BACKGROUND: The proline-rich tyrosine kinase Pyk2 is a focal adhesion kinase expressed in blood platelets, and is activated downstream of G-protein coupled receptors as well as integrin α2β1. OBJECTIVE: In this study we have investigated the involvement of Pyk2 in integrin αIIbβ3 outside-in signaling in human and murine platelets. METHODS: We analyzed the stimulation of intracellular signaling pathways in platelets from Pyk2 knockout mice adherent to immobilized fibrinogen. RESULTS: Pyk2 was rapidly phosphorylated and activated in human and murine platelets adherent to fibrinogen through integrin αIIbβ3. Activation of Pyk2 was Src-dependent, but did not require phospholipase Cγ2 activity. Platelets from Pyk2 knockout mice showed a defective ability to adhere and spread on fibrinogen, in association with a dramatic reduction of phosphatidylinositol 3-kinase (PI3K) activation and Akt phosphorylation. Pharmacological and genetic analysis demonstrated that integrin αIIbβ3 engagement selectively stimulated the β-isoform of PI3K (PI3Kβ), and that, as for Pyk2, PI3Kβ activation required Src family kinases activity, but not phospholipase Cγ2. In fibrinogen-adherent platelets, both Pyk2 and PI3Kβ were necessary for stimulation of the small GTPase Rap1b, a regulator of cell adhesion and spreading. Integrin αIIbβ3 engagement triggered the association of the PI3Kβ regulatory subunit p85 with the adaptor protein c-Cbl, which was mediated by the p85 SH3 domain, and was independent of c-Cbl tyrosine phosphorylation. However, p85-associated c-Cbl was tyrosine phosphorylated by activated Pyk2 in fibrinogen adherent platelets. CONCLUSIONS: These results identify a novel pathway of integrin αIIbβ3 outside-in signaling and recognize the tyrosine kinase Pyk2 as a major regulator of platelet adhesion and spreading on fibrinogen.
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