Skip to main content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Biochem J. 1996 May 1; 315(Pt 3): 709–713.
doi: 10.1042/bj3150709
PMCID: PMC1217264
PMID: 8645147

Specific binding of the Akt-1 protein kinase to phosphatidylinositol 3,4,5-trisphosphate without subsequent activation.

S R James, C P Downes, R Gigg, S J Grove, A B Holmes, and D R Alessi
Author information Copyright and License information PMC Disclaimer

Abstract

Recent evidence has suggested that activation of phosphoinositide 3-kinase (PI 3-kinase) is required for the activation of Akt-1 by growth factors and insulin. Here we demonstrate by two independent methods that Akt-1 from L6 myotubes binds to PtdIns(3,4,5)P3, PtdIns(3,4)P2 and PtdIns(4,5)P2 when presented against a background of phosphatidylserine (PtdSer) or a 1:1 mixture of PtdSer and phosphatidylcholine (PtdCho). No binding was observed with the lipids PtdIns(3,5)P2, PtdIns4P and PtdIns3P or background lipids. Activated, hyperphosphorylated forms of Akt-1 from insulin-stimulated L6 myotubes bound to PtdIns(3,4,5)P3 in a similar manner as inactive Akt-1. Quantitative analysis using surface plasmon resonance showed that the equilibrium association constant for the binding of Akt-1 to PtdIns(3,4,5)P3 was submicromolar and that PtdIns(3,4)P2 and PtdIns(4,5)P2 bound to Akt-1 with 3- and 6-fold lower affinities respectively. Interaction of Akt-1 with PtdIns(3,4,5)P3 did not activate the protein kinase activity, either before or after incubation with MgATP. A model is presented in which PtdIns(3,4,5)P3 may prime Akt-1 for activation by another protein kinase, perhaps by recruiting it to the plasma membrane.

Full Text

The Full Text of this article is available as a PDF (360K).

