title: Tyrosine dephosphorylation, but not phosphorylation, of p130Cas is dependent on integrin alpha IIb beta 3-mediated aggregation in platelets: implication of p130Cas involvement in pathways unrelated to cytoskeletal reorganization.
abstract: The newly described adapter molecule p130 Crk-associated substrate (Cas) has been reported to contribute to cytoskeletal organization through assembly of actin filaments and to be pivotal in embryonic development and in oncogene-mediated transformation. We characterized the regulation of Cas tyrosine phosphorylation in highly differentiated, anucleate platelets. Phospholipase C-activating receptor agonists, including collagen, thrombin receptor-activating peptide (TRAP), and U46619 (a thromboxane A2 analogue), and A23187 (a Ca2+ ionophore) induced rapid Cas tyrosine phosphorylation in platelets. 12-O-Tetradecanoylphorbol 13-acetate and 1-oleoyl-2-acetyl-sn-glycerol, protein kinase C (PKC) activators, also induced Cas tyrosine phosphorylation, albeit sluggishly. Cas tyrosine phosphorylation induced by collagen or TRAP was transient in aggregating platelets; Cas became dephosphorylated in a manner dependent on integrin alpha IIb beta 3-mediated aggregation. While BAPTA-AM (an intracellular Ca2+ chelator) inhibited Cas phosphorylation induced by collagen or TRAP, Ro31-8220 (a PKC inhibitor) rather prolonged it. 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 prolongation of stimulation-dependent Cas tyrosine phosphorylation but in suppression of FAK tyrosine phosphorylation. Furthermore, TRAP-induced Cas phosphorylation was insensitive to cytochalasin D, an actin polymerization inhibitor. The failure of FAK to associate with Cas in immunoprecipitation studies also suggests that Cas tyrosine phosphorylation is independent of FAK activation. Of the signaling molecules investigated in this study, Src seemed to associate with Cas. Finally, Cas existed mainly in cytosol and membrane cytoskeleton fractions in the resting state, and remained unchanged during platelet aggregation, when FAK translocated to the cytoskeletal fraction. Our findings on platelet Cas suggest that (i) rapid Cas tyrosine phosphorylation occurs following phosphoinositide turnover by receptor-mediated agonists and may be mediated by intracellular Ca2+ mobilization; (ii) PKC activation, by itself, may elicit sluggish Cas phosphorylation; (iii) Cas tyrosine dephosphorylation, but not phosphorylation, is dependent on integrin alpha IIb beta 3-mediated aggregation; and (iv) Cas is not involved in cytoskeletal reorganization. Anucleate platelets seem to provide a unique model system to fully elucidate the functional role(s) of Cas.
authors.affiliation: Department of Laboratory Medicine, Yamanashi Medical University, Nakakoma, Yamanashi 409-3898, Japan.
authors.forename: T
authors.initials: T
authors.lastname: Ohmori
authors.forename: Y
authors.initials: Y
authors.lastname: Yatomi
authors.forename: K
authors.initials: K
authors.lastname: Inoue
authors.forename: K
authors.initials: K
authors.lastname: Satoh
authors.forename: Y
authors.initials: Y
authors.lastname: Ozaki
mesh_terms: D001792:Blood Platelets / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism* / Q000502:physiology; D001798:Blood Proteins / Q000378:metabolism; D002118:Calcium / Q000097:blood; D002352:Carrier Proteins; D050719:Crk-Associated Substrate Protein; D000242:Cyclic AMP / Q000819:agonists / Q000097:blood; D002373:Cyclic AMP Receptor Protein / Q000819:agonists / Q000097:blood; D015638:Cytochalasin D / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D004789:Enzyme Activation; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D010750:Phosphoproteins / Q000097:blood* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D015539:Platelet Activation / Q000187:drug effects; D010974:Platelet Aggregation / Q000187:drug effects; D019039:Platelet Glycoprotein GPIIb-IIIa Complex / Q000502:physiology*; D011493:Protein Kinase C / Q000097:blood; D011505:Protein-Tyrosine Kinases / Q000097:blood; D011506:Proteins; D011956:Receptors, Cell Surface / Q000819:agonists; D050717:Retinoblastoma-Like Protein p130; D015398:Signal Transduction / Q000187:drug effects; D013347:Subcellular Fractions / Q000201:enzymology / Q000378:metabolism; D014443:Tyrosine / Q000097:blood*; D018909:src Homology Domains
chemical_list: C493841:BCAR1 protein, human; D001798:Blood Proteins; D002352:Carrier Proteins; D050719:Crk-Associated Substrate Protein; D002373:Cyclic AMP Receptor Protein; D010750:Phosphoproteins; D019039:Platelet Glycoprotein GPIIb-IIIa Complex; D011506:Proteins; D011956:Receptors, Cell Surface; D050717:Retinoblastoma-Like Protein p130; D015638:Cytochalasin D; D014443:Tyrosine; D000242:Cyclic AMP; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051418:Focal Adhesion Kinase 2; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D011493:Protein Kinase C; D002118:Calcium