Serine / Threonine Phosphatases and Platelet Physiology

丝氨酸/苏氨酸磷酸酶和血小板生理学

• 批准号: 7096797
• 负责人: K. Vinod VIJAYAN
• 金额: $ 26.25万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-03-01 至 2011-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7096797
• 关键词: actin binding protein; binding sites; biological signal transduction; cell adhesion; cell line; clinical research; enzyme activity; enzyme mechanism; genetically modified animals; human subject; immunoprecipitation; integrins; laboratory mouse; lipid raft; megakaryocytes; phosphatase inhibitor; phosphoprotein phosphatase; platelets; protein binding; protein isoforms; protein protein interaction; small interfering RNA; western blottings

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.

描述（由申请方提供）：血小板血栓形成依赖于由内而外的信号传导至整联蛋白allbb 3（调节配体结合）和由外向内的信号传导（通过allbbS控制血小板细胞骨架重排）。由内向外和由外向内信号传导事件涉及多种蛋白质上的酪氨酸（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Tyr或Ser/Thr磷酸化受蛋白激酶和磷酸酶两者的活性调节。从历史上看，激酶和磷酸化事件在整合素介导的信号传导过程中占据中心地位，而磷酸酶的作用知之甚少。我们的初步研究表明，蛋白磷酸酶1（PP 1c）和蛋白磷酸酶2A（PP 2Ac）的催化亚基，而不是蛋白磷酸酶2C（PP 2Cc）组成型关联的整合素allbbS和调节allbbS粘附功能。我们假设PP 1c和PP 2Ac协调整合素allbbS信号传导的时间和空间调节，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的激活，信号和功能，使用人血小板，血小板从PP 1c null小鼠，小鼠巨核细胞和细胞系凝血酶激活allbbS（DT 40 PAR 1/allbb 3）的机制。目的1将检验以下假设：PP 1c a）使静息血小板中整联蛋白bS上的Ser/Thr残基去磷酸化，B）使cofilin去磷酸化，从而激活细胞骨架重组，以及c）通过allbbS参与由内向外和由外向内的信号传导。目的2将检查a）PP 1c-all B相互作用是否调节其他all B结合蛋白的结合，B）血小板脂筏是否在整合素-磷酸酶结合的空间调节中起作用。将评价PP 1c与其他带有PP 1c结合位点的整合素相互作用的可能性。目的3将确定PP 2Ac在整合素allbbS生物学中的作用。值得注意的是，我们将绘制PP 2Ac在整合素allbbS上的结合位点，研究PP 2Ac在allbbS由内向外和由外向内信号传导中的作用。因此，通过阐明allbbS-磷酸酶相互作用的机制和后果，这些研究应该提供新的见解，研究不足的方面血小板功能，并可能提供潜在的新的抗血栓治疗的治疗靶点。