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. 2000 Nov 1;351(Pt 3):669–676.

Platelet alpha IIb-beta 3 integrin engagement induces the tyrosine phosphorylation of Cbl and its association with phosphoinositide 3-kinase and Syk.

A Saci 1, F Rendu 1, C Bachelot-Loza 1
PMCID: PMC1221406  PMID: 11042121

Abstract

Agonist-induced platelet activation triggers 'inside-out' signalling which activates alpha IIb-beta 3, the most abundant integrin in platelet membranes. The engagement of activated alpha IIb-beta 3 integrin by linking fibrinogen is necessary for platelet aggregation, and this induces subsequent outside-in signalling, which enhances platelet activation. Here we studied the involvement of Cbl during alpha IIb-beta 3-integrin-mediated signal transduction. During thrombin-induced platelet activation, Cbl was tyrosine phosphorylated, and phosphoinositide 3-kinase (PI 3-kinase) activity measured in Cbl immunoprecipitates was increased. Both Cbl phosphorylation and its association with PI 3-kinase were dependent on alpha IIb-beta 3 engagement by linking fibrinogen. The P256 and anti-LIBS6 (ligand-induced binding site 6) antibodies, which activate platelets directly through alpha IIb-beta 3, induced Cbl phosphorylation and increased the PI 3-kinase activity associated with Cbl. Both thrombin and antibodies to alpha IIb-beta 3 induced association of Cbl with the tyrosine kinase, Syk. Experiments performed with inhibitors of tyrosine kinases indicated that both Src-family kinases and Syk contribute to phosphorylation of Cbl and its consequent association with PI 3-kinase. The results show that, following integrin alpha IIb-beta 3 engagement, Cbl is tyrosine phosphorylated, recruits PI 3-kinase to this integrin signalling pathway and possibly enhances PI 3-kinase activity, downstream of Src-family tyrosine kinases and Syk activation.

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Selected References

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  1. Andrews R. K., López J. A., Berndt M. C. Molecular mechanisms of platelet adhesion and activation. Int J Biochem Cell Biol. 1997 Jan;29(1):91–105. doi: 10.1016/s1357-2725(96)00122-7. [DOI] [PubMed] [Google Scholar]
  2. Asazuma N., Ozaki Y., Satoh K., Yatomi Y., Handa M., Fujimura Y., Miura S., Kume S. Glycoprotein Ib-von Willebrand factor interactions activate tyrosine kinases in human platelets. Blood. 1997 Dec 15;90(12):4789–4798. [PubMed] [Google Scholar]
  3. Bachelot C., Rendu F., Gulino D. Anti-GPIIb/IIIa antibodies: powerful tools to investigate function and regulation of an integrin. Semin Thromb Hemost. 1995;21(1):23–36. doi: 10.1055/s-2007-1000376. [DOI] [PubMed] [Google Scholar]
  4. Bachelot C., Saffroy R., Gandrille S., Aiach M., Rendu F. Role of Fc gamma RIIA gene polymorphism in human platelet activation by monoclonal antibodies. Thromb Haemost. 1995 Dec;74(6):1557–1563. [PubMed] [Google Scholar]
  5. Beitz L. O., Fruman D. A., Kurosaki T., Cantley L. C., Scharenberg A. M. SYK is upstream of phosphoinositide 3-kinase in B cell receptor signaling. J Biol Chem. 1999 Nov 12;274(46):32662–32666. doi: 10.1074/jbc.274.46.32662. [DOI] [PubMed] [Google Scholar]
  6. Bertagnolli M. E., Hudson L. A., Stetsenko G. Y. Selective association of the tyrosine kinases Src, Fyn, and Lyn with integrin-rich actin cytoskeletons of activated, nonaggregated platelets. Biochem Biophys Res Commun. 1999 Jul 14;260(3):790–798. doi: 10.1006/bbrc.1999.0985. [DOI] [PubMed] [Google Scholar]
  7. Blake T. J., Shapiro M., Morse H. C., 3rd, Langdon W. Y. The sequences of the human and mouse c-cbl proto-oncogenes show v-cbl was generated by a large truncation encompassing a proline-rich domain and a leucine zipper-like motif. Oncogene. 1991 Apr;6(4):653–657. [PubMed] [Google Scholar]
