Functional PKC isoforms in platelets

描述(由申请人提供)：蛋白激酶是细胞功能的关键调节器。来自该实验室和其他实验室的工作证实，蛋白激酶C(PKC)途径调节激动剂诱导的血小板中纤维蛋白原受体的激活和分泌。然而，PKC亚型的特性和潜在的机制还不完全清楚。例如，本实验室以前的研究表明，PKCd在GPVI介导的致密颗粒释放中起负面调节作用，但它促进凝血酶受体下游的分泌。同样，血小板新型蛋白激酶C(NPKC)亚型酪氨酸磷酸化的生理意义还需要进一步阐明。我们的总体假设是，不同的nPKC亚型在血小板功能中发挥不同的作用，不同的酪氨酸磷酸化的PKCd亚型触发不同的信号级联反应，导致不同的功能反应。我们将使用互补细胞生物学、药理学、生化和分子遗传学方法来检验这一总体假设。我们的具体目的1是评估不同的PKC亚型在血小板纤维蛋白原受体激活和分泌中的功能作用。我们将验证这样的假设：“血栓烷A2和凝血酶激活调节致密颗粒释放的特定PKC亚型；然而，ADP未能激活这些亚型”。为了支持这一观点，我们最近证明了不被ADP激活的PKCd亚型在致密颗粒释放中发挥着重要作用。目的2阐明PKCd对血小板致密颗粒释放的差异性调控的分子基础。我们假设，GPVI和PARS下游的PKCd的不同调控是由于它与SHIP1的不同关联。初步研究表明，SHIP1与PKCd选择性相关，位于GPVI下游，但不与PARs相关，支持这一假说。目的3是研究PKCd和SHIP1在血小板中差异相互作用的分子机制。我们假设酪氨酸磷酸化的PKCd触发不同的信号级联反应。PKC亚型有几个可被磷酸化的酪氨酸残基。我们假设G蛋白偶联受体和酪氨酸激酶相关受体下游的不同信号通路使PKCd上不同的酪氨酸残基磷酸化，这些差异磷酸化改变了这些异构体的功能。我们的初步研究表明，G蛋白偶联的PARs和酪氨酸激酶连接的胶原受体GPVI分别差异磷酸化Y-311和Y-155残基。我们建议用分子细胞生物学的方法测试这些磷酸化酪氨酸残基在与SHIP1相互作用中的作用。最后，我们将通过生化和蛋白质组学方法鉴定与差异磷酸化PKCd相关的其他信号分子。这项申请中提出的研究将确定血栓形成治疗的新治疗靶点。公共卫生相关性：血小板激活对止血至关重要，并可能导致血栓事件。激动剂受体下游的信号转导机制对于了解血小板活化的分子基础非常重要。这项拟议的研究通过结合生化、药理学和遗传学的方法来研究血小板表面受体下游的信号事件的调节和功能。深入了解这些机制将有助于确定抗血栓治疗的新靶点。

DESCRIPTION (provided by applicant): Protein kinases are critical regulators of cellular functions. Work from this and other laboratories established that protein kinase C (PKC)-pathways modulate agonist-induced fibrinogen receptor activation and secretion in platelets. However, the identity of PKC isoforms and the underlying mechanisms are incompletely understood. For example, previous studies from this lab have shown that PKCd plays a negative regulatory role in GPVI- mediated dense granule release whereas it promotes secretion downstream of thrombin receptors. Similarly, the physiological significance of tyrosine phosphorylation of platelet novel class PKC (nPKC) isoforms requires further elucidation. Our overall hypothesis is that different nPKC isoforms play different roles in platelet functions and differentially tyrosine phosphorylated PKCd isoforms trigger distinct signaling cascades leading to diverse functional responses. We will test this overall hypothesis using complimentary cell biological, pharmacological, biochemical, and molecular genetic approaches. Our specific aim 1 is to evaluate the functional role of different PKC isoforms in platelet fibrinogen receptor activation and secretion. We will test the hypothesis that "thromboxane A2 and thrombin activate specific PKC isoforms that regulate dense granule release; ADP, however, fails to activate these isoforms". In support of this idea, we have recently demonstrated the PKCd isoform, which is not activated by ADP, plays an important role in dense granule release. Aim 2 is to delineate the molecular basis for differential regulation of dense granule release by PKCd in platelets. We hypothesize that differential regulation of PKCd, downstream of GPVI and PARS, occurs due to its differential association with SHIP1. Preliminary studies that show selectively association of SHIP1 with PKCd, downstream of GPVI but not PARs, supports this hypothesis. The aim 3 is to investigate the molecular mechanism of differential interaction of PKCd and SHIP1 in platelets. We hypothesize that tyrosine phosphorylated PKCd triggers different signaling cascades. PKC isoforms have several tyrosine residues that can be phosphorylated. We hypothesize that diverse signaling pathways downstream of G protein-coupled receptors and tyrosine kinase-linked receptors phosphorylate different tyrosine residues on PKCd and these differential phosphorylations modify the functional implications of these isoforms. Our preliminary studies indicate that G protein-coupled PARs and tyrosine kinase-linked collagen receptor GPVI differentially phosphorylate Y-311 and Y-155 residues, respectively. We propose to test the role of these phospho-tyrosine residues in the interaction with SHIP1 by molecular cell biological approaches. Finally, we will identify additional signaling molecules associated with differentially phosphorylated PKCd by biochemical and proteomic approaches. The studies proposed in this application will identify novel therapeutic targets towards treatment of thrombosis. PUBLIC HEALTH RELEVANCE: Platelet activation is critical for hemostasis and can lead to thrombotic events. The signal transduction mechanisms downstream of agonist receptors are important to understand the molecular basis of platelet activation. The proposed research examines the regulation and function of signaling events downstream of platelet surface receptors through a combination of biochemical, pharmacological and genetic approaches. An in-depth understanding of these mechanisms will aid in identifying novel targets of antithrombotic therapeutics.