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因子。本实验室和其他实验室的工作证实，蛋白激酶C （PKC）通路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同工异构体的身份和潜在的机制尚不完全清楚。例如，本实验室之前的研究表明PKCd在GPVI介导的致密颗粒释放中发挥负调控作用，同时促进凝血酶受体下游的分泌。同样，血小板新型PKC （nPKC）异构体酪氨酸磷酸化的生理意义需要进一步阐明。我们的总体假设是，不同的nPKC亚型在血小板功能中起着不同的作用，不同的酪氨酸磷酸化PKCd亚型触发不同的信号级联，导致不同的功能反应。我们将使用互补的细胞生物学、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。我们将验证以下假设：“血栓素A2和凝血酶激活调节致密颗粒释放的特异性PKC亚型；然而，ADP不能激活这些同工异构体。为了支持这一观点，我们最近证明了PKCd异构体，它不被ADP激活，在致密颗粒释放中起重要作用。目的2是描述PKCd在血小板中致密颗粒释放差异调节的分子基础。我们假设GPVI和PARS下游PKCd的差异调控是由于其与SHIP1的差异关联而发生的。初步研究表明SHIP1与GPVI下游的PKCd选择性关联，而不是PARs，支持了这一假设。目的3是探讨血小板中PKCd和SHIP1差异相互作用的分子机制。我们假设酪氨酸磷酸化PKCd触发不同的信号级联反应。PKC异构体有几个可以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连接受体下游的不同信号通路磷酸化PKCd上不同的酪氨酸残基，这些差异磷酸化改变了这些同工异构体的功能意义。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分别对Y-311和Y-155残基进行不同的磷酸化。我们建议用分子细胞生物学方法测试这些磷酸酪氨酸残基在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确定与PKCd差异磷酸化相关的其他信号分子。在本应用中提出的研究将确定治疗血栓形成的新治疗靶点。公共卫生相关性：血小板活化对止血至关重要，可导致血栓事件。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。本研究拟通过结合生化、药理学和遗传学的方法，探讨血小板表面受体下游信号事件的调控和功能。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.