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. 1999 Mar 1;18(5):1292–1302. doi: 10.1093/emboj/18.5.1292

Autophosphorylation of p110delta phosphoinositide 3-kinase: a new paradigm for the regulation of lipid kinases in vitro and in vivo.

B Vanhaesebroeck 1, K Higashi 1, C Raven 1, M Welham 1, S Anderson 1, P Brennan 1, S G Ward 1, M D Waterfield 1
PMCID: PMC1171219  PMID: 10064595

Abstract

Phosphoinositide 3-kinases (PI3Ks) are lipid kinases which also possess an in vitro protein kinase activity towards themselves or their adaptor proteins. The physiological relevance of these phosphorylations is unclear at present. Here, the protein kinase activity of the tyrosine kinase-linked PI3K, p110delta, is characterized and its functional impact assessed. In vitro autophosphorylation of p110delta completely down-regulates its lipid kinase activity. The single site of autophosphorylation was mapped to Ser1039 at the C-terminus of p110delta. Antisera specific for phospho-Ser1039 revealed a very low level of phosphorylation of this residue in cell lines. However, p110delta that is recruited to activated receptors (such as CD28 in T cells) shows a time-dependent increase in Ser1039 phosphorylation and a concomitant decrease in associated lipid kinase activity. Treatment of cells with okadaic acid, an inhibitor of Ser/Thr phosphatases, also dramatically increases the level of Ser1039-phosphorylated p110delta. LY294002 and wortmannin blocked these in vivo increases in Ser1039 phosphorylation, consistent with the notion that PI3Ks, and possibly p110delta itself, are involved in the in vivo phosphorylation of p110delta. In summary, we show that PI3Ks are subject to regulatory phosphorylations in vivo similar to those identified under in vitro conditions, identifying a new level of control of these signalling molecules.

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Selected References

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  1. Antonetti D. A., Algenstaedt P., Kahn C. R. Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain. Mol Cell Biol. 1996 May;16(5):2195–2203. doi: 10.1128/mcb.16.5.2195. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Backer J. M., Myers M. G., Jr, Shoelson S. E., Chin D. J., Sun X. J., Miralpeix M., Hu P., Margolis B., Skolnik E. Y., Schlessinger J. Phosphatidylinositol 3'-kinase is activated by association with IRS-1 during insulin stimulation. EMBO J. 1992 Sep;11(9):3469–3479. doi: 10.1002/j.1460-2075.1992.tb05426.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Banin S., Moyal L., Shieh S., Taya Y., Anderson C. W., Chessa L., Smorodinsky N. I., Prives C., Reiss Y., Shiloh Y. Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science. 1998 Sep 11;281(5383):1674–1677. doi: 10.1126/science.281.5383.1674. [DOI] [PubMed] [Google Scholar]
  4. Bondeva T., Pirola L., Bulgarelli-Leva G., Rubio I., Wetzker R., Wymann M. P. Bifurcation of lipid and protein kinase signals of PI3Kgamma to the protein kinases PKB and MAPK. Science. 1998 Oct 9;282(5387):293–296. doi: 10.1126/science.282.5387.293. [DOI] [PubMed] [Google Scholar]
  5. Brunn G. J., Hudson C. C., Sekulić A., Williams J. M., Hosoi H., Houghton P. J., Lawrence J. C., Jr, Abraham R. T. Phosphorylation of the translational repressor PHAS-I by the mammalian target of rapamycin. Science. 1997 Jul 4;277(5322):99–101. doi: 10.1126/science.277.5322.99. [DOI] [PubMed] [Google Scholar]
