Serine / Threonine Phosphatases and Platelet Physiology

血小板血栓的形成依赖于调节配体的整合素allbb3的内向外信号 通过allbbs结合和由外向内的信号控制血小板细胞骨架重排。里面- Out和Outside-In信号事件涉及酪氨酸(Tyr)或丝氨酸/苏氨酸的可逆磷酸化 多个蛋白质上的(丝氨酸/苏氨酸)残基。蛋白质底物的净Tyr或Ser/Thr磷酸化是 受蛋白激酶和磷酸酶活性的调节。从历史上看，激酶和 在整合素介导的信号转导过程中，磷酸化事件占据了中心舞台，而整合素介导的信号转导过程中 人们对磷酸酶知之甚少。我们的初步研究首次证明了催化剂 蛋白磷酸酶1亚基(PP1c)和蛋白磷酸酶2A亚基(PP2Ac)，但不包括蛋白磷酸酶 2C(PP2Cc)与整合素allbbS结合，调节allbbS的黏附功能。我们 假设PP1c和PP2Ac协调整合素allbbS信号的时间和空间调节 并参与血小板功能。 我们的目标是研究丝氨酸/苏氨酸磷酸酶调节整合素allbbS激活的机制。 利用人血小板、PP1c基因缺失小鼠的血小板、小鼠巨核细胞和 凝血酶激活的allbbS(DT40 PAR1/allbb3)细胞株。目标1将检验PP1c a)的假设 使静息血小板整合素B上的丝氨酸/苏氨酸残基去磷酸化，b)从而使粘附素去磷酸化 激活细胞骨架重组，c)参与由内而外和由外而内的信号传递。 一切都好。AIM 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。这个 将评估PP1c与其他带有PP1c结合位点的整合素相互作用的可能性。目标3将 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结合位点映射到 整合素allbbS，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 研究应该提供对未被研究的血小板功能方面的新见解，并可能提供 抗血栓治疗的潜在新治疗靶点。

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 rearrangment. 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.