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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)

Microtubule-associated protein 2 kinase (MAP kinase), which exists in several forms, is a protein serine/threonine kinase that participates in a growth factor-activated protein kinase cascade in which it activates a ribosomal protein S6 kinase (pp90rsk) while being regulated itself by a cytoplasmic factor (MAP kinase activator). Experiments with recombinant MAP kinase, ERK2, purified from Escherichia coli in a nonactivated form revealed a self-catalyzed phosphate incorporation into both tyrosine and threonine residues. Another MAP kinase, ERK1, purified from insulin-stimulated cells also autophosphorylated on tyrosine and threonine residues. Autophosphorylation of ERK2 correlated with its autoactivation, although both autophosphorylation and autoactivation were slow compared to that occurring in the presence of MAP kinase activator. Therefore, we propose that autophosphorylation is probably involved in the MAP kinase activation process in vitro, but it may not be sufficient for full activation. The specificity toward tyrosine and threonine residues indicates that the MAP kinases ERK1 and ERK2 are members of a group of kinases with specificity for tyrosine as well as serine and threonine residues.
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PMID:Microtubule-associated protein 2 kinases, ERK1 and ERK2, undergo autophosphorylation on both tyrosine and threonine residues: implications for their mechanism of activation. 171 80

We have approached the functioning of a MAP kinase, which is thought to be a "switch kinase" in the phosphorylation cascade initiated from various receptor tyrosine kinases including the insulin receptor. To do so, antipeptide antibodies were raised against the C-terminal portion of ERK1 (extracellular signal-regulated kinase 1), a protein kinase belonging to the family of MAP kinases. With these antipeptide antibodies, we observed the following: (i) a 44-kDa protein can be specifically recognized both under native and denaturing conditions; (ii) a 44-kDa phosphoprotein can be revealed in 32P-labeled cells; its phosphorylation is stimulated by insulin, sodium orthovanadate, and okadaic acid; (iii) a MBP kinase activity can be precipitated, which phosphorylates MBP on threonine residues, and which is stimulated by insulin, sodium orthovanadate, okadaic acid, and fetal calf serum; (iv) this MBP kinase activity appears to be correlated with the in vivo induced phosphorylation of the 44-kDa protein. We next studied the in vitro phosphorylation of this 44-kDa/ERK1-immunoreactive protein. A time- and manganese-dependent phosphorylation was stimulated by the in vitro addition of sodium orthovanadate. Phosphoamino acid analysis of the in vitro phosphorylated 44-kDa protein revealed both threonine and tyrosine phosphorylation. Importantly, this in vitro phosphorylation of MAP kinase results in activation of phosphorylation of added MBP substrate. As a whole, our data indicate that the 44-kDa phosphoprotein identified by our antipeptide antibodies very likely corresponds to a MAP kinase.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Tyrosine and threonine phosphorylation of an immunoaffinity-purified 44-kDa MAP kinase. 171 57

The Raf-1 proto-oncogene protein kinase can be phosphorylated and activated after stimulation of cells with insulin and a variety of other growth factors and mitogens. We recently presented evidence that insulin and certain other growth factors activated one or more Raf-1 kinase kinase activities (Lee, R.M., Rapp, U. R., and Blackshear, P.J. (1991) J. Biol. Chem. 266, 10351-10357). In the present study, four peaks of Raf-1 kinase kinase activity were identified after anion-exchange chromatography of cell lysates, and two of these were activated by insulin. Further chromatographic characterization of these two peaks of insulin-activated kinase activity indicated that they contained three apparently distinct kinase activities. Two of these activities comigrated with immunoreactive extracellular signal-regulated kinases (ERK) 1 and 2 (mitogen-activated protein kinase) through three different chromatographic separations. Both ERK1 and ERK2 phosphorylated Raf-1 with reasonably high affinity (Km for ERK1 = 90 nM; Km for ERK2 = 120 nM), and produced similar, complex phosphopeptide maps; both kinases also phosphorylated myelin basic protein. The third kinase activity also phosphorylated Raf-1 and myelin basic protein but did not comigrate exactly with either immunoreactive ERK1 or ERK2. We conclude that two and possibly three insulin-activated Raf-1 kinase kinases are members of the ERK family.
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PMID:Evidence that extracellular signal-regulated kinases are the insulin-activated Raf-1 kinase kinases. 173 Jun 37

