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)

Nck is a 47-kDa cytosolic protein devoid of intrinsic catalytic activity and consisting of Src homology 2 and 3 (SH2 and SH3) domains organized as follows: SH3-SH3-SH3-SH2. Nck is believed to act as an adaptor protein mediating signal transduction initiated by receptor tyrosine kinases (RTKs). Through its SH2 domain, Nck recognizes a specific phosphotyrosine residue on RTKs or on protein substrates of RTKs like insulin receptor substrate-1, the major substrate of the insulin receptor, and through its SH3 domains it interacts with poorly characterized effector molecules. To identify novel proteins that might interact with Nck, we have used the amino-terminal segment of Nck encompassing its three SH3 domains in the yeast two-hybrid system. Among the polypeptides that associate with Nck, we have identified the gamma2 isoform of the serine/threonine casein kinase I (CKI-gamma2). In transformed rat hepatocytes overexpressing the insulin receptor (HTC-IR cells), serine/threonine protein kinase activity coimmunoprecipitates with Nck, an interaction mediated mainly by the third SH3 domain of Nck. This kinase activity is not apparently modulated by insulin, nor is it sensitive to staurosporine or heparin, and it does not use GTP as a phosphate donor. However the kinase activity coimmunoprecipitated with Nck is completely abolished by N-(2-aminoethyl)-5-chloroisoquinoline-8-sulfonamide, a specific inhibitor of casein kinase I. In an in vitro renaturation gel kinase assay, a protein kinase of 70-75 kDa was detected associated with the SH3 domains of Nck. Far Western analysis demonstrated that the SH3 domains of Nck bound directly to a cytosolic protein of 70-75 kDa. A rabbit polyclonal antibody raised against the C-terminal region of CKI-gamma2 protein kinase immunoprecipitated a single specific protein of 70-75 kDa from HTC-IR cell lysates and detected CKI-gamma2 among the proteins coimmunoprecipitated with Nck. These results support an in vivo interaction between Nck and CKI-gamma2 and suggest that CKI-gamma2 could be involved in signaling pathways downstream of RTKs.
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PMID:A casein kinase I activity is constitutively associated with Nck. 900 5

During engagement of the type I IFN receptor, IRS-1 is phosphorylated on tyrosine and associates with the p85 regulatory subunit of the phosphatidylinositol (PI) 3'-kinase, which is a dual-specificity enzyme possessing both lipid and serine kinase activities. We sought to determine whether treatment of cells with IFN-alpha activates the PI 3'-kinase serine kinase. 32P-labeling experiments and phosphoaminoacid analysis of immunoprecipitated IRS-1 protein demonstrated that, in addition to tyrosine phosphorylation, IFN-alpha induces its phosphorylation on serine residues. In vitro kinase assays on alphaIRS-1 immunoprecipitates also demonstrated IFN-alpha-dependent serine phosphorylation of IRS-1, suggesting that the protein associates with an IFN-alpha-regulated serine kinase. Furthermore, IFN-alpha-dependent phosphorylation of IRS-1 was detected in in vitro kinase assays on alpha p85 immunoprecipitates, and was inhibited by pretreatment of cells with the specific PI 3'-kinase inhibitor wortmannin, consistent with a regulatory role of the PI 3'-kinase serine kinase on the phosphorylation of the protein. Treatment of cells with wortmannin also inhibited the phosphorylation of the p85 subunit of PI 3'-kinase and the type I IFN-regulated activation of the Map kinase, but had no inhibitory effect on the IFN-alpha-induced activation of Tyk-2 and Jak-1 kinases nor on the activation of Stat-1, Stat-2, and Stat-3. Taken all together, these data establish that the PI 3'-kinase serine kinase is activated by IFN-alpha and may play an important role in the transmission of type I IFN receptor-generated signals.
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PMID:Activation of the phosphatidylinositol 3-kinase serine kinase by IFN-alpha. 903 89

Insulin and insulin-like growth factor-1 (IGF-1) are two structurally related hormones which produce similar biological activities such as metabolic and growth promoting actions. Their receptors, insulin and IGF-1 receptors, also share similarities in both structure and functions such as tyrosine-specific protein kinase. We identified insulin receptor substrate-1 (IRS-1) as a common substrate for insulin and IGF-1 receptor tyrosine kinases. We generated IRS-1 knockout mice and showed that IRS-1 plays a physiological role in signal transduction and biological actions of insulin and IGF-1. We also identified pp190 (IRS-2) as an alternative substrate for IRS-1.
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PMID:Signal transduction mechanism of insulin and insulin-like growth factor-1. 907 40

