Tyrosine dephosphorylation, but not phosphorylation, of p130(Cas) is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: Implication of p130(Cas) involvement in pathways unrelated to cytoskeletal reorganization

The newly described adapter molecule p130 Crk-associated substrate (Cas) ha s been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncog ene-mediated transformation. We characterized the regulation of Cas tyrosin e phosphorylation in highly differentiated, anucleate platelets. Phospholip ase C-activating receptor agonists, including collagen, thrombin receptor-a ctivating peptide (TRAP), and U46619 (a thromboxane A(2) analogue), and A23 187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platel ets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol , protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylat ion, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated i n a manner dependent on integrin alpha IIb beta 3-mediated aggregation. Whi le BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged i t. Under the conditions, this PKC inhibitor suppressed platelet aggregation but not intracellular Ca2+ mobilization. In contrast to Cas involvement in focal adhesions in other cells, platelet Cas phosphorylation preceded the activation of focal adhesion kinase (FAK), and blockage of alpha IIb beta 3 -mediated platelet aggregation with a GRGDS peptide resulted in prolongatio n of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylat ion was insensitive to cytochalasin D, an actin polymerization inhibitor. T he failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activatio n. Of the signaling molecules investigated in this study, Src seemed to ass ociate with Gas. Finally, Cas existed mainly in cytosol and membrane cytosk eleton fractions in the resting state, and remained unchanged during platel et aggregation, when FAK translocated to the cytoskeletal fraction. Our fin dings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation o ccurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, b y itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine depho sphorylation, but not phosphorylation, is dependent on integrin alpha IIb b eta 3-mediated aggregation, and (iv) Cas is not involved in cytoskeletal re organization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.