  6. Brunn G. J., Williams J., Sabers C., Wiederrecht G., Lawrence J. C., Jr, Abraham R. T. Direct inhibition of the signaling functions of the mammalian target of rapamycin by the phosphoinositide 3-kinase inhibitors, wortmannin and LY294002. EMBO J. 1996 Oct 1;15(19):5256–5267. [PMC free article] [PubMed] [Google Scholar]
  7. Canman C. E., Lim D. S., Cimprich K. A., Taya Y., Tamai K., Sakaguchi K., Appella E., Kastan M. B., Siliciano J. D. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53. Science. 1998 Sep 11;281(5383):1677–1679. doi: 10.1126/science.281.5383.1677. [DOI] [PubMed] [Google Scholar]
  8. Carpenter C. L., Auger K. R., Chanudhuri M., Yoakim M., Schaffhausen B., Shoelson S., Cantley L. C. 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]
  9. Carpenter C. L., Auger K. R., Duckworth B. C., Hou W. M., Schaffhausen B., Cantley L. C. A tightly associated serine/threonine protein kinase regulates phosphoinositide 3-kinase activity. Mol Cell Biol. 1993 Mar;13(3):1657–1665. doi: 10.1128/mcb.13.3.1657. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Cohen B., Liu Y. X., Druker B., Roberts T. M., Schaffhausen B. S. Characterization of pp85, a target of oncogenes and growth factor receptors. Mol Cell Biol. 1990 Jun;10(6):2909–2915. doi: 10.1128/mcb.10.6.2909. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dhand R., Hara K., Hiles I., Bax B., Gout I., Panayotou G., Fry M. J., Yonezawa K., Kasuga M., Waterfield M. D. PI 3-kinase: structural and functional analysis of intersubunit interactions. EMBO J. 1994 Feb 1;13(3):511–521. doi: 10.1002/j.1460-2075.1994.tb06289.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Dhand R., Hiles I., Panayotou G., Roche S., Fry M. J., Gout I., Totty N. F., Truong O., Vicendo P., Yonezawa K. PI 3-kinase is a dual specificity enzyme: autoregulation by an intrinsic protein-serine kinase activity. EMBO J. 1994 Feb 1;13(3):522–533. doi: 10.1002/j.1460-2075.1994.tb06290.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Domin J., Dhand R., Waterfield M. D. Binding to the platelet-derived growth factor receptor transiently activates the p85alpha-p110alpha phosphoinositide 3-kinase complex in vivo. J Biol Chem. 1996 Aug 30;271(35):21614–21621. doi: 10.1074/jbc.271.35.21614. [DOI] [PubMed] [Google Scholar]
  14. End P., Gout I., Fry M. J., Panayotou G., Dhand R., Yonezawa K., Kasuga M., Waterfield M. D. A biosensor approach to probe the structure and function of the p85 alpha subunit of the phosphatidylinositol 3-kinase complex. J Biol Chem. 1993 May 15;268(14):10066–10075. [PubMed] [Google Scholar]
  15. Evans D. R., Hemmings B. A. Signal transduction. What goes up must come down. Nature. 1998 Jul 2;394(6688):23–24. doi: 10.1038/27782. [DOI] [PubMed] [Google Scholar]
  16. Franke T. F., Kaplan D. R., Cantley L. C. PI3K: downstream AKTion blocks apoptosis. Cell. 1997 Feb 21;88(4):435–437. doi: 10.1016/s0092-8674(00)81883-8. [DOI] [PubMed] [Google Scholar]
  17. Fruman D. A., Meyers R. E., Cantley L. C. Phosphoinositide kinases. Annu Rev Biochem. 1998;67:481–507. doi: 10.1146/annurev.biochem.67.1.481. [DOI] [PubMed] [Google Scholar]
  18. He Z., He Y. S., Kim Y., Chu L., Ohmstede C., Biron K. K., Coen D. M. The human cytomegalovirus UL97 protein is a protein kinase that autophosphorylates on serines and threonines. J Virol. 1997 Jan;71(1):405–411. doi: 10.1128/jvi.71.1.405-411.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Holt K. H., Olson L., Moye-Rowley W. S., Pessin J. E. Phosphatidylinositol 3-kinase activation is mediated by high-affinity interactions between distinct domains within the p110 and p85 subunits. Mol Cell Biol. 1994 Jan;14(1):42–49. doi: 10.1128/mcb.14.1.42. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hu Q., Klippel A., Muslin A. J., Fantl W. J., Williams L. T. Ras-dependent induction of cellular responses by constitutively active phosphatidylinositol-3 kinase. Science. 1995 Apr 7;268(5207):100–102. doi: 10.1126/science.7701328. [DOI] [PubMed] [Google Scholar]
