p21rasGAP association with Fyn, Lyn, and Yes in thrombin-activated platelets.
Activation of platelets by thrombin and other physiological agonists leads to a dramatic increase in tyrosine phosphorylation of multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3606-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). To date, none of the tyrosine kinases that are involved in platelet activation, nor the substrates that are phosphorylated in response to agonists, have been identified. A "kinase trapping" strategy, designed to take advantage of the stability of known tyrosine kinase-substrate interactions, was employed to address both issues. p21rasGAP antibodies were used to examine the phosphorylated state of GAP in agonist-treated platelets and to isolate potential GAP-kinase complexes. We show that GAP and two proteins of 59 and 68 kDa are phosphorylated on tyrosine after thrombin stimulation and that three Src-related protein tyrosine kinases, Fyn, Lyn and Yes, are associated with GAP in complexes, detectable only after agonist stimulation. The thrombin-dependent detection of these kinases in GAP immunoprecipitates suggests that thrombin may either induce the formation of these complexes or activate kinases that are associated with GAP prior to, or following, agonist stimulation. This approach of "trapping" kinases bound to their substrates will be useful in identifying non-receptor tyrosine kinases involved in signaling pathways. Furthermore, although GAP phosphorylation has been previously implicated in growth factor signaling pathways, this is the first example of its involvement downstream from a G-protein-coupled receptor.
Cichowski
K
K
Howard Hughes Medical Institute, Department of Microbiology, University of Pennsylvania School of Medicine, Philadelphia 19104-6148.
McCormick
F
F
Brugge
J S
JS
D001792:Blood Platelets / Q000187:drug effects / Q000502:physiology*; D019204:GTP-Binding Proteins / Q000378:metabolism; D020690:GTPase-Activating Proteins; D006801:Humans; D015151:Immunoblotting; D010766:Phosphorylation; D015539:Platelet Activation; D011505:Protein-Tyrosine Kinases / Q000097:blood*; D011506:Proteins / Q000302:isolation & purification / Q000378:metabolism*; D011518:Proto-Oncogene Proteins / Q000097:blood*; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; D015398:Signal Transduction; D013917:Thrombin / Q000494:pharmacology*; D019061:src-Family Kinases
D020690:GTPase-Activating Proteins; D011506:Proteins; D011518:Proto-Oncogene Proteins; D011505:Protein-Tyrosine Kinases; C494957:FYN protein, human; D051076:Proto-Oncogene Proteins c-fyn; D051575:Proto-Oncogene Proteins c-yes; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D013917:Thrombin; D019204:GTP-Binding Proteins