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:2.7.1.137 (phosphatidylinositol 3-kinase)
11,064 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The rate-limiting step in the uptake and metabolism of D-glucose by insulin target cells is thought to be glucose transport mediated by glucose transporters (primarily the GLUT4 isoform) localized to the plasma membrane. However, subcellular fractionation, photolabelling and immunocytochemical studies have shown that the pool of GLUT4 present in the plasma membrane is only one of many subcellular pools of this protein. GLUT4 has been found in occluded vesicles at the plasma membrane, clathrin-coated pits and vesicles, early endosomes, and tubulo-vesicular structures; the latter are analogous to known specialized secretory compartments. Tracking the movement of GLUT4 through these compartments, and defining the mechanism and site of action of insulin in stimulating this subcellular trafficking, are major topics of current investigation. Recent evidence focuses attention on the exocytosis of GLUT4 as the major site of insulin action. Increased exocytosis may be due to decreased retention of glucose transporters in an intracellular pool, or possibly to increased assembly of a vesicle docking and fusion complex. Although details are unknown, the presence in GLUT4 vesicles of a synaptobrevin homologue leads us to propose that a process analogous to that occurring in synaptic vesicle trafficking is involved in the assembly of GLUT4 vesicles into a form suitable for fusion with the plasma membrane. Evidence that the pathways of signalling from the insulin receptor and of GLUT4 vesicle exocytosis may converge at the level of the key signalling enzyme, phosphatidylinositol 3-kinase, is discussed.
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PMID:Subcellular localization and trafficking of the GLUT4 glucose transporter isoform in insulin-responsive cells. 798 Apr 79

Brain-derived neurotrophic factor (BDNF) interacts with the TrkB receptor tyrosine kinase, the tyrosine kinase domain of which has homology with the insulin receptor subfamily of protein kinases. This includes the conservation of three regulatory tyrosines (residues 670, 674, and 675) known to play a crucial role in signal transmission by the insulin receptor (tyrosines 1158, 1162, and 1163). Wild-type TrkB and TrkB mutants with Y670F, Y674F/Y675F, Y751F (the tyrosine reported to be important in phosphatidylinositol 3-kinase binding (Obermeier, A., Lammers, R., Wiesmuller, K. H., June, G., Schlessinger, J., and Ullrich, A. (1993) J. Biol. Chem. 268, 22963-22966)), and K540R (consensus ATP binding lysine) substitutions were transiently expressed in COS cells for analysis of phosphorylation sites by two-dimensional phosphopeptide mapping. TrkB phosphorylation sites were also studied in MG86 cells stably expressing wild-type TrkB. In addition, the mutants were expressed in Chinese hamster ovary cells for analysis of the ability of the receptor to mediate BDNF-stimulated transcription from a 12-O-tetradecanoylphorbol-13-acetate response element (TRE). BDNF stimulated the phosphorylation of wild-type TrkB on multiple tyrosine and serine residues. This phosphorylation occurred on tyrosines 670, 674, and 675 plus two other tyrosines and at least two serines that were not unequivocally identified. Wild-type TrkB mediated a pronounced stimulation of TRE-dependent transcription. A Y674F/Y675F, but not Y670F, substitution dramatically inhibited this response. Surprisingly, in COS cells, a Y751F substitution induced dramatically lower tyrosine and serine phosphorylation at all sites but mediated a normal BDNF-stimulated activation of a TRE. Our results demonstrate a critical role for the phosphorylation of tyrosines 674 and 675 in BDNF-dependent signaling by wild-type TrkB.
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PMID:Identification of in vivo brain-derived neurotrophic factor-stimulated autophosphorylation sites on the TrkB receptor tyrosine kinase by site-directed mutagenesis. 798 51

