, which encodes a serine/threonine protein kinase that is activated by many extracellular stimuli. The physiological substrates of the protein c-Raf-1 are unknown. The mitogen-activated protein (MAP) kinases Erk1 and 2 are also activated by mitogens through phosphorylation of Erk tyrosine and threonine residues catalysed by a protein kinase of relative molecular mass 50,000, MAP kinase-kinase (MAPK-K). Here we report that MAPK-K as well as Erk1 and 2 are constitutively active in v-raf-transformed cells. MAPK-K partially purified from v-raf-transformed cells or from mitogen-treated cells can be deactivated by phosphatase 2A. c-Raf-1 purified after mitogen stimulation can reactivate the phosphatase 2A-inactivated MAPK-K over 30-fold in vitro. c-Raf-1 reactivation of MAPK-K coincides with the selective phosphorylation at serine/threonine residues of a polypeptide with M(r) 50,000 which coelutes precisely on cation-exchange chromatography with the MAPK-K activatable by c-Raf-1. These results indicate that c-Raf-1 is an immediate upstream activator of MAPK-K in vivo. To our knowledge, MAPK-K is the first physiological substrate of the c-raf-1 protooncogene product to be identified.
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PMID:Raf-1 activates MAP kinase-kinase. 132

As detected by coimmunoprecipitation from PC12 cells, NGF induces rapid association between ERK1 (a growth factor-activated serine/threonine protein kinase) and gp140prototrk NGF receptors. In contrast, no such association is found with the closely related ERK2. Anti-trk immunocomplexes generated from NGF-treated cells also contain protein kinase activity that shares many properties with soluble ERK1. The association of both ERK1 protein and ERK-like kinase activity with gp140prototrk is maximal by 5 min of NGF treatment, persists for approximately 1 hr, and subsequently declines by 18 hr. Treatment with either basic fibroblast growth factor, epidermal growth factor, or orthovanadate also leads to association of ERK1 with gp140prototrk without tyrosine phosphorylation of the latter. The interaction between ERK1 and gp140prototrk may prove relevant to the NGF mechanism.
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PMID:NGF and other growth factors induce an association between ERK1 and the NGF receptor, gp140prototrk. 146 7

The small GTP-binding protein Ras appears to be required for transformation and differentiation induced by tyrosine kinases. The Ras requirement may be limited to a few tyrosine kinase-regulated signaling pathways or may be universal for all tyrosine kinase actions. Because both Ras and the microtubule-associated protein 2 kinases ERK1 and ERK2 have been implicated in events that lead to neurite outgrowth, we explored the possibility that Ras and ERKs may lie on the same signaling pathway. Utilizing PC-12 rat adrenal pheochromocytoma cell lines that contain a dominant inhibitory Ras mutant (S17N-Ras(H)), we found that Ras was required for stimulation of the ERK cascade by nerve growth factor but apparently not by the heterotrimeric G protein activator AlF4-. Within this cascade, Ras appears to be upstream of an ERK activator, raising the intriguing possibility that Ras may directly regulate a serine/threonine protein kinase.
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PMID:Evidence for a Ras-dependent extracellular signal-regulated protein kinase (ERK) cascade. 149 81

Mitogen-activated protein kinase (MAP kinase) is a serine/threonine protein kinase that becomes enzymatically activated and phosphorylated on tyrosine and threonine following treatment of quiescent cells with a variety of stimulatory agonists. Phosphorylation on both tyrosine and threonine is necessary to maintain full activity, and these two regulatory phosphorylations occur close to each other, separated by a single glutamate. To study the mechanisms by which MAP kinase becomes phosphorylated and activated, we have cloned a full-length cDNA encoding MAP kinase and have expressed the enzyme in Escherichia coli as a soluble nonfusion protein. We find that the enzyme displays a basal, intramolecular autophosphorylation on tyrosine-185 that is accompanied by activation of the enzyme's kinase activity towards an exogenous substrate. The tyrosine-phosphorylated protein displays a small fraction of the activity seen with the fully activated, doubly phosphorylated enzyme isolated from mammalian cells but is activated 10- to 20-fold relative to the unphosphorylated enzyme. These findings raise the possibility that regulation of MAP kinase activity in response to agonist stimulation could occur in part through the enhancement of autophosphorylation on tyrosine.
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PMID:Autophosphorylation in vitro of recombinant 42-kilodalton mitogen-activated protein kinase on tyrosine. 183 84