  8. Clark E. A., Brugge J. S. Integrins and signal transduction pathways: the road taken. Science. 1995 Apr 14;268(5208):233–239. doi: 10.1126/science.7716514. [DOI] [PubMed] [Google Scholar]
  9. Clark E. A., Shattil S. J., Ginsberg M. H., Bolen J., Brugge J. S. Regulation of the protein tyrosine kinase pp72syk by platelet agonists and the integrin alpha IIb beta 3. J Biol Chem. 1994 Nov 18;269(46):28859–28864. [PubMed] [Google Scholar]
  10. Fitzer-Attas C. J., Schindler D. G., Waks T., Eshhar Z. Direct T cell activation by chimeric single chain Fv-Syk promotes Syk-Cbl association and Cbl phosphorylation. J Biol Chem. 1997 Mar 28;272(13):8551–8557. doi: 10.1074/jbc.272.13.8551. [DOI] [PubMed] [Google Scholar]
  11. Hartwig J. H., Kung S., Kovacsovics T., Janmey P. A., Cantley L. C., Stossel T. P., Toker A. D3 phosphoinositides and outside-in integrin signaling by glycoprotein IIb-IIIa mediate platelet actin assembly and filopodial extension induced by phorbol 12-myristate 13-acetate. J Biol Chem. 1996 Dec 20;271(51):32986–32993. doi: 10.1074/jbc.271.51.32986. [DOI] [PubMed] [Google Scholar]
  12. Jackson S. P., Schoenwaelder S. M., Yuan Y., Rabinowitz I., Salem H. H., Mitchell C. A. Adhesion receptor activation of phosphatidylinositol 3-kinase. von Willebrand factor stimulates the cytoskeletal association and activation of phosphatidylinositol 3-kinase and pp60c-src in human platelets. J Biol Chem. 1994 Oct 28;269(43):27093–27099. [PubMed] [Google Scholar]
  13. Joazeiro C. A., Wing S. S., Huang H., Leverson J. D., Hunter T., Liu Y. C. The tyrosine kinase negative regulator c-Cbl as a RING-type, E2-dependent ubiquitin-protein ligase. Science. 1999 Oct 8;286(5438):309–312. doi: 10.1126/science.286.5438.309. [DOI] [PubMed] [Google Scholar]
  14. Keely P. J., Parise L. V. The alpha2beta1 integrin is a necessary co-receptor for collagen-induced activation of Syk and the subsequent phosphorylation of phospholipase Cgamma2 in platelets. J Biol Chem. 1996 Oct 25;271(43):26668–26676. [PubMed] [Google Scholar]
  15. Kovacsovics T. J., Bachelot C., Toker A., Vlahos C. J., Duckworth B., Cantley L. C., Hartwig J. H. Phosphoinositide 3-kinase inhibition spares actin assembly in activating platelets but reverses platelet aggregation. J Biol Chem. 1995 May 12;270(19):11358–11366. doi: 10.1074/jbc.270.19.11358. [DOI] [PubMed] [Google Scholar]
  16. Laffargue M., Ragab-Thomas J. M., Ragab A., Tuech J., Missy K., Monnereau L., Blank U., Plantavid M., Payrastre B., Raynal P. Phosphoinositide 3-kinase and integrin signalling are involved in activation of Bruton tyrosine kinase in thrombin-stimulated platelets. FEBS Lett. 1999 Jan 22;443(1):66–70. doi: 10.1016/s0014-5793(98)01680-9. [DOI] [PubMed] [Google Scholar]
  17. Langdon W. Y., Hartley J. W., Klinken S. P., Ruscetti S. K., Morse H. C., 3rd v-cbl, an oncogene from a dual-recombinant murine retrovirus that induces early B-lineage lymphomas. Proc Natl Acad Sci U S A. 1989 Feb;86(4):1168–1172. doi: 10.1073/pnas.86.4.1168. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Law D. A., DeGuzman F. R., Heiser P., Ministri-Madrid K., Killeen N., Phillips D. R. Integrin cytoplasmic tyrosine motif is required for outside-in alphaIIbbeta3 signalling and platelet function. Nature. 1999 Oct 21;401(6755):808–811. doi: 10.1038/44599. [DOI] [PubMed] [Google Scholar]
  19. Law D. A., Nannizzi-Alaimo L., Ministri K., Hughes P. E., Forsyth J., Turner M., Shattil S. J., Ginsberg M. H., Tybulewicz V. L., Phillips D. R. Genetic and pharmacological analyses of Syk function in alphaIIbbeta3 signaling in platelets. Blood. 1999 Apr 15;93(8):2645–2652. [PubMed] [Google Scholar]
  20. Law D. A., Nannizzi-Alaimo L., Phillips D. R. Outside-in integrin signal transduction. Alpha IIb beta 3-(GP IIb IIIa) tyrosine phosphorylation induced by platelet aggregation. J Biol Chem. 1996 May 3;271(18):10811–10815. doi: 10.1074/jbc.271.18.10811. [DOI] [PubMed] [Google Scholar]
