Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.11.13 (protein kinase C)
 49,245 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cytosolic insulin-sensitive serine kinase has been purified to apparent homogeneity in parallel from livers of control or acutely insulin-treated rats. The kinase is labile and requires rapid purification for stability. The kinase migrates as a band of apparent Mr = 90,000 on denaturing gels and elutes as a monomer on Superose 12 gel filtration. After sodium dodecyl sulfate-polyacrylamide gel electrophoresis and renaturation, the 90-kDa band presumed to be the kinase shows kinase activity toward myelin basic protein in situ. Substrates of the kinase include Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide), ribosomal protein S6, S6 peptide, a proline-rich peptide substrate, microtubule-associated protein 2, and myelin basic protein. The kinase also phosphorylates histones H1 and H2B, but does not autophosphorylate to a significant stoichiometry. The activity of the kinase is inhibited by fluoride, glycerophosphate, p-nitrophenyl phosphate, p-nitrophenol, heparin, quercetin, poly-L-lysine, and potassium phosphate, but is unaffected by calcium, cAMP, spermine, protein kinase inhibitor peptide, phorbol myristate acetate, calcium plus phosphatidylserine, or vanadate. The kinase will utilize magnesium (10 mM) as well as manganese (1 mM) as a cofactor for maximal phosphotransferase activity. The kinase is not detected by immunoblotting with antibodies directed against protein kinase C or type II S6 kinase. Taken together, these properties distinguish this kinase from other insulin-sensitive kinases that have been described previously. The purified kinase from livers of insulin-treated rats shows a 5-20-fold higher specific activity compared to enzyme prepared from control rats, suggesting a covalent modification as the mechanism of activation. Incubation of purified, insulin-stimulated kinase with purified phosphatase 2A leads to deactivation of the kinase activity, and the phosphatase inhibitor nitrophenyl phosphate blocks this deactivation. The insulin-activated kinase fails to immunoblot with anti-tyrosine phosphate antibodies. Taken together, these results indicate that insulin activates this novel cytosolic protein kinase by a mechanism that causes its phosphorylation on serine or threonine residues.
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PMID:Purification and characterization of a cytosolic insulin-stimulated serine kinase from rat liver. 153 38

A serine protein kinase that phosphorylates the beta-subunit of the insulin receptor has been partially purified 5,000-fold from HeLa cell membranes. The enzyme has been purified by ion-exchange and hydroxylapatite chromatography and sucrose gradient centrifugation; it has an apparent molecular weight of 36,000-43,000 daltons. It exhibits the following properties: (a) it catalyzes the phosphorylation of the autophosphorylated insulin receptor more efficiently than the nonautophosphorylated insulin receptor, (b) it decreases insulin receptor phosphorylation of tubulin but has no effect on insulin receptor phosphorylation of microtubule-associated proteins or reduced and carboxyamidomethylated lysozyme. The enzyme also phosphorylates casein and ribosomal protein S6 and shares many properties with casein kinase I: (a) similar molecular weight, (b) utilization of ATP but not GTP as phosphoryl donor, and (c) sensitivity to inhibition by heparin. Based on several criteria the receptor serine kinase is neither protein kinase C nor the cAMP-dependent protein kinase.
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PMID:Phosphorylation of the insulin receptor by a casein kinase I-like enzyme. 164 67

Previous work (Gandino, L., Di Renzo, M. F., Giordano, S., Bussolino, F., and Comoglio, P.M. (1990) Oncogene 5, 721-725) has shown that the tyrosine kinase activity of the receptor encoded by the MET protooncogene is negatively modulated by protein kinase C (PKC). We now show that an increase of intracellular Ca2+ has a similar inhibitory effect in vivo, via a PKC-independent mechanism. In GTL-16 cells the p145MET kinase is overexpressed and constitutively phosphorylated on tyrosine. A rapid and reversible decrease of p145MET tyrosine phosphorylation was induced by treatment with the calcium ionophores A23187 or ionomycin. Experiments performed with the ionophores in absence of extracellular calcium showed that a rise in cytoplasmic Ca2+ concentration to 450 nM (due to release from intracellular stores) resulted in a similar effect. These Ca2+ concentrations had no effect on p145MET autophosphorylation in an in vitro kinase assay. This suggests that the effect of Ca2+ on p145MET tyrosine phosphorylation is not direct but may be mediated by Ca(2+)-activated proteins(s). Involvement of Ca(2+)-dependent tyrosine phosphatases was ruled out by experiments carried out in presence of Na2VO4. In vivo labeling with [32P]orthophosphate showed that the rise of intracellular Ca2+ induces serine phosphorylation of p145MET on a specific phosphopeptide. This suggests that Ca2+ negatively modulates p145MET kinase through the phosphorylation of a critical serine residue by a Ca(2+)-activated serine kinase distinct from PKC.
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PMID:Intracellular calcium regulates the tyrosine kinase receptor encoded by the MET oncogene. 165 34

