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. 1992 Nov 2;119(4):905–912. doi: 10.1083/jcb.119.4.905

Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets

PMCID: PMC2289696  PMID: 1385445

Abstract

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune- complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin- mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.

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Selected References

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  1. Albelda S. M., Buck C. A. Integrins and other cell adhesion molecules. FASEB J. 1990 Aug;4(11):2868–2880. [PubMed] [Google Scholar]
  2. Banga H. S., Simons E. R., Brass L. F., Rittenhouse S. E. Activation of phospholipases A and C in human platelets exposed to epinephrine: role of glycoproteins IIb/IIIa and dual role of epinephrine. Proc Natl Acad Sci U S A. 1986 Dec;83(23):9197–9201. doi: 10.1073/pnas.83.23.9197. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baumgartner H. R. Platelet interaction with collagen fibrils in flowing blood. I. Reaction of human platelets with alpha chymotrypsin-digested subendothelium. Thromb Haemost. 1977 Feb 28;37(1):1–16. [PubMed] [Google Scholar]
  4. Bennett J. S., Vilaire G. Exposure of platelet fibrinogen receptors by ADP and epinephrine. J Clin Invest. 1979 Nov;64(5):1393–1401. doi: 10.1172/JCI109597. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Burridge K., Nuckolls G., Otey C., Pavalko F., Simon K., Turner C. Actin-membrane interaction in focal adhesions. Cell Differ Dev. 1990 Dec 2;32(3):337–342. doi: 10.1016/0922-3371(90)90048-2. [DOI] [PubMed] [Google Scholar]
  6. Carlsson L., Markey F., Blikstad I., Persson T., Lindberg U. Reorganization of actin in platelets stimulated by thrombin as measured by the DNase I inhibition assay. Proc Natl Acad Sci U S A. 1979 Dec;76(12):6376–6380. doi: 10.1073/pnas.76.12.6376. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Carroll R. C., Butler R. G., Morris P. A., Gerrard J. M. Separable assembly of platelet pseudopodal and contractile cytoskeletons. Cell. 1982 Sep;30(2):385–393. doi: 10.1016/0092-8674(82)90236-7. [DOI] [PubMed] [Google Scholar]
  8. Coller B. S., Beer J. H., Scudder L. E., Steinberg M. H. Collagen-platelet interactions: evidence for a direct interaction of collagen with platelet GPIa/IIa and an indirect interaction with platelet GPIIb/IIIa mediated by adhesive proteins. Blood. 1989 Jul;74(1):182–192. [PubMed] [Google Scholar]
  9. 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]
  10. Ferrell J. E., Jr, Martin G. S. Tyrosine-specific protein phosphorylation is regulated by glycoprotein IIb-IIIa in platelets. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2234–2238. doi: 10.1073/pnas.86.7.2234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fox J. E., Phillips D. R. Inhibition of actin polymerization in blood platelets by cytochalasins. Nature. 1981 Aug 13;292(5824):650–652. doi: 10.1038/292650a0. [DOI] [PubMed] [Google Scholar]
  12. Golden A., Brugge J. S., Shattil S. J. Role of platelet membrane glycoprotein IIb-IIIa in agonist-induced tyrosine phosphorylation of platelet proteins. J Cell Biol. 1990 Dec;111(6 Pt 2):3117–3127. doi: 10.1083/jcb.111.6.3117. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Grondin P., Plantavid M., Sultan C., Breton M., Mauco G., Chap H. Interaction of pp60c-src, phospholipase C, inositol-lipid, and diacyglycerol kinases with the cytoskeletons of thrombin-stimulated platelets. J Biol Chem. 1991 Aug 25;266(24):15705–15709. [PubMed] [Google Scholar]
  14. Guan J. L., Trevithick J. E., Hynes R. O. Fibronectin/integrin interaction induces tyrosine phosphorylation of a 120-kDa protein. Cell Regul. 1991 Nov;2(11):951–964. doi: 10.1091/mbc.2.11.951. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hemler M. E. VLA proteins in the integrin family: structures, functions, and their role on leukocytes. Annu Rev Immunol. 1990;8:365–400. doi: 10.1146/annurev.iy.08.040190.002053. [DOI] [PubMed] [Google Scholar]
  16. Hynes R. O. Integrins: versatility, modulation, and signaling in cell adhesion. Cell. 1992 Apr 3;69(1):11–25. doi: 10.1016/0092-8674(92)90115-s. [DOI] [PubMed] [Google Scholar]