The functional role of the oligosaccharide chains linked to the insulin receptor (IR) beta subunit was investigated by site-directed mutagenesis of each of the 4 acceptor asparagines (N1 to N4 from the amino to the carboxyl terminus) and stable expression of the receptors in CHO cells. All mutant receptors are expressed normally at the cell surface, bind insulin with similar affinity, but have a beta subunit of smaller molecular mass, and a defect in ligand-induced internalization as compared to wild type receptor. In terms of receptor activation and signal transduction, the N1 and N2 mutants function normally, whereas the N4 mutant exhibits major alterations in in vitro tyrosine kinase activity and autophosphorylation and is unable to transduce the signal for either glycogen or DNA synthesis. By contrast, in vivo autophosphorylation and IRS-1 phosphorylation appear quantitatively normal, and only partial alterations of phosphatidylinositol 3-kinase and mitogen-activated protein kinase activation are observed. Mutation of the N3 site results in partial defect of IR activation. These data provide evidence for (i) glycosylation of each N-linked glycosylation site of the IR beta subunit, (ii) absence of correlation between internalization and transmembrane signaling, and (iii) a major role for oligosaccharide side chain(s) located close to the cell membrane in IR activation and transmembrane signaling.
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PMID:The functions of the human insulin receptor are affected in different ways by mutation of each of the four N-glycosylation sites in the beta subunit. 802 66

Insulin stimulation drives the formation of a complex between tyrosine-phosphorylated insulin receptor substrate 1 (IRS-1) and 1-phosphatidylinositol 3-kinase (PI 3-kinase; ATP:1-phosphatidyl-1D-myo-inositol 3-phosphotransferase, EC 2.7.1.137), a heterodimer consisting of regulatory 85-kDa (p85) and catalytic 110-kDa (p110) subunits. This interaction takes place via the phosphorylated YMXM motifs of IRS-1 and the Src homology region 2 (SH2) domains of p85. In this study, the stable overexpression in a Chinese hamster ovary (CHO) cell line of a mutant p85 alpha (delta p85) protein, which lacks a binding site for p110, disrupted the complex formation between IRS-1 and the catalytic subunit of PI 3-kinase in intact cells during insulin stimulation. Activation of insulin receptor kinase and the tyrosine phosphorylation of IRS-1 remained unaffected. In this cell line, both insulin-stimulated accumulation of phosphatidylinositol 3,4,5-trisphosphate and the insulin-stimulated glucose uptake due to the translocation of GLUT1 glucose transporters were markedly impaired, whereas neither phorbol 12-myristate 13-acetate-stimulated glucose uptake nor the insulin-stimulated activation of RAS was impaired. These results suggest that PI 3-kinase is required for glucose transport in insulin signaling in CHO cells.
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PMID:1-Phosphatidylinositol 3-kinase activity is required for insulin-stimulated glucose transport but not for RAS activation in CHO cells. 805 99

The role of insulin receptor tyrosine kinase activity in stimulation of intracellular enzymes linked to insulin action [phosphatidylinositol 3-kinase (PtdIns 3-kinase), microtubule-associated protein (MAP) kinase, and S6 kinases] was studied in Chinese hamster ovary cells which overexpress wild type human insulin receptors, receptors with reduced kinase activity due to substitution of Phe for Tyr1146 (single-Phe), Tyr1150,1151 (double-Phe), and Tyr1146,1150,1151 (triple-Phe), or kinase-inactive receptors with a substitution of Ala for Lys1018 in the ATP binding site (A1018). We have previously shown that receptor autophosphorylation and kinase activity of these mutants were reduced by approximately 50, 65, 85, and 100%, respectively. Glycogen and DNA synthesis parallel the level of receptor autophosphorylation and kinase activity; however, receptor serine and threonine phosphorylation was independent of receptor tyrosine kinase activity and receptor internalization was completely dependent on maximal receptor kinase activity. Overexpression of the wild type insulin receptor increased both maximal insulin receptor substrate-1-associated and total insulin-stimulated PtdIns 3-kinase activity, as well as S6 and MAP kinase activities 2.0- to 3.6-fold. In addition there was a leftward shift of the dose-response curves for PtdIns 3-kinase and S6 kinases by approximately 10-fold. Expression of the single- and double-Phe mutant receptors also enhanced maximal PtdIns 3-kinase activity, but had no effect on insulin sensitivity, whereas expression of either the triple-Phe or kinase-inactive receptors did not enhance insulin stimulation or increase insulin sensitivity as compared to the control cells. When comparing the mutant and wild type receptors, differences in insulin sensitivity were least for insulin-stimulated MAP kinase and greatest for S6 kinase; with the latter there was greater than a 1000-fold difference in insulin sensitivity when cells that overexpress wild type vs. kinase-inactive insulin receptors were compared. Thus, the level of insulin receptor tyrosine autophosphorylation and kinase activity regulate both maximal activation and insulin sensitivity of these intracellular kinases in the insulin action pathway which may lead to glycogen and/or DNA synthesis. The differential sensitivity of these enzymes to changes in receptor activation suggests that they may be differently coupled to the receptor kinase.
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PMID:The level of insulin receptor tyrosine kinase activity modulates the activities of phosphatidylinositol 3-kinase, microtubule-associated protein, and S6 kinases. 805 65