Selected References

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

  • Stephens LR, Jackson TR, Hawkins PT. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta. 1993 Oct 7;1179(1):27–75. [PubMed] [Google Scholar]
  • Chung J, Grammer TC, Lemon KP, Kazlauskas A, Blenis J. PDGF- and insulin-dependent pp70S6k activation mediated by phosphatidylinositol-3-OH kinase. Nature. 1994 Jul 7;370(6484):71–75. [PubMed] [Google Scholar]
  • Baxter RM, Cohen P, Obermeier A, Ullrich A, Downes CP, Doza YN. Phosphotyrosine residues in the nerve-growth-factor receptor (Trk-A). Their role in the activation of inositolphospholipid metabolism and protein kinase cascades in phaeochromocytoma (PC12) cells. Eur J Biochem. 1995 Nov 15;234(1):84–91. [PubMed] [Google Scholar]
  • Cross DA, Alessi DR, Vandenheede JR, McDowell HE, Hundal HS, Cohen P. The inhibition of glycogen synthase kinase-3 by insulin or insulin-like growth factor 1 in the rat skeletal muscle cell line L6 is blocked by wortmannin, but not by rapamycin: evidence that wortmannin blocks activation of the mitogen-activated protein kinase pathway in L6 cells between Ras and Raf. Biochem J. 1994 Oct 1;303(Pt 1):21–26. [PMC free article] [PubMed] [Google Scholar]
  • Welsh GI, Foulstone EJ, Young SW, Tavaré JM, Proud CG. Wortmannin inhibits the effects of insulin and serum on the activities of glycogen synthase kinase-3 and mitogen-activated protein kinase. Biochem J. 1994 Oct 1;303(Pt 1):15–20. [PMC free article] [PubMed] [Google Scholar]
  • Karnitz LM, Burns LA, Sutor SL, Blenis J, Abraham RT. Interleukin-2 triggers a novel phosphatidylinositol 3-kinase-dependent MEK activation pathway. Mol Cell Biol. 1995 Jun;15(6):3049–3057. [PMC free article] [PubMed] [Google Scholar]
  • Hawkins PT, Eguinoa A, Qiu RG, Stokoe D, Cooke FT, Walters R, Wennström S, Claesson-Welsh L, Evans T, Symons M, et al. PDGF stimulates an increase in GTP-Rac via activation of phosphoinositide 3-kinase. Curr Biol. 1995 Apr 1;5(4):393–403. [PubMed] [Google Scholar]
  • Parker PJ. Intracellular signalling. PI 3-kinase puts GTP on the Rac. Curr Biol. 1995 Jun 1;5(6):577–579. [PubMed] [Google Scholar]
  • Burgering BM, Coffer PJ. Protein kinase B (c-Akt) in phosphatidylinositol-3-OH kinase signal transduction. Nature. 1995 Aug 17;376(6541):599–602. [PubMed] [Google Scholar]
  • Franke TF, Yang SI, Chan TO, Datta K, Kazlauskas A, Morrison DK, Kaplan DR, Tsichlis PN. The protein kinase encoded by the Akt proto-oncogene is a target of the PDGF-activated phosphatidylinositol 3-kinase. Cell. 1995 Jun 2;81(5):727–736. [PubMed] [Google Scholar]
  • Kohn AD, Kovacina KS, Roth RA. Insulin stimulates the kinase activity of RAC-PK, a pleckstrin homology domain containing ser/thr kinase. EMBO J. 1995 Sep 1;14(17):4288–4295. [PMC free article] [PubMed] [Google Scholar]
  • Coffer PJ, Woodgett JR. Molecular cloning and characterisation of a novel putative protein-serine kinase related to the cAMP-dependent and protein kinase C families. Eur J Biochem. 1991 Oct 15;201(2):475–481. [PubMed] [Google Scholar]
  • Jones PF, Jakubowicz T, Pitossi FJ, Maurer F, Hemmings BA. Molecular cloning and identification of a serine/threonine protein kinase of the second-messenger subfamily. Proc Natl Acad Sci U S A. 1991 May 15;88(10):4171–4175. [PMC free article] [PubMed] [Google Scholar]
  • Cheng JQ, Godwin AK, Bellacosa A, Taguchi T, Franke TF, Hamilton TC, Tsichlis PN, Testa JR. AKT2, a putative oncogene encoding a member of a subfamily of protein-serine/threonine kinases, is amplified in human ovarian carcinomas. Proc Natl Acad Sci U S A. 1992 Oct 1;89(19):9267–9271. [PMC free article] [PubMed] [Google Scholar]
  • Konishi H, Kuroda S, Tanaka M, Matsuzaki H, Ono Y, Kameyama K, Haga T, Kikkawa U. Molecular cloning and characterization of a new member of the RAC protein kinase family: association of the pleckstrin homology domain of three types of RAC protein kinase with protein kinase C subspecies and beta gamma subunits of G proteins. Biochem Biophys Res Commun. 1995 Nov 13;216(2):526–534. [PubMed] [Google Scholar]
  • Harlan JE, Hajduk PJ, Yoon HS, Fesik SW. Pleckstrin homology domains bind to phosphatidylinositol-4,5-bisphosphate. Nature. 1994 Sep 8;371(6493):168–170. [PubMed] [Google Scholar]
  • Hyvönen M, Macias MJ, Nilges M, Oschkinat H, Saraste M, Wilmanns M. Structure of the binding site for inositol phosphates in a PH domain. EMBO J. 1995 Oct 2;14(19):4676–4685. [PMC free article] [PubMed] [Google Scholar]