This study characterizes the insulin-activated serine/threonine protein kinases in H4 hepatoma cells active on a 37-residue synthetic peptide (called the SKAIPS peptide) corresponding to a putative autoinhibitory domain in the carboxyl-terminal tail of the p70 S6 kinase as well as on recombinant p70 S6 kinase. Three peaks of insulin-stimulated protein kinase active on both these substrates are identified as two (possibly three) isoforms of the 40-45-kDa erk/microtubule-associated protein (MAP)-2 kinase family and a 150-kDa form of cdc2. Although distinguishable in their substrate specificity, these protein kinases together with the p54 MAP-2 kinase share a major common specificity determinant reflected in the SKAIPS peptide: the requirement for a proline residue immediately carboxyl-terminal to the site of Ser/Thr phosphorylation. In addition, however, at least one peak of insulin-stimulated protein kinase active on recombinant p70, but not on the SKAIPS peptide, is present although not yet identified. MFP/cdc2 phosphorylates both rat liver p70 S6 kinase and recombinant p70 S6 kinase exclusively at a set of Ser/Thr residues within the putative autoinhibitory (SKAIPS peptide) domain. erk/MAP kinase does not phosphorylate rat liver p70 S6 kinase, but readily phosphorylates recombinant p70 S6 kinase at sites both within and in addition to those encompassed by the SKAIPS peptide sequences. Although the tryptic 32P-peptides bearing the cdc2 and erk/MAP kinase phosphorylation sites co-migrate with a subset of the sites phosphorylated in situ in insulin-stimulated cells, phosphorylation of the p70 S6 kinase by these proline-directed protein kinases in vitro does not reproducibly activate p70 S6 kinase activity. Thus, one or more erk/MAP kinases and cdc2 are likely to participate in the insulin-induced phosphorylation of the p70 S6 kinase. In addition to these kinases, however, phosphorylation of the p70 S6 kinase by other as yet unidentified protein kinases is necessary to recapitulate the multisite phosphorylation required for activation of the p70 S6 kinase.
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PMID:An array of insulin-activated, proline-directed serine/threonine protein kinases phosphorylate the p70 S6 kinase. 173 88

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

Staurosporine, a potent inhibitor of protein kinase C, arrests fission yeast cell elongation specifically at a stage immediately after cell division. We isolated two genes, which, when carried on multicopy plasmids, confer drug resistance in fission yeast. One, spk1+, encodes a protein kinase highly similar (54% identity) to those encoded by the mammalian ERK1/MAP2 kinase and the budding yeast KSS1 and FUS3 genes. It is not essential for vegetative growth of Schizosaccharomyces pombe cells but is required for conjugation. The spk1+ gene product is a 45-kD protein enriched in the nucleus, and its level increases 10-fold after addition of staurosporine. The other gene pap1+ encodes an AP-1-like transcription factor that contains a region rich in basic amino acids followed by a "leucine zipper" motif. The pap1+ gene is required for spk1(+)-conferred staurosporine resistance. These two genes appear to function as a part of the fission yeast growth control pathway.
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PMID:Fission yeast genes that confer resistance to staurosporine encode an AP-1-like transcription factor and a protein kinase related to the mammalian ERK1/MAP2 and budding yeast FUS3 and KSS1 kinases. 189 30

