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)

The technique of radiation inactivation has been used on a highly purified insulin receptor in order to determine the functional molecular size responsible for tyrosine-specific protein kinase activity. When both insulin binding and kinase activities were analyzed with the same receptor preparations, the functional size for kinase activity was found to be larger than that for insulin binding activity. The radiation inactivation curve for kinase activity was multiphasic. This indicates that at least two components contribute to total kinase activity. The average minimal functional size for the kinase was 370,000 +/- 60,000 daltons (n = 7) which corresponds to the alpha 2 beta 2 form of the insulin receptor. The average functional size for larger forms was estimated to be approximately 4 X 10(6) daltons. (To minimize the complexity of the model used in this analysis, we have analyzed the radiation inactivation curves of the insulin receptor kinase activity with a two-component model. However, we believe that the larger component, greater than 1 X 10(6) daltons, is probably not a single molecular weight species but rather represents a continuum of sizes or aggregates of the alpha 2 beta 2 form of the receptor.) These larger forms contributed 93% of the total activity. Mild reduction of the insulin receptor preparation with dithiothreitol (DTT) activated the total kinase activity by 3.5-fold. Under this condition, the minimal functional kinase size was 380,000 +/- 30,000 daltons (n = 6) while the average functional size for the larger forms was approximately 3 X 10(6) daltons.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Radiation inactivation experiments predict that a large aggregate form of the insulin receptor is a highly active tyrosine-specific protein kinase. 266 59

The insulin receptor contains in its beta-subunit a tyrosine (-) specific protein kinase. It is believed that transmission of an insulin signal across the plasma membrane of target cells of insulin action occurs through activation of this kinase, autophosphorylation of the insulin receptor beta-subunit and subsequent phosphorylation of other cellular substrates. We studied the insulin receptor kinase in a number of insulin resistant cell systems in order to elucidate if defects of this kinase are a possible cause of cellular insulin resistance. Three different patterns of kinase abnormalities were found, in different insulin resistant cells: 1. In an insulin resistance melanoma cell line a reduced receptor kinase autophosphorylation was found apparently due to a defect of the tyrosine autophosphorylation sites of this receptor; 2. Catecholamine and phorbol ester induced insulin resistance of isolated rat fat cells as well as human fat cells was associated with a decreased activity of the insulin receptor tyrosine kinase which was apparently due to a modulation of the ATP binding site of the insulin receptor tyrosine kinase; 3. The receptor kinase isolated from the skeletal muscle of diabetic Zucker rats (fa/fa) was found to be insulin insensitive with no major alteration of maximal responsiveness. These results suggested that different forms of kinase defects exist which can contribute to the pathogenesis of cellular insulin resistance. Based on these data studies in skeletal muscle from type II diabetic patients were started. Results from five patients so far suggest that, here as well, an abnormality of the insulin receptor kinase exists which might be involved in the pathogenesis of insulin resistance in type II diabetes.
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PMID:Insulin receptor kinase defects as a possible cause of cellular insulin resistance. 282 Aug 11

The beta-subunit of the insulin receptor contains a tyrosine-specific protein kinase. Insulin binding activates this kinase and causes phosphorylation of the beta-subunit of the insulin receptor. It is believed that phosphorylation of other proteins might transmit the insulin signal from the receptor to the cell. In the present study we used a polyclonal anti-phosphotyrosine antibody to detect other proteins that become tyrosine phosphorylated upon insulin stimulation. Glycoproteins from human placenta membranes were enriched by wheat germ agglutinin chromatography and phosphorylation was studied with [gamma-32P]ATP and insulin in vitro. Phosphorylated proteins were immunoprecipitated by antibodies against the insulin receptor and by serum containing the anti-phosphotyrosine antibody. Beside the insulin-stimulated phosphorylation of the 95 kDa beta-subunit of the insulin receptor, an insulin-stimulated phosphorylation of a 180 kDa protein was found. The phosphorylation of both proteins occurred only on tyrosine residues. Insulin increased 32P incorporation into the 180 kDa band 2.7-fold (S.E.M. +/- 0.3, n = 5). The 180 kDa protein was not precipitated by antibodies against the insulin receptor. H.p.l.c. chromatograms of tryptic fragments of the phosphorylated 180 kDa protein and of the beta-subunit of the insulin receptor revealed different patterns for both proteins. Insulin-stimulated phosphorylation of the 180 kDa protein was also detectable in unfractionated detergent-solubilized membranes. The phosphorylation of the 180 kDa protein was stimulated by insulin with the same dose-response curve as the phosphorylation of the beta-subunit, suggesting that this protein might be another endogenous substrate of the insulin receptor kinase.
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PMID:An Mr 180,000 protein is an endogenous substrate for the insulin-receptor-associated tyrosine kinase in human placenta. 282 93

