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)

Insulin receptor was co-purified from human placenta together with insulin-stimulated kinase activity that phosphorylates the insulin receptor on serine residues. By using this 'in vitro' system, the mechanism of activation of the serine kinase by insulin was explored. Peptide 1150, histone, poly(Glu-Tyr), eliminating Mn2+ (Mg2+ only), treatment at 37 degrees C (1 h), N-ethylmaleimide, phosphate, beta-glycerol phosphate and anti-phosphotyrosine antibody all inhibited insulin-receptor tyrosine kinase activity and the ability of insulin to stimulate phosphorylation of the insulin receptor on serine. Additionally, direct stimulation of the receptor tyrosine kinase by vanadate increased serine phosphorylation of the insulin receptor. Insulin-stimulated tyrosine phosphorylation preceded insulin-stimulated serine phosphorylation of the insulin receptor. The activity of the insulin-sensitive receptor serine kinase was not augmented by cyclic AMP, cyclic GMP, Ca2+, Ca2+ + calmodulin, Ca2+ + phosphatidylserine + diolein or spermine, or inhibited appreciably by heparin. Additionally, the serine kinase phosphorylated casein or phosvitin poorly and was active with Mn2+. This indicates that it is distinct from Ca2+, Ca2+/phospholipid, Ca2+/calmodulin, cyclic AMP- and cyclic GMP-dependent protein kinases, casein kinases I and II and insulin-activated ribosomal S6 kinase. Taken together, these data indicate that a novel species of serine kinase catalyses the insulin-dependent phosphorylation of the insulin receptor and that activation of this receptor serine kinase by insulin requires an active insulin-receptor tyrosine kinase.
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PMID:Evidence that a novel serine kinase catalyses phosphorylation of the insulin receptor in an insulin-dependent and tyrosine kinase-dependent manner. 297 46

Native, cell-surface insulin receptor consists of two glycoprotein subunit types with apparent masses of about 125,000 daltons (alpha subunit) and 90,000 daltons (beta subunit). The alpha and beta subunits are derived from a single polypeptide precursor by one or more proteolytic cleavages. The predominant subunit configuration in the native insulin receptor is a disulfide-linked heterotetrameric structure containing two alpha and two beta subunits. The alpha and beta insulin-receptor subunits seem to have distinct functions such that alpha appears to bind hormone whereas beta appears to possess intrinsic tyrosine kinase activity. In detergent extracts, insulin activates receptor autophosphorylation of tyrosine residues on its beta subunit, whereas in the presence of reductant, the alpha subunit is also phosphorylated. Other physiologically relevant substrates of the insulin receptor tyrosine kinase in target cells, if any, have not yet been identified. In intact cells, insulin activates serine/threonine phosphorylation of insulin receptor beta subunit as well as tyrosine phosphorylation. The biological role of the receptor-associated tyrosine kinase is not known. Tyrosine phosphorylation, catalyzed by either autophosphorylation or purified src kinase, of insulin receptor beta subunit in vitro activates the receptor kinase activity, whereas dephosphorylation with alkaline phosphatase deactivates the receptor kinase. The insulin receptor kinase is regulated by beta-adrenergic agonists and other agents that elevate cAMP in adipocytes, presumably via the cAMP-dependent protein kinase. Such agents decrease receptor affinity for insulin and partially uncouple receptor tyrosine kinase activity from activation by insulin. These effects appear to contribute to the biological antagonism between insulin and beta-agonists. The insulin receptor kinase is also inhibited in intact cells by phorbol esters that mediate serine/threonine phosphorylation of the insulin receptor, presumably via the Ca++-phospholipid-dependent protein kinase. These data suggest the hypothesis that a complex network of tyrosine and serine/threonine phosphorylations on the insulin receptor modulate its binding and kinase activities in an antagonistic manner.
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PMID:The nature and regulation of the insulin receptor: structure and function. 298 34