  21. Liu Y. C., Altman A. Cbl: complex formation and functional implications. Cell Signal. 1998 Jun;10(6):377–385. doi: 10.1016/s0898-6568(97)00179-4. [DOI] [PubMed] [Google Scholar]
  22. Lupher M. L., Jr, Andoniou C. E., Bonita D., Miyake S., Band H. The c-Cbl oncoprotein. Int J Biochem Cell Biol. 1998 Apr;30(4):439–444. doi: 10.1016/s1357-2725(97)00075-7. [DOI] [PubMed] [Google Scholar]
  23. Lupher M. L., Jr, Rao N., Lill N. L., Andoniou C. E., Miyake S., Clark E. A., Druker B., Band H. Cbl-mediated negative regulation of the Syk tyrosine kinase. A critical role for Cbl phosphotyrosine-binding domain binding to Syk phosphotyrosine 323. J Biol Chem. 1998 Dec 25;273(52):35273–35281. doi: 10.1074/jbc.273.52.35273. [DOI] [PubMed] [Google Scholar]
  24. Manié S. N., Sattler M., Astier A., Phifer J. S., Canty T., Morimoto C., Druker B. J., Salgia R., Griffin J. D., Freedman A. S. Tyrosine phosphorylation of the product of the c-cbl protooncogene is [corrected] induced after integrin stimulation. Exp Hematol. 1997 Jan;25(1):45–50. [PubMed] [Google Scholar]
  25. McNicol A., Gerrard J. M. Post-receptor events associated with thrombin-induced platelet activation. Blood Coagul Fibrinolysis. 1993 Dec;4(6):975–991. [PubMed] [Google Scholar]
  26. Meng F., Lowell C. A. A beta 1 integrin signaling pathway involving Src-family kinases, Cbl and PI-3 kinase is required for macrophage spreading and migration. EMBO J. 1998 Aug 3;17(15):4391–4403. doi: 10.1093/emboj/17.15.4391. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Meng W., Sawasdikosol S., Burakoff S. J., Eck M. J. Structure of the amino-terminal domain of Cbl complexed to its binding site on ZAP-70 kinase. Nature. 1999 Mar 4;398(6722):84–90. doi: 10.1038/18050. [DOI] [PubMed] [Google Scholar]
  28. Miranti C. K., Leng L., Maschberger P., Brugge J. S., Shattil S. J. Identification of a novel integrin signaling pathway involving the kinase Syk and the guanine nucleotide exchange factor Vav1. Curr Biol. 1998 Dec 3;8(24):1289–1299. doi: 10.1016/s0960-9822(07)00559-3. [DOI] [PubMed] [Google Scholar]
  29. Miyake S., Lupher M. L., Jr, Andoniou C. E., Lill N. L., Ota S., Douillard P., Rao N., Band H. The Cbl protooncogene product: from an enigmatic oncogene to center stage of signal transduction. Crit Rev Oncog. 1997;8(2-3):189–218. doi: 10.1615/critrevoncog.v8.i2-3.30. [DOI] [PubMed] [Google Scholar]
  30. Nadal F., Lévy-Toledano S., Grelac F., Caen J. P., Rosa J. P., Bryckaert M. Negative regulation of mitogen-activated protein kinase activation by integrin alphaIIbbeta3 in platelets. J Biol Chem. 1997 Sep 5;272(36):22381–22384. doi: 10.1074/jbc.272.36.22381. [DOI] [PubMed] [Google Scholar]
  31. Oda A., Ozaki K., Druker B. J., Miyakawa Y., Miyazaki H., Handa M., Morita H., Ohashi H., Ikeda Y. p120c-cbl is present in human blood platelets and is differentially involved in signaling by thrombopoietin and thrombin. Blood. 1996 Aug 15;88(4):1330–1338. [PubMed] [Google Scholar]
  32. Ojaniemi M., Langdon W. Y., Vuori K. Oncogenic forms of Cbl abrogate the anchorage requirement but not the growth factor requirement for proliferation. Oncogene. 1998 Jun 18;16(24):3159–3167. doi: 10.1038/sj.onc.1201859. [DOI] [PubMed] [Google Scholar]
  33. Ojaniemi M., Martin S. S., Dolfi F., Olefsky J. M., Vuori K. The proto-oncogene product p120(cbl) links c-Src and phosphatidylinositol 3'-kinase to the integrin signaling pathway. J Biol Chem. 1997 Feb 7;272(6):3780–3787. doi: 10.1074/jbc.272.6.3780. [DOI] [PubMed] [Google Scholar]
  34. Ota Y., Samelson L. E. The product of the proto-oncogene c-cbl: a negative regulator of the Syk tyrosine kinase. Science. 1997 Apr 18;276(5311):418–420. doi: 10.1126/science.276.5311.418. [DOI] [PubMed] [Google Scholar]
