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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 (MAP) kinases form a group of serine/threonine kinases stimulated by various growth factors such as nerve growth factor (NGF) and hormones such as insulin. Interestingly, MAP kinases are thought to participate in a protein kinase cascade leading to cell growth as they have been shown to phosphorylate and activate ribosomal protein S6 kinase. To further evaluate the interactions between the different components of this cascade, we looked at the possible coprecipitation of MAP kinase activator(s) or MAP kinase substrate(s) with MAP kinase. Using antipeptides to the C terminus of the M(r) 44,000 MAP kinase, ERK1, and cell extracts from unstimulated or NGF-treated PC12 cells, we obtained in addition to MAP kinase itself coprecipitation of a protein with a M(r) in the 90,000 range. We further show that this protein is a protein kinase since it becomes phosphorylated on serine residues, after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transfer to a polyvinylidene difluoride membrane. In vitro phosphorylation performed before sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrates NGF-sensitive phosphorylation of this 90-kDa protein on both serine and threonine; the serine phosphorylation is likely to be due to autophosphorylation, and the threonine phosphorylation due to phosphorylation by the copurifying MAP kinase. Furthermore, immunoprecipitation of this 90-kDa protein was obtained with antibodies to S6 kinase II. Finally, using in situ chemical cross-linking, we were able to demonstrate in intact cells the occurrence of an anti-ERK1 immunoreactive species with a molecular mass of approximately 125,000 compatible with a complex between ERK1 and a 90-kDa S6 kinase. Taken together, our observations demonstrate that the 44-kDa MAP kinase is associated, in intact PC12 cells, with a protein kinase which is very likely to be S6 kinase II. In conclusion, our data represent strong evidence for a physiological role of the MAP kinase-S6 kinase cascade in PC12 cells. Finally, our antipeptides provide us with a powerful tool to search for additional physiologically relevant substrates for MAP kinase, a key integrator enzyme for growth factors and hormones.
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PMID:Nerve growth factor-induced phosphorylation cascade in PC12 pheochromocytoma cells. Association of S6 kinase II with the microtubule-associated protein kinase, ERK1. 132 33

The activation of insulin-stimulated protein-serine/threonine kinases has been investigated in CHO cell lines transfected with cDNAs encoding either wild-type or mutant human insulin receptors. (1) Insulin treatment of CHO cells over-expressing wild-type insulin receptors resulted in the rapid and substantial (5-10-fold) activation of cytosolic protein kinases which phosphorylated myelin basic protein, Kemptide and two peptide substrates based on sites phosphorylated on ribosomal protein S6 in vivo. (2) Further fractionation of cytosolic extracts by MonoQ chromatography revealed two peaks of insulin-stimulated myelin basic protein kinase activity which were highly related to the previously described mitogen-activated protein (MAP) kinases ERK1 and ERK2. In addition, at least two major peaks of S6 kinase activity were resolved, which exhibited properties similar to the 70 kDa and 90 kDa S6 kinases described by others; the predominant effect of insulin was on the activity of the 90 kDa enzyme and was in excess of 10-fold. (3) MonoQ fractionation of extracts from parental CHO cells, or cells expressing kinase-deficient receptors, showed all insulin-stimulated peaks of activity to be almost completely absent. (4) Further studies demonstrated that substitution of tyrosine residues 1162 and 1163 (or 1162 alone) with phenylalanine led to a substantial reduction in the ability of insulin to stimulate these protein kinase activities when assayed in cytosolic extracts. In contrast, deletion of 69 amino acids from the C-terminus of the insulin receptor beta-subunit caused a leftward shift in the insulin dose-response curve of the MAP kinase activity, but apparently not in that of the 90 kDa S6 kinase activity.
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PMID:Characterization of insulin-stimulated protein serine/threonine kinases in CHO cells expressing human insulin receptors with point and deletion mutations. 132 27