The insulin receptor appears as a tetrameric glycoprotein consisting of two Mr 130,000 subunits (alpha), and two Mr 95,000 subunits (beta) in a disulfide-linked complex. Insulin bound to its specific cell surface receptors in its target cells leads to a complex array of molecular events resulting in insulin effects. It is now generally believed that protein phosphorylation-dephosphorylation reactions represent an important mechanism by which a variety of extracellular stimuli regulate cellular functions. Insulin mediates such reactions, but it is not known whether these are the biochemical link between the binding of insulin to its receptor and its final cellular effects. In search of initial post-binding events which might play a role in insulin action, we looked for phosphorylation of insulin receptors. We show that the insulin receptor displays two functional domains, an insulin binding alpha-subunit, and an insulin responsive protein kinase contained in the beta-subunit. We envisage the insulin receptor as an integrated system for transmembrane signal transmission in which hormone binding to the alpha-subunit leads to activation of the beta-subunit via conformational changes.
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PMID:The insulin receptor kinase. 300 Apr 60

The receptor for both insulin and epidermal growth factor (EGF) from human placental membranes, after crosslink labeling with 125I-labeled insulin and EGF, can be absorbed to an organomercurial-agarose derivative (Affi-Gel 501) and can be recovered from the gel by elution with dithiothreitol (DTT). Pretreatment of crosslink-labeled membranes with N-ethylmaleimide (NEM) blocks the ability of the receptor to react with the organomercurial column. NEM also abolishes the protein kinase activity of both receptors. Under appropriate conditions, insulin can promote the reaction of the insulin receptor with the organomercurial-agarose derivative. For both the insulin and EGF receptors, our results provide an avenue for the isolation of the sulfhydryl-containing receptor domains that may play a role in the control of receptor function.
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PMID:Receptors for insulin and epidermal growth factor: interaction with organomercurial agarose. 300 Nov 6

Insulin causes rapid phosphorylation of the beta subunit (Mr = 95,000) of its receptor in broken cell preparations. This occurs on tyrosine residues and is due to activation of a protein kinase which is contained in the receptor itself. In the intact cell, insulin also stimulates the phosphorylation of the receptor and other cellular proteins on serine and threonine residues. In an attempt to find a protein that might link the receptor tyrosine kinase to these serine/threonine phosphorylation reactions, we have studied the interaction of a partially purified preparation of insulin receptor with purified preparations of serine/threonine kinases known to phosphorylate glycogen synthase. No insulin-dependent phosphorylation was observed when casein kinases I and II, phosphorylase kinase, or glycogen synthase kinase 3 was incubated in vitro with the insulin receptor. These kinases also failed to phosphorylate the receptor. By contrast, the insulin receptor kinase catalyzed the phosphorylation of the calmodulin-dependent kinase and addition of insulin in vitro resulted in a 40% increase in this phosphorylation. In the presence of calmodulin-dependent kinase and the insulin receptor kinase, insulin also stimulated the phosphorylation of calmodulin. Phosphoamino acid analysis showed an increase of phosphotyrosine content in both calmodulin and calmodulin-dependent protein kinase. These data suggest that the insulin receptor kinase may interact directly and specifically with the calmodulin-dependent kinase and calmodulin. Further studies will be required to determine if these phosphorylations modify the action of these regulatory proteins.
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PMID:Interaction of the insulin receptor kinase with serine/threonine kinases in vitro. 300 Nov 7

