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. 1990 Dec;87(23):9396–9400. doi: 10.1073/pnas.87.23.9396

Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.

C A Mitchell 1, A B Jefferson 1, B E Bejeck 1, J S Brugge 1, T F Deuel 1, P W Majerus 1
PMCID: PMC55172  PMID: 2174561

Abstract

Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated with a time course and concentration dependence consistent with a role for these lipids in platelet activation. We now report that when human platelets are stimulated with thrombin, a complex forms between phosphatidylinositol 3-kinase, a protein-serine/threonine kinase, and an uncharacterized platelet membrane protein. The complex is immunoprecipitated from detergent lysates of thrombinstimulated platelets by a rabbit antiserum prepared against a peptide from the cytoplasmic domain of the mouse platelet-derived growth factor (PDGF) receptor. The antigen is not the PDGF receptor, since complex formation is not stimulated by PDGF and thrombin-induced complexes are not precipitated by another rabbit antiserum against the same peptide or by monoclonal anti-human PDGF receptor antibodies. Formation of the complex is rapid (within 30 sec) and occurs at thrombin concentrations that stimulate platelet aggregation and secretion (50% of maximal complex formation at 0.03 unit of thrombin per ml). We propose that the complex initiates formation of 3-phosphate-containing phosphatidylinositols that may function in platelet activation.