Mitogen-activated protein kinase (MAP kinase) is a 42 kd serine/threonine protein kinase whose enzymatic activity requires phosphorylation of both tyrosyl and threonyl residues. As a step in elucidating the mechanism(s) for activation of this enzyme, we have determined the sites of regulatory phosphorylation. Following proteolytic digestion of 32P-labeled pp42/MAP kinase with trypsin, only a single phosphopeptide was detected by two-dimensional peptide mapping, and this peptide contained both phosphotyrosine and phosphothreonine. The amino acid sequence of the peptide, including the phosphorylation sites, was determined using a combination of Fourier transform mass spectrometry and collision-activated dissociation tandem mass spectrometry with electrospray ionization. The sequence for the pp42/MAP kinase tryptic phosphopeptide is similar (but not identical) to a sequence present in the ERK1- and KSS1-encoded kinases. The two phosphorylation sites are separated by only a single residue. The regulation of activity by dual phosphorylations at closely spaced threonyl and tyrosyl residues has a functional correlate in p34cdc2, and may be characteristic of a family of protein kinases regulating cell cycle transitions.
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PMID:Identification of the regulatory phosphorylation sites in pp42/mitogen-activated protein kinase (MAP kinase). 184 75

The cellular actions of nerve growth factor (NGF) and epidermal growth factor (EGF) may be mediated by changes in protein phosphorylation. The tyrosine phosphorylation of two predominant proteins of molecular mass 40 and 42 kDa is seen in PC-12 cells treated with NGF or EGF, correlating with activation of a previously identified serine/threonine protein kinase that phosphorylates microtubule-associated protein (MAP). Stimulation of phosphoprotein (pp) 40 and 42 phosphorylation and MAP kinase activity by NGF but not EGF is selectively attenuated by staurosporine and K-252A. Moreover, the time courses of pp40/42 phosphorylation and MAP kinase activation produced by NGF or EGF are identical. Chromatography of lysates from growth factor-treated cells on ion-exchange or hydrophobic-interaction HPLC resolves MAP kinase into two peaks, neither of which precisely coelutes with pp40 or pp42. One of these peaks (II) exhibits no detectable phosphotyrosine. The other peak (I) has some overlap with pp40. However, the activity residing in both peaks is almost completely inhibited after treatment with alkaline phosphatase, suggesting that, at least, serine/threonine phosphorylation is required for the activity of these enzymes. These data indicate that while tyrosine phosphorylation appears to be a critical early event in NGF action, the role of this modification in activation of MAP kinases remains unclear.
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PMID:Nerve growth factor stimulates protein tyrosine phosphorylation in PC-12 pheochromocytoma cells. 184 70

The dominant insulin-stimulated ribosomal protein S6 kinase activity was purified to near homogeneity from insulin-treated 32P-labeled rat H4 hepatoma cells and found to copurify with a 70-kDa 32P-labeled polypeptide. The dominant S6 kinase purified from livers of cycloheximide-treated rats is also a 70-kDa polypeptide. Antiserum raised against rat liver S6 kinase specifically immunoprecipitates the purified 32P-labeled H4 hepatoma insulin-stimulated S6 kinase. This antiserum also specifically precipitates insulin-stimulated S6 kinase activity directly from cytosolic extracts of H4 cells. Immune complexes prepared from the cytosol of 32P-labeled H4 cells contain several 32P-labeled polypeptides; only a 70-kDA 32P-labeled peptide, however, is specifically displaced by preadsorption of the antiserum with nonradioactive rat liver S6 kinase. Insulin treatment increases the 32P content of the immunoprecipitated 70-kDa S6 kinase polypeptide 3- to 4-fold over basal levels; 32P-labeled serine, some 32P-labeled threonine, but no 32P-labeled tyrosine are detected after partial acid hydrolysis. Tryptic peptide maps indicate that the insulin-stimulated S6 kinase purified from 32P-labeled H4 cells is phosphorylated at multiple sites distinct from those which participate in autophosphorylation in vitro. Autophosphorylation of rat liver S6 kinase in vitro does not modify S6 kinase activity. The S6 kinases purified from liver of cycloheximide-treated rat and H4 hepatoma insulin-stimulated enzyme are each completely deactivated by incubation with protein phosphatase type 2A in both autophosphorylating and 40S S6 phosphorylating activities. The phosphatase 2A-deactivated 70-kDa S6 kinase is neither reactivated nor phosphorylated by partially purified insulin-stimulated microtubule-associated protein 2 kinase, in experiments where Xenopus S6 kinase II undergoes phosphorylation and partial reactivation. Thus insulin activates the 70-kDa S6 kinase by promoting phosphorylation of specific serine/threonine residues on the enzyme polypeptide, probably through activating an as-yet-unidentified serine/threonine protein kinase distinct from microtubule-associated protein 2 kinase.
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PMID:Insulin activates a 70-kDa S6 kinase through serine/threonine-specific phosphorylation of the enzyme polypeptide. 212 50