  17. Hynes R. O., Lander A. D. Contact and adhesive specificities in the associations, migrations, and targeting of cells and axons. Cell. 1992 Jan 24;68(2):303–322. doi: 10.1016/0092-8674(92)90472-o. [DOI] [PubMed] [Google Scholar]
  18. Jennings L. K., Fox J. E., Edwards H. H., Phillips D. R. Changes in the cytoskeletal structure of human platelets following thrombin activation. J Biol Chem. 1981 Jul 10;256(13):6927–6932. [PubMed] [Google Scholar]
  19. Kanner S. B., Reynolds A. B., Vines R. R., Parsons J. T. Monoclonal antibodies to individual tyrosine-phosphorylated protein substrates of oncogene-encoded tyrosine kinases. Proc Natl Acad Sci U S A. 1990 May;87(9):3328–3332. doi: 10.1073/pnas.87.9.3328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kieffer N., Phillips D. R. Platelet membrane glycoproteins: functions in cellular interactions. Annu Rev Cell Biol. 1990;6:329–357. doi: 10.1146/annurev.cb.06.110190.001553. [DOI] [PubMed] [Google Scholar]
  21. Kornberg L. J., Earp H. S., Turner C. E., Prockop C., Juliano R. L. Signal transduction by integrins: increased protein tyrosine phosphorylation caused by clustering of beta 1 integrins. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8392–8396. doi: 10.1073/pnas.88.19.8392. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Phillips D. R., Charo I. F., Scarborough R. M. GPIIb-IIIa: the responsive integrin. Cell. 1991 May 3;65(3):359–362. doi: 10.1016/0092-8674(91)90451-4. [DOI] [PubMed] [Google Scholar]
  23. Plow E. F., Ginsberg M. H. Cellular adhesion: GPIIb-IIIa as a prototypic adhesion receptor. Prog Hemost Thromb. 1989;9:117–156. [PubMed] [Google Scholar]
  24. Santoro S. A. Identification of a 160,000 dalton platelet membrane protein that mediates the initial divalent cation-dependent adhesion of platelets to collagen. Cell. 1986 Sep 12;46(6):913–920. doi: 10.1016/0092-8674(86)90073-5. [DOI] [PubMed] [Google Scholar]
  25. Schaller M. D., Borgman C. A., Cobb B. S., Vines R. R., Reynolds A. B., Parsons J. T. pp125FAK a structurally distinctive protein-tyrosine kinase associated with focal adhesions. Proc Natl Acad Sci U S A. 1992 Jun 1;89(11):5192–5196. doi: 10.1073/pnas.89.11.5192. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Sweatt J. D., Connolly T. M., Cragoe E. J., Limbird L. E. Evidence that Na+/H+ exchange regulates receptor-mediated phospholipase A2 activation in human platelets. J Biol Chem. 1986 Jul 5;261(19):8667–8673. [PubMed] [Google Scholar]
  27. Sweatt J. D., Johnson S. L., Cragoe E. J., Limbird L. E. Inhibitors of Na+/H+ exchange block stimulus-provoked arachidonic acid release in human platelets. Selective effects on platelet activation by epinephrine, ADP, and lower concentrations of thrombin. J Biol Chem. 1985 Oct 25;260(24):12910–12919. [PubMed] [Google Scholar]
  28. Tangen O., Berman H. J., Marfey P. Gel filtration. A new technique for separation of blood platelets from plasma. Thromb Diath Haemorrh. 1971 Jun 30;25(2):268–278. [PubMed] [Google Scholar]
  29. Wang J. Y., Baltimore D. Localization of tyrosine kinase-coding region in v-abl oncogene by the expression of v-abl-encoded proteins in bacteria. J Biol Chem. 1985 Jan 10;260(1):64–71. [PubMed] [Google Scholar]
  30. Weiss H. J., Hawiger J., Ruggeri Z. M., Turitto V. T., Thiagarajan P., Hoffmann T. Fibrinogen-independent platelet adhesion and thrombus formation on subendothelium mediated by glycoprotein IIb-IIIa complex at high shear rate. J Clin Invest. 1989 Jan;83(1):288–297. doi: 10.1172/JCI113871. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Weiss H. J., Turitto V. T., Baumgartner H. R. Platelet adhesion and thrombus formation on subendothelium in platelets deficient in glycoproteins IIb-IIIa, Ib, and storage granules. Blood. 1986 Feb;67(2):322–330. [PubMed] [Google Scholar]
  32. Zhang J., Fry M. J., Waterfield M. D., Jaken S., Liao L., Fox J. E., Rittenhouse S. E. Activated phosphoinositide 3-kinase associates with membrane skeleton in thrombin-exposed platelets. J Biol Chem. 1992 Mar 5;267(7):4686–4692. [PubMed] [Google Scholar]

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