The mechanisms by which growth factors and oncogenic agents activate phosphatidylinositol 3-kinase (PI3 kinase) are unknown. Previously, we reported that the 85-kDa regulatory subunit of PI3 kinase is tyrosine-phosphorylated both in vitro by the platelet-derived growth factor beta-receptor (PDGFR) tyrosine kinase and in fibroblasts in response to PDGF. As a first step in determining the role of tyrosine phosphorylation in PDGF signaling through PI3 kinase, we investigated which tyrosines on p85 are phosphorylated by the PDGFR. Recombinant p85 was phosphorylated with recombinant PDGF receptors, and tryptic phosphopeptides were purified by HPLC and analyzed by Edman degradation. By this approach and by mutational analysis, Y508 was identified as the major in vitro phosphorylation site. Tryptic phosphopeptide mapping demonstrated Y508 to also be phosphorylated in vivo in COS cells. Comparison of these data with a previous report [Hayashi, H., Nishioka, Y., Kamohara, S., Kanai, F., Ishii, K., Fukui, Y., Shibasaki, F., Takenawa, T., Kido, H., Katsunuma, N., & Ebina, Y. (1993) J. Biol. Chem. 268, 7107-7117] suggests that p85 is phosphorylated differently by the PDGF and insulin receptor tyrosine kinases. Therefore, p85 may be regulated differently by PDGF and insulin. Mapping of phosphorylation sites on p85 may lead to new insights into the regulation of signal transduction through PI3 kinase.
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PMID:Tyrosine 508 of the 85-kilodalton subunit of phosphatidylinositol 3-kinase is phosphorylated by the platelet-derived growth factor receptor. 808 21

The insulin receptor tyrosine kinase is required for insulin to elicit subsequent biological signalling. Recent studies have identified several endogenous substrates of the insulin receptor kinase, including one called insulin receptor substrate 1 (IRS-1). Tyrosine phosphorylation of this substrate results in its being bound by various proteins containing src homology 2 (SH2) sites, including a phosphatidylinositol 3-kinase and a ras activator complex containing GRB2 and son of sevenless (SOS) 1. Decreases in the insulin receptor tyrosine kinase activity have been observed in various insulin-resistant states, such as non-insulin-dependent diabetes mellitus. A model of insulin resistance has recently been described in which the insulin receptor is expressed in Chinese hamster ovary cells along with the phospholipid- and calcium-activated serine/threonine kinase called protein kinase C. In this model system, activation of protein kinase C is shown to interfere with insulin receptor signalling by inhibiting tyrosine phosphorylation of IRS-1 and its subsequent binding by phosphatidylinositol 3-kinase. Such a model system may be further utilized to determine the detailed biochemical basis for insulin resistance.
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PMID:Biochemical mechanisms of insulin resistance. 808 4