The immunosuppressant rapamycin inhibited proliferation of the H4IIEC hepatoma cell line. Rapamycin, but not its structural analog FK506, also inhibited the basal and insulin-stimulated activity of the p70 ribosomal protein S6 kinase. By contrast, insulin stimulation of the p85 Rsk S6 kinase and mitogen-activated protein (MAP) kinase activity were unaffected by drug. Rapamycin treatment of COS cells transfected with recombinant p70 S6 kinase completely inhibited the appearance of the hyperphosphorylated form of p70 S6 kinase concomitant with the inhibition of enzyme activity toward 40S subunits. Thus, rapamycin inhibits a signal transduction element that is necessary for the activation of p70 S6 kinase and mitogenesis but unnecessary for activation of p85 Rsk S6 kinase or MAP kinase.
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PMID:Rapamycin-induced inhibition of the 70-kilodalton S6 protein kinase. 138 Jan 82

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

Bacterial expression of mouse gene Erk-1 yielded an active kinase with the same substrate specificity shown for ERK1 protein purified from rat cells. Although rat gene ERK1 is believed to encode a serine/threonine kinase based on sequence data and known ERK1 substrate phosphorylation sites, bacterially-produced mouse Erk-1 (bt-Erk-1) autophosphorylated on tyrosine in addition to serine and threonine residues. The bt-Erk-1 protein also had the capacity to reactivate the ribosomal protein S6 kinase (S6KII). Furthermore, treatment of bt-Erk-1 with either serine/threonine-specific phosphatase 2A or tyrosine-specific phosphatase 1B significantly decreased its kinase activity. These findings predict that autophosphorylation may play an important role in Erk-1/ERK1 regulation.
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PMID:Mouse Erk-1 gene product is a serine/threonine protein kinase that has the potential to phosphorylate tyrosine. 171 89

In rat 1 fibroblasts, insulin has little or no stimulatory effect on the activities of either MAP2 protein kinase or ribosomal protein S6 kinase. In contrast, in rat 1 cells that overexpress the normal human insulin receptor (rat 1 HIRc B; McClain et al. (1987) J. Biol. Chem. 262, 14663-14671), insulin activates both MAP2 and S6 kinase activities close to 5-fold. A MAP2 kinase has been purified from insulin-treated rat 1 HIRc B cells over 6300-fold by chromatography on Q-Sepharose, phenyl-Sepharose, S-Sepharose, phosphocellulose, QAE-Sepharose, UltrogelAcA54, DEAE-cellulose, and a second Q-Sepharose. Its specific activity is approximately 0.8-1 mumol.min-1.mg-1 with MAP2 and 3 mumol.min-1.mg-1 with myelin basic protein. The enzyme preparation contains one major band of Mr = 43,000 upon SDS-polyacrylamide gel electrophoresis, which is immunoblotted by antibodies to phosphotyrosine. A sequence from the 43-kDa band led to the isolation of a cDNA encoding the enzyme, which we have named ERK1 for extracellular signal-regulated kinase (Boulton et al. (1990) Science 249, 64-67).
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PMID:Purification and properties of extracellular signal-regulated kinase 1, an insulin-stimulated microtubule-associated protein 2 kinase. 184 91

