Functional PKC isoforms in platelets

蛋白激酶是细胞功能的关键调节因子。从这个实验室和其他实验室建立的工作 蛋白激酶C(PKC)通路调节激动剂诱导的纤维蛋白原受体激活和分泌 血小板。然而，PKC亚型的特性和潜在的机制还不完全清楚。 例如，该实验室以前的研究表明，PKCd在GPVI-1中起着负面调节作用。 介导致密颗粒释放，促进凝血酶受体下游分泌。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需要 进一步澄清。我们的总体假设是，不同的nPKC亚型在血小板中发挥不同的作用。 功能和差异酪氨酸磷酸化的PKCd亚型触发不同的信号级联 不同的功能反应。我们将使用互补细胞生物学来检验这一总体假设， 药理学、生化和分子遗传学方法。我们的具体目标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。我们将测试 血栓素A2和凝血酶激活调节致密颗粒的特定蛋白激酶C亚型的假说 然而，ADP未能激活这些亚型。为了支持这一想法，我们最近展示了 不被ADP激活的PKCd亚型在致密颗粒释放中起重要作用。目标2是 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的不同 与SHIP1关联。初步研究显示SHIP1与PKCd选择性地关联， GPVI下游，但不是PARS，支持这一假说。目标3是研究分子 PKCd和SHIP1在血小板中的差异相互作用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亚型有几个酪氨酸残基， 可以被磷酸化。我们假设G蛋白下游的不同信号通路偶联 受体和酪氨酸激酶相关受体磷酸化PKCd上不同的酪氨酸残基 差异磷酸化修饰了这些异构体的功能含义。我们的初步研究 提示G蛋白偶联的PARs和酪氨酸激酶型胶原受体GPVI的差异 分别磷酸化Y-311和Y-155残基。我们建议测试这些磷酸化酪氨酸的作用 用分子细胞生物学方法研究SHIP1相互作用中的残基。最后，我们将确定 其他与差异磷酸化PKCd相关的信号分子通过生化和 蛋白质组学方法。本申请中提出的研究将确定新的治疗靶点 血栓形成的治疗。

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.