  • Lemmon MA, Ferguson KM, O'Brien R, Sigler PB, Schlessinger J. Specific and high-affinity binding of inositol phosphates to an isolated pleckstrin homology domain. Proc Natl Acad Sci U S A. 1995 Nov 7;92(23):10472–10476. [PMC free article] [PubMed] [Google Scholar]
  • Pitcher JA, Touhara K, Payne ES, Lefkowitz RJ. Pleckstrin homology domain-mediated membrane association and activation of the beta-adrenergic receptor kinase requires coordinate interaction with G beta gamma subunits and lipid. J Biol Chem. 1995 May 19;270(20):11707–11710. [PubMed] [Google Scholar]
  • Cross DA, Alessi DR, Cohen P, Andjelkovich M, Hemmings BA. Inhibition of glycogen synthase kinase-3 by insulin mediated by protein kinase B. Nature. 1995 Dec 21;378(6559):785–789. [PubMed] [Google Scholar]
  • Downward J. Signal transduction. A target for PI(3) kinase. Nature. 1995 Aug 17;376(6541):553–554. [PubMed] [Google Scholar]
  • Bos JL. A target for phosphoinositide 3-kinase: Akt/PKB. Trends Biochem Sci. 1995 Nov;20(11):441–442. [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]
  • James SR, Demel RA, Downes CP. Interfacial hydrolysis of phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate by turkey erythrocyte phospholipase C. Biochem J. 1994 Mar 1;298(Pt 2):499–506. [PMC free article] [PubMed] [Google Scholar]
  • Hiraga A, Kemp BE, Cohen P. Further studies on the structure of the glycogen-bound form of protein phosphatase-1 from rabbit skeletal muscle. Eur J Biochem. 1987 Mar 2;163(2):253–258. [PubMed] [Google Scholar]
  • Mitsumoto Y, Klip A. Development regulation of the subcellular distribution and glycosylation of GLUT1 and GLUT4 glucose transporters during myogenesis of L6 muscle cells. J Biol Chem. 1992 Mar 5;267(7):4957–4962. [PubMed] [Google Scholar]
  • James SR, Paterson A, Harden TK, Downes CP. Kinetic analysis of phospholipase C beta isoforms using phospholipid-detergent mixed micelles. Evidence for interfacial catalysis involving distinct micelle binding and catalytic steps. J Biol Chem. 1995 May 19;270(20):11872–11881. [PubMed] [Google Scholar]
  • Fägerstam LG, Frostell-Karlsson A, Karlsson R, Persson B, Rönnberg I. Biospecific interaction analysis using surface plasmon resonance detection applied to kinetic, binding site and concentration analysis. J Chromatogr. 1992 Apr 24;597(1-2):397–410. [PubMed] [Google Scholar]
  • Rebecchi M, Peterson A, McLaughlin S. Phosphoinositide-specific phospholipase C-delta 1 binds with high affinity to phospholipid vesicles containing phosphatidylinositol 4,5-bisphosphate. Biochemistry. 1992 Dec 29;31(51):12742–12747. [PubMed] [Google Scholar]
  • Rameh LE, Chen CS, Cantley LC. Phosphatidylinositol (3,4,5)P3 interacts with SH2 domains and modulates PI 3-kinase association with tyrosine-phosphorylated proteins. Cell. 1995 Dec 1;83(5):821–830. [PubMed] [Google Scholar]
  • Nakanishi H, Brewer KA, Exton JH. Activation of the zeta isozyme of protein kinase C by phosphatidylinositol 3,4,5-trisphosphate. J Biol Chem. 1993 Jan 5;268(1):13–16. [PubMed] [Google Scholar]
  • Toker A, Meyer M, Reddy KK, Falck JR, Aneja R, Aneja S, Parra A, Burns DJ, Ballas LM, Cantley LC. Activation of protein kinase C family members by the novel polyphosphoinositides PtdIns-3,4-P2 and PtdIns-3,4,5-P3. J Biol Chem. 1994 Dec 23;269(51):32358–32367. [PubMed] [Google Scholar]
  • Palmer RH, Dekker LV, Woscholski R, Le Good JA, Gigg R, Parker PJ. Activation of PRK1 by phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate. A comparison with protein kinase C isotypes. J Biol Chem. 1995 Sep 22;270(38):22412–22416. [PubMed] [Google Scholar]
  • Leevers SJ, Paterson HF, Marshall CJ. Requirement for Ras in Raf activation is overcome by targeting Raf to the plasma membrane. Nature. 1994 Jun 2;369(6479):411–414. [PubMed] [Google Scholar]
  • Stokoe D, Macdonald SG, Cadwallader K, Symons M, Hancock JF. Activation of Raf as a result of recruitment to the plasma membrane. Science. 1994 Jun 3;264(5164):1463–1467. [PubMed] [Google Scholar]
  • Staal SP, Hartley JW, Rowe WP. Isolation of transforming murine leukemia viruses from mice with a high incidence of spontaneous lymphoma. Proc Natl Acad Sci U S A. 1977 Jul;74(7):3065–3067. [PMC free article] [PubMed] [Google Scholar]
Articles from Biochemical Journal are provided here courtesy of The Biochemical Society
-