Serine/Threonine Phosphatases and Platelet Physiology

描述（由申请人提供）：血小板血栓的形成依赖于激动剂诱导的调节可溶性纤维蛋白原结合的整合素α ib ß3的内向信号，以及通过α ib ß3控制血小板细胞骨架重排的内向信号。由内到外的信号是由几种激动剂结合G蛋白偶联受体（GPCR）产生的。内在的由内向外和由外向内的信号，是可逆的酪氨酸（Tyr）和丝氨酸/苏氨酸（Ser/Thr）磷酸化依赖于多个效应物的组装。几种效应物的磷酸化和活性受蛋白激酶和磷酸酶的调控。虽然在由内到外和由外到内的信号传导过程中激酶介导的磷酸化事件已经得到了深入的研究，但Ser/Thr蛋白磷酸酶1 （PP1c）和蛋白磷酸酶2A （PP2Ac）的催化亚基的作用却相对未被探索。在目前的资助期内，我们注意到在缺乏蛋白磷酸酶1 γ (PP1cγ)催化亚基的小鼠中，凝血酶诱导的内外信号传导减少，体内血栓形成延迟。外向内信号传导不受PP1cγ缺失的影响，但在PP2Ac缺失的情况下增加。我们的总体假设是，丝氨酸/苏氨酸磷酸酶的特定亚型协调了由内向外和由外向内信号传导的空间调节。我们的目标是在对损伤的生理反应中，破解PP1c与G蛋白信号和PP2Ac与整合素信号功能偶联的分子细节。Aim 1将定义PP1c及其相互作用蛋白Gß1在内向外信号传导中的作用。Gß1是异源三聚体G蛋白的一个组成部分，与GPCR偶联，在静息血小板中与PP1c相互作用，而激动剂治疗解离该复合物。小鼠巨核细胞中Gb1的缺失或血小板中Gßγ信号的阻断，凝血酶受体激活肽减少，诱导纤维蛋白原结合和聚集。利用人类和小鼠缺乏PP1cγ、PP1cα和Gß1的血小板，我们的目标是测试Gß1是否将PP1c靶向GPCR复合体并积极调节内外信号传导。Aim 2将确定PP2Ac在α ib ß3介导的外内信号传导中的作用。Src的激活对于外向内信号传导至关重要，我们发现PP2Ac的耗尽激活了Src。CIN85是一种与血小板中PP2Ac和外向内信号分离的复合物相关的接头蛋白。CIN85缺失降低了Src活化和α ib ß3粘附性。使用血小板和α ib ß3模型细胞，我们将检测PP2Ac是否通过CIN85负性调节outside-in信号传导。拟议的研究具有创新性，因为它代表了血小板活化过程中激酶介导的磷酸化事件向磷酸酶介导的去磷酸化事件的转变。这项研究具有重要意义，因为它将促进我们对血小板活化的分子机制的理解，并为确定磷酸酶相互作用蛋白作为未来抗血栓治疗的新治疗靶点奠定基础。

DESCRIPTION (provided by applicant): Formation of platelet thrombi is dependent on the agonist-induced inside-out signaling to integrin αIIbß3 that regulates soluble fibrinogen binding, and outside-in signaling through αIIbß3 that controls the platelet cytoskeletal rearrangement. Inside-out signaling is generated by several agonists that engage the G protein coupled receptors (GPCR). Intrinsic to inside-out and outside-in signaling, is the reversible tyrosine (Tyr and serine/threonine (Ser/Thr) phosphorylation-dependent assembly of multiple effectors. The phosphorylation and the activity of several effectors are regulated by protein kinases and phosphatases. While kinase mediated phosphorylation events during inside-out and outside-in signaling has been intensely investigated, the contribution of the catalytic subunits of Ser/Thr protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) is relatively unexplored. In the current funding period, we noticed decreased thrombin-induced inside- out signaling and delayed in vivo thrombus formation in mice lacking the catalytic subunit of protein phosphatase 1 γ (PP1cγ). Outside-in signaling was unaffected by the loss of PP1cγ, but increased in the absence of PP2Ac. Our overarching hypothesis is that the specific subtypes of Ser/Thr phosphatases orchestrate a spatial regulation of inside-out and outside-in signaling. Our goal is to decipher the molecular details underpinning the functional coupling of PP1c with the G protein signaling and PP2Ac with the integrin signaling, during physiological responses to injury. Aim 1 will define the role of PP1c and its interacting protein Gß1 during inside-out signaling. Gß1, a component of the heterotrimeric G proteins that couple to GPCR, interacted with PP1c in resting platelets, while agonist treatment dissociated this complex. Depletion of Gb1 in murine megakaryocytes or blockade of Gßγ signaling in platelets, decreased thrombin receptor activating peptide induced fibrinogen binding and aggregation. Using platelets from human and mice deficient in PP1cγ, PP1cα and Gß1, our goal is to test if Gß1 targets PP1c to the GPCR complex and positively regulates inside- out signaling. Aim 2 will define the role of PP2Ac during αIIbß3 mediated outside-in signaling. Src activation is critical for outside-in signaling and we showed that PP2Ac depletion activates Src. CIN85 is an adaptor protein that associated with PP2Ac and outside-in signaling dissociated this complex in platelets. CIN85 depletion reduced Src activation and αIIbß3 adhesiveness. Using platelets and αIIbß3 model cells, we will test if PP2Ac negatively regulates outside-in signaling via CIN85. The proposed research is innovative because it represents a departure from the kinase-mediated phosphorylation events to a phosphatase mediated dephosporylation events during platelet activation. The proposed research is significant because it will advance our understanding of the molecular mechanisms of platelet activation, and lay the basic groundwork for identifying phosphatase-interacting proteins as the new therapeutic targets for future anti-thrombotic therapy.