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Selected References

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

  1. Auger K. R., Serunian L. A., Soltoff S. P., Libby P., Cantley L. C. PDGF-dependent tyrosine phosphorylation stimulates production of novel polyphosphoinositides in intact cells. Cell. 1989 Apr 7;57(1):167–175. doi: 10.1016/0092-8674(89)90182-7. [DOI] [PubMed] [Google Scholar]
  2. Baenziger N. L., Majerus P. W. Isolation of human platelets and platelet surface membranes. Methods Enzymol. 1974;31:149–155. doi: 10.1016/0076-6879(74)31015-4. [DOI] [PubMed] [Google Scholar]
  3. Berridge M. J. Inositol trisphosphate and diacylglycerol: two interacting second messengers. Annu Rev Biochem. 1987;56:159–193. doi: 10.1146/annurev.bi.56.070187.001111. [DOI] [PubMed] [Google Scholar]
  4. Berridge M. J., Irvine R. F. Inositol trisphosphate, a novel second messenger in cellular signal transduction. Nature. 1984 Nov 22;312(5992):315–321. doi: 10.1038/312315a0. [DOI] [PubMed] [Google Scholar]
  5. Bjorge J. D., Chan T. O., Antczak M., Kung H. J., Fujita D. J. Activated type I phosphatidylinositol kinase is associated with the epidermal growth factor (EGF) receptor following EGF stimulation. Proc Natl Acad Sci U S A. 1990 May;87(10):3816–3820. doi: 10.1073/pnas.87.10.3816. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bryckaert M. C., Rendu F., Tobelem G., Wasteson A. Collagen-induced binding to human platelets of platelet-derived growth factor leading to inhibition of P43 and P20 phosphorylation. J Biol Chem. 1989 Mar 15;264(8):4336–4341. [PubMed] [Google Scholar]
  7. Cama A., Marcus-Samuels B., Taylor S. I. Immunological abnormalities in insulin receptors on cultured EBV-transformed lymphocytes from insulin-resistant patient with leprechaunism. Diabetes. 1988 Jul;37(7):982–988. doi: 10.2337/diab.37.7.982. [DOI] [PubMed] [Google Scholar]
  8. Cleveland D. W., Fischer S. G., Kirschner M. W., Laemmli U. K. Peptide mapping by limited proteolysis in sodium dodecyl sulfate and analysis by gel electrophoresis. J Biol Chem. 1977 Feb 10;252(3):1102–1106. [PubMed] [Google Scholar]
  9. Cohen B., Yoakim M., Piwnica-Worms H., Roberts T. M., Schaffhausen B. S. Tyrosine phosphorylation is a signal for the trafficking of pp85, an 85-kDa phosphorylated polypeptide associated with phosphatidylinositol kinase activity. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4458–4462. doi: 10.1073/pnas.87.12.4458. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Coughlin S. R., Escobedo J. A., Williams L. T. Role of phosphatidylinositol kinase in PDGF receptor signal transduction. Science. 1989 Mar 3;243(4895):1191–1194. doi: 10.1126/science.2466336. [DOI] [PubMed] [Google Scholar]
  11. Courtneidge S. A., 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. doi: 10.1016/0092-8674(87)90169-3. [DOI] [PubMed] [Google Scholar]
  12. Druker B. J., Mamon H. J., Roberts T. M. Oncogenes, growth factors, and signal transduction. N Engl J Med. 1989 Nov 16;321(20):1383–1391. doi: 10.1056/NEJM198911163212007. [DOI] [PubMed] [Google Scholar]
  13. Fukui Y., Hanafusa H. Phosphatidylinositol kinase activity associates with viral p60src protein. Mol Cell Biol. 1989 Apr;9(4):1651–1658. doi: 10.1128/mcb.9.4.1651. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Glenney J. R., Jr, Zokas L., Kamps M. P. Monoclonal antibodies to phosphotyrosine. J Immunol Methods. 1988 May 9;109(2):277–285. doi: 10.1016/0022-1759(88)90253-0. [DOI] [PubMed] [Google Scholar]
  15. Golden A., Nemeth S. P., Brugge J. S. Blood platelets express high levels of the pp60c-src-specific tyrosine kinase activity. Proc Natl Acad Sci U S A. 1986 Feb;83(4):852–856. doi: 10.1073/pnas.83.4.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gutkind J. S., Lacal P. M., Robbins K. C. Thrombin-dependent association of phosphatidylinositol-3 kinase with p60c-src and p59fyn in human platelets. Mol Cell Biol. 1990 Jul;10(7):3806–3809. doi: 10.1128/mcb.10.7.3806. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Hart C. E., Seifert R. A., Ross R., Bowen-Pope D. F. Synthesis, phosphorylation, and degradation of multiple forms of the platelet-derived growth factor receptor studied using a monoclonal antibody. J Biol Chem. 1987 Aug 5;262(22):10780–10785. [PubMed] [Google Scholar]
  18. Hokin L. E. Receptors and phosphoinositide-generated second messengers. Annu Rev Biochem. 1985;54:205–235. doi: 10.1146/annurev.bi.54.070185.001225. [DOI] [PubMed] [Google Scholar]
  19. Horak I. D., Corcoran M. L., Thompson P. A., Wahl L. M., Bolen J. B. Expression of p60fyn in human platelets. Oncogene. 1990 Apr;5(4):597–602. [PubMed] [Google Scholar]
  20. Kai M., Salway J. G., Hawthorne J. N. The diphosphoinositide kinase of rat brain. Biochem J. 1968 Feb;106(4):791–801. doi: 10.1042/bj1060791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kaplan D. R., Morrison D. K., Wong G., McCormick F., Williams L. T. PDGF beta-receptor stimulates tyrosine phosphorylation of GAP and association of GAP with a signaling complex. Cell. 1990 Apr 6;61(1):125–133. doi: 10.1016/0092-8674(90)90220-9. [DOI] [PubMed] [Google Scholar]
  22. 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]
  23. Kaplan D. R., Whitman M., Schaffhausen B., Raptis L., Garcea R. L., Pallas D., Roberts T. M., Cantley L. Phosphatidylinositol metabolism and polyoma-mediated transformation. Proc Natl Acad Sci U S A. 1986 Jun;83(11):3624–3628. doi: 10.1073/pnas.83.11.3624. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Kucera G. L., Rittenhouse S. E. 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]
  25. Kumjian D. A., Wahl M. I., Rhee S. G., Daniel T. O. Platelet-derived growth factor (PDGF) binding promotes physical association of PDGF receptor with phospholipase C. Proc Natl Acad Sci U S A. 1989 Nov;86(21):8232–8236. doi: 10.1073/pnas.86.21.8232. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  27. Lips D. L., Majerus P. W., Gorga F. R., Young A. T., Benjamin T. L. 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]
