Study of Tyrosine Kinases and Protein Tyrosine Phosphorylation

  • Jonathan M. Gibbins
Part of the Methods in Molecular Biology™ book series (MIMB, volume 273)


In recent years, our increased understanding of the complex signal transduction mechanisms that regulate cellular function has fueled huge advances in all aspects of biomedical science and cell biology. Platelet and megakaryocyte function is no exception to this. In the last 10 yr our understanding of the receptor biochemistry and the systems that they control has been pivotal in the development of new strategies to inhibit platelet function and thereby prevent thrombosis. Experimental techniques have become more and more elegant, however; the basic toolbox that a researcher requires to study signaling in platelets and megakaryoctes is described in this and several subsequent chapters.


Tyrosine Phosphorylation HEPES Buffer Kinase Assay Protein Tyrosine Phosphorylation Platelet Suspension 
These keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.


  1. 1.
    Gibbins, J., Asselin, J., Farndale, R., Barnes, M., Law, C. L., and Watson, S. P. (1996) Tyrosine phosphorylation of the Fc receptor gamma-chain in collagen-stimulated platelets. J. Biol. Chem. 271, 18,095–18,099.PubMedCrossRefGoogle Scholar
  2. 2.
    Gibbins, J. M., Okuma, M., Farndale, R., Barnes, M., and Watson, S. P. (1997) Glycoprotein VI is the collagen receptor in platelets which underlies tyrosine phosphorylation of the Fc receptor γ-chain. FEBS Lett. 413, 255–259.PubMedCrossRefGoogle Scholar
  3. 3.
    Poole, A., Gibbins, J. M., Turner, M., van Vugt, M. J., van de Winkel, J. G. J., Saito, T., et al. (1997) The Fc receptor γ-chain and the tyrosine kinase Syk are essential for activation of mouse platelets by collagen. EMBO J. 16, 2333–2341.PubMedCrossRefGoogle Scholar
  4. 4.
    Tsuji, M., Ezumi, Y., Arai, M., and Takayama, H. (1997) A novel association of Fc receptor γ-chain with glycoprotein VI and their co-expression as a collagen receptor in human platelets. J. Biol. Chem. 272, 23,528–23,531.PubMedCrossRefGoogle Scholar
  5. 5.
    Yanaga, F., Poole, A., Asselin, J., Blake, R., Schieven, G., Clark, E. A., et al. (1995) Syk interacts with tyrosine-phosphorylated proteins in human platelets activated by collagen and cross-linking of the Fcg-IIA receptor. Biochem. J. 311, 471–478.PubMedGoogle Scholar
  6. 6.
    Robinson, A., Gibbins, J., Rodriguez-Linares, B., Finan, P. M., Wilson, L., Kellie, S., et al. (1996) Characterization of Grb2-binding proteins in human platelets activated by Fc gamma RIIA cross-linking. Blood 88, 522–530.PubMedGoogle Scholar
  7. 7.
    Law, D. A., DeGuzman, F R., Heiser, P., Ministri-Madrid, K., Killeen, N., and Phillips, D. R. (1999) Integrin cytoplasmic tyrosine motif is required for outside-in alpha IIb beta 3 signalling and platelet function. Nature 401, 808–811.PubMedCrossRefGoogle Scholar
  8. 8.
    Cicmil, M., Thomas, J. M., Leduc, M., Bon, C., and Gibbins, J. M. (2002) PECAM-1 signalling inhibits the activation of human platelets. Blood 99, 137–144.PubMedCrossRefGoogle Scholar
  9. 9.
    Cicmil, M., Thomas, J. M., Sage, T, Barry, F. A., Leduc, M., Bon, C., et al. (2000) Collagen, convulxin, and thrombin stimulate aggregation-independent tyrosine phosphorylation of CD31 in platelets. Evidence for the involvement of Src family kinases. J. Biol. Chem. 275, 27,339–27,347.PubMedGoogle Scholar
  10. 10.
    Jones, K. L., Hughan, S. C., Dopheide, S. M., Farndale, R. W., Jackson, S. P., and Jackson, D. E. (2001) Platelet endothelial cell adhesion molecule-1 is a negative regulator of platelet-collagen interactions. Blood 98, 1456–1463.PubMedCrossRefGoogle Scholar