Engagement of membrane IgM on a number of human and murine B-cell lines induced activation of a Mn(2+)-preferring serine/threonine kinase that phosphorylated microtubule-associated protein-2 (MAP-2) in vitro. B-cell MAP-2 kinase (MAP-2K) activity could be fractionated into two peaks by sequential DEAE and hydrophobic chromatography. Although peak I included two tyrosine phosphoproteins of molecular mass 36 and 38 kDa, peak II showed a single 42-kDa tyrosine phosphoprotein (pp42). Since all kinase activity could be removed from peak II material over an antiphosphotyrosine immune affinity column, it suggests that pp42 is identical with lymphoid MAP-2K. Although peak I activity showed a similarity to peak II with regard to its preference for Mn2+, sensitivity to phosphatase exposure, and resistance to a range of common serine kinase inhibitors, it is not clear whether these activities are related. MAP-2 kinase activity could also be induced by treatment with the phorbol ester, phorbol myristate 13-acetate, suggesting that protein kinase C may also be involved with MAP-2K regulation. Although MAP-2K activity reached a peak response within minutes of receptor ligation, there were differences in the rates of dephosphorylation of pp42 and decline of MAP-2K activity in different B-cell lines. The tyrosine phosphatase inhibitor, vanadate, transformed a rapidly reversible MAP-2K response in BAL 17.2 cells into a sustained state of activation that resembled the kinetics of activation in WEHI-231 cells. The latter finding implies involvement of a tyrosine phosphatase, which opposes the effect of an inducing tyrosine kinase.
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PMID:Stimulation of B-cells via the membrane immunoglobulin receptor or with phorbol myristate 13-acetate induces tyrosine phosphorylation and activation of a 42-kDa microtubule-associated protein-2 kinase. 165 69

The pathways depicted in Figure 1 summarize the data discussed in this article. In neurons, the binding of insulin and IGF-I to their respective receptors triggers autophosphorylation of the receptor beta-subunits. IGF-II binds to both neuronal insulin and IGF-I receptors and can stimulate autophosphorylation of either receptor type. In addition to enhancing insulin and IGF-I receptor autophosphorylation, all 3 peptides stimulate the tyrosine phosphorylation of a 70 kDa protein with a similar time course and dose response to receptor phosphorylation. The identity of pp70 is unknown, although the close temporal relationship between pp70 phosphorylation and neurite outgrowth suggests a potential role for this protein. Subsequent to these very early events, two neuronal serine kinases are activated by insulin. One has S6 kinase activity and may represent either the pp90rsk or pp70 class of S6 kinases. Since S6 kinases are activated by direct phosphorylation rather than by second messengers, it is likely that a neuronal S6 kinase kinase exists. The activation of S6 kinase is likely to mediate insulin's effects on neuronal protein synthesis or other growth-related processes. The second serine kinase that is activated by insulin is PKC epsilon. This enzyme is largely restricted to the nervous system, so this signalling pathway may be neuronal-specific. The mechanism of activation of PKC epsilon is unknown, although preliminary data suggests that enhanced phosphorylation of the enzyme is involved. Studies are currently underway to investigate the potential role of diacylglycerol, a potential second messenger generated from either phosphotidylinositol or phosphotidylcholine hydrolysis, in the activation of PKC epsilon by insulin.
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PMID:Regulation of protein phosphorylation by insulin and insulin-like growth factors in cultured fetal neurons. 166 64

