Tyrosine Phosphorylation of the Novel Protein-tyrosine Kinase RAFTK during an Early Phase of Platelet Activation by an Integrin Glycoprotein IIb-IIIa-independent Mechanism

Sandhya Raja,Shalom Avraham,Hava Avraham

doi:10.1074/jbc.272.16.10941

Abstract

A key regulatory event controlling platelet activation is mediated through the phosphorylation of several cellular proteins by protein-tyrosine kinases. The related adhesion focal tyrosine kinase (RAFTK) is a novel cytoplasmic tyrosine kinase and a member of the focal adhesion kinase (FAK) gene family. FAK phosphorylation in platelets is integrin-dependent, occurs in a late stage of platelet activation, and is dependent on platelet aggregation. In this study, we have investigated the involvement of RAFTK phosphorylation during different stages of platelet activation. Treatment of platelets with thrombin induced, in as early as 10 s, a rapid tyrosine phosphorylation of RAFTK in a time- and concentration-dependent manner. Treatment of platelets with thrombin in the absence of stirring or pretreatment of platelets with RGDS peptide prevented platelet aggregation, but not RAFTK phosphorylation. Furthermore, phosphorylation of RAFTK did not require integrin engagement since platelets treated with the 7E3 inhibitory antibodies that block fibrinogen binding to glycoprotein IIb-IIIa did not inhibit RAFTK phosphorylation. Similarly, platelets treated with LIBS6 antibodies, which specifically activate glycoprotein IIb-IIIa, did not induce RAFTK phosphorylation. Stimulation of platelets by several agonists such as collagen, ADP, epinephrine, and calcium ionophore A23187 induced RAFTK phosphorylation. Tyrosine phosphorylation of RAFTK in platelets is regulated by calcium and is mediated through the protein kinase C pathway. Phosphorylation of RAFTK is dependent upon the formation of actin cytoskeleton as disruption of actin polymerization by cytochalasin D significantly inhibited this phosphorylation. The RAFTK protein appears to be proteolytically cleaved by calpain in an aggregation dependent manner upon thrombin stimulation. These results demonstrate that RAFTK is tyrosine-phosphorylated during an early phase of platelet activation by an integrin- independent mechanism and is not dependent on platelet aggregation, suggesting different mechanisms of regulation for FAK and RAFTK phosphorylation during platelet activation.

Highlights

This work is dedicated to the memory of Dananagoud Hiregowdara. ‡ To whom correspondence should be addressed: Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center (West Campus), Harvard Institutes of Medicine, One Deaconess Rd., Boston, MA 02215
We have examined the signaling mechanisms involved in the tyrosine phosphorylation of the newly identified protein-tyrosine kinase, related adhesion focal tyrosine kinase (RAFTK), a member of the tyrosine kinase focal adhesion kinase (FAK) family
We have demonstrated that thrombin induced a rapid tyrosine phosphorylation of RAFTK in a timeand concentration-dependent manner

Summary

Introduction

This work is dedicated to the memory of Dananagoud Hiregowdara. ‡ To whom correspondence should be addressed: Divisions of Experimental Medicine and Hematology/Oncology, Beth Israel Deaconess Medical Center (West Campus), Harvard Institutes of Medicine, One Deaconess Rd., Boston, MA 02215. Agonist-induced platelet tyrosine phosphorylation has been divided into three temporal waves based upon their dependence on integrin (GPIIb-IIIa) binding to fibrinogen or on subsequent platelet aggregation [2]. We have investigated the role of RAFTK during platelet activation by studying the integrin-mediated regulation of the tyrosine phosphorylation of RAFTK in human platelets.

Results

Conclusion