  21. Hunter T. When is a lipid kinase not a lipid kinase? When it is a protein kinase. Cell. 1995 Oct 6;83(1):1–4. doi: 10.1016/0092-8674(95)90225-2. [DOI] [PubMed] [Google Scholar]
  22. Isakov N., Wange R. L., Watts J. D., Aebersold R., Samelson L. E. Purification and characterization of human ZAP-70 protein-tyrosine kinase from a baculovirus expression system. J Biol Chem. 1996 Jun 28;271(26):15753–15761. doi: 10.1074/jbc.271.26.15753. [DOI] [PubMed] [Google Scholar]
  23. Jimenez C., Jones D. R., Rodríguez-Viciana P., Gonzalez-García A., Leonardo E., Wennström S., von Kobbe C., Toran J. L., R-Borlado L., Calvo V. Identification and characterization of a new oncogene derived from the regulatory subunit of phosphoinositide 3-kinase. EMBO J. 1998 Feb 2;17(3):743–753. doi: 10.1093/emboj/17.3.743. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kaplan D. R., Whitman M., Schaffhausen B., Pallas D. C., White M., Cantley L., Roberts T. M. Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity. Cell. 1987 Sep 25;50(7):1021–1029. doi: 10.1016/0092-8674(87)90168-1. [DOI] [PubMed] [Google Scholar]
  25. Keith C. T., Schreiber S. L. PIK-related kinases: DNA repair, recombination, and cell cycle checkpoints. Science. 1995 Oct 6;270(5233):50–51. doi: 10.1126/science.270.5233.50. [DOI] [PubMed] [Google Scholar]
  26. Klippel A., Escobedo J. A., Hirano M., Williams L. T. The interaction of small domains between the subunits of phosphatidylinositol 3-kinase determines enzyme activity. Mol Cell Biol. 1994 Apr;14(4):2675–2685. doi: 10.1128/mcb.14.4.2675. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Lam K., Carpenter C. L., Ruderman N. B., Friel J. C., Kelly K. L. The phosphatidylinositol 3-kinase serine kinase phosphorylates IRS-1. Stimulation by insulin and inhibition by Wortmannin. J Biol Chem. 1994 Aug 12;269(32):20648–20652. [PubMed] [Google Scholar]
  28. Lees-Miller S. P., Sakaguchi K., Ullrich S. J., Appella E., Anderson C. W. Human DNA-activated protein kinase phosphorylates serines 15 and 37 in the amino-terminal transactivation domain of human p53. Mol Cell Biol. 1992 Nov;12(11):5041–5049. doi: 10.1128/mcb.12.11.5041. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pleiman C. M., Hertz W. M., Cambier J. C. Activation of phosphatidylinositol-3' kinase by Src-family kinase SH3 binding to the p85 subunit. Science. 1994 Mar 18;263(5153):1609–1612. doi: 10.1126/science.8128248. [DOI] [PubMed] [Google Scholar]
  30. Reif K., Gout I., Waterfield M. D., Cantrell D. A. Divergent regulation of phosphatidylinositol 3-kinase P85 alpha and P85 beta isoforms upon T cell activation. J Biol Chem. 1993 May 25;268(15):10780–10788. [PubMed] [Google Scholar]
  31. Rodriguez-Viciana P., Warne P. H., Vanhaesebroeck B., Waterfield M. D., Downward J. Activation of phosphoinositide 3-kinase by interaction with Ras and by point mutation. EMBO J. 1996 May 15;15(10):2442–2451. [PMC free article] [PubMed] [Google Scholar]
  32. Rubio I., Rodriguez-Viciana P., Downward J., Wetzker R. Interaction of Ras with phosphoinositide 3-kinase gamma. Biochem J. 1997 Sep 15;326(Pt 3):891–895. doi: 10.1042/bj3260891. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Schinkmann K., Blenis J. Cloning and characterization of a human STE20-like protein kinase with unusual cofactor requirements. J Biol Chem. 1997 Nov 7;272(45):28695–28703. doi: 10.1074/jbc.272.45.28695. [DOI] [PubMed] [Google Scholar]
