Functional PKC Isoforms in Platelets

描述(申请人提供)：蛋白激酶C(PKC)亚型是丝氨酸苏氨酸激酶，在激动剂刺激下被激活，并调节几种功能。在之前的资助期间，我们展示了nPKC亚型在血小板中的功能作用以及PKC？用SHIP1和LYN调节颗粒的差异释放。我们有强有力的初步数据表明，PKC？Y155和Y311上的酪氨酸是磷酸化的，但这两种酪氨酸在刺激下被不同的磷酸化 胶原蛋白或凝血酶受体。我们已经为PKC培育出了敲入小鼠？Y155F突变和我们的初步数据表明，PKC？通过Y155残留物。我们也有强有力的初步数据表明，Y311可能调节cPLA2的激活和血栓素的产生。最后，我们确定了PKC的负面调节作用？巨核细胞的生成和血小板的生成。因此，我们将重点研究PKC？调节血小板功能和巨核细胞生成的分子机制。在这一批款期内。我们的总体假设是PKC？通过Y155和Y311残基的酪氨酸磷酸化在血小板功能和巨核细胞生成中起关键作用。我们认为这些磷酸化的酪氨酸通过与其他信号分子的相互作用形成信号转导的结点，从而调节功能反应。1)我们假设不同的信号 G蛋白偶联受体和酪氨酸激酶连接受体下游的通路使PKC上不同的酪氨酸残基磷酸化？这些不同的磷酸化 修饰该异构体的功能作用。我们假设PKC？Y155通过与SHIP1和Lyn结合来调节致密颗粒的释放，而PKC？Y311的磷酸化调节cPLA2的激活和血栓素的产生。我们将研究Y155和Y311的磷酸化对PKC？使用PKC的血小板？敲入特定突变Y155F或Y311F的小鼠。我们假设Y155和Y311在PKC上发生了磷酸化？在PKC上启动不同于其他酪氨酸残基的信号通路？通过不同的信号分子的联合，我们将通过生化/蛋白质组学的方法来验证这一假说。2)我们假设PKC？在巨核细胞分化和血小板生成中起着明显的作用。我们建议使用缺乏PKC？的小鼠骨髓巨核细胞来验证这一假说。以及Y155F和Y311F基因敲入小鼠。本申请中提出的研究将增进我们对PKC？信号网络及其对血小板功能反应和血小板产生的调节。这些研究将帮助我们确定治疗血栓性疾病和血小板减少症的靶点。

DESCRIPTION (provided by applicant): Protein kinase C (PKC) isoforms are serine threonine kinases that are activated upon agonist stimulation and regulate several functions. In the previous grant period, we demonstrated the functional role of nPKC isoforms in platelets and the molecular interactions of PKC? with SHIP1 and Lyn to regulate differential granule release. We have strong preliminary data indicating that PKC? is phosphorylated on Y155 and Y311, but these two tyrosines are differentially phosphorylated upon stimulation of Collagen or Thrombin receptors. We have generated knock-in mice for PKC? Y155F mutation and our preliminary data point to the regulation of GPVI-mediated functional events by PKC? through the Y155 residue. We also have strong preliminary data suggesting that Y311 might regulate cPLA2 activation and thromboxane generation. Finally, we have identified a negative regulatory role for PKC? in megakaryopoiesis and platelet production. Hence we will focus our efforts on the molecular mechanisms of regulation of platelet function and megakaryopoiesis by PKC? in this grant period. Our overall hypothesis is that PKC? plays a crucial role in platelet function and megakaryopoiesis through tyrosine phosphorylation of Y155 and Y311 residues. We propose that these phosphorylated tyrosines form nodal points for signal transduction through interaction with other signaling molecules thus regulating functional responses. 1) We hypothesize that diverse signaling pathways downstream of G protein-coupled receptors and tyrosine kinase-linked receptors phosphorylate different tyrosine residues on PKC? and these differential phosphorylations modify the functional role of this isoform. We hypothesize that PKC? Y155 phosphorylation regulates dense granule release through association with SHIP1 and Lyn, whereas PKC? Y311 phosphorylation regulates cPLA2 activation and thromboxane generation. We will investigate the functional role of phosphorylation of Y155 and Y311 on PKC? in platelets using PKC? knock-in mice with the specific mutation Y155F or Y311F. We hypothesize that phosphorylated Y155 and Y311 on PKC? initiate different signaling pathways than other tyrosine residues on PKC? through association of different signaling molecules and we will test this hypothesis by biochemical/proteomic approaches. 2) We hypothesize that PKC? plays a distinct role in megakaryocyte differentiation and platelet production. We propose to test this hypothesis using bone marrow megakaryocytes from mice lacking PKC? as well as from Y155F and Y311F knock-in mice. The studies proposed in this application will enhance our understanding of PKC? signaling networks and their regulation of functional responses in platelets and the production of platelets. These studies will then help us identify targets to trea thrombotic disorders as well as thrombocytopenia.