  35. Pain S., Falet H., Saci A., Bachelot-Loza C., Rendu F. Tyrosine phosphorylation and association of FcgammaRII and p72(Syk) are not limited to the FcgammaRII signalling pathway. Cell Signal. 2000 Mar;12(3):165–171. doi: 10.1016/s0898-6568(99)00079-0. [DOI] [PubMed] [Google Scholar]
  36. Polgár J., Clemetson J. M., Kehrel B. E., Wiedemann M., Magnenat E. M., Wells T. N., Clemetson K. J. Platelet activation and signal transduction by convulxin, a C-type lectin from Crotalus durissus terrificus (tropical rattlesnake) venom via the p62/GPVI collagen receptor. J Biol Chem. 1997 May 23;272(21):13576–13583. doi: 10.1074/jbc.272.21.13576. [DOI] [PubMed] [Google Scholar]
  37. Saci A., Pain S., Rendu F., Bachelot-Loza C. Fc receptor-mediated platelet activation is dependent on phosphatidylinositol 3-kinase activation and involves p120(Cbl). J Biol Chem. 1999 Jan 22;274(4):1898–1904. doi: 10.1074/jbc.274.4.1898. [DOI] [PubMed] [Google Scholar]
  38. Sattler M., Salgia R., Shrikhande G., Verma S., Uemura N., Law S. F., Golemis E. A., Griffin J. D. Differential signaling after beta1 integrin ligation is mediated through binding of CRKL to p120(CBL) and p110(HEF1). J Biol Chem. 1997 May 30;272(22):14320–14326. doi: 10.1074/jbc.272.22.14320. [DOI] [PubMed] [Google Scholar]
  39. Scaife R. M., Langdon W. Y. c-Cbl localizes to actin lamellae and regulates lamellipodia formation and cell morphology. J Cell Sci. 2000 Jan;113(Pt 2):215–226. doi: 10.1242/jcs.113.2.215. [DOI] [PubMed] [Google Scholar]
  40. Shattil S. J., Kashiwagi H., Pampori N. Integrin signaling: the platelet paradigm. Blood. 1998 Apr 15;91(8):2645–2657. [PubMed] [Google Scholar]
  41. Songyang Z., Shoelson S. E., McGlade J., Olivier P., Pawson T., Bustelo X. R., Barbacid M., Sabe H., Hanafusa H., Yi T. Specific motifs recognized by the SH2 domains of Csk, 3BP2, fps/fes, GRB-2, HCP, SHC, Syk, and Vav. Mol Cell Biol. 1994 Apr;14(4):2777–2785. doi: 10.1128/mcb.14.4.2777. [DOI] [PMC free article] [PubMed] [Google Scholar]
  42. Sultan C., Plantavid M., Bachelot C., Grondin P., Breton M., Mauco G., Lévy-Toledano S., Caen J. P., Chap H. Involvement of platelet glycoprotein IIb-IIIa (alpha IIb-beta 3 integrin) in thrombin-induced synthesis of phosphatidylinositol 3',4'-bisphosphate. J Biol Chem. 1991 Dec 15;266(35):23554–23557. [PubMed] [Google Scholar]
  43. Thien C. B., Langdon W. Y. c-Cbl: a regulator of T cell receptor-mediated signalling. Immunol Cell Biol. 1998 Oct;76(5):473–482. doi: 10.1046/j.1440-1711.1998.00768.x. [DOI] [PubMed] [Google Scholar]
  44. Ueno H., Sasaki K., Honda H., Nakamoto T., Yamagata T., Miyagawa K., Mitani K., Yazaki Y., Hirai H. c-Cbl is tyrosine-phosphorylated by interleukin-4 and enhances mitogenic and survival signals of interleukin-4 receptor by linking with the phosphatidylinositol 3'-kinase pathway. Blood. 1998 Jan 1;91(1):46–53. [PubMed] [Google Scholar]
  45. Zhang J., Banfić H., Straforini F., Tosi L., Volinia S., Rittenhouse S. E. A type II phosphoinositide 3-kinase is stimulated via activated integrin in platelets. A source of phosphatidylinositol 3-phosphate. J Biol Chem. 1998 Jun 5;273(23):14081–14084. doi: 10.1074/jbc.273.23.14081. [DOI] [PubMed] [Google Scholar]
  46. Zhang J., Zhang J., Shattil S. J., Cunningham M. C., Rittenhouse S. E. Phosphoinositide 3-kinase gamma and p85/phosphoinositide 3-kinase in platelets. Relative activation by thrombin receptor or beta-phorbol myristate acetate and roles in promoting the ligand-binding function of alphaIIbbeta3 integrin. J Biol Chem. 1996 Mar 15;271(11):6265–6272. doi: 10.1074/jbc.271.11.6265. [DOI] [PubMed] [Google Scholar]
  47. 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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