Synthetic peptides have been used to define the consensus amino acid sequence for substrate recognition by the meiosis-activated myelin basic protein (MBP) kinase (p44mpk), which was purified from maturing sea star oocytes. This protein kinase shares many properties with the mitogen-activated microtubule-associated protein-2 kinase (p42mapk) in vertebrates. Recently, Thr-97 in the tryptic fragment KNIVTPRTPPPSQGK of bovine MBP was identified as the major site of phosphorylation by p44mpk (Sanghera, J. S., Aebersold, R., Morrison, H. D., Bures, E. J., and Pelech, S. L. (1990) FEBS Lett. 273, 223-226). Synthetic peptides modeled after this sequence revealed that the presence of a proline residue C-terminal (+1 position) to the phosphorylatable threonine (or serine) residue was critical for recognition by p44mpk. Although not essential, a proline residue located at the -2 position enhanced the Vmax of peptide phosphorylation. Basic, acidic, and non-polar residues were equally tolerated at the -1 position. The presence of an amino acid residue at position -3 also increased peptide phosphorylation. Thus, the optimum consensus sequence for phosphorylation by p44mpk was defined as Pro-X-(Ser/Thr)-Pro, where X is a variable amino acid residue, but ideally not a Pro. Peptides that included this sequence were phosphorylated by p44mpk with Vmax values approaching 1 mumol.min-1.mg-1 and with apparent Km values of approximately 1 mM). Pseudosubstrate peptides in which the phosphorylatable residue was replaced by valine or alanine were weak inhibitors of p44mpk (apparent Ki values of approximately 3 mM). Over 40 distinct protein kinases contain Pro-X-(Ser/Thr)-Pro sequences including the human receptors for insulin and epidermal growth factor, and kinases encoded by the human proto-oncogenes abl, neu, and raf-1, and Schizosaccharomyces pombe cell cycle control genes ran-1 and wee-1. Multiple putative sites were also identified in rat microtubule-associated protein-2, human retinoblastoma protein, human tau protein, and Drosophila myb protein and RNA polymerase II.
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PMID:Definition of a consensus sequence for peptide substrate recognition by p44mpk, the meiosis-activated myelin basic protein kinase. 190 71

We have previously described the purification of a myelin basis protein (MBP) kinase from maturing sea star oocytes (Sanghera, J. S., Paddon, H. B., Bader, S. A., and Pelech, S. L. (1990) J. Biol. Chem. 265, 52-57). The ability of the purified 44-kDa protein to bind azido-ATP and undergo autophosphorylation on the serine residue implied that it is a protein kinase. Furthermore, partial amino acid sequence data has revealed that it is a novel protein kinase, which we have provisionally designated p44mpk. Autophosphorylation of p44mpk to 0.7 mol of phosphate/mol of enzyme was correlated with a modest (approximately 17%) increase in the MBP-phosphorylating activity of the kinase. Rabbit polyclonal antibody raised against purified p44mpk recognized on immunoblots the protein in highly purified preparations as well as crude oocyte extracts. The affinity-purified anti-p44mpk antibody could immunoprecipitate active kinase, but a subpopulation of the antibody also appeared to be inhibitory. Using this antibody, we have demonstrated that the up to 12-fold stimulation of the cytosolic MBP-phosphorylating activity of this kinase that occurs during sea star oocyte maturation is not due to an increase in the amount of enzyme protein, either from a redistribution within the oocyte or protein synthesis. A slight retardation of the migration of the activated p44mpk on sodium dodecyl sulfate-polyacrylamide gels and its tighter interaction with a MonoQ column is consistent with phosphorylation of the kinase during maturation. p44mpk underwent enhanced phosphorylation when oocytes prelabeled with [32P]orthophosphate were induced to mature with 1-methyladenine. The stimulated MBP-phosphorylating activity of p44mpk in cytosols from maturing oocytes was partly stabilized by the presence of the phosphatase inhibitor beta-glycerol phosphate. Furthermore, treatment of purified p44mpk with protein phosphatase 2A and alkaline phosphatase resulted in 56 and 86% decreases, respectively, in the activity of the kinase. Together, these findings strongly implicate a role for phosphorylation of p44mpk in its activation during sea star oocyte maturation.
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PMID:Role of protein phosphorylation in the maturation-induced activation of a myelin basic protein kinase from sea star oocytes. 201 85


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