Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Mol Cell Biol. 1993 Dec; 13(12): 7677–7688.
doi: 10.1128/mcb.13.12.7677
PMCID: PMC364839
PMID: 8246984

Cloning of a novel, ubiquitously expressed human phosphatidylinositol 3-kinase and identification of its binding site on p85.

P Hu, A Mondino, E Y Skolnik, and J Schlessinger
Author information Copyright and License information PMC Disclaimer

Abstract

Phosphatidylinositol 3-kinase (PI 3-kinase) has been implicated as a participant in signaling pathways regulating cell growth by virtue of its activation in response to various mitogenic stimuli. Here we describe the cloning of a novel and ubiquitously expressed human PI 3-kinase. The 4.8-kb cDNA encodes a putative translation product of 1,070 amino acids which is 42% identical to bovine PI 3-kinase and 28% identical to Vps34, a Saccharomyces cerevisiae PI 3-kinase involved in vacuolar protein sorting. Human PI 3-kinase is also similar to Tor2, a yeast protein required for cell cycle progression. Northern (RNA) analysis demonstrated expression of human PI 3-kinase in all tissues and cell lines tested. Protein synthesized from an epitope-tagged cDNA had intrinsic PI 3-kinase activity and associated with the adaptor 85-kDa subunit of PI 3-kinase (p85) in intact cells, as did endogenous human PI 3-kinase. Coprecipitation assays showed that a 187-amino-acid domain between the two src homology 2 domains of p85 mediates interaction with PI 3-kinase in vitro and in intact cells. These results demonstrate the existence of different PI 3-kinase isoforms and define a family of genes encoding distinct PI 3-kinase catalytic subunits that can associate with p85.

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 (3.0M), 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 7677
7677
icon of scanned page 7678
7678
icon of scanned page 7679
7679
icon of scanned page 7680
7680
icon of scanned page 7681
7681
icon of scanned page 7682
7682
icon of scanned page 7683
7683
icon of scanned page 7684
7684
icon of scanned page 7685
7685
icon of scanned page 7686
7686
icon of scanned page 7687
7687
icon of scanned page 7688
7688
 

Images in this article

Image

Image
on p.7683

Image

Image
on p.7684

Image

Image
on p.7684

Image

Image
on p.7685

Image

Image
on p.7686

Image

Image
on p.7686

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.

