Lacabaratz-Porret C, Corvazier E, Kovàcs T, Bobe R, Bredoux R, Launay S, Papp B, Enouf J
Institut National de la Santé et de la Recherche Médicale U 348, IFR Circulation Lariboisière, Hôpital Lariboisière, 8 rue Guy Patin, 75475 Paris Cedex 10, France.
Biochem J. 1998 May 15;332 ( Pt 1)(Pt 1):173-81. doi: 10.1042/bj3320173.
Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein-protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.
血小板钙离子信号传导涉及细胞内钙离子池，其含量由肌浆网/内质网钙离子ATP酶（SERCAs）控制。其中，与SERCAs 3b亚型相关的三磷酸肌醇敏感钙离子池起关键作用。我们研究了通过GTP结合蛋白Rap（Ras近端）1b的环磷酸腺苷（cAMP）依赖性磷酸化对该钙离子池的调控。我们首先通过蛋白质免疫印迹法和逆转录聚合酶链反应研究人血小板及各种细胞系中不同SERCAs和Rap 1蛋白的表达，寻找该钙离子池的调控靶点。由于获得了Rap 1b和SERCAs 3b的共表达，我们研究了作为Rap 1b的cAMP依赖性磷酸化功能的蛋白质-蛋白质相互作用。在无cAMP依赖性蛋白激酶催化亚基（csPKA）诱导的磷酸化情况下，发现SERCAs 3b和Rap 1b蛋白存在共免疫沉淀。相反，用csPKA预处理血小板膜后，SERCAs 3b与Rap 1b蛋白解离，这与其磷酸化状态在它们相互作用中的作用一致。最后，我们在高血压血小板病理模型中研究了该复合物的适应性变化。我们研究了对照正常血压的Wistar-Kyoto大鼠和自发性高血压大鼠（SHRs）血小板中这两种蛋白的表达、Rap 1b的cAMP依赖性磷酸化以及SERCAs 3b活性。SHRs血小板中SERCAs 3b活性降低与Rap 1b内源性磷酸化降低相关，这与SERCAs 3b相关钙离子池调控中的功能作用一致。