In fetal brown adipocyte primary cultures, insulin rapidly (at 5 min) induced tyrosine phosphorylation of the insulin receptor beta-subunit; this effect was maximal at physiological concentrations (1 nM). Insulin also stimulated insulin receptor substrate-1 tyrosine phosphorylation and subsequently activated phosphatidylinositol 3-kinase. Moreover, a 3-fold increase in the Ras.GTP active form and a 6-fold increase in Raf-1 kinase activity were induced after insulin stimulation. An immortalized brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo cotransfection) showed a reduced maximal responsiveness to insulin in the same range of insulin concentrations studied (1-100 nM). Transformed brown adipocyte cell line (by permanent simian virus 40 large T antigen and pMEXneo H-ras(lys12) cotransfection) developed insulin resistance upstream from Ras, showing an impairment in the insulin receptor autophosphorylation, and in insulin receptor substrate-1 tyrosine phosphorylation and its association with phosphatidylinositol 3-kinase upon treatment with 1 nM insulin, although insulin receptor number and affinity (Kd) remained unaltered. This lack of effect was ameliorated upon treatment with higher insulin concentrations, in a dose-dependent manner. However, downstream from Ras, events such as formation of the Ras.GTP active form, and Raf-1 kinase and 12-O-tetradecanoylphorbol-13-acetate response element-chloramphenicol transferase (transiently transfected) activities were overstimulated, compared with those in primary and immortalized cells, in an insulin-independent manner. Wheat-germ lectin-purified receptors from H-ras(lys12)-transformed brown adipocytes showed a marked phosphorylation in the basal state, which was suppressed by serine-threonine phosphatase pretreatment. Moreover, alkaline phosphatase pretreatment restored the tyrosine kinase activity of the receptor in response to insulin. We conclude that the decreased tyrosine autophosphorylation rate of the insulin receptor from H-ras(lys12)-transformed brown adipocytes is a consequence of its basal serine/threonine phosphorylation, resulting in severe insulin resistance.
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PMID:Alterations in the insulin signaling pathway induced by immortalization and H-ras transformation of brown adipocytes. 923 68

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an "unregulated" mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.
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PMID:Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action. 927 79

Activation of the endogenous protein kinase Cs in human kidney fibroblast (293) cells was found in the present study to inhibit the subsequent ability of insulin to stimulate the tyrosine phosphorylation of an expressed insulin receptor substrate-1. This inhibition was also observed in an in vitro phosphorylation reaction if the insulin receptor and its substrate were both isolated from cells in which the protein kinase C had been activated. To test whether serine phosphorylation of the insulin receptor substrate-1 was contributing to this process, serine 612 of this molecule was changed to an alanine. The insulin-stimulated tyrosine phosphorylation and the associated phosphatidylinositol 3-kinase activity of the expressed mutant were found to be comparable to those of the expressed wild-type substrate. However, unlike the wild-type protein, activation of protein kinase C did not inhibit the insulin-stimulated tyrosine phosphorylation of the S612A mutant nor its subsequent association with phosphatidylinositol 3-kinase. Tryptic peptide mapping of in vivo labeled IRS-1 and the S612A mutant revealed that PMA stimulates the phosphorylation of a peptide from wild-type IRS-1 that is absent from the tryptic peptide maps of the S612A mutant. Moreover, a synthetic peptide containing this phosphoserine and its nearby tyrosine was found to be phosphorylated by the insulin receptor to a much lower extent than the same peptide without the phosphoserine. Activation of protein kinase C was found to stimulate by 10-fold the ability of a cytosolic kinase to phosphorylate this synthetic peptide as well as the intact insulin receptor substrate-1. Finally, cytosolic extracts from the livers of ob/ob mice showed an 8-fold increase in a kinase activity capable of phosphorylating this synthetic peptide, compared to extracts of livers from lean litter mates. These results indicate that activation of protein kinase C stimulates a kinase which can phosphorylate insulin receptor substrate-1 at serine 612, resulting in an inhibition of insulin signaling in the cell, posing a potential mechanism for insulin resistance in some models of obesity.
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PMID:Protein kinase C modulation of insulin receptor substrate-1 tyrosine phosphorylation requires serine 612. 933 53

1321N1 astrocytoma cells have proved a valuable model system in which to study interactions between two major PtdIns (4,5) P2-utilizing signaling pathways, since they possess receptor populations which elicit independent activation of PI 3-kinase and a G-protein-dependent PLC respectively. Activation of PLC down-regulates PI 3-kinase by at least two mechanisms involving inhibition of IRS-1-associated PI 3-kinase and acute activation of a PtdIns (3,4,5) P3 5-phosphatase. PKB, which is an important early PI 3-kinase-dependent component of insulin signalling pathways, is also down-regulated by PLC-coupled agonists. The activation of PKB by insulin appears to involve a novel PtdIns (3,4,5) P3-dependent protein kinase, which we have named PDK1. The molecular mechanisms underlying PtdIns (3,4,5) P3-stimulated phosphorylation and activation of PKB by PDK1 are currently under investigation.
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PMID:Cross-talk between phospholipase C and phosphoinositide 3-kinase signalling pathways. 944 62