The insulin receptor is an insulin-activated, tyrosine-specific protein kinase. Previous studies have shown that autophosphorylation of tyrosine residues on the Mr 95,000 is associated with an activation of the protein kinase activity toward exogenous protein substrates. We have employed the highly purified insulin receptor, immobilized on insulin-Sepharose or eluted in an active form, to define the metal/ATP requirements for kinase activation, the relationship of receptor autophosphorylation to activation, and the kinetic properties of the autophosphorylated, activated receptor kinase. Prior incubation of the immobilized receptor with 2 mM ATP, 10 mM Mg (or 10 mM Mn), followed by removal of these reactants, served to abolish the upward curvilinearity in the rate of histone 2b (tyrosine) phosphorylation measured subsequently. This treatment also markedly increased the rate of histone 2b phosphorylation as compared to that observed with the unmodified, immobilized receptor, as estimated under conditions that per se minimized further activation. The extents of maximal activation of receptor histone 2b (tyrosine) kinase obtained on preincubation with MgATP or MnATP are identical; however, the affinity of the receptor for MnATP is approximately 10-fold higher than that for MgATP. The higher affinity of the receptor for MnATP is observed for both autophosphorylation/autoactivation and histone 2b tyrosine kinase activity (Km MnATP approximately 0.01 mM; Km MgATP approximately 0.1 mM). Autophosphorylation/autoactivation per se does not significantly alter the apparent affinity for MeATP (or protein substrate, as previously reported) but increases Vmax. Activation of receptor histone 2b (tyrosine) kinase is due to tyrosine-specific autophosphorylation of the Mr 95,000 (beta) subunit; thus the extent of total 32P incorporation into the beta subunit correlates precisely with the extent of kinase activation, both over time and at a wide variety of Me2+ ATP concentrations. Sequential treatment of the autophosphorylated receptor with elastase and trypsin yields a single, basically charged 32P-peptide, Mr less than 2000. The functional properties of the unphosphorylated and fully phosphorylated receptor were compared after elution from insulin-Sepharose. The insulin binding characteristics of the two forms of the receptor were indistinguishable; the kinase properties differed greatly; whereas the histone 2b activity of the unphosphorylated receptor was low in the basal state, and activated 10-fold by insulin, the fully autophosphorylated receptor exhibits maximal histone 2b kinase in the basal state and is unaffected by insulin addition.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Kinetic properties of the insulin receptor tyrosine protein kinase: activation through an insulin-stimulated tyrosine-specific, intramolecular autophosphorylation. 300 34

Insulin-like growth factor (IGF) I receptor was purified from Triton X-100-solubilized human placental membranes by wheat germ agglutinin-Sepharose chromatography followed by immunoaffinity chromatography using alpha IR-3, a monoclonal antibody directed against the IGF-I receptor. Purification of 3200-fold and 2800-fold was achieved from wheat germ agglutinin-Sepharose eluates with regard to IGF-I binding and kinase activities. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions revealed two major protein bands corresponding to the alpha and beta subunits of the receptor, which accounted for at least 90% of the protein content. The purified receptor bound 10-20 micrograms of IGF-I/mg of protein and was more than 95% free of contamination by insulin receptor. It sedimented in glycerol gradients as a single species with a sedimentation coefficient of 13.7 S and gave three protein bands with Mr = approximately 300,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, indicating that alpha 2 beta 2 is an intact form of the IGF-I receptor. The purified receptor, when incubated with [gamma-32P] ATP, became phosphorylated at tyrosine residues of its beta subunit. This was stimulated 3-fold by IGF-I. It also had IGF-I-stimulated tyrosine kinase activity (5264 pmol of 32P incorporated/min/mg of protein) toward a synthetic peptide corresponding to the autophosphorylation site of pp60src. These data strongly suggest that it is a tyrosine-specific protein kinase.
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PMID:Purification of insulin-like growth factor I receptor from human placental membranes. 301 95

The beta-subunit of the insulin receptor possesses a tyrosine-specific protein kinase activity which may play a role in coupling insulin binding to insulin action. Previously, we have identified a substrate for the receptor-associated protein kinase in a cell-free system. This endogenous substrate (pp120), which appeared to be a glycoprotein with an apparent mol wt of 120,000, was detected in rat liver microsomes. In the present work, we have demonstrated that pp120 is localized to a highly purified preparation of rat liver plasma membranes (Neville preparation). Moreover, pp120 appears to be specific to liver, having been detected in liver from rat, monkey, and rabbit, but not in rat brain, skeletal muscle, heart, kidney, or adipocytes. As a preliminary to addressing the question of whether insulin stimulates phosphorylation of pp120 in intact cells, we have sought to identify tissue culture cell lines that contain both insulin receptors and pp120. We have succeeded in identifying pp120 in two cell lines derived from rat liver: 1) H35 hepatoma cells (Reuber hepatoma) and 2) rat hepatocytes transformed with a temperature-sensitive mutant form of SV-40 (cultivated at both permissive and nonpermissive temperatures). In conclusion, pp120 appears to be a liver-specific plasma membrane glycoprotein which serves as a substrate for phosphorylation by the insulin receptor-associated protein kinase in a soluble cell-free system. The presence of pp120 in cultured cell lines will facilitate investigation of whether the phosphorylation of pp120 in intact cells is physiologically regulated in response to insulin.
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PMID:Tissue distribution and subcellular localization of an endogenous substrate (pp 120) for the insulin receptor-associated tyrosine kinase. 301 74

