Department of Medical Biotechnology and Laboratory Science, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan, Taiwan.
J Thromb Haemost. 2017 Oct;15(10):2029-2044. doi: 10.1111/jth.13785. Epub 2017 Sep 6.
Essentials Disabled-2 (Dab2) phosphorylation status in thrombin signaling of human platelet was investigated. Ser723 was the major Dab2 phosphorylation site in human platelets stimulated by thrombin. Dab2 S723 phosphorylation (pS723) caused the dissociation of Dab2-CIN85 protein complex. Dab2-pS723 regulated ADP release and integrin αIIbβ3 activation in thrombin-treated platelets.
Background Disabled-2 (Dab2) is a platelet protein that is functionally involved in thrombin signaling in mice. It is unknown whether or not Dab2 undergoes phosphorylation during human platelet activation. Objectives To investigate the phosphorylation status of Dab2 and its functional consequences in thrombin-stimulated human platelets. Methods Dab2 was immunoprecipitated from resting and thrombin-stimulated platelet lysates for differential isotopic labeling. After enrichment of the phosphopeptides, the phosphorylation sites were analyzed by mass spectrometry. The corresponding phospho-specific antibody was generated. The protein kinases responsible for and the functional significance of Dab2 phosphorylation were defined by the use of signaling pathway inhibitors/activators, protein kinase assays, and various molecular approaches. Results Dab2 was phosphorylated at Ser227, Ser394, Ser401 and Ser723 in thrombin-stimulated platelets, with Ser723 phosphorylation being the most significantly increased by thrombin. Dab2 was phosphorylated by protein kinase C at Ser723 in a G -dependent manner. ADP released from the stimulated platelets further activated the G -dependent pathway to sustain Ser723 phosphorylation. The Cbl-interacting protein of 85 kDa (CIN85) bound to Dab2 at a motif adjacent to Ser723 in resting platelets. The consequence of Ser723 phosphorylation was the dissociation of CIN85 from the Dab2-CIN85 complex. These molecular events led to increases in fibrinogen binding and platelet aggregation in thrombin-stimulated platelets by regulating α β activation and ADP release. Conclusions Dab2 Ser723 phosphorylation is a key molecular event in thrombin-stimulated inside-out signaling and platelet activation, contributing to a new function of Dab2 in thrombin signaling.
研究了人血小板中凝血酶信号转导中Essentials Disabled-2（Dab2）的磷酸化状态。凝血酶刺激的人血小板中主要的 Dab2 磷酸化位点是 Ser723。Dab2 S723 磷酸化（pS723）导致 Dab2-CIN85 蛋白复合物的解离。Dab2-pS723 调节凝血酶处理血小板中 ADP 的释放和整合素 αIIbβ3 的激活。
背景：Disabled-2（Dab2）是一种血小板蛋白，在小鼠的凝血酶信号转导中具有功能作用。尚不清楚人血小板激活过程中 Dab2 是否发生磷酸化。目的：研究 Dab2 的磷酸化状态及其在凝血酶刺激的人血小板中的功能后果。方法：从静止和凝血酶刺激的血小板裂解物中免疫沉淀 Dab2 进行差异同位素标记。磷酸肽富集后，通过质谱分析磷酸化位点。生成相应的磷酸特异性抗体。通过使用信号转导途径抑制剂/激活剂、蛋白激酶测定和各种分子方法，确定负责 Dab2 磷酸化的蛋白激酶及其功能意义。结果：凝血酶刺激的血小板中 Dab2 磷酸化于 Ser227、Ser394、Ser401 和 Ser723，凝血酶最显著地增加了 Ser723 磷酸化。蛋白激酶 C 以 G 依赖性方式使 Dab2 磷酸化 Ser723。从刺激的血小板中释放的 ADP 进一步激活 G 依赖性途径以维持 Ser723 磷酸化。Cbl 相互作用蛋白 85kDa（CIN85）在静止血小板中与 Dab2 结合于紧邻 Ser723 的一个基序上。Ser723 磷酸化的后果是 CIN85 从 Dab2-CIN85 复合物中解离。这些分子事件通过调节 αβ 激活和 ADP 释放，导致凝血酶刺激的血小板中纤维蛋白原结合和聚集增加。结论：Dab2 Ser723 磷酸化是凝血酶刺激的内-外信号转导和血小板激活中的关键分子事件，为 Dab2 在凝血酶信号转导中的新功能做出贡献。