  28. Lips D. L., Majerus P. W. The discovery of a 3-phosphomonoesterase that hydrolyzes phosphatidylinositol 3-phosphate in NIH 3T3 cells. J Biol Chem. 1989 Nov 25;264(33):19911–19915. [PubMed] [Google Scholar]
  29. Lipsich L. A., Lewis A. J., Brugge J. S. Isolation of monoclonal antibodies that recognize the transforming proteins of avian sarcoma viruses. J Virol. 1983 Nov;48(2):352–360. doi: 10.1128/jvi.48.2.352-360.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Lou L. L., Schulman H. Distinct autophosphorylation sites sequentially produce autonomy and inhibition of the multifunctional Ca2+/calmodulin-dependent protein kinase. J Neurosci. 1989 Jun;9(6):2020–2032. doi: 10.1523/JNEUROSCI.09-06-02020.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Majerus P. W., Connolly T. M., Deckmyn H., Ross T. S., Bross T. E., Ishii H., Bansal V. S., Wilson D. B. The metabolism of phosphoinositide-derived messenger molecules. Science. 1986 Dec 19;234(4783):1519–1526. doi: 10.1126/science.3024320. [DOI] [PubMed] [Google Scholar]
  32. McEver R. P., Baenziger N. L., Majerus P. W. Isolation and quantitation of the platelet membrane glycoprotein deficient in thrombasthenia using a monoclonal hybridoma antibody. J Clin Invest. 1980 Dec;66(6):1311–1318. doi: 10.1172/JCI109983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Miletich J. P., Jackson C. M., Majerus P. W. Properties of the factor Xa binding site on human platelets. J Biol Chem. 1978 Oct 10;253(19):6908–6916. [PubMed] [Google Scholar]
  34. Morrison D. K., Kaplan D. R., Escobedo J. A., Rapp U. R., Roberts T. M., Williams L. T. Direct activation of the serine/threonine kinase activity of Raf-1 through tyrosine phosphorylation by the PDGF beta-receptor. Cell. 1989 Aug 25;58(4):649–657. doi: 10.1016/0092-8674(89)90100-1. [DOI] [PubMed] [Google Scholar]
  35. Morrison D. K., Kaplan D. R., Rhee S. G., Williams L. T. Platelet-derived growth factor (PDGF)-dependent association of phospholipase C-gamma with the PDGF receptor signaling complex. Mol Cell Biol. 1990 May;10(5):2359–2366. doi: 10.1128/mcb.10.5.2359. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Nishizuka Y. Studies and perspectives of protein kinase C. Science. 1986 Jul 18;233(4761):305–312. doi: 10.1126/science.3014651. [DOI] [PubMed] [Google Scholar]
  37. Nolan R. D., Lapetina E. G. Thrombin stimulates the production of a novel polyphosphoinositide in human platelets. J Biol Chem. 1990 Feb 15;265(5):2441–2445. [PubMed] [Google Scholar]
  38. Pignataro O. P., Ascoli M. Epidermal growth factor increases the labeling of phosphatidylinositol 3,4-bisphosphate in MA-10 Leydig tumor cells. J Biol Chem. 1990 Jan 25;265(3):1718–1723. [PubMed] [Google Scholar]
  39. Stephens L., Hawkins P. T., Downes C. P. Metabolic and structural evidence for the existence of a third species of polyphosphoinositide in cells: D-phosphatidyl-myo-inositol 3-phosphate. Biochem J. 1989 Apr 1;259(1):267–276. doi: 10.1042/bj2590267. [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Talmage D. A., Freund R., Young A. T., Dahl J., Dawe C. J., Benjamin T. L. Phosphorylation of middle T by pp60c-src: a switch for binding of phosphatidylinositol 3-kinase and optimal tumorigenesis. Cell. 1989 Oct 6;59(1):55–65. doi: 10.1016/0092-8674(89)90869-6. [DOI] [PubMed] [Google Scholar]
  41. Tollefsen S. E., Thompson K. The structural basis for insulin-like growth factor I receptor high affinity binding. J Biol Chem. 1988 Nov 5;263(31):16267–16273. [PubMed] [Google Scholar]
  42. Traynor-Kaplan A. E., Harris A. L., Thompson B. L., Taylor P., Sklar L. A. An inositol tetrakisphosphate-containing phospholipid in activated neutrophils. Nature. 1988 Jul 28;334(6180):353–356. doi: 10.1038/334353a0. [DOI] [PubMed] [Google Scholar]
  43. Traynor-Kaplan A. E., Thompson B. L., Harris A. L., Taylor P., Omann G. M., Sklar L. A. 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]
  44. Vadnal R. E., Parthasarathy R. The identification of a novel inositol lipid, phosphatidylinositol trisphosphate (PIP3), in rat cerebrum using in vivo techniques. Biochem Biophys Res Commun. 1989 Sep 15;163(2):995–1001. doi: 10.1016/0006-291x(89)92320-6. [DOI] [PubMed] [Google Scholar]
  45. 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. doi: 10.1038/342699a0. [DOI] [PubMed] [Google Scholar]
  46. Whitman M., Cantley L. Phosphoinositide metabolism and the control of cell proliferation. Biochim Biophys Acta. 1989 Feb;948(3):327–344. doi: 10.1016/0304-419x(89)90005-x. [DOI] [PubMed] [Google Scholar]
  47. Whitman M., Downes C. P., 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. doi: 10.1038/332644a0. [DOI] [PubMed] [Google Scholar]
  48. Whitman M., Kaplan D. R., Schaffhausen B., Cantley L., Roberts T. M. Association of phosphatidylinositol kinase activity with polyoma middle-T competent for transformation. Nature. 1985 May 16;315(6016):239–242. doi: 10.1038/315239a0. [DOI] [PubMed] [Google Scholar]
  49. 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. doi: 10.1042/bj2470165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Williams L. T. Signal transduction by the platelet-derived growth factor receptor. Science. 1989 Mar 24;243(4898):1564–1570. doi: 10.1126/science.2538922. [DOI] [PubMed] [Google Scholar]
  51. Yarden Y., Escobedo J. A., Kuang W. J., Yang-Feng T. L., Daniel T. O., Tremble P. M., Chen E. Y., Ando M. E., Harkins R. N., Francke U. Structure of the receptor for platelet-derived growth factor helps define a family of closely related growth factor receptors. Nature. 1986 Sep 18;323(6085):226–232. doi: 10.1038/323226a0. [DOI] [PubMed] [Google Scholar]

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