Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Mol Cell Biol. 1994 Nov; 14(11): 7466–7475.
doi: 10.1128/mcb.14.11.7466
PMCID: PMC359282
PMID: 7935461

Microinjection of the SH2 domain of the 85-kilodalton subunit of phosphatidylinositol 3-kinase inhibits insulin-induced DNA synthesis and c-fos expression.

B H Jhun, D W Rose, B L Seely, L Rameh, L Cantley, A R Saltiel, and J M Olefsky
Author information Copyright and License information PMC Disclaimer

Abstract

We have investigated the functional role of the SH2 domain of the 85-kDa subunit (p85) of the phosphatidylinositol 3-kinase in the insulin signal transduction pathway. Microinjection of a bacterial fusion protein containing the N-terminal SH2 domain of p85 inhibited insulin- and other growth factor-induced DNA synthesis by 90% and c-fos protein expression by 80% in insulin-responsive rat fibroblasts. The specificity of the fusion protein was examined by in vitro precipitation experiments, which showed that the SH2 domain of p85 can independently associate with both insulin receptor substrate 1 and the insulin receptor itself in the absence of detectable binding to other phosphoproteins. The microinjection results were confirmed through the use of an affinity-purified antibody directed against p85, which gave the same phenotype. Additional studies were carried out in another cell line expressing mutant insulin receptors which lack the cytoplasmic tyrosine residues with which p85 interacts. Microinjection of the SH2 domain fusion protein also inhibited insulin signaling in these cells, suggesting that association of p85 with insulin receptor substrate 1 is a key element in insulin-mediated cell cycle progression. In addition, coinjection of purified p21ras protein with the p85 fusion protein or the antibody restored DNA synthesis, suggesting that ras function is either downstream or independent of p85 SH2 domain interaction.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (3.2M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

 
icon of scanned page 7466
7466
icon of scanned page 7467
7467
icon of scanned page 7468
7468
icon of scanned page 7469
7469
icon of scanned page 7470
7470
icon of scanned page 7471
7471
icon of scanned page 7472
7472
icon of scanned page 7473
7473
icon of scanned page 7474
7474
icon of scanned page 7475
7475
 