The purified human placental insulin-receptor beta-subunit autophosphorylating activity was found to be inhibited, in a time- and concentration-dependent manner, by the specific thiol-alkylating agents N-ethylmaleimide and 5,5'-dithiobis-(2-nitrobenzoic acid). The insulin-receptor kinase was observed to be more sensitive to inhibition by N-ethylmaleimide in the presence [IC50 (concn, giving 50% inhibition) = 25 +/- 3 microM] than in the absence (IC50 = 73 +/- 6 microM) of insulin. Similarly, inhibition by 5,5'-dithiobis-(2-nitrobenzoic acid) occurred with IC50 = 30 +/- 6 microM in the presence and 155 +/- 35 microM in the absence of insulin. Examination of the exogenous-substrate protein kinase activity demonstrated that the differential sensitivity to N-ethylmaleimide was due to direct inhibition of protein kinase activity, as opposed to blockade of the phospho-acceptor properties of the insulin receptor. In contrast, iodoacetamide had essentially no effect on the insulin-receptor beta-subunit autophosphorylating activity and was able to protect partially against the N-ethylmaleimide inhibition in both the presence and the absence of insulin. Consistent with these findings, none of the thiol-specific agents were able to alter significantly insulin binding at concentrations which maximally inhibited the beta-subunit autophosphorylation. Further, in the presence of insulin, the insulin-receptor kinase activity was also observed to be more sensitive to oxidation by H2O2 and FeCl3/ascorbate compared with insulin receptors in the absence of insulin. These results indicate that there is a critical thiol group(s) necessary for the beta-subunit autophosphorylating activity of the insulin-receptor kinase and that in the presence of insulin is more susceptible to exogenously added thiol and oxidizing agents.
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PMID:Differential sensitivity of the insulin-receptor kinase to thiol and oxidizing agents in the absence and presence of insulin. 282 20

The ability of insulin to activate the insulin receptor protein kinase is shown to be completely dependent on prior beta subunit tyrosine autophosphorylation. Autophosphorylation in the presence of insulin is a highly concerted reaction; tryptic digestion of insulin receptor beta subunits derived from preparations whose kinase activation ranges from under 5% to 100% of maximal yields the same array of [32P]Tyr(P)-containing peptides over the entire range. Of special note is the significant contribution of multiply phosphorylated forms of tryptic peptides corresponding to proreceptor residues 1144-1152 (from the "tyrosine kinase" domain) and 1314-1329 (near the carboxyl terminus) to overall beta subunit phosphorylation at kinase activations of 5% and under. Thus, partially activated/autophosphorylated receptor preparations consist of mixtures of unactivated unphosphorylated receptors and activated fully (or nearly fully) phosphorylated receptors. The latter can be selectively removed by adsorption to antiphosphotyrosine antibodies. This abrupt multiple phosphorylation of individual receptor molecules explains why, in the presence of insulin, overall beta subunit tyrosine phosphorylation tracks closely with kinase, up to approximately 90% activation. Insulin stimulates phosphorylation into all domains (involving at least 6 of the 13 tyrosines on the intracellular portion of the beta subunit) but does not cause the appearance of "new" 32P-labeled species. Rather, insulin directs 32P incorporation preferentially into those domains most productive of kinase activation. Phosphorylation of the tyrosine residues at 1146, 1150, and 1151 correlates most closely with kinase activation. These residues show the largest 32P incorporation during rapid kinase activation; moreover, in comparisons of receptors with similar overall autophosphorylation but very different activations (or similar activations but different extents of autophosphorylation), achieved by omitting insulin or varying [ATP], the phosphorylation of peptide 1144-1152 tracks closely with kinase activation, and phosphorylation of sites and Mr 4000-5000 tryptic peptide (presumably Tyr 953 and/or 960) tract nearly as well. By contrast the extent of phosphorylation of the carboxy-terminal peptide is frequently dissociated from the extent of kinase activation. Phosphorylation of this latter domain probably underlies a beta subunit function other than tyrosine kinase activity.
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PMID:Relationship of site-specific beta subunit tyrosine autophosphorylation to insulin activation of the insulin receptor (tyrosine) protein kinase activity. 283 99