  34. Shepherd P. R., Withers D. J., Siddle K. Phosphoinositide 3-kinase: the key switch mechanism in insulin signalling. Biochem J. 1998 Aug 1;333(Pt 3):471–490. doi: 10.1042/bj3330471. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Shieh S. Y., Ikeda M., Taya Y., Prives C. DNA damage-induced phosphorylation of p53 alleviates inhibition by MDM2. Cell. 1997 Oct 31;91(3):325–334. doi: 10.1016/s0092-8674(00)80416-x. [DOI] [PubMed] [Google Scholar]
  36. Stack J. H., Emr S. D. Vps34p required for yeast vacuolar protein sorting is a multiple specificity kinase that exhibits both protein kinase and phosphatidylinositol-specific PI 3-kinase activities. J Biol Chem. 1994 Dec 16;269(50):31552–31562. [PubMed] [Google Scholar]
  37. Stephens L. R., Jackson T. R., Hawkins P. T. Agonist-stimulated synthesis of phosphatidylinositol(3,4,5)-trisphosphate: a new intracellular signalling system? Biochim Biophys Acta. 1993 Oct 7;1179(1):27–75. doi: 10.1016/0167-4889(93)90072-w. [DOI] [PubMed] [Google Scholar]
  38. Stoyanova S., Bulgarelli-Leva G., Kirsch C., Hanck T., Klinger R., Wetzker R., Wymann M. P. Lipid kinase and protein kinase activities of G-protein-coupled phosphoinositide 3-kinase gamma: structure-activity analysis and interactions with wortmannin. Biochem J. 1997 Jun 1;324(Pt 2):489–495. doi: 10.1042/bj3240489. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Tanti J. F., Grémeaux T., Van Obberghen E., Le Marchand-Brustel Y. Insulin receptor substrate 1 is phosphorylated by the serine kinase activity of phosphatidylinositol 3-kinase. Biochem J. 1994 Nov 15;304(Pt 1):17–21. doi: 10.1042/bj3040017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Toker A., Cantley L. C. Signalling through the lipid products of phosphoinositide-3-OH kinase. Nature. 1997 Jun 12;387(6634):673–676. doi: 10.1038/42648. [DOI] [PubMed] [Google Scholar]
  41. Uddin S., Fish E. N., Sher D. A., Gardziola C., White M. F., Platanias L. C. Activation of the phosphatidylinositol 3-kinase serine kinase by IFN-alpha. J Immunol. 1997 Mar 1;158(5):2390–2397. [PubMed] [Google Scholar]
  42. Ueda Y., Levine B. L., Huang M. L., Freeman G. J., Nadler L. M., June C. H., Ward S. G. Both CD28 ligands CD80 (B7-1) and CD86 (B7-2) activate phosphatidylinositol 3-kinase, and wortmannin reveals heterogeneity in the regulation of T cell IL-2 secretion. Int Immunol. 1995 Jun;7(6):957–966. doi: 10.1093/intimm/7.6.957. [DOI] [PubMed] [Google Scholar]
  43. Vanhaesebroeck B., Leevers S. J., Panayotou G., Waterfield M. D. Phosphoinositide 3-kinases: a conserved family of signal transducers. Trends Biochem Sci. 1997 Jul;22(7):267–272. doi: 10.1016/s0968-0004(97)01061-x. [DOI] [PubMed] [Google Scholar]
  44. Vanhaesebroeck B., Welham M. J., Kotani K., Stein R., Warne P. H., Zvelebil M. J., Higashi K., Volinia S., Downward J., Waterfield M. D. P110delta, a novel phosphoinositide 3-kinase in leukocytes. Proc Natl Acad Sci U S A. 1997 Apr 29;94(9):4330–4335. doi: 10.1073/pnas.94.9.4330. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Volinia S., Dhand R., Vanhaesebroeck B., MacDougall L. K., Stein R., Zvelebil M. J., Domin J., Panaretou C., Waterfield M. D. A human phosphatidylinositol 3-kinase complex related to the yeast Vps34p-Vps15p protein sorting system. EMBO J. 1995 Jul 17;14(14):3339–3348. doi: 10.1002/j.1460-2075.1995.tb07340.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Ward S. G., June C. H., Olive D. PI 3-kinase: a pivotal pathway in T-cell activation? Immunol Today. 1996 Apr;17(4):187–197. doi: 10.1016/0167-5699(96)80618-9. [DOI] [PubMed] [Google Scholar]