Images in this article

Image

Image
on p.7468

Image

Image
on p.7469

Image

Image
on p.7470

Image

Image
on p.7472

Image

Image
on p.7473

Click on the image to see a larger version.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  • Backer JM, Myers MG, Jr, Shoelson SE, Chin DJ, Sun XJ, Miralpeix M, Hu P, Margolis B, Skolnik EY, Schlessinger J, et al. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. [PMC free article] [PubMed] [Google Scholar]
  • Backer JM, Schroeder GG, Kahn CR, Myers MG, Jr, Wilden PA, Cahill DA, White MF. Insulin stimulation of phosphatidylinositol 3-kinase activity maps to insulin receptor regions required for endogenous substrate phosphorylation. J Biol Chem. 1992 Jan 15;267(2):1367–1374. [PubMed] [Google Scholar]
  • Burgering BM, Medema RH, Maassen JA, van de Wetering ML, van der Eb AJ, McCormick F, Bos JL. Insulin stimulation of gene expression mediated by p21ras activation. EMBO J. 1991 May;10(5):1103–1109. [PMC free article] [PubMed] [Google Scholar]
  • Cantley LC, Auger KR, Carpenter C, Duckworth B, Graziani A, Kapeller R, Soltoff S. Oncogenes and signal transduction. Cell. 1991 Jan 25;64(2):281–302. [PubMed] [Google Scholar]
  • Carpenter CL, Duckworth BC, Auger KR, Cohen B, Schaffhausen BS, Cantley LC. Purification and characterization of phosphoinositide 3-kinase from rat liver. J Biol Chem. 1990 Nov 15;265(32):19704–19711. [PubMed] [Google Scholar]
  • Chou CK, Dull TJ, Russell DS, Gherzi R, Lebwohl D, Ullrich A, Rosen OM. Human insulin receptors mutated at the ATP-binding site lack protein tyrosine kinase activity and fail to mediate postreceptor effects of insulin. J Biol Chem. 1987 Feb 5;262(4):1842–1847. [PubMed] [Google Scholar]
  • Chuang LM, Myers MG, Jr, Backer JM, Shoelson SE, White MF, Birnbaum MJ, Kahn CR. Insulin-stimulated oocyte maturation requires insulin receptor substrate 1 and interaction with the SH2 domains of phosphatidylinositol 3-kinase. Mol Cell Biol. 1993 Nov;13(11):6653–6660. [PMC free article] [PubMed] [Google Scholar]
  • Coughlin SR, Escobedo JA, Williams LT. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science. 1989 Mar 3;243(4895):1191–1194. [PubMed] [Google Scholar]
  • Ebina Y, Araki E, Taira M, Shimada F, Mori M, Craik CS, Siddle K, Pierce SB, Roth RA, Rutter WJ. Replacement of lysine residue 1030 in the putative ATP-binding region of the insulin receptor abolishes insulin- and antibody-stimulated glucose uptake and receptor kinase activity. Proc Natl Acad Sci U S A. 1987 Feb;84(3):704–708. [PMC free article] [PubMed] [Google Scholar]
  • Ellis L, Clauser E, Morgan DO, Edery M, Roth RA, Rutter WJ. Replacement of insulin receptor tyrosine residues 1162 and 1163 compromises insulin-stimulated kinase activity and uptake of 2-deoxyglucose. Cell. 1986 Jun 6;45(5):721–732. [PubMed] [Google Scholar]
  • Endemann G, Yonezawa K, Roth RA. Phosphatidylinositol kinase or an associated protein is a substrate for the insulin receptor tyrosine kinase. J Biol Chem. 1990 Jan 5;265(1):396–400. [PubMed] [Google Scholar]
  • Escobedo JA, Kaplan DR, Kavanaugh WM, Turck CW, Williams LT. A phosphatidylinositol-3 kinase binds to platelet-derived growth factor receptors through a specific receptor sequence containing phosphotyrosine. Mol Cell Biol. 1991 Feb;11(2):1125–1132. [PMC free article] [PubMed] [Google Scholar]
  • Escobedo JA, Navankasattusas S, Kavanaugh WM, Milfay D, Fried VA, Williams LT. cDNA cloning of a novel 85 kd protein that has SH2 domains and regulates binding of PI3-kinase to the PDGF beta-receptor. Cell. 1991 Apr 5;65(1):75–82. [PubMed] [Google Scholar]
  • Fantl WJ, Escobedo JA, Martin GA, Turck CW, del Rosario M, McCormick F, Williams LT. Distinct phosphotyrosines on a growth factor receptor bind to specific molecules that mediate different signaling pathways. Cell. 1992 May 1;69(3):413–423. [PubMed] [Google Scholar]
  • Fukui Y, Hanafusa H. Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol. 1989 Apr;9(4):1651–1658. [PMC free article] [PubMed] [Google Scholar]
