Activation of phosphatidylinositol 3-kinase β by the platelet collagen receptors integrin α2β1 and GPVI: The role of Pyk2 and c-Cbl

Phosphatidylinositol 3-kinaseβ (PI3Kβ) plays a predominant role in integrin outside-in signaling and in platelet activation by GPVI engagement. We have shown that the tyrosine kinase Pyk2 mediates PI3Kβ activation downstream of integrin αIIbβ3, and promotes the phosphorylation of the PI3K-associated adaptor protein c-Cbl. In this study, we compared the functional correlation between Pyk2 and PI3Kβ upon recruitment of the two main platelet collagen receptors, integrin α2β1 and GPVI. PI3Kβ-mediated phosphorylation of Akt was inhibited in Pyk2-deficient platelets adherent to monomeric collagen through integrin α2β1, but occurred normally upon GPVI ligation. Integrin α2β1 engagement led to Pyk2-independent association of c-Cbl with PI3K. However, c-Cbl was not phosphorylated in adherent platelets, and phosphorylation of Akt occurred normally in c-Cbl-deficient platelets, indicating that the c-Cbl is dispensable for Pyk2-mediated PI3Kβ activation. Stimulation of platelets with CRP, a selective GPVI ligand, induced c-Cbl phosphorylation in the absence of Pyk2, but failed to promote its association with PI3K. Pyk2 activation was completely abrogated in PI3KβKD, but not in PI3KγKD platelets, and was strongly inhibited by Src kinases and phospholipase C inhibitors, and by BAPTA-AM. The absence of PI3Kβ activity also hampered GPVI-induced tyrosine-phosphorylation and activation of PLCγ2, preventing intracellular Ca(2+) increase and phosphorylation of pleckstrin. Moreover, GPVI-induced intracellular Ca(2+) increase and pleckstrin phosphorylation were also strongly inhibited in human platelets treated with the PI3Kβ inhibitor TGX-221. These results outline important differences in the regulation of PI3Kβ by GPVI and integrin α2β1 and suggest that inhibition of Pyk2 may target PI3Kβ activation in a selective context of platelet stimulation.