Studies investigating the effects of beta-naphthoflavone (beta NF) on insulin receptor binding and its intrinsic protein kinase activity in rat liver and placenta were performed. Membranes were prepared from maternal liver and placenta on gestation day 11 and used for [125I]insulin radioreceptor assay. Scatchard analysis showed that association constants (Ka) for high affinity binding sites were similar for placental and liver membrane. The administration of beta NF, 15 mg/kg, 1 day before study did not alter the specific binding of insulin to liver membranes, whereas ligand binding to placental preparations was decreased 40% from control. Scatchard analysis of binding to placental membranes suggests that beta NF treatment was associated with a change in the number of high affinity binding sites. In further studies membrane receptors were solubilized and partially purified by wheat germ agglutinin affinity chromatography for protein kinase assay. Insulin stimulated the phosphorylation of the Mr 95,000 subunit of the receptor in lectin-purified membrane proteins from liver and placenta. In liver receptor preparations, beta NF treatment was associated with a nearly 3-fold increase in the insulin-stimulated phosphorylation of the 95-kD protein. In contrast, placental receptor preparations showed a 40% decrease in the extent of autophosphorylation following beta NF treatment. Insulin-stimulated phosphorylation of an exogenous substrate poly(Glu4, Tyr) also showed a divergent pattern of changes in liver and placental receptors following beta NF treatment. In studies during late gestation (day 18), beta NF treatment was also associated with an increase in liver receptor kinase activity, whereas placental receptors showed a decrease in autophosphorylation. Thus, acute treatment with beta NF during mid and late gestation was associated with significant alterations in insulin receptor protein kinase activity, and data suggest that fetal insulin receptors may respond in a different manner than maternal receptors to polyaromatic compounds like beta NF. The observed effects of beta NF on liver and placental receptor kinase activity may be related to alterations in insulin function in the regulation of pregnancy and fetoplacental growth.
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PMID:Effects of beta-naphthoflavone on insulin receptor binding and protein kinase activity in rat liver and placenta. 283 20

We have previously shown that a mutant human insulin receptor with a COOH-terminal 43-amino acid deletion (HIR delta CT), when expressed in Rat 1 fibroblasts, binds insulin normally, autophosphorylates, and undergoes endocytosis after insulin binding in a manner comparable to the normal human insulin receptor (HIRc). In this paper we have examined the biologic activity of the truncated and normal insulin receptors. In vitro, the HIR delta CT receptors caused a 1.8-fold greater phosphorylation of a Glu4/Tyr1 polypeptide than did the HIRc receptors, but the two receptor types were nearly equivalent in their ability to phosphorylate a src-derived peptide. Furthermore, insulin preactivation of HIRc and HIR delta CT receptors in intact cells led to equivalent stimulation of tyrosine kinase activity as subsequently determined for histone in vitro. Expression of HIRc receptors in cells led to enhanced sensitivity to insulin of 2-deoxy-D-glucose uptake and glycogen synthase activation. This increased sensitivity was proportional to receptor number at low (Ro = 6400) but not at high (Ro = 1.25 X 10(6] levels of receptor expression. However, expression of HIR delta CT receptors (Ro = 2.5 X 10(5] led to little, if any, increase in insulin sensitivity of either 2-deoxy-D-glucose uptake or glycogen synthase activation. Furthermore, compared with HIRc cells, HIR delta CT cells respond poorly to an agonistic monoclonal antibody specific for the human insulin receptor. In conclusion, the HIR delta CT receptor retains intact protein kinase activity in vitro. Despite this, however, the receptor displays low activity in mediating the metabolic effects of insulin.
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PMID:Properties of a human insulin receptor with a COOH-terminal truncation. II. Truncated receptors have normal kinase activity but are defective in signaling metabolic effects. 283 68

