Summary
Phosphoinositide 3-kinase (PI3-kinase) plays a crucial role in insulin signal transduction. We studied the molecular mechanism of the insulin-induced activation of PI3-kinase in rat hepatoma Fao cells using an antibody against the 110-kDa catalytic subunit (p110) and two against the 85-kDa regulatory subunit (p85α). PI3-kinase activity increased 1.6-fold in anti-p85 immunoprecipitates after insulin stimulation, whereas it did not increase when cell lysates were first immunoprecipitated with anti-phosphotyrosine or anti-insulin receptor substrate-1 (IRS-1), then with anti-p85, suggesting that the PI3-kinase which associates with tyrosyl phosphoproteins including IRS-1 is responsible for the increase in kinase activity. The activated PI3-kinase molecules constituted 4–6% of the total PI3-kinase, and their specific activity was 11–14 times higher than that of the basal state. Anti-p110 recognized the catalytically active form of p110, and immunoprecipitated p110 only after exposure to insulin. Hence, the epitope of anti-p110, P200-C215, seems to be included in the portion of p110, the conformation of which is changed by insulin stimulation. We conclude that, in response to insulin stimulation, only a small fraction of p85 in the PI3-kinase pool associates with tyrosyl phosphoproteins including IRS-1, and that the specific activity of p110 is increased presumably through a conformational change including the P200-C215 region.
Similar content being viewed by others
Abbreviations
- PI3-kinase:
-
Phosphoinositide 3-kinase
- p85:
-
85-kDa subunit of PI3-kinase
- p110:
-
110-kDa subunit of PI3-kinase
- IRS-1:
-
insulin receptor substrate-1
- SH2:
-
src homology 2
- SH3:
-
src homology 3
- BCR:
-
breakpoint cluster region
- PMSF:
-
phenylmethylsulphonyl fluoride
- HEPES:
-
4-(2-hydroxyethyl)-1-piperazineethanesulphonic acid
- PIP:
-
phosphatidylinositol phosphate
- TLC:
-
thin layer chromatography
- IP (in figures):
-
immunoprecipitation with the indicated antibody
- TBS:
-
Tris-buffered saline
References
Sun XJ, Rothenberg P, Kahn CR et al. (1991) Structure of the insulin receptor substrate IRS-1 defines a unique signal transduction protein. Nature 352: 73–77
Myers MG Jr, White MF (1993) The new elements of insulin signaling: insulin receptor substrate-1 and proteins with SH2 domains. Diabetes 42: 643–650
White MF, Kahn CR (1994) The insulin signaling system. J Biol Chem 269: 1–4
Escobedo JA, Navankasattusas S, Kavanaugh WM, Milfay D, Fried VA, Williams LT (1991) cDNA cloning of a novel 85 kD protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF Β-receptor. Cell 65: 75–82
Skolnik EY, Margolis B, Mohammadi M et al. (1991) Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell 65: 83–90
Otsu M, Hiles I, Gout I et al. (1991) Characterization of two 85 kD proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell 65: 91–104
Hiles ID, Otsu M, Volinia S et al. (1992) Phosphatidylinositol 3-kinase: structure and expression of the 110kD catalytic subunit. Cell 70: 419–429
End P, Gout I, Fry MJ et al. (1993) A biosensor approach to probe the structure and function of the p85α subunit of the phosphatidylinositol 3-kinase complex. J Biol Chem 268: 10 066–10 075
Dhand R, Hara K, Hiles I et al. (1994) PI3-kinase: structural and functional analysis of intersubunit interactions. EMBO J 13: 511–521
Whitman M, Downes CP, Keeler M, Keller T, Cantley L (1988) Type 1 phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature 332: 644–646
Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC (1989) PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell 57: 167–175
Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS, Cantley LC (1990) Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem 265: 19 704–19 711
Cheatham B, Vlahos CJ, Cheatham L, Wang L, Blenis J, Kahn CR (1994) Phosphatidylinositol 3-kinase activation is required for insulin stimulation of pp70 S6 kinase, DNA synthesis, and glucose transporter translocation. Mol Cell Biol 14: 4902–4911
Clarke JF, Young PW, Yonezawa K, Kasuga M, Holman GD (1994) Inhibition of the translocation of GLUT1 and GLUT4 in 3T3-L1 cells by the phosphatidylinositol 3-kinase inhibitor, wortmannin. Biochem J 300: 631–635
Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M (1994) Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. J Biol Chem 269: 3568–3573
Kanai F, Ito K, Todaka M et al. (1994) Insulin-stimulated GLUT4 translocation is relevant to the phosphorylation of IRS-1 and the activity of PI3-kinase. Biochem Biophys Res Commun 195: 762–768
Kotani K, Carozzi AJ, Sakaue H et al. (1995) Requirement for phosphoinositide 3-kinase in insulin-stimulated GLUT4 translocation in 3T3-L1 adipocytes. Biochem Biophys Res Commun 209: 343–348