  • Auger KR, Carpenter CL, Cantley LC, Varticovski L. Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae. J Biol Chem. 1989 Dec 5;264(34):20181–20184. [PubMed] [Google Scholar]
  • Auger KR, Serunian LA, Soltoff SP, Libby P, Cantley LC. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. [PubMed] [Google Scholar]
  • 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]
  • Berridge MJ. Inositol trisphosphate and calcium signalling. Nature. 1993 Jan 28;361(6410):315–325. [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, Auger KR, Chanudhuri M, Yoakim M, Schaffhausen B, Shoelson S, Cantley LC. Phosphoinositide 3-kinase is activated by phosphopeptides that bind to the SH2 domains of the 85-kDa subunit. J Biol Chem. 1993 May 5;268(13):9478–9483. [PubMed] [Google Scholar]
  • Carpenter CL, Auger KR, Duckworth BC, Hou WM, Schaffhausen B, Cantley LC. A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity. Mol Cell Biol. 1993 Mar;13(3):1657–1665. [PMC free article] [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]
  • Carter AN, Downes CP. Phosphatidylinositol 3-kinase is activated by nerve growth factor and epidermal growth factor in PC12 cells. J Biol Chem. 1992 Jul 25;267(21):14563–14567. [PubMed] [Google Scholar]
  • Chen C, Okayama H. High-efficiency transformation of mammalian cells by plasmid DNA. Mol Cell Biol. 1987 Aug;7(8):2745–2752. [PMC free article] [PubMed] [Google Scholar]
  • Cohen B, Liu YX, Druker B, Roberts TM, Schaffhausen BS. Characterization of pp85, a target of oncogenes and growth factor receptors. Mol Cell Biol. 1990 Jun;10(6):2909–2915. [PMC free article] [PubMed] [Google Scholar]
  • Cooper JA, Kashishian A. In vivo binding properties of SH2 domains from GTPase-activating protein and phosphatidylinositol 3-kinase. Mol Cell Biol. 1993 Mar;13(3):1737–1745. [PMC free article] [PubMed] [Google Scholar]
  • Corey S, Eguinoa A, Puyana-Theall K, Bolen JB, Cantley L, Mollinedo F, Jackson TR, Hawkins PT, Stephens LR. Granulocyte macrophage-colony stimulating factor stimulates both association and activation of phosphoinositide 3OH-kinase and src-related tyrosine kinase(s) in human myeloid derived cells. EMBO J. 1993 Jul;12(7):2681–2690. [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]
  • Courtneidge SA, Heber A. An 81 kd protein complexed with middle T antigen and pp60c-src: a possible phosphatidylinositol kinase. Cell. 1987 Sep 25;50(7):1031–1037. [PubMed] [Google Scholar]
  • Devereux J, Haeberli P, Smithies O. A comprehensive set of sequence analysis programs for the VAX. Nucleic Acids Res. 1984 Jan 11;12(1 Pt 1):387–395. [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]
  • Field J, Nikawa J, Broek D, MacDonald B, Rodgers L, Wilson IA, Lerner RA, Wigler M. Purification of a RAS-responsive adenylyl cyclase complex from Saccharomyces cerevisiae by use of an epitope addition method. Mol Cell Biol. 1988 May;8(5):2159–2165. [PMC free article] [PubMed] [Google Scholar]
  • Hawkins PT, Jackson TR, Stephens LR. Platelet-derived growth factor stimulates synthesis of PtdIns(3,4,5)P3 by activating a PtdIns(4,5)P2 3-OH kinase. Nature. 1992 Jul 9;358(6382):157–159. [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]
  • Jackson TR, Stephens LR, Hawkins PT. Receptor specificity of growth factor-stimulated synthesis of 3-phosphorylated inositol lipids in Swiss 3T3 cells. J Biol Chem. 1992 Aug 15;267(23):16627–16636. [PubMed] [Google Scholar]
  • Kaplan DR, Whitman M, Schaffhausen B, Pallas DC, White M, Cantley L, Roberts TM. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. [PubMed] [Google Scholar]
  • Kaplan DR, Whitman M, Schaffhausen B, Raptis L, Garcea RL, Pallas D, Roberts TM, Cantley L. Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3624–3628. [PMC free article] [PubMed] [Google Scholar]
  • Kavanaugh WM, Klippel A, Escobedo JA, Williams LT. Modification of the 85-kilodalton subunit of phosphatidylinositol-3 kinase in platelet-derived growth factor-stimulated cells. Mol Cell Biol. 1992 Aug;12(8):3415–3424. [PMC free article] [PubMed] [Google Scholar]
  • Kazlauskas A, Kashishian A, Cooper JA, Valius M. GTPase-activating protein and phosphatidylinositol 3-kinase bind to distinct regions of the platelet-derived growth factor receptor beta subunit. Mol Cell Biol. 1992 Jun;12(6):2534–2544. [PMC free article] [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]
  • Kozak M. The scanning model for translation: an update. J Cell Biol. 1989 Feb;108(2):229–241. [PMC free article] [PubMed] [Google Scholar]
  • Kucera GL, Rittenhouse SE. Human platelets form 3-phosphorylated phosphoinositides in response to alpha-thrombin, U46619, or GTP gamma S. J Biol Chem. 1990 Apr 5;265(10):5345–5348. [PubMed] [Google Scholar]
  • Kunz J, Henriquez R, Schneider U, Deuter-Reinhard M, Movva NR, Hall MN. Target of rapamycin in yeast, TOR2, is an essential phosphatidylinositol kinase homolog required for G1 progression. Cell. 1993 May 7;73(3):585–596. [PubMed] [Google Scholar]
  • Lavan BE, Kuhné MR, Garner CW, Anderson D, Reedijk M, Pawson T, Lienhard GE. The association of insulin-elicited phosphotyrosine proteins with src homology 2 domains. J Biol Chem. 1992 Jun 5;267(16):11631–11636. [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]
  • Lips DL, Majerus PW, Gorga FR, Young AT, Benjamin TL. Phosphatidylinositol 3-phosphate is present in normal and transformed fibroblasts and is resistant to hydrolysis by bovine brain phospholipase C II. J Biol Chem. 1989 May 25;264(15):8759–8763. [PubMed] [Google Scholar]