  • Herman PK, Emr SD. Characterization of VPS34, a gene required for vacuolar protein sorting and vacuole segregation in Saccharomyces cerevisiae. Mol Cell Biol. 1990 Dec;10(12):6742–6754. [PMC free article] [PubMed] [Google Scholar]
  • Hiles ID, Otsu M, Volinia S, Fry MJ, Gout I, Dhand R, Panayotou G, Ruiz-Larrea F, Thompson A, Totty NF, et al. Phosphatidylinositol 3-kinase: structure and expression of the 110 kd catalytic subunit. Cell. 1992 Aug 7;70(3):419–429. [PubMed] [Google Scholar]
  • Hu P, Margolis B, Skolnik EY, Lammers R, Ullrich A, Schlessinger J. Interaction of phosphatidylinositol 3-kinase-associated p85 with epidermal growth factor and platelet-derived growth factor receptors. Mol Cell Biol. 1992 Mar;12(3):981–990. [PMC free article] [PubMed] [Google Scholar]
  • Jhun BH, Meinkoth JL, Leitner JW, Draznin B, Olefsky JM. Insulin and insulin-like growth factor-I signal transduction requires p21ras. J Biol Chem. 1994 Feb 25;269(8):5699–5704. [PubMed] [Google Scholar]
  • Kahn CR, White MF. The insulin receptor and the molecular mechanism of insulin action. J Clin Invest. 1988 Oct;82(4):1151–1156. [PMC free article] [PubMed] [Google Scholar]
  • Kazlauskas A, Cooper JA. Autophosphorylation of the PDGF receptor in the kinase insert region regulates interactions with cell proteins. Cell. 1989 Sep 22;58(6):1121–1133. [PubMed] [Google Scholar]
  • Kelly KL, Ruderman NB. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J Biol Chem. 1993 Feb 25;268(6):4391–4398. [PubMed] [Google Scholar]
  • Klippel A, Escobedo JA, Fantl WJ, Williams LT. The C-terminal SH2 domain of p85 accounts for the high affinity and specificity of the binding of phosphatidylinositol 3-kinase to phosphorylated platelet-derived growth factor beta receptor. Mol Cell Biol. 1992 Apr;12(4):1451–1459. [PMC free article] [PubMed] [Google Scholar]
  • Ling LE, Druker BJ, Cantley LC, Roberts TM. Transformation-defective mutants of polyomavirus middle T antigen associate with phosphatidylinositol 3-kinase (PI 3-kinase) but are unable to maintain wild-type levels of PI 3-kinase products in intact cells. J Virol. 1992 Mar;66(3):1702–1708. [PMC free article] [PubMed] [Google Scholar]
  • McClain DA, Maegawa H, Lee J, Dull TJ, Ulrich A, Olefsky JM. A mutant insulin receptor with defective tyrosine kinase displays no biologic activity and does not undergo endocytosis. J Biol Chem. 1987 Oct 25;262(30):14663–14671. [PubMed] [Google Scholar]
  • McGlade CJ, Ellis C, Reedijk M, Anderson D, Mbamalu G, Reith AD, Panayotou G, End P, Bernstein A, Kazlauskas A, et al. SH2 domains of the p85 alpha subunit of phosphatidylinositol 3-kinase regulate binding to growth factor receptors. Mol Cell Biol. 1992 Mar;12(3):991–997. [PMC free article] [PubMed] [Google Scholar]
  • Myers MG, Jr, Backer JM, Sun XJ, Shoelson S, Hu P, Schlessinger J, Yoakim M, Schaffhausen B, White MF. IRS-1 activates phosphatidylinositol 3'-kinase by associating with src homology 2 domains of p85. Proc Natl Acad Sci U S A. 1992 Nov 1;89(21):10350–10354. [PMC free article] [PubMed] [Google Scholar]
  • Okada T, Kawano Y, Sakakibara T, Hazeki O, Ui M. Essential role of phosphatidylinositol 3-kinase in insulin-induced glucose transport and antilipolysis in rat adipocytes. Studies with a selective inhibitor wortmannin. J Biol Chem. 1994 Feb 4;269(5):3568–3573. [PubMed] [Google Scholar]
  • Olefsky JM. The insulin receptor. A multifunctional protein. Diabetes. 1990 Sep;39(9):1009–1016. [PubMed] [Google Scholar]
  • Otsu M, Hiles I, Gout I, Fry MJ, Ruiz-Larrea F, Panayotou G, Thompson A, Dhand R, Hsuan J, Totty N, et al. Characterization of two 85 kd proteins that associate with receptor tyrosine kinases, middle-T/pp60c-src complexes, and PI3-kinase. Cell. 1991 Apr 5;65(1):91–104. [PubMed] [Google Scholar]
  • Pawson T, Schlessingert J. SH2 and SH3 domains. Curr Biol. 1993 Jul 1;3(7):434–442. [PubMed] [Google Scholar]
  • Reedijk M, Liu X, van der Geer P, Letwin K, Waterfield MD, Hunter T, Pawson T. Tyr721 regulates specific binding of the CSF-1 receptor kinase insert to PI 3'-kinase SH2 domains: a model for SH2-mediated receptor-target interactions. EMBO J. 1992 Apr;11(4):1365–1372. [PMC free article] [PubMed] [Google Scholar]