  11. 11.
    Briddon, S. J. and Watson, S. P. (1999) Evidence for the involvement of p59(fyn) and p53/56(lyn) in collagen receptor signalling in human platelets. Biochem. J. 338, 203–209.PubMedCrossRefGoogle Scholar
  12. 12.
    Quek, L. S., Pasquet, J. M., Hers, I., Cornall, R., Knight, G., Barnes, M., et al. (2000) Fyn and Lyn phosphorylate the Fc receptor gamma chain downstream of glycoprotein VI in murine platelets, and Lyn regulates a novel feedback pathway. Blood 96, 4246–4253.PubMedGoogle Scholar
  13. 13.
    Quek, L. S., Bolen, J., and Watson, S. P. (1998) A role for Bruton’s tyrosine kinase (Btk) in platlet activation by collagen. Curr. Biol. 8, 1137–1140.PubMedCrossRefGoogle Scholar
  14. 14.
    Achison, M., Knight, G. G., Barnes, M. J., and Farndale, R. W. (1997) Activation by collagen and a collagen-related peptide of the intracellular tyrosine kinase, P125(fak), in human platelets which is independent of the integrin α2β1. Thromb. Haemost. PD637–PD637.Google Scholar
  15. 15.
    Achison, M., Elton, C. M., Hargreaves, P. G., Knight, C. G., Barnes, M. J., and Farndale, R. W. (2001) Integrin-independent tyrosine phosphorylation of p125(fak) in human platelets stimulated by collagen. J. Biol. Chem. 276, 3167–3174.PubMedCrossRefGoogle Scholar
  16. 16.
    Gibbins, J. M., Briddon, S., Shutes, A., van Vugt, M. J., van de Winkel, J. G., Saito, T., et al. (1998) The p85 subunit of phosphatidylinositol 3-kinase associates with the Fc receptor gamma-chain and linker for activitor of T cells (LAT) in platelets stimulated by collagen and convulxin. J. Biol. Chem. 273, 34,437–34,443.PubMedCrossRefGoogle Scholar
  17. 17.
    Cichowski, K., Brugge, J. S., and Brass, L. F. (1996) Thrombin receptor activation and integrin engagement stimulate tyrosine phosphorylation of the proto-oncogene product, p95(vav), in platelets. J. Biol. Chem. 271, 7544–7550.PubMedCrossRefGoogle Scholar
  18. 18.
    Gross, B. S., Melford, S. K., and Watson, S. P. (1999) Evidence that phospholipase C-γ2 interacts with SLP-76, syk, lyn, LAT and the Fc receptor γ-chain after stimulation of the collagen receptor glycoprotein VI in human platelets. Eur. J. Biochem. 263, 612–623.PubMedCrossRefGoogle Scholar
  19. 19.
    Pasquet, J. M., Gross, B., Quek, L., Asazuma, N., Zhang, W. G., Sommers, C. L., et al. (1999) LAT is required for tyrosine phosphorylation of phospholipase C gamma 2 and platelet activation by the collagen receptor GPVI. Mol. Cell. Biol. 19, 8326–8334.PubMedGoogle Scholar
  20. 20.
    Zhang, J., Zhang, J., Shattil, S. J., Cunningham, M. C., and Rittenhouse, S. E. (1996) Phos-phoinositide 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. 271, 6265–6572.PubMedCrossRefGoogle Scholar
  21. 21.
    Cho, M. J., Pestina, T. I., Steward, S. A., Lowell, C. A., Jackson, C. W., and Gartner, T. K. (2002) Role of the Src family kinase Lyn in TxA2 production, adenosine diphosphate secretion, Akt phosphorylation, and irreversible aggregation in platelets stimulated with γ-thrombin. Blood 99, 2442–2447.PubMedCrossRefGoogle Scholar
  22. 22.
    Davies, S. P., Reddy, H., Caivano, M., and Cohen, P. (2000) Specificity and mechanism of action of some commonly used protein kinase inhibitors. Biochem. J. 351, 95–105.PubMedCrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2004

Authors and Affiliations

  • Jonathan M. Gibbins
    • 1
  1. 1.School of Animal & Microbial SciencesThe University of ReadingReadingUK

Personalised recommendations