The insulin receptor is an integral membrane glycoprotein (Mr approximately 300,000) composed of two alpha-subunits (Mr approximately 130,000) and two beta-subunits (Mr approximately 95,000) linked by disulphide bonds. This oligomeric structure divides the receptor into two functional domains such that alpha-subunits bind insulin and beta-subunits possess tyrosine kinase activity. The amino acid sequence deduced from cDNA of the single polypeptide chain precursor of human placental insulin receptor revealed that alpha- and beta-subunits consist of 735 and 620 residues, respectively. The alpha-subunit is hydrophilic, disulphide-bonded, glycosylated and probably extracellular. The beta-subunit consists of a short extracellular region which links the alpha-subunit through disulphide bridges, a hydrophobic transmembrane region and a longer cytoplasmic region which is structurally homologous with other tyrosine kinases like the src oncogene product and EGF receptor kinases. The cellular function of insulin receptors is dual: transmembrane signalling and endocytosis of hormone. The binding of insulin to its receptor on the cell membrane induces transfer of signal from extracellular to cytoplasmic receptor domains leading to activation of cell metabolism and growth. In addition, hormone-receptor complexes are internalized leading to intracellular proteolysis of insulin, whereas receptors are recycled to the membrane. These phenomena are kinetically well-characterized, but their molecular mechanisms remain obscure. Insulin receptor in different tissues and animal species are homologous in their structure and function, but show also significant differences regarding size of alpha-subunits, binding kinetics, insulin specificity and receptor-mediated degradation. We suggest that this heterogeneity of receptors may be linked to the diversity in insulin effects on metabolism and growth in various cell types. The purified insulin receptor phosphorylates its own beta-subunit and exogenous protein and peptide substrates on tyrosine residues, a reaction which is insulin-sensitive, Mn2+-dependent and specific for ATP. Tyrosine phosphorylation of the beta-subunit activates receptor kinase activity, and dephosphorylation with alkaline phosphatase deactivates the kinase. In intact cells or impure receptor preparations, a serine kinase is also activated by insulin. The cellular role of two kinase activities associated with the insulin receptor is not known, but we propose that the tyrosine- and serine-specific kinases mediate insulin actions on metabolism and growth either through dual-signalling or sequential pathways.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Protein kinase activity of the insulin receptor. 301 97

Insulin responsive protein kinase activities of wheat germ purified glycoproteins were examined. Glycoproteins were first incubated without or with insulin, and then exposed to a serum containing antibodies to insulin receptor. Thereafter, both immunoprecipitates and supernatants were studied for their kinase activity toward histone. Incubation with anti receptor antibodies promoted insulin receptor beta subunit and histone phosphorylation. More important insulin receptor depleted extract contained a kinase activity toward histone, that was increased by preincubation with insulin. This stimulation was observed only when insulin was added before the immunoprecipitation of insulin receptors. Alkali treatment and phosphoamino acids analysis revealed that the kinase activity remaining in the supernatant is serine specific. These findings suggest, that a serine kinase activity is associated with the insulin receptor, that it can be separated from the insulin receptor with anti receptor antibodies, that the serine kinase is activated by the hormone-receptor complex.
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PMID:Presence of an insulin-stimulated serine kinase in cell extracts from IM-9 cells. 302 Nov 23


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