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: 8435171
• 负责人: K. Vinod VIJAYAN
• 金额: $ 39.13万
• 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 2017-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8435171
• 关键词: Adaptor Signaling Protein; Adhesions; Adhesiveness; Affinity; Agonist; Binding; Binding Sites; Blood Platelets; Blood Vessels; Catalytic Domain; Cell model; Cells; Collagen; Complex; Couples; Coupling; Cytoskeleton; DNA Sequence Rearrangement; Dose; Event; Fibrinogen; Funding; Future; G-Protein-Coupled Receptors; G-substrate; GTP-Binding Proteins; Goals; Guanosine Triphosphate; Heterotrimeric GTP-Binding Proteins; Human; Injury; Integrins; Ligand Binding; Mediating; Megakaryocytes; Molecular; Mus; Myocardial Infarction; Pathway interactions; Phospholipase; Phosphoric Monoester Hydrolases; Phosphorylation; Phosphotransferases; Physiological; Physiology; Platelet Activation; Process; Proline-Rich Domain; Protein Isoforms; Protein Kinase; Protein Phosphatase 2A Regulatory Subunit PR53; Protein Serine/Threonine Phosphatase; Protein phosphatase; Proteins; Publishing; Regulation; Reporting; Research; Rest; Role; SRC gene; Serine; Serine/Threonine Phosphorylation; Signal Transduction; Site; Stroke; TRAP Peptide; Testing; Threonine; Thrombin; Thrombus; Tubulin; Tyrosine; Yeasts; expectation; in vivo; innovation; new therapeutic target; phospholipase C beta; public health relevance; receptor coupling; response; response to injury; src-Family Kinases; yeast two hybrid system

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.

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由内向外信号传导至整联蛋白<$IIb <$3（调节可溶性纤维蛋白原结合），以及由外向内信号传导至aIIb 3（控制血小板细胞骨架重排）。由内而外的信号传导是由几种激动剂产生的，这些激动剂与G蛋白偶联受体（GPCR）结合。由内向外和由外向内信号传导的内在特征是多种效应物的可逆酪氨酸（Tyr）和丝氨酸/苏氨酸（Ser/Thr）磷酸化依赖性组装。几种效应物的磷酸化和活性由蛋白激酶和磷酸酶调节。虽然激酶介导的磷酸化事件在由内向外和由外向内的信号传导过程中已被深入研究，但Ser/Thr蛋白磷酸酶1（PP 1c）和蛋白磷酸酶2A（PP 2Ac）的催化亚基的贡献相对未被探索。在目前的资助期间，我们注意到减少凝血酶诱导的由内而外的信号传导和延迟体内血栓形成的小鼠缺乏蛋白磷酸酶1的催化亚基？(PP1c?).由外向内信号传导不受PP 1c？缺失的影响，但在不存在PP 2Ac的情况下增加。我们的总体假设是，特定亚型的丝氨酸/苏氨酸磷酸酶编排的空间调节的由内而外和由外而内的信号。我们的目标是破译PP 1c与G蛋白信号传导和PP 2Ac与整合素信号传导的功能耦合的分子细节，在对损伤的生理反应过程中。目的1将定义PP 1c及其相互作用蛋白G？1在由内而外信号传导过程中的作用。G？1，异源三聚体G蛋白的一个组成部分，耦合到GPCR，与PP 1c在静息血小板相互作用，而激动剂治疗解离这种复合物。小鼠巨核细胞中Gb 1的耗尽或G？血小板中的信号传导，减少凝血酶受体激活肽诱导的纤维蛋白原结合和聚集。使用缺乏PP 1c？的人和小鼠的血小板，PP 1ca和G <$1，我们的目标是测试G <$1是否将PP 1c靶向GPCR复合物并积极调节由内而外的信号传导。目的2将定义PP 2Ac在aIIbb 3介导的由外向内信号传导过程中的作用。Src激活对于由外向内信号传导是至关重要的，并且我们表明PP 2Ac耗尽激活Src。CIN 85是一种与PP 2Ac结合的衔接蛋白，它通过由外向内的信号传导使PP 2Ac在血小板中解离。CIN 85耗竭减少Src活化和<$IIb <$3 β。使用血小板和aIIbb 3模型细胞，我们将测试PP 2Ac是否通过CIN 85负调节由外向内的信号传导。这项研究是创新性的，因为它代表了血小板活化过程中从激酶介导的磷酸化事件到磷酸酶介导的去磷酸化事件的偏离。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，并为识别磷酸酶相互作用蛋白作为未来抗血栓治疗的新靶点奠定基础。