Raja S, Avraham S, Avraham H
Division of Experimental Medicine, Beth Israel Deaconess Medical Center, Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215, USA.
J Biol Chem. 1997 Apr 18;272(16):10941-7. doi: 10.1074/jbc.272.16.10941.
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.
控制血小板活化的一个关键调节事件是通过蛋白酪氨酸激酶对几种细胞蛋白的磷酸化来介导的。相关黏附灶酪氨酸激酶（RAFTK）是一种新型的细胞质酪氨酸激酶，属于黏附斑激酶（FAK）基因家族的成员。血小板中的FAK磷酸化是整合素依赖性的，发生在血小板活化的后期，并且依赖于血小板聚集。在本研究中，我们调查了RAFTK磷酸化在血小板活化不同阶段的参与情况。用凝血酶处理血小板，早在10秒时就以时间和浓度依赖性方式诱导了RAFTK的快速酪氨酸磷酸化。在不搅拌的情况下用凝血酶处理血小板或用RGDS肽预处理血小板可防止血小板聚集，但不影响RAFTK磷酸化。此外，RAFTK的磷酸化不需要整合素参与，因为用阻断纤维蛋白原与糖蛋白IIb-IIIa结合的7E3抑制性抗体处理血小板不会抑制RAFTK磷酸化。同样，用特异性激活糖蛋白IIb-IIIa的LIBS6抗体处理血小板也不会诱导RAFTK磷酸化。几种激动剂如胶原、ADP、肾上腺素和钙离子载体A23187刺激血小板可诱导RAFTK磷酸化。血小板中RAFTK的酪氨酸磷酸化受钙调节，并通过蛋白激酶C途径介导。RAFTK的磷酸化依赖于肌动蛋白细胞骨架的形成，因为细胞松弛素D破坏肌动蛋白聚合会显著抑制这种磷酸化。RAFTK蛋白在凝血酶刺激后似乎以聚集依赖性方式被钙蛋白酶蛋白水解切割。这些结果表明，RAFTK在血小板活化的早期阶段通过整合素非依赖性机制被酪氨酸磷酸化，且不依赖于血小板聚集，提示血小板活化过程中FAK和RAFTK磷酸化的调节机制不同。
