Serine / Threonine Phosphatases and Platelet Physiology

描述(申请人提供)：血小板血栓的形成依赖于整合素allbb3的内向外信号，整合素allb3通过allbbS调节配体结合和外向内信号，allbbS控制血小板细胞骨架重排。由内而外和由外而内的信号事件涉及多种蛋白质上酪氨酸(Tyr)或丝氨酸/苏氨酸(Ser/Thr)残基的可逆磷酸化。蛋白质底物的净酪氨酸或丝氨酸/苏氨酸磷酸化受蛋白激酶和磷酸酶活性的调节。历史上，在整合素介导的信号转导过程中，激酶和磷酸化事件一直占据着中心阶段，而磷酸酶的作用却鲜为人知。我们的初步研究首次证明蛋白磷酸酶1(PP1c)和蛋白磷酸酶2A(PP2Ac)的催化亚基(PP1c)而不是蛋白磷酸酶2c(PP2Cc)与整合素allbbS发生结构性结合并调节allbbS的黏附功能。我们假设PP1c和PP2Ac协调整合素allbbS信号的时空调节，并参与血小板功能。我们的目标是利用人血小板、PP1c缺失小鼠的血小板、小鼠巨核细胞和凝血酶激活的allbbS细胞系(DT40PAR1/allbb3)，研究Ser/Thr磷酸酶调节整合素allbbS激活、信号和功能的机制。目的1验证PP1c a)去磷酸化静息血小板整合素B上的Ser/Thr残基，b)去磷酸化cofilin从而激活细胞骨架重组，c)通过allbbS参与由内而外和由外而内的信号转导。目的2将检查a)PP1c-Al1b相互作用是否调节其他Al1b结合蛋白的结合，b)血小板脂筏在整合素-磷酸酶结合的空间调节中起作用。PP1c与其他带有PP1c结合位点的整合素相互作用的可能性将被评估。目的3确定PP2Ac在整合素allbbS生物学中的作用。值得注意的是，我们将PP2Ac结合位点定位在整合素allbbS上，研究PP2Ac在allbbS由内向外和由外向内信号转导中的作用。因此，通过阐明allbbS-磷酸酶相互作用的机制和后果，这些研究应该为血小板功能未被研究的方面提供新的见解，并可能为抗血栓治疗提供潜在的新的治疗靶点。

DESCRIPTION (provided by applicant): Platelet thrombus formation is dependent on inside-out signaling to integrin allbb3 that regulates ligand binding and outside-in signaling through allbbS that controls the platelet cytoskeletal rearrangement. Inside- out and outside-in signaling events involve reversible phosphorylation of tyrosine (Tyr) or serine/threonine (Ser/Thr) residues on multiple proteins. The net Tyr or Ser/Thr phosphorylation of a protein substrate is regulated by the activities of both protein kinases and phosphatases. Historically, kinases and phosphorylation events have held the center stage during integrin-mediated signaling, while a role for the phosphatases is poorly understood. Our preliminary studies demonstrate for the first time that the catalytic subunits of protein phosphatase 1 (PP1c) and protein phosphatase 2A (PP2Ac) but not protein phosphatase 2c (PP2Cc) associate constitutively with the integrin allbbS and regulates allbbS adhesive function. We hypothesize that PP1c and PP2Ac orchestrate a temporal and spatial regulation of integrin allbbS signaling and participate in platelet function. Our goal is to study the mechanisms by which Ser/Thr phosphatases regulate integrin allbbS activation, signaling and function using human platelets, platelets from PP1c null mice, murine megakaryocytes and a cell line with thrombin activatable allbbS (DT40 PAR1/allbb3). Aim 1 will test the hypothesis that PP1c a) dephosphorylates Ser/Thr residues on integrin bS in resting platelets, b) dephosphorylates cofilin thereby activating the cytoskeletal reorganization, and c) is involved in inside-out and outside-in signaling through allbbS. Aim 2 will examine if a) the PP1c-allb interaction modulates the binding of other allb binding proteins, b) the platelet lipid rafts have a role in the spatial regulation of the integrin-phosphatase association. The possibility that PP1c interacts with other integrins bearing the PP1c binding site will be evaluated. Aim 3 will establish the role for PP2Ac in the biology of integrin allbbS. Notably, we will map the PP2Ac binding site on integrin allbbS, study the role of PP2Ac in allbbS inside-out and outside-in signaling. Thus, by elucidating the mechanisms and consequences of allbbS-phosphatase interactions, these studies should provide novel insights into the understudied aspects of platelet function and may provide potential new therapeutic targets for anti-thrombotic therapy.