Taniguchi T, Kitagawa H, Yasue S, Yanagi S, Sakai K, Asahi M, Ohta S, Takeuchi F, Nakamura S, Yamamura H
Department of Biochemistry, Fukui Medical School, Japan.
J Biol Chem. 1993 Feb 5;268(4):2277-9.
Activation of platelets by thrombin results in a dramatic increase in tyrosine phosphorylation on multiple cellular proteins (Ferrell, J. E., and Martin, G. S. (1988) Mol. Cell. Biol. 8, 3603-3610; Golden, A., and Brugge, J. S. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 901-905; Nakamura, S., and Yamamura, H. (1989) J. Biol. Chem. 264, 7089-7091). However, none of the responsible protein-tyrosine kinase has been reported so far. We report here that p72syk, one of the non-receptor-type protein-tyrosine kinases, is activated following thrombin stimulation in blood platelets. Washed porcine platelets were stimulated by thrombin, and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 5 s, reached a maximum at 10 s, and decreased to a basal level within 60 s after 0.5 unit/ml thrombin stimulation. The amount of immunoprecipitated p72syk was not altered throughout the time course. This activation was greatly enhanced in a dose-dependent manner and was completely canceled by the pretreatment of platelet suspension with hirudin, a specific antagonist of thrombin. In the Ca(2+)-depleted condition both extra- and intracellularly, the activation of p72syk was still persistent; in contrast, the deactivation process was completely abrogated even at 120 s after thrombin stimulation. In addition, the replenishment of Ca2+ resulted in a similar deactivation pattern as seen in the Ca(2+)-rich condition. Furthermore, this deactivation was also canceled by the pretreatment of platelets with W7, a calmodulin antagonist, as well as ML9, a myosin-light-chain kinase inhibitor. These results indicate that p72syk can be a responsible enzyme to the protein-tyrosine phosphorylation events following the platelet activation by thrombin and may be negatively regulated by Ca2+ in a calmodulin-dependent manner, inter alia myosin light-chain kinase, in thrombin-stimulated platelets.
凝血酶激活血小板会导致多种细胞蛋白的酪氨酸磷酸化显著增加（费雷尔，J.E.，和马丁，G.S.（1988年）《分子与细胞生物学》8卷，3603 - 3610页；戈尔登，A.，和布鲁格，J.S.（1989年）《美国国家科学院院刊》86卷，901 - 905页；中村，S.，和山村，H.（1989年）《生物化学杂志》264卷，7089 - 7091页）。然而，迄今为止尚未报道任何相关的蛋白酪氨酸激酶。我们在此报告，非受体型蛋白酪氨酸激酶之一的p72syk在凝血酶刺激血液血小板后被激活。用凝血酶刺激洗涤过的猪血小板，并在免疫沉淀激酶测定中评估p72syk的激活情况。在0.5单位/毫升凝血酶刺激后，p72syk的活性在5秒内增加，在10秒时达到最大值，并在60秒内降至基础水平。在整个时间过程中，免疫沉淀的p72syk量没有改变。这种激活以剂量依赖性方式大大增强，并且通过用凝血酶的特异性拮抗剂水蛭素预处理血小板悬浮液而完全被消除。在细胞外和细胞内均缺乏Ca(2+)的条件下，p72syk的激活仍然持续；相反，即使在凝血酶刺激后120秒，失活过程也完全被消除。此外，补充Ca2+导致了与富含Ca(2+)条件下类似的失活模式。此外，用钙调蛋白拮抗剂W7以及肌球蛋白轻链激酶抑制剂ML9预处理血小板也消除了这种失活。这些结果表明，p72syk可能是凝血酶激活血小板后蛋白酪氨酸磷酸化事件的相关酶，并且在凝血酶刺激的血小板中可能以钙调蛋白依赖性方式受到Ca2+的负调控，尤其是通过肌球蛋白轻链激酶。
