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:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Two-dimensional analysis of 32P-labelled ribosomal proteins revealed three proteins which are phosphorylated in vaccinia-virus-infected HeLa cells. All three proteins belong to the 40-S ribosomal subunits and were identified as S2, S6 and S16. The ribosomal protein S6 is phosphorylated also in uninfected HeLa cells. Phosphoserine was detected in all three proteins, phosphothreonine only in the protein S2. Phosphorylation of these ribosomal proteins in infected cells is dependent on the multiplicity of the viral infection and increases during the first six hours of infection. All three proteins are also phosphorylated in virus-infected cells treated with cycloheximide and in cells infected with ultraviolet-irradiated virus. This suggests that the phosphorylation reaction involves a vaccinia virion-associated protein kinase.
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PMID:Identification and characterization of ribosomal proteins phosphorylated in vaccinia-virus-infected HeLa cells. 71 Apr 42

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 small heat-shock protein hsp25 of the Ehrlich ascites tumor exists in one non-phosphorylated (hsp25/1) and two phosphorylated (hsp25/2, hsp25/3) isoforms. In stationary phase tumor cells, a protein kinase activity was detected which phosphorylates hsp25/1, resulting in the formation of several phosphorylated hsp25 isoforms, including those occurring naturally in the tumor. Cell-free phosphorylation of hsp25 required Mg2+ and ATP and was independent of Ca2+, phosphatidylserine, cAMP and cGMP. Polymyxin B inhibited, specifically, hsp25 phosphorylation, whereas trifluoperazine, staurosporine and the protein inhibitor of protein kinase A had no effect. In its properties, the hsp25 phosphorylating kinase differs from other common kinases such as protein kinases A and C, calcium/calmodulin-dependent kinases, and the ribosomal protein S6 kinase.
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PMID:Cell-free phosphorylation of the murine small heat-shock protein hsp25 by an endogenous kinase from Ehrlich ascites tumor cells. 150 5

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

Spontaneous S6 phosphatase activities dephosphorylating Ser(P)-235 and Ser(P)-236 of the ribosomal protein S6 were measured and compared in microsomes and cytosol of rat liver. The substrate used, small (40S) ribosomal subunits 32P-labelled in vitro by protein kinase A, contained phosphorylated S6 (mainly in the diphosphorylated form) and some minor phosphorylated species. The microsomal and cytosolic S6 phosphatase activities displayed a number of distinct properties. The microsomal activity, representing ca 20% of the S6 phosphatase activity in the post-mitochondrial supernatant, was mainly due to a type-1 phosphatase and dephosphorylated only S6. The remaining post-mitochondrial S6 phosphatase activity, which was fully recovered in the cytosol, and appeared to result from a combination of type-1 (43%) and type 2 (57%) phosphatases, acted on S6 as well as on the minor phosphorylated species. The microsomal activity was 50% inhibited by MgCl2 (10 mM) and was stimulated at least 4.3 fold by MnCl2 (1 mM), while the cytosolic activity was inhibited only 18% by Mg2+ (10 mM) and was increased 2.2 fold by Mn2+ (1 mM). The microsomal activity was increased 10% (P less than 0.06) by lower doses of insulin (25 U/Kg) and 14% (P less than 0.05) by vanadate, but was not significantly (P greater than 0.10) affected by larger doses of insulin (100 U/kg), hepatectomy or cycloheximide. By comparison the cytosolic S6 phosphatase activity was unresponsive to insulin and vanadate, but was decreased 14% and 17% (P less than 0.05) by hepatectomy and cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:A comparative study of microsomal and cytosolic S6 phosphatase activities in rat liver. 166 99

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

Rat brain plasma membranes were solubilized in detergent and a glycoprotein-enriched fraction was obtained by lectin affinity chromatography. This glycoprotein fraction contained insulin receptors, as well as protein kinases capable of phosphorylating some exogenously added substrates such as MAP2 (microtubule associated protein 2) and MBP (myelin basic protein), but not ribosomal protein S6. Phosphoamino acid analysis of MAP2 and MBP showed that phosphotyrosine residues, as well as phosphoserine/phosphotheronine residues, were present in both proteins under basal conditions. Whereas the addition of insulin to the rat brain membrane glycoprotein fraction in vitro had no effect on MAP2 phosphorylation, MBP phosphorylation was stimulated 2.7-fold in response to insulin. This phenomenon was dose-dependent, with half-maximal stimulation of MBP phosphorylation observed with 2 nM insulin. Phosphoamino acid analysis of MBP indicated that insulin stimulated the phosphorylation of tyrosine residues nearly three-fold, whereas the phosphorylation of serine or threonine residues was not increased. These results identify MBP as a substrate for the rat brain insulin receptor tyrosine-specific protein kinase in vitro.
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PMID:Insulin-sensitive myelin basic protein phosphorylation on tyrosine residues. 171 93

The substrate specificity determinants of a protease-activated protein kinase from rat liver, termined PAK-1, have been investigated using peptide analogues of the ribosomal protein S6 sequence: Ala229-Lys-Arg-Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala-Ser-Thr-Ser-Lys- Ser244. Low peptide substrate Km's and a preference for Ser236 were attributed to a combination of sequence determinants located in the vicinity of this site. Thus, Km's are increased appreciably with analogues in which the N-terminal cluster of basic residues is reduced or where alanine is substituted for Arg238. Even more dramatic effects are elicited by alanine substitution of one of the adjacent serine residues, resulting in 20-fold to 800-fold increases in the Km's for the [Ala235] and [Ala236] S6(229-239) variants, respectively. Arg238 is the major specificity determinant of the Ser236 site, with little detectable phosphorylation of Ser236 occurring in the [Ala238] S6(229-239) substrate. Ser235 phosphorylation is also selectively enhanced by the addition of N-terminal basic residues to the Arg-Arg-Leu-Ser-Ser-Leu-Arg-Ala analogue. Finally, multiple phosphorylation events are influenced by negative cooperativity between the Ser235 and Ser236 sites and positive cooperativity between the Ser236 and Ser240 sites. The general S6 peptide substrate determinants for liver PAK-1 resembled those for brain protein kinase C and another major liver PAK, termed PAK-2. However, subtle differences observed between the kinetic properties with individual S6 peptide substrates distinguished PAK-1 from the other enzymes. More striking differences were observed between the liver PAKs and cyclic AMP-dependent protein kinase (cAK), particularly with respect to the cAK's relatively poor S6 peptide substrate kinetics, its preference for Ser235 and its ability to more extensively phosphorylate multiple sites in the S6 peptides.
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PMID:Determinants of multi-site phosphorylation of peptide analogues of ribosomal protein S6 by novel protease-activated protein kinases. 182 86


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