  • Macara IG, Marinetti GV, Balduzzi PC. Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis. Proc Natl Acad Sci U S A. 1984 May;81(9):2728–2732. [PMC free article] [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]
  • Morgan SJ, Smith AD, Parker PJ. Purification and characterization of bovine brain type I phosphatidylinositol kinase. Eur J Biochem. 1990 Aug 17;191(3):761–767. [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]
  • 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]
  • Panayotou G, Bax B, Gout I, Federwisch M, Wroblowski B, Dhand R, Fry MJ, Blundell TL, Wollmer A, Waterfield MD. 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. 1992 Dec;11(12):4261–4272. [PMC free article] [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]
  • Remillard B, Petrillo R, Maslinski W, Tsudo M, Strom TB, Cantley L, Varticovski L. Interleukin-2 receptor regulates activation of phosphatidylinositol 3-kinase. J Biol Chem. 1991 Aug 5;266(22):14167–14170. [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]
  • Schu PV, Takegawa K, Fry MJ, Stack JH, Waterfield MD, Emr SD. Phosphatidylinositol 3-kinase encoded by yeast VPS34 gene essential for protein sorting. Science. 1993 Apr 2;260(5104):88–91. [PubMed] [Google Scholar]
  • Serunian LA, Auger KR, Roberts TM, Cantley LC. Production of novel polyphosphoinositides in vivo is linked to cell transformation by polyomavirus middle T antigen. J Virol. 1990 Oct;64(10):4718–4725. [PMC free article] [PubMed] [Google Scholar]
  • Serunian LA, Haber MT, Fukui T, Kim JW, Rhee SG, Lowenstein JM, Cantley LC. Polyphosphoinositides produced by phosphatidylinositol 3-kinase are poor substrates for phospholipases C from rat liver and bovine brain. J Biol Chem. 1989 Oct 25;264(30):17809–17815. [PubMed] [Google Scholar]
  • Shibasaki F, Homma Y, Takenawa T. Two types of phosphatidylinositol 3-kinase from bovine thymus. Monomer and heterodimer form. J Biol Chem. 1991 May 5;266(13):8108–8114. [PubMed] [Google Scholar]
  • Shoelson SE, Sivaraja M, Williams KP, Hu P, Schlessinger J, Weiss MA. Specific phosphopeptide binding regulates a conformational change in the PI 3-kinase SH2 domain associated with enzyme activation. EMBO J. 1993 Feb;12(2):795–802. [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]
  • Soltoff SP, Rabin SL, Cantley LC, Kaplan DR. Nerve growth factor promotes the activation of phosphatidylinositol 3-kinase and its association with the trk tyrosine kinase. J Biol Chem. 1992 Aug 25;267(24):17472–17477. [PubMed] [Google Scholar]
  • Stephens LR, Hughes KT, Irvine RF. Pathway of phosphatidylinositol(3,4,5)-trisphosphate synthesis in activated neutrophils. Nature. 1991 May 2;351(6321):33–39. [PubMed] [Google Scholar]
  • Sugimoto Y, Whitman M, Cantley LC, Erikson RL. Evidence that the Rous sarcoma virus transforming gene product phosphorylates phosphatidylinositol and diacylglycerol. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2117–2121. [PMC free article] [PubMed] [Google Scholar]
  • Traynor-Kaplan AE, Harris AL, Thompson BL, Taylor P, Sklar LA. An inositol tetrakisphosphate-containing phospholipid in activated neutrophils. Nature. 1988 Jul 28;334(6180):353–356. [PubMed] [Google Scholar]
  • Traynor-Kaplan AE, Thompson BL, Harris AL, Taylor P, Omann GM, Sklar LA. Transient increase in phosphatidylinositol 3,4-bisphosphate and phosphatidylinositol trisphosphate during activation of human neutrophils. J Biol Chem. 1989 Sep 15;264(26):15668–15673. [PubMed] [Google Scholar]
  • Tyers M, Tokiwa G, Nash R, Futcher B. The Cln3-Cdc28 kinase complex of S. cerevisiae is regulated by proteolysis and phosphorylation. EMBO J. 1992 May;11(5):1773–1784. [PMC free article] [PubMed] [Google Scholar]
  • Ulug ET, Hawkins PT, Hanley MR, Courtneidge SA. Phosphatidylinositol metabolism in cells transformed by polyomavirus middle T antigen. J Virol. 1990 Aug;64(8):3895–3904. [PMC free article] [PubMed] [Google Scholar]
  • Valius M, Kazlauskas A. Phospholipase C-gamma 1 and phosphatidylinositol 3 kinase are the downstream mediators of the PDGF receptor's mitogenic signal. Cell. 1993 Apr 23;73(2):321–334. [PubMed] [Google Scholar]
  • Varticovski L, Daley GQ, Jackson P, Baltimore D, Cantley LC. Activation of phosphatidylinositol 3-kinase in cells expressing abl oncogene variants. Mol Cell Biol. 1991 Feb;11(2):1107–1113. [PMC free article] [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]
  • Vennström B, Bishop JM. Isolation and characterization of chicken DNA homologous to the two putative oncogenes of avian erythroblastosis virus. Cell. 1982 Jan;28(1):135–143. [PubMed] [Google Scholar]
  • Whitman M, Downes CP, Keeler M, Keller T, Cantley L. Type I phosphatidylinositol kinase makes a novel inositol phospholipid, phosphatidylinositol-3-phosphate. Nature. 1988 Apr 14;332(6165):644–646. [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]
  • Yu JC, Heidaran MA, Pierce JH, Gutkind JS, Lombardi D, Ruggiero M, Aaronson SA. Tyrosine mutations within the alpha platelet-derived growth factor receptor kinase insert domain abrogate receptor-associated phosphatidylinositol-3 kinase activity without affecting mitogenic or chemotactic signal transduction. Mol Cell Biol. 1991 Jul;11(7):3780–3785. [PMC free article] [PubMed] [Google Scholar]
  • Zippel R, Sturani E, Toschi L, Naldini L, Alberghina L, Comoglio PM. In vivo phosphorylation and dephosphorylation of the platelet-derived growth factor receptor studied by immunoblot analysis with phosphotyrosine antibodies. Biochim Biophys Acta. 1986 Mar 19;881(1):54–61. [PubMed] [Google Scholar]
Articles from Molecular and Cellular Biology are provided here courtesy of Taylor & Francis
-