Mitogen-activated protein (MAP) kinase is a serine/threonine-specific protein kinase which is activated in response to various mitogenic agonists (e.g., epidermal growth factor, insulin, and the tumor promoter tetradecanoyl phorbol acetate [TPA]) and requires both threonine and tyrosine phosphorylation for activity. This enzyme has recently been shown to be identical or closely related to pp42, a protein which becomes tyrosine phosphorylated in response to mitogenic stimulation. Neither the kinases which regulate MAP kinase/pp42 nor the in vivo substrates for this enzyme are known. Because MAP MAP kinase is activated and phosphorylated in response both to agents which stimulate tyrosine kinase receptors and to agents which stimulate protein kinase C, a serine/threonine kinase, we have examined the regulation and phosphorylation of this enzyme in 3T3-TNR9 cells, a variant cell line partially defective in protein kinase C-mediated signalling. In this communication, we show that in the 3T3-TNR9 variant cell line, TPA does not cause the characteristically rapid phosphorylation of pp42 or the activation and phosphorylation of MAP kinase. This defective response is not due to the absence of the MAP kinase/pp42 protein itself because both tyrosine phosphorylation of MAP kinase/pp42 and its enzymatic activation could be induced by platelet-derived growth factor in the 3T3-TNR9 cells. Thus, the defect in these variant cells apparently resides in some aspect of the regulation of MAP kinase phosphorylation. Since the 3T3-TNR9 cells are also defective with respect to the TPA-induced increase in ribosomal protein S6 kinase, these in vivo results reinforce the earlier in vitro finding that MAP kinase can regulate S6 kinase activity. These findings suggest a key role for MAP kinase in a kinase cascade cascade involved in the control of cell proliferation.
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PMID:Defective regulation of mitogen-activated protein kinase activity in a 3T3 cell variant mitogenically nonresponsive to tetradecanoyl phorbol acetate. 199 Feb 61

Ribosomal protein S6 kinase I has been purified from unfertilized Xenopus eggs to near homogeneity as a Mr = 90,000 protein. S6 kinase I is phosphorylated when activated in vivo and can be phosphorylated by mitogen-activated protein kinase in vitro. The purified enzyme is inactivated upon treatment with protein phosphatase 2A. Immunological data and analysis of substrate specificity demonstrate that S6 kinase I is related to, but distinct from, the previously characterized S6 kinase II. Both enzymes are members of the ribosomal protein S6 kinase (rsk) gene family.
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PMID:Purification and characterization of ribosomal protein S6 kinase I from Xenopus eggs. 200 57

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

The control of cell proliferation involves both regulatory events initiated at the plasma membrane that control reentry into the cell cycle and intracellular biochemical changes that direct the process of cell division itself. Both of these aspects of cell growth control can be studied in Xenopus oocytes undergoing meiotic maturation in response to mitogenic stimulation. All mitogenic signaling pathways so far identified lead to the phosphorylation of ribosomal protein S6 on serine residues, and the biochemistry of this event has been investigated. Insulin and other mitogens activate ribosomal protein S6 kinase II, which has been cloned and sequences in oocytes and other cells. This enzyme is activated by phosphorylation on serine and threonine residues by an insulin-stimulated protein kinase known as MAP-2 kinase. MAP kinase itself is also activated by direct phosphorylation on threonine and tyrosine residues in vivo. These results reconstitute one step of the insulin signaling pathway evident shortly after insulin receptor binding at the membrane. Several hours after mitogenic stimulation, a cell cycle cytoplasmic control element is activated that is sufficient to cause entry into M phase. This control element, known as maturation-promoting factor or MPF, has been purified to near homogeneity and shown to consist of a complex between p34cdc2 protein kinase and cyclin B2. In addition to apparent phosphorylation of cyclin, regulation of MPF activity involves synthesis of the cyclin subunit and its periodic degradation at the metaphase----anaphase transition. The p34cdc2 kinase subunit is regulated by phosphorylation/dephosphorylation on threonine and tyrosine residues, being inactive when phosphorylated and active when dephosphorylated. Analysis of phosphorylation sides in histone H1 for p34cdc2 has revealed a consensus sequence of (K/R)S/TP(X)K/R, where the elements in parentheses are present in some but not all sites. Sites with such a consensus are specifically phosphorylated in mitosis and by MPF in the protooncogene pp60c-src. These results provide a link between cell cycle control and cell growth control and suggest that changes in cell adhesion and the cytoskeleton in mitosis may be regulated indirectly by MPF via protooncogene activation. S6 kinase II is also activated upon expression of MPF in cells, indicating that MPF is upstream of S6 kinase on the mitogenic signaling pathway. Further study both of the signaling events that lead to MPF activation and of the substrates for phosphorylation by MPF should lead to a comprehensive understanding of the biochemistry of cell division.
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PMID:Xenopus oocytes and the biochemistry of cell division. 215 26


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