Interleukin 1 (IL1) increased phosphorylation of the small heat-shock protein (hsp 27) in MRC5 fibroblasts. The increase was maintained for at least 30 min, but levels had returned to pre-stimulation values by 2 h. When hsp 27 was metabolically labelled with [3H]leucine, about 15% was phosphorylated in resting confluent cells; this rose to 90% upon stimulation by IL1. Peptide maps of the three differently charged phosphorylated forms were consistent with their arising by phosphorylation of increasing numbers of serine residues. IL1 had the same effect on hsp 27 in pig articular chondrocytes, endothelial cells from human umbilical vein and an epidermoid carcinoma cell line (KB). Certain other agents were found selectively to increase phosphorylation of hsp 27 in MRC5 cells besides IL1 [and tumour necrosis factor (TNF)]. Platelet-derived growth factor had a similar effect to that of IL1; bradykinin, acid fibroblast growth factor and ATP caused an intermediate effect; phorbol myristate acetate (PMA) and 1-oleoyl-2-acetylglycerol had smaller effects. Dibutyryl cyclic AMP and forskolin had no effects on hsp 27 phosphorylation. When cells had been depleted of protein kinase C (PKC) by prolonged treatment with PMA, stimulation by IL1, TNF or bradykinin still increased hsp 27 phosphorylation. The stimulation by all three agents was also unaffected by the PKC inhibitor staurosporine. IL1, TNF and bradykinin each caused hsp 27 phosphorylation by a pathway independent of PKC. The results are consistent with IL1 activating a serine kinase which remains to be identified.
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PMID:Phosphorylation of the small heat-shock protein is regulated by interleukin 1, tumour necrosis factor, growth factors, bradykinin and ATP. 187 99

In these studies we demonstrate that insulin stimulates both tyrosine and serine phosphorylation of the insulin receptor after its partial purification on wheat germ-agarose, and after affinity purification on insulin-agarose. Analysis of the serine phosphate incorporated into partially purified or highly purified insulin receptor suggests that an insulin-sensitive serine kinase (IRSK) copurifies with the insulin receptor. Following trypsin digestion, reversed-phase high pressure liquid chromatography (HPLC) analysis of the phosphorylated, affinity-purified insulin receptor preparation reveals phosphopeptide profiles similar to those of trypsin-digested receptors immunoprecipitated from 32P-labeled fibroblasts overexpressing the human insulin receptor. The major insulin-stimulated HPLC phosphopeptide peak from insulin receptors labeled in intact cells contains a hydrophilic phosphoserine-containing peptide which rapidly elutes from a C18 column. HPLC and two-dimensional separation indicate that the same phosphopeptide is obtained when affinity-purified insulin receptors are phosphorylated by IRSK. The serine containing tryptic peptide within the cytoplasmic domain of the human insulin receptor predicted to elute most rapidly upon HPLC had the sequence SSHCQR corresponding to residues 1293-1298. A synthetic peptide containing this sequence is phosphorylated by the insulin receptor/IRSK preparation. After alkylation and trypsin digestion, the synthetic phosphopeptide comigrates with the alkylated, tryptic phosphopeptide derived from insulin receptor phosphorylated in vitro by IRSK. We propose that serine 1293 or 1294 of the human insulin receptor is a major site(s) phosphorylated on the insulin receptor in intact cells and is phosphorylated by IRSK. Furthermore, insulin added directly to affinity-purified insulin receptor/IRSK preparations stimulates the phosphorylation of synthetic peptides corresponding to this receptor phosphorylation site and another containing threonine 1336. Kemptide phosphorylation is not stimulated by insulin under these conditions. No phosphorylation of peptide substrates for Ca2+/calmodulin-dependent protein kinase, protein kinase C, casein kinase II, or cGMP-dependent protein kinase by IRSK is detected. These data indicate that IRSK exhibits specificity for the insulin receptor and may be activated by the insulin receptor tyrosine kinase in an insulin-dependent manner.
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PMID:Insulin-sensitive phosphorylation of serine 1293/1294 on the human insulin receptor by a tightly associated serine kinase. 213 51