The ability of ethanol to interfere with insulin-like growth factor 1 (IGF-1)-mediated cell survival was examined in primary cultured cerebellar granule neurons. Cells underwent apoptosis when switched from medium containing 25 mM K+ to one containing 5 mM K+. IGF-1 protected granule neurons from apoptosis in medium containing 5 mM K+. Ethanol inhibited IGF-1-mediated neuronal survival but did not inhibit IGF-1 receptor binding or the neurotrophic action of elevated K+, and failed to potentiate cell death in the presence of 5 mM K+. Inhibition of neuronal survival by ethanol was not reversed by increasing the concentration of IGF-1. Significant inhibition by ethanol (15-20%) was observed at 1 mM and was half-maximal at 45 mM. The inhibition of IGF-1 protection by ethanol corresponded to a marked reduction in the phosphorylation of insulin receptor substrate 1, the binding of phosphatidylinositol 3-kinase (PI 3-kinase), and a block of IGF-1-stimulated PI 3-kinase activity. The neurotrophic response of IGF-1 was also inhibited by the PI 3-kinase inhibitor LY294002, the protein kinase C inhibitor chelerythrine chloride, and the protein kinase A inhibitor KT5720, but unaffected by the mitogen-activated protein kinase kinase inhibitor PD 98059. These data demonstrate that ethanol promotes cell death in cerebellar granule neurons by inhibiting the antiapoptotic action of IGF-1.
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PMID:Ethanol induces apoptosis in cerebellar granule neurons by inhibiting insulin-like growth factor 1 signaling. 964 66

Serine phosphorylation of insulin receptor substrate-1 (IRS-1) has been proposed as a counter-regulatory mechanism in insulin and cytokine signalling. Here we report that IRS-1 is phosphorylated by a wortmannin insensitive phosphatidylinositol 3'-kinase (PI 3-kinase)-associated serine kinase (PAS kinase) distinct from PI 3-kinase serine kinase. We found that PI 3-kinase immune complexes contain 5-fold more wortmannin-insensitive serine kinase activity than SH2-containing protein tyrosine phosphatase-2 (SHP2) and IRS-1 immune complexes. Affinity chromatography of cell lysates with a glutathione S-transferase fusion protein for the p85 subunit of PI 3-kinase showed that PAS kinase associated with the p85 subunit of PI 3-kinase. This interaction required unoccupied SH2 domain(s) but did not require the PI 3-kinase p110 subunit binding domain. In terms of function, PAS kinase phosphorylated IRS-1 and, after insulin stimulation, PAS kinase phosphorylated IRS-1 in PI 3-kinase-IRS-1 complexes. Phosphopeptide mapping showed that insulin-dependent in vivo sites of IRS-1 serine phosphorylation were comparable to those of PAS kinase phosphorylated IRS-1. More importantly, PAS kinase-dependent phosphorylation of IRS-1 reduced by 4-fold the ability of IRS-1 to act as an insulin receptor substrate. Taken together, these findings indicate that: (a) PAS kinase is distinct from the intrinsic serine kinase activity of PI 3-kinase, (b) PAS kinase associates with the p85 subunit of PI 3-kinase through SH2 domain interactions, and (c) PAS kinase is an IRS-1 serine kinase that can reduce the ability of IRS-1 to serve as an insulin receptor substrate.
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PMID:Phosphatidylinositol 3'-kinase associates with an insulin receptor substrate-1 serine kinase distinct from its intrinsic serine kinase. 976 40

Both p21ras and phosphatidylinositol 3-kinase (PI 3-k) are critical elements in signaling pathways mediating insulin/IGF-I induced cell cycle progression. For example, microinjection of antibodies, peptides, or recombinant proteins which block the interaction of the SH2 domains of the PI 3-k p85alpha subunit with tyrosine phosphorylated intracellular targets blocks insulin mediated DNA synthesis. We report here that this inhibitory phenotype is observed whether the injections are made into quiescent cells (the standard approach), or at any time point during G1 phase subsequent to stimulation. This observation is not true, however, for the major substrate of the insulin/IGF-I receptor (IRS-1) despite the well known interaction of p85 with IRS-1. Antibodies to IRS-1 are inhibitory only when injected during the first 15 min of G1 phase, as are antibodies to another major IRS-1 binding protein, the tyrosine phosphatase SHP2. We also have microinjected reagents which target proteins involved in the formation of rasGTP and which mediate some of the downstream effects of ras activation. Reagents which target the formation of rasGTP (Shc and dominant negative ras protein) inhibit DNA synthesis only at points early in G1, as do reagents which target components of the MAP kinase pathway. Injection of antibodies to p21ras itself, or a recombinant Raf-1 protein domain which binds to the effector region of ras in a GTP-dependent manner, results in the inhibition of cell cycle progression throughout G1 phase. The results point to a continuous requirement for both PI 3-k and ras activity until cellular commitment to DNA synthesis, although some of the molecules which are both upstream and downstream of these activities are only required transiently. Our results are also consistent with a Raf-1 independent ras activity late in G1, as well as IRS-1 independent effects of PI 3-kinase.
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PMID:Prolonged vs transient roles for early cell cycle signaling components. 978 5


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