Folli F, Saad MJA, Backer JM, Kahn CR (1993) Regulation of phosphatidylinositol 3-kinase activity in liver and muscle of animal models of insulin-resistant and insulin-deficient diabetes mellitus. J Clin Invest 92: 1787–1794
Saad MJA, Folli F, Kahn JA, Kahn CR (1993) Modulation of insulin receptor, insulin receptor substrate-1, and phosphatidylinositol 3-kinase in liver and muscle of dexamethasone-treated rats. J Clin Invest 92: 2065–2072
Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, Emr SD (1993) Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science 260: 88–91
White MF, Maron R, Kahn CR (1985) Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature 318: 183–186
Crettaz M, Kahn CR (1984) Insulin receptor regulation and desensitization in rat hepatoma cells. Diabetes 33: 477–485
Pang DT, Sharma BR, Shafer JA, White MF, Kahn CR (1985) Predominance of tyrosine phosphorylation of insulin receptors during the initial response of intact cells to insulin. J Biol Chem 260: 7131–7136
Okamoto M, Okamoto M, Kono S et al. (1992) Effects of a high-fat diet on insulin receptor kinase and the glucose transporter in rats. J Nutr Biochem 3: 241–250
Okamoto M, Hayashi T, Kono S et al. (1993) Specific activity of phosphatidylinositol 3-kinase is increased by insulin stimulation. Biochem J 290: 327–333
Yonezawa K, Ueda H, Hara K et al. (1992) Insulin-dependent formation of a complex containing an 85-kDa subunit of phosphatidylinositol 3-kinase and tyrosine-phosphorylated insulin receptor substrate 1. J Biol Chem 267: 25 958–25 966
Whitman M, Kaplan DR, Schaffhausen B, Cantley L, Roberts TM (1985) Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature 315: 239–242
White MF, Backer JM (1991) Preparation and use of antiphosphotyrosine antibodie to study structure and function of insulin receptor. Methods Enzymol 201: 65–79
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680–685
Okamoto M, Karasik A, White MF, Kahn CR (1990) Epidermal growth factor stimulated phosphorylation of a 120-kilodalton endogenous substrate protein in rat hepatocytes. Biochemistry 29: 9489–9494
Endemann G, Yonezawa K, Roth RA (1990) Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem 265: 396–400
Okamoto M, Hayashi T, Kono S, Okamoto M, Nakao K (1993) A 29kDa protein associates with phosphatidylinositol 3-kinase (PI3K) and insulin receptor substrate-1 (IRS-1) complex. Diabetes 42 (Suppl 1):2A (Abstract)
Wang LM, Myers MG Jr, Sun XJ, Aaronson SA, White M, Pierce JH (1993) IRS-1: essential for insulin- and IL-4-stimulated mitogenesis in hematopoietic cells. Science 261: 1591–1594
Cohen B, Yoakim M, Piwnica-Worms H, Roberts TM, Schaffhausen BS (1990) Tyrosine phosphorylation is a signal for the trafficking of pp85, an 85-kDa phosphorylated polypeptide associated with phosphatidylinositol kinase activity. Proc Natl Acad Sci USA 87: 4458–4462
Giorgetti S, Ballotti R, Kowalski-Chauvel A, Cormont M, Van Obberghen E (1992) Insulin stimulates phosphatidylinositol-3-kinase activity in rat adipocytes. Eur J Biochem 207: 599–606
Kelly KL, Ruderman NB, Chen KS (1992) Phosphatidylinositol-3-kinase in isolated rat adipocytes. J Biol Chem 267: 3423–3428
Bar-Sagi D, Rotin D, Batzer A, Mandiyan V, Schlessinger J (1993) SH3 domains direct cellular localization of signaling molecules. Cell 74: 83–91
Del Vecchio RL, Pilch PF (1989) Insulin stimulates the tyrosine phosphorylation of a 165 kDa protein that is associated with microsomal membranes of rat adipocytes. Biochem Biophys Acta 986: 41–46
Madoff DH, Martensen TM, Lane MD (1988) Insulin and insulin-like growth factor 1 stimulate the phosphorylation on tyrosine of a 160 kDa cytosolic protein in 3T3-L1 adipocytes. Biochem J 252: 7–15
Panayotou G, Bax B, Gout I et al. (1992) Interaction of the p85 subunit of PI 3-kinase and its N-terminal SH2 domain with a PDGF receptor phosphorylation site: structural features and analysis of conformational changes. EMBO J 11: 4261–4272
Shoelson SE, Sivaraja M, Williams KP, Hu P, Schlessinger J, Weiss MA (1993) Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation. EMBO J 12: 795–802
Baltensperger K, Kozma LM, Jaspers SR, Czech MP (1994) Regulation by insulin of phosphatidylinositol 3′-kinase bound to α- and β-isoforms of p85 regulatory subunit. J Biol Chem 269: 28 937–28 946
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Hayashi, T., Okamoto, M., Yoshimasa, Y. et al. Insulin-induced activation of phosphoinositide 3-kinase in Fao cells. Diabetologia 39, 515–522 (1996). https://doi.org/10.1007/BF00403297
Received:
Revised:
Issue Date:
DOI: https://doi.org/10.1007/BF00403297