Five protein kinases are shown to serve as specific phosphatases in the absence of ADP. Although the rates of hydrolysis are very slow compared to the forward phosphorylation rates under optimal conditions, they are of the same order as the reverse reaction in the presence of ADP. Because cells contain approximately equal to 3 mM ATP, neither the reverse reaction nor the phosphatase is likely to play a physiological role. beta-casein B phosphorylated by the catalytic subunit of cAMP-dependent protein kinase (protein kinase A) is specifically dephosphorylated by protein kinase A but not by polypeptide-dependent protein kinase (protein kinase P). beta-casein B phosphorylated by protein kinase P is specifically dephosphorylated by protein kinase P but not by protein kinase A. Histone H1 phosphorylated by protein kinase C is dephosphorylated by the same enzyme in the absence of ADP. In all cases tested addition of ADP and F1-ATPase accelerates moderately the rate of dephosphorylation. Native H+-ATPase from yeast plasma membranes is isolated mainly in the phosphorylated form. It is dephosphorylated and rephosphorylated by protein kinase P but not by protein kinase A. Protein-tyrosine kinase of the epidermal growth factor receptor phosphorylates the random synthetic polypeptide poly(Glu80Tyr20). The phosphorylated polymer is specifically dephosphorylated in the absence of ADP by epidermal growth factor receptor preparations but not by insulin receptor preparations. The same polymer phosphorylated by insulin receptor is dephosphorylated by insulin receptor but not by epidermal growth factor receptor preparations. By using a cycle of dephosphorylation-rephosphorylation, it is possible to identify proteins that are phosphorylated by these protein kinases in vivo. Should this method be applicable to additional protein kinases, it should be possible to estimate the quantitative contribution of each protein kinase to a single phosphoprotein.
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PMID:Specific dephosphorylation of phosphoproteins by protein-serine and -tyrosine kinases. 290 Oct 92

Insulin-like growth factor (IGF)-I receptor purified from human placental membranes as previously described (LeBon, T. R., Jacobs, S., Cuatrecasas, P., Kathuria, S., and Fujita-Yamaguchi, Y. (1986) J. Biol. Chem. 261, 7685-7689) was characterized. The IGF-I receptor was similar to the insulin receptor with respect to subunit structure (beta-alpha-alpha-beta), apparent sizes of deglycosylated alpha (Mr = approximately 88,000) and beta (Mr = approximately 67,000) subunits, and amino acid composition of the subunits. Monoclonal antibody specific to each receptor recognized its own receptor whereas polyclonal anti-human insulin receptor antibody cross-reacted with the IGF-I receptor, indicating that the receptors share one or more antigenic sites. Further characterization of the purified IGF-I receptor tyrosine-protein kinase activity indicated that by analogy with the insulin receptor the monomeric alpha beta form of the IGF-I receptor appears to have higher kinase activity than the intact receptor in the alpha 2 beta 2 form. The most significant difference between the two receptors was found in the N-terminal amino acid sequences of their alpha subunits, which apparently show 60% identity. The IGF-I receptor alpha subunit lacks residues corresponding to the N-terminal 4 amino acids of the insulin receptor alpha subunit. These results provide the first direct proof that the IGF-I receptor is a molecule distinct from the insulin receptor despite numerous similarities.
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PMID:Comparison of insulin-like growth factor I receptor and insulin receptor purified from human placental membranes. 294 89

Two systems in vitro are described that show insulin-stimulated phosphorylation of the insulin receptor on serine residues. In the first system, insulin receptor was purified partially from Fao rat hepatoma cells by direct solubilization of the cells in Triton X-100 and chromatography on wheat-germ-agglutinin-agarose. Phosphorylation of these preparations with [gamma-32P]ATP in the presence or absence of insulin resulted in 32P incorporation exclusively into phosphotyrosine residues. Serine kinase activity towards the insulin receptor was reconstituted by adding extracts of Fao cells. Prior exposure of the cells to insulin stimulated serine kinase activity towards the insulin receptor in extracts 7.2-fold. A receptor serine kinase activity enhanced by treatment of cells with cyclic AMP analogues was also retained in the reconstituted system. In the second system, insulin receptor and insulin-sensitive serine kinase activity towards the insulin receptor were co-purified from human placenta. The protocol involved preparation of membranes, before solubilization and chromatography on wheat-germ-agglutinin-agarose, by using gentle procedures designed not to disrupt a potentially labile association between the insulin receptor and the serine kinase. Serine kinase activity in these preparations towards the insulin receptor was stimulated up to 10-fold by insulin, and the stoicheiometry of serine phosphorylation was estimated to be approx 0.8 mol/mol of insulin receptor for phosphorylations performed in the presence of insulin. Thus a preparation of insulin receptor is described for the first time that is phosphorylated to high stoicheiometry on serine in an insulin-dependent manner. Conditions that facilitate recovery and assay of serine kinase activity are defined and discussed. These systems provide a basis for characterizing the nature of the insulin-sensitive serine kinase that phosphorylates the insulin receptor, and defining its role in insulin action and control of receptor function.
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PMID:Two systems in vitro that show insulin-stimulated serine kinase activity towards the insulin receptor. 296 79


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