pp42, a low-abundance 42-kDa protein, becomes transiently phosphorylated on tyrosine after stimulation of fibroblasts by a variety of mitogens, including epidermal growth factor, platelet-derived growth factor, phorbol 12-myristate 13-acetate, thrombin, and insulin-like growth factor II. The induction of pp42 phosphorylation on tyrosine by such diverse mitogenic agents suggests an important role for pp42 in the cascade of events necessary for cell transition from G0 into the cell cycle. However, as with most proteins identified on the basis of their tyrosine phosphorylation, the function of pp42 in cellular regulation is unknown. In this manuscript we report evidence that suggests that pp42 is a serine/threonine-specific protein kinase. Stimulation of 3T3-L1 cells with insulin has been shown to activate a cytosolic serine/threonine kinase capable of phosphorylating microtubule-associated protein 2 (MAP-2) and ribosomal protein S6 kinase II. This cytosolic serine/threonine protein kinase, which itself is phosphorylated on tyrosine, has been termed "MAP kinase". We now report that pp42 phosphorylation and MAP kinase activation occur in fibroblasts in response to similar mitogens, that the two proteins comigrate on one- and two-dimensional polyacrylamide gels, and that the two proteins copurify chromatographically. The major peptides generated from purified MAP kinase by V8 protease digestion are present as a subset of the peptides in digests of pp42 excised from two-dimensional gels. Thus, the results suggest that MAP kinase is tyrosine-phosphorylated pp42.
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PMID:Evidence that pp42, a major tyrosine kinase target protein, is a mitogen-activated serine/threonine protein kinase. 255 Sep 26

Mos is a germ cell-specific serine/threonine protein kinase that activates mitogen-activated protein kinase (MAPK) through MAPK kinase (MKK). In Xenopus oocytes, Mos synthesis is required for progesterone-induced activation of MAPK and maturation promoting factor. Injection of Mos or active MAPK causes mitotic arrest in early embryos, suggesting that Mos also acts via MKK and MAPK to induce the arrest of unfertilized eggs in metaphase of meiosis II. We have investigated whether Mos activity is regulated by phosphorylation. Previous studies have identified Ser-3 as the principal autophosphorylation site. We show that Mos interacts with the catalytic domain of MKK in a Saccharomyces cerevisiae two-hybrid test. Acidic substitutions of the sites phosphorylated by Mos in MKK reduce the interaction, implying that the complex may dissociate after phosphorylation of MKK by Mos. Furthermore, the Mos-MKK interaction requires Mos kinase activity, suggesting that Mos autophosphorylation may be involved in the interaction. Substitution of Ser-3 of Mos with Ala reduces the interaction with MKK and also reduces both the activation of MKK by Mos in vitro and cleavage arrest induced by Mos fusion protein in Xenopus embryos. By contrast, substitution of Ser-3 by Glu, an acidic amino acid that mimics phosphoserine, fosters the Mos interaction with MKK and permits activation of MKK in vitro and Mos-induced cleavage arrest. Moreover, the Glu-3 substitution increases the interaction of a kinase-inactive Mos mutant with MKK. Taken together, these results suggest that an important step in Mos activation involves the phosphorylation at Ser-3, which promotes Mos interaction with and activation of MKK.
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PMID:Ser-3 is important for regulating Mos interaction with and stimulation of mitogen-activated protein kinase kinase. 765 90

Gq is the heterotrimeric guanine nucleotide-binding protein that activates the beta isoforms of phosphatidyl-inositol-specific phospholipase C (PI-PLC). The Gq alpha-subunit polypeptide (alpha qa) was N-terminally modified by addition of a 9-aa sequence, YPYDVPDYA. Placement of the 9-aa epitope tag at the N terminus allowed expression of functional alpha q polypeptides and selective identification of plasmid-expressed wild-type and mutant G-protein alpha subunits. Mutation of glutamine-209 to leucine in the N-terminally epitope-tagged alpha q (N(epi) alpha qQ209L) inhibited GTPase activity and persistently activated PI-PLC, resulting in high steady-state levels of inositol phosphates. The elevated levels of inositol phosphates resulting from N(epi) alpha qQ209L expression were similar to those obtained with carbachol activation of the M1 muscarinic acetylcholine receptor. The Gq-coupled M1 receptor, which stimulates PI-PLC activity, and phorbol esters, acting via protein kinase C, activate the cytoplasmic mitogen-activated protein kinase in COS cells. However, the constitutive activation of PI-PLC enzymatic activity resulting from expression of GTPase-deficient alpha q was unable to persistently activate this kinase. The results indicate that persistent PI-PLC activation is insufficient to sustain the stimulation of a cytoplasmic serine/threonine protein kinase regulated by Gq-coupled receptor signal-transduction pathways.
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PMID:Epitope-tagged Gq alpha subunits: expression of GTPase-deficient alpha subunits persistently stimulates phosphatidylinositol-specific phospholipase C but not mitogen-activated protein kinase activity regulated by the M1 muscarinic acetylcholine receptor. 768 19


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