Significant activity of phosphatidylinositol 3-kinase (PI 3-kinase) was detected in the membrane fractions, or in the immunoprecipitates prepared with anti-phosphotyrosine antibodies, from rat adipocytes that had been incubated with insulin for 20 min. The PI 3-kinase activity in these preparations as well as in the whole cell lysates of adipocytes not treated with insulin was inhibited by the addition of wortmannin, a fungal metabolite, to the enzyme assay mixture. The inhibition was dependent on the inhibitor concentration with IC50 being less than 10 nM and perfect inhibition at 100 nM. The effect of insulin to induce membrane PI 3-kinase activity was mostly abolished, but its effects to tyrosine-phosphorylate the beta-subunit of the insulin receptor or other cellular substrate proteins including insulin-receptor-substrate-1 were not at all antagonized, by wortmannin added to the cell incubation medium. Insulin stimulation of cellular 2-deoxyglucose uptake and inhibition of isoproterenol-induced lipolysis observable in adipocytes under the same conditions were also antagonized by wortmannin added in the same concentration range as used for the inhibition of insulin-susceptible PI 3-kinase. It is concluded, therefore, that activation of wortmannin-sensitive PI 3-kinase plays a pivotal role in the intracellular signaling pathways arising from the insulin receptor autophosphorylation and leading to certain metabolic responses.
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PMID:Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. 810

Treatment of cells with okadaic acid, a protein phosphatase inhibitor, leads to an insulin-resistant state without modification in the tyrosine kinase activity of the receptor toward exogenous substrates. In 3T3-L1 adipocytes, okadaic acid induced a similar dose-dependent inhibition of the insulin effect on deoxyglucose uptake, phosphatidylinositol 3-kinase (PI 3-kinase) activation, and insulin receptor substrate (IRS) 1 tyrosine phosphorylation. Simultaneously, in okadaic acid-treated 3T3-L1 adipocytes, the reduced IRS 1 tyrosine phosphorylation was linked to a decrease in its electrophoretic mobility due to phosphorylation on serine/threonine residues. This phosphorylation appeared to result from the activation of cytosolic kinase(s). Furthermore, using in vitro reconstitution, we show that, compared to IRS 1 immunopurified from untreated cells, the IRS 1 obtained from okadaic acid-treated cells had a reduced capacity to be phosphorylated by insulin receptors and, concomitantly, to bind PI 3-kinase. Taken together these data suggest that serine/threonine phosphorylation of IRS 1 induced by okadaic acid reduces the ability of the insulin receptor to phosphorylate IRS 1 and to dock one of its interacting molecules, i.e. PI 3-kinase. Finally, the inhibitory effect of okadaic acid on the stimulatory action of insulin on glucose transport suggests that the serine/threonine phosphorylation of IRS 1 might represent a key regulatory mechanism of insulin action.
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PMID:Serine/threonine phosphorylation of insulin receptor substrate 1 modulates insulin receptor signaling. 811 50

A line of Chinese hamster ovary cells overexpressing protein kinase C alpha was transfected with cDNAs encoding either the wild-type human insulin receptor or one of two mutant insulin receptors with either Ser-967 and -968 or -974 and -976 in the juxtamembrane region changed to alanine. Both mutant receptors exhibited normal insulin-activated tyrosine kinase activity as assessed by either autophosphorylation or insulin-stimulated increases in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase. The wild-type and mutant insulin receptors were also examined for serine and threonine phosphorylation in response to insulin and activation of protein kinase C. To visualize Ser/Thr-phosphorylation sites of the receptor better in response to insulin, the receptor from in vivo-labelled insulin-treated cells was first treated with a tyrosine-specific phosphatase to remove all tyrosine phosphorylation. Phosphopeptides from the three receptors were analysed by high-percentage polyacrylamide/urea gel electrophoresis and two-dimensional t.l.c. The mutant receptor lacking Ser-967 and -968 but not the mutant lacking Ser-974 and -976 was found to be missing phosphorylated peptides in response to insulin and, to a lesser extent, after activation of protein kinase C. However, the insulin-stimulated increase in anti-phosphotyrosine-precipitable phosphatidylinositol 3-kinase was inhibited to the same extent by activation of protein kinase C in cells expressing the two mutant receptors as in cells expressing the wild-type receptor. These results indicate that these four serine residues in the juxtamembrane region are not major regulatory sites of the intrinsic tyrosine kinase activity of the insulin receptor by protein kinase C, although Ser-967 and/or -968 appear to be phosphorylated in response to insulin.
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PMID:Identification of serines-967/968 in the juxtamembrane region of the insulin receptor as insulin-stimulated phosphorylation sites. 813 57


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