Signaling via the alpha-beta T cell Ag receptor (Ti)-CD3 complex is a complicated event that implicates several protein kinases, most notably protein kinase C (PKC). We have recently identified a serine kinase in T lymphocytes with the following characteristics: molecular mass 43 kDa, in vitro substrate affinity for microtubule associated protein 2 (MAP-2) with a preference for Mn2+ during the catalytic reaction, and elution from DEAE resin over a salt range 100 to 200 mM NaCl. This kinase is activated in a rapidly reversible fashion during ligation of CD3/Ti by a process which involves prior phosphorylation; in vitro exposure of activated 43-kDa MAP-2 kinase (MAP-K) to an immobilized phosphatase abrogated its kinase activity. We now show that a MAP-2K response could also be obtained during treatment with mAb to Ti and the specific PKC agonist, PMA. Although the kinetics of the former response was rapidly reversible, PMA elicited a more prolonged response. The dose responsiveness for PMA was similar to the requirements for PKC activation in intact lymphocytes. Moreover, as with PKC, we found that the CD3-induced MAP-2K response could be further enhanced by using a second layer cross-linking antibody. The specificity of CD3/Ti in the Jurkat cell response is demonstrated by the fact that OKT-11(CD2) and anti-CD4 mAb did not stimulate a MAP-2K response. It was also not possible to elicit a response in a Jurkat cell mutant that lacks surface expression of CD3 and Ti. The specificity of PKC in these events was further explored with the cell permeant diacylglycerol, 1-oleoyl-2-acetylglycerol, and the nonagonist phorbol ester, 4 alpha-phorbol 12,13-didecanoate: whereas the former was an effective inducer of the MAP-2K response, the latter failed to yield any stimulation. Prior exposure of Jurkat cells to 100 mM PMA for 24 h eliminated greater than 60% of the MAP-2K response during anti-CD3 treatment. This response could also be inhibited in dose-dependent fashion by prior treatment of Jurkat cells with the potent PKC inhibitor 1-(5-isoquinolinesulfonyl) 2-methylpiperazine dihydrochloride. Although a Ca2(+)-ionophore failed to synergize with PMA at inducing a MAP-2K response, depletion of extracellular Ca2+ by EGTA abrogated anti-CD3 responsiveness. The events culminating in MAP-2K activation were slightly inhibited in the presence of cholera toxin but not pertussis toxin.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Stimulation of MAP-2 kinase activity in T lymphocytes by anti-CD3 or anti-Ti monoclonal antibody is partially dependent on protein kinase C. 215 31

The authors have previously observed that glucocorticoids dramatically increase the number of interleukin 1 (IL-1) receptors on normal human peripheral blood mononuclear cells (PBMC) (from approximately 100 to 2000 receptors/cell) without significant change in the binding affinity (Kd = approximately 2.6 x 10(-10) M). We, therefore, used such a receptor-enriched glucocorticoid-pretreated PBMC to investigate whether IL-1 induces/increases the phosphorylation of any cell-associated proteins, including possible autophosphorylation of IL-1 receptors. Extraction of 125I-labeled IL-1 alpha cross-linked to IL-1 receptor on steroid-treated PBMC yielded two bands estimated to be 60 and 70 kDa in molecular mass. No molecules were significantly cross-linked with 125I-labeled IL-1 alpha on untreated PBMC. Carrier-free recombinant human IL-1 alpha induced phosphorylation of an acidic 65-kDa protein (pp65) at serine residues within 5 min more effectively in glucocorticoid-treated PBMC than in untreated PBMC. Fractionation of extracts of IL-1-stimulated prednisolone-pretreated PBMC by ultracentrifugation showed that pp65 is located in the cytosol, suggesting that pp65 is not the IL-1 receptor itself. Protein kinase inhibitors, HA1004 and W-7, but not H-7, significantly inhibited the induction of the phosphorylation of 65-kDa protein by IL-1. These data indicate that the glucocorticoid-induced IL-1 receptor is functional and either contains or is closely associated with a serine kinase that is distinct from protein kinase C.
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PMID:Phosphorylation of a cytosolic 65-kDa protein induced by interleukin 1 in glucocorticoid pretreated normal human peripheral blood mononuclear leukocytes. 296 Jul 34

Stimulation of serine protein kinase activity (referred to as S6 kinase) occurs within minutes of addition of nerve growth factor (NGF) to PC12 rat pheochromocytoma cells. This enzyme activity is not related to the cAMP-dependent protein kinase (protein kinase A) or the Ca2+- and phospholipid-dependent protein kinase (protein kinase C), two other protein kinases potentially involved in signal transduction. Two peaks of NGF-stimulated S6 phosphotransferase activity are observed upon ion exchange chromatography; one that comigrates with the serine kinase previously described in chicken embryo fibroblasts and another with distinct elution properties. Several other factors are also found to regulate S6 phosphotransferase activity in PC12 cells including epidermal growth factor, insulin, and phorbol myristate acetate. Dibutyryl cAMP stimulates S6 phosphotransferase activity; however, this activity is strongly inhibited by the protein kinase A heat stable inhibitor. At least two mechanisms exist through which the NGF-stimulated S6 kinase activity can be regulated, one that apparently can use protein kinase C whereas the other(s) does not. The potential roles of these protein kinase activities in signal transduction and regulation of cell growth and differentiation is discussed.
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PMID:Regulation of protein kinase activities in PC12 pheochromocytoma cells. 303 Jul 27


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