Sada K, Minami Y, Yamamura H
Department of Biochemistry, Kobe University School of Medicine, Japan.
Eur J Biochem. 1997 Sep 15;248(3):827-33. doi: 10.1111/j.1432-1033.1997.00827.x.
Previous studies demonstrated that Syk protein-tyrosine kinase (Syk) is activated by thrombin in platelets. To elucidate the function of Syk in platelets, we have biochemically examined the intracellular location of Syk and the molecules associated with Syk, following platelet activation. In human platelets, thrombin induces the relocation of Syk to the cytoskeletal fraction presumably via Syk tyrosine phosphorylation. Relocated Syk is associated with the actin filament network, and the early phase (10-90 s) of this association can be partially inhibited by the pretreatment of platelets with cytochalasin D, an inhibitor of actin polymerization. Upon thrombin stimulation, Syk becomes associated with Fak as demonstrated by co-immunoprecipitation. The association of both kinases can be inhibited by pretreatment of platelets with cytochalasin D. Interestingly, reconstitution experiments, using COS cells transfected with various porcine Syk mutants, revealed that the kinase domain, but not the kinase activity, of Syk is required for the association of Syk with the actin filament network. These findings suggest that thrombin-induced association of Syk with Fak correlates with the state of actin polymerization, and may play an important role in platelet activation.
先前的研究表明，脾酪氨酸激酶（Syk）在血小板中可被凝血酶激活。为阐明Syk在血小板中的功能，我们在血小板激活后，对Syk的细胞内定位以及与Syk相关的分子进行了生化检测。在人血小板中，凝血酶大概通过Syk酪氨酸磷酸化诱导Syk重新定位到细胞骨架部分。重新定位的Syk与肌动蛋白丝网络相关，这种关联的早期阶段（10 - 90秒）可通过用细胞松弛素D（一种肌动蛋白聚合抑制剂）预处理血小板而部分受到抑制。凝血酶刺激后，通过共免疫沉淀证明Syk与粘着斑激酶（Fak）相关。两种激酶的关联可通过用细胞松弛素D预处理血小板而受到抑制。有趣的是，使用转染了各种猪Syk突变体的COS细胞进行的重组实验表明，Syk与肌动蛋白丝网络的关联需要Syk的激酶结构域，但不需要激酶活性。这些发现表明，凝血酶诱导的Syk与Fak的关联与肌动蛋白聚合状态相关，并且可能在血小板激活中起重要作用。