  47. Ward S. G., Reif K., Ley S., Fry M. J., Waterfield M. D., Cantrell D. A. Regulation of phosphoinositide kinases in T cells. Evidence that phosphatidylinositol 3-kinase is not a substrate for T cell antigen receptor-regulated tyrosine kinases. J Biol Chem. 1992 Nov 25;267(33):23862–23869. [PubMed] [Google Scholar]
  48. Ward S. G., Westwick J., Hall N. D., Sansom D. M. Ligation of CD28 receptor by B7 induces formation of D-3 phosphoinositides in T lymphocytes independently of T cell receptor/CD3 activation. Eur J Immunol. 1993 Oct;23(10):2572–2577. doi: 10.1002/eji.1830231029. [DOI] [PubMed] [Google Scholar]
  49. Weinkove D., Leevers S. J., MacDougall L. K., Waterfield M. D. p60 is an adaptor for the Drosophila phosphoinositide 3-kinase, Dp110. J Biol Chem. 1997 Jun 6;272(23):14606–14610. doi: 10.1074/jbc.272.23.14606. [DOI] [PubMed] [Google Scholar]
  50. Welham M. J., Duronio V., Schrader J. W. Interleukin-4-dependent proliferation dissociates p44erk-1, p42erk-2, and p21ras activation from cell growth. J Biol Chem. 1994 Feb 25;269(8):5865–5873. [PubMed] [Google Scholar]
  51. Withers D. J., Ouwens D. M., Nave B. T., van der Zon G. C., Alarcon C. M., Cardenas M. E., Heitman J., Maassen J. A., Shepherd P. R. Expression, enzyme activity, and subcellular localization of mammalian target of rapamycin in insulin-responsive cells. Biochem Biophys Res Commun. 1997 Dec 29;241(3):704–709. doi: 10.1006/bbrc.1997.7878. [DOI] [PubMed] [Google Scholar]
  52. Woo R. A., McLure K. G., Lees-Miller S. P., Rancourt D. E., Lee P. W. DNA-dependent protein kinase acts upstream of p53 in response to DNA damage. Nature. 1998 Aug 13;394(6694):700–704. doi: 10.1038/29343. [DOI] [PubMed] [Google Scholar]
  53. Woscholski R., Kodaki T., McKinnon M., Waterfield M. D., Parker P. J. A comparison of demethoxyviridin and wortmannin as inhibitors of phosphatidylinositol 3-kinase. FEBS Lett. 1994 Apr 4;342(2):109–114. doi: 10.1016/0014-5793(94)80482-6. [DOI] [PubMed] [Google Scholar]
  54. Wymann M. P., Bulgarelli-Leva G., Zvelebil M. J., Pirola L., Vanhaesebroeck B., Waterfield M. D., Panayotou G. Wortmannin inactivates phosphoinositide 3-kinase by covalent modification of Lys-802, a residue involved in the phosphate transfer reaction. Mol Cell Biol. 1996 Apr;16(4):1722–1733. doi: 10.1128/mcb.16.4.1722. [DOI] [PMC free article] [PubMed] [Google Scholar]
  55. Wymann M. P., Pirola L. Structure and function of phosphoinositide 3-kinases. Biochim Biophys Acta. 1998 Dec 8;1436(1-2):127–150. doi: 10.1016/s0005-2760(98)00139-8. [DOI] [PubMed] [Google Scholar]
  56. Yu J., Zhang Y., McIlroy J., Rordorf-Nikolic T., Orr G. A., Backer J. M. Regulation of the p85/p110 phosphatidylinositol 3'-kinase: stabilization and inhibition of the p110alpha catalytic subunit by the p85 regulatory subunit. Mol Cell Biol. 1998 Mar;18(3):1379–1387. doi: 10.1128/mcb.18.3.1379. [DOI] [PMC free article] [PubMed] [Google Scholar]
  57. Zvelebil M. J., MacDougall L., Leevers S., Volinia S., Vanhaesebroeck B., Gout I., Panayotou G., Domin J., Stein R., Pages F. Structural and functional diversity of phosphoinositide 3-kinases. Philos Trans R Soc Lond B Biol Sci. 1996 Feb 29;351(1336):217–223. doi: 10.1098/rstb.1996.0019. [DOI] [PubMed] [Google Scholar]

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