Serine/Threonine Phosphatases and Platelet Physiology

说明(申请人提供)：血小板血栓的形成依赖于激动剂诱导的由内向外的整合素IIb 3信号(调节可溶性纤维蛋白原结合)，以及通过aIIbb3控制血小板细胞骨架重排的自外向内信号。由内向外的信号是由几种激动剂产生的，它们与G蛋白偶联受体(GPCR)结合。内向外和外向内信号转导的本质是依赖于酪氨酸(Tyr)和丝氨酸/苏氨酸(Ser/Thr)磷酸化的多个效应器的组装。几种效应器的磷酸化和活性受蛋白激酶和磷酸酶的调节。虽然在由内向外和由外向内的信号转导过程中，KK介导的磷酸化事件已经被深入研究，但Ser/Thr蛋白磷酸酶1(PP1c)和蛋白磷酸酶2A(PP2Ac)的催化亚基的作用相对较少。在目前的资助期，我们注意到凝血酶诱导的内向外信号减少，体内血栓形成延迟，缺乏蛋白磷酸酶1？催化亚单位的小鼠。(PP1c？)外向内信号转导不受PP1c？缺失的影响，但在PP2Ac缺失的情况下增强。我们的主要假设是，Ser/Thr磷酸酶的特定亚型协调了由内向外和由外向内信号的空间调节。我们的目标是破译PP1c与G蛋白信号和PP2Ac与整合素信号在生理反应中的功能耦合的分子细节。目标1将确定PP1c及其相互作用蛋白G？1在内向外信号转导中的作用。G？1是与gpr偶联的异源三聚体G蛋白的一种成分，在静息状态下与PP1c相互作用，而激动剂处理使这一复合体解离。小鼠巨核细胞中GB1的耗竭还是G？在血小板信号转导中，凝血酶受体激活肽减少引起纤维蛋白原结合和聚集。使用缺乏PP1c？、PP1ca和G？1的人和小鼠的血小板，我们的目标是测试G？1是否针对GPCR复合体中的PP1c，并积极地调节内向外信号。目标2将确定PP2Ac在aIIbb3介导的由外向内信号转导中的作用。SRC的激活是由外向内信号转导的关键，我们证明了PP2Ac的耗竭激活了Src。CIN85是一种与PP2Ac相关的接头蛋白，外向内信号在血小板中解离了这一复合体。CIN85缺失降低了Src的活性和IIb 3的粘附性。利用血小板和aIIbb3模型细胞，我们将测试PP2Ac是否通过CIN85负向调节外向内信号。这项拟议的研究是创新的，因为它代表了在血小板激活过程中从激酶介导的磷酸化事件到磷酸酶介导的去磷孢子化事件的转变。这项研究具有重要意义，因为它将促进我们对血小板激活的分子机制的理解，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 ¿IIb¿3 that regulates soluble fibrinogen binding, and outside-in signaling through aIIbb3 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 ? (PP1c?). Outside-in signaling was unaffected by the loss of PP1c?, 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 G¿1 during inside-out signaling. G¿1, 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 G¿? signaling in platelets, decreased thrombin receptor activating peptide induced fibrinogen binding and aggregation. Using platelets from human and mice deficient in PP1c?, PP1ca and G¿1, our goal is to test if G¿1 targets PP1c to the GPCR complex and positively regulates inside- out signaling. Aim 2 will define the role of PP2Ac during aIIbb3 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 ¿IIb¿3 adhesiveness. Using platelets and aIIbb3 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.