  • Rose DW, Saltiel AR, Majumdar M, Decker SJ, Olefsky JM. Insulin receptor substrate 1 is required for insulin-mediated mitogenic signal transduction. Proc Natl Acad Sci U S A. 1994 Jan 18;91(2):797–801. [PMC free article] [PubMed] [Google Scholar]
  • Ruderman NB, Kapeller R, White MF, Cantley LC. Activation of phosphatidylinositol 3-kinase by insulin. Proc Natl Acad Sci U S A. 1990 Feb;87(4):1411–1415. [PMC free article] [PubMed] [Google Scholar]
  • Sasaoka T, Rose DW, Jhun BH, Saltiel AR, Draznin B, Olefsky JM. Evidence for a functional role of Shc proteins in mitogenic signaling induced by insulin, insulin-like growth factor-1, and epidermal growth factor. J Biol Chem. 1994 May 6;269(18):13689–13694. [PubMed] [Google Scholar]
  • Shoelson SE, Chatterjee S, Chaudhuri M, White MF. YMXM motifs of IRS-1 define substrate specificity of the insulin receptor kinase. Proc Natl Acad Sci U S A. 1992 Mar 15;89(6):2027–2031. [PMC free article] [PubMed] [Google Scholar]
  • Skolnik EY, Margolis B, Mohammadi M, Lowenstein E, Fischer R, Drepps A, Ullrich A, Schlessinger J. Cloning of PI3 kinase-associated p85 utilizing a novel method for expression/cloning of target proteins for receptor tyrosine kinases. Cell. 1991 Apr 5;65(1):83–90. [PubMed] [Google Scholar]
  • Smith DB, Johnson KS. Single-step purification of polypeptides expressed in Escherichia coli as fusions with glutathione S-transferase. Gene. 1988 Jul 15;67(1):31–40. [PubMed] [Google Scholar]
  • Songyang Z, Shoelson SE, Chaudhuri M, Gish G, Pawson T, Haser WG, King F, Roberts T, Ratnofsky S, Lechleider RJ, et al. SH2 domains recognize specific phosphopeptide sequences. Cell. 1993 Mar 12;72(5):767–778. [PubMed] [Google Scholar]
  • Sun XJ, Crimmins DL, Myers MG, Jr, Miralpeix M, White MF. Pleiotropic insulin signals are engaged by multisite phosphorylation of IRS-1. Mol Cell Biol. 1993 Dec;13(12):7418–7428. [PMC free article] [PubMed] [Google Scholar]
  • Sun XJ, Miralpeix M, Myers MG, Jr, Glasheen EM, Backer JM, Kahn CR, White MF. Expression and function of IRS-1 in insulin signal transmission. J Biol Chem. 1992 Nov 5;267(31):22662–22672. [PubMed] [Google Scholar]
  • Takata Y, Webster NJ, Olefsky JM. Intracellular signaling by a mutant human insulin receptor lacking the carboxyl-terminal tyrosine autophosphorylation sites. J Biol Chem. 1992 May 5;267(13):9065–9070. [PubMed] [Google Scholar]
  • Ugi S, Maegawa H, Olefsky JM, Shigeta Y, Kashiwagi A. Src homology 2 domains of protein tyrosine phosphatase are associated in vitro with both the insulin receptor and insulin receptor substrate-1 via different phosphotyrosine motifs. FEBS Lett. 1994 Mar 7;340(3):216–220. [PubMed] [Google Scholar]
  • Varticovski L, Druker B, Morrison D, Cantley L, Roberts T. The colony stimulating factor-1 receptor associates with and activates phosphatidylinositol-3 kinase. Nature. 1989 Dec 7;342(6250):699–702. [PubMed] [Google Scholar]
  • White MF, Maron R, Kahn CR. Insulin rapidly stimulates tyrosine phosphorylation of a Mr-185,000 protein in intact cells. Nature. 1985 Nov 14;318(6042):183–186. [PubMed] [Google Scholar]
  • Whitman M, Kaplan D, Roberts T, Cantley L. Evidence for two distinct phosphatidylinositol kinases in fibroblasts. Implications for cellular regulation. Biochem J. 1987 Oct 1;247(1):165–174. [PMC free article] [PubMed] [Google Scholar]
  • Whitman M, Kaplan DR, Schaffhausen B, Cantley L, Roberts TM. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. [PubMed] [Google Scholar]
  • Yamamoto K, Lapetina EG, Moxham CP. Insulin like growth factor-I induces limited association of phosphatidylinositol 3-kinase to its receptor. Endocrinology. 1992 Mar;130(3):1490–1498. [PubMed] [Google Scholar]
  • Yamauchi K, Holt K, Pessin JE. Phosphatidylinositol 3-kinase functions upstream of Ras and Raf in mediating insulin stimulation of c-fos transcription. J Biol Chem. 1993 Jul 15;268(20):14597–14600. [PubMed] [Google Scholar]
Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis
-