Integrin-dependent phosphorylation and activation of the protein tyrosine kinase pp125FAK in platelets.

TLDR

The protein tyrosine kinase pp125FAK is localized to focal adhesions in fibroblasts and is phosphorylated on tyrosine in normal and Src‑transformed cells. The study aims to elucidate integrin‑mediated signal transduction in platelets by examining phosphorylation and activation of pp125FAK. The authors evaluated pp125FAK phosphorylation and activation in platelets stimulated with thrombin and collagen, assessing dependence on fibrinogen binding to GP IIb‑IIIa and actin polymerization. Thrombin and collagen trigger tyrosine phosphorylation of pp125FAK in platelets, an event that requires fibrinogen‑mediated GP IIb‑IIIa engagement and platelet aggregation, is blocked by cytochalasin D, and correlates with enhanced pp125FAK kinase activity in vitro, providing the first genetic evidence that pp125FAK phosphorylation is integrin‑dependent.

Abstract

We have investigated mechanisms involved in integrin-mediated signal transduction in platelets by examining integrin-dependent phosphorylation and activation of a newly identified protein tyrosine kinase, pp125FAK (FAK, focal adhesion kinase). This kinase was previously shown to be localized in focal adhesions in fibroblasts, and to be phosphorylated on tyrosine in normal and Src-transformed fibroblasts. We show that thrombin and collagen activation of platelets causes an induction of tyrosine phosphorylation of pp125FAK and that pp125FAK molecules isolated from activated platelets display enhanced levels of phosphorylation in immune-complex kinase assays. pp125FAK was not phosphorylated on tyrosine after thrombin or collagen treatment of Glanzmann's thrombasthenic platelets deficient in the fibrinogen receptor GPIIb-IIIa, or of platelets pretreated with an inhibitory monoclonal antibody to GP IIb-IIIa. Fibrinogen binding to GP IIb-IIIa was not sufficient to induce pp125FAK phosphorylation because pp125FAK was not phosphorylated on tyrosine in thrombin-treated platelets that were not allowed to aggregate. These results indicate that tyrosine phosphorylation of pp125FAK is dependent on platelet aggregation mediated by fibrinogen binding to the integrin receptor GP IIb-IIIa. The induction of tyrosine phosphorylation of pp125FAK was inhibited in thrombin- and collagen-treated platelets preincubated with cytochalasin D, which prevents actin polymerization following activation. Under all of these conditions, there was a strong correlation between the induction of tyrosine phosphorylation of pp125FAK in vivo and stimulation of the phosphorylation of pp125FAK in vitro in immune-complex kinase assays. This study provides the first genetic evidence that tyrosine phosphorylation of pp125FAK is dependent on integrin-mediated events, and demonstrates that there is a strong correlation between tyrosine phosphorylation of pp125FAK in platelets, and the activation of pp125FAK-associated phosphorylating activity in vitro.