Functional PKC Isoforms in Platelets

描述（由申请人提供）：蛋白激酶C（PKC）同种型是丝氨酸苏氨酸激酶，在激动剂刺激后被激活并调节多种功能。在上一个资助期，我们证明了nPKC亚型在血小板中的功能作用和PKC？与SHIP 1和林恩调节差异颗粒释放。我们有强有力的初步数据表明，PKC？在Y155和Y311上被磷酸化，但这两个酪氨酸在刺激后被差异磷酸化 胶原蛋白或凝血酶受体。 我们已经产生了PKC基因敲入小鼠？Y155 F突变和我们的初步数据点的调控GPVI介导的功能事件的PKC？通过Y155残基。 我们也有强有力的初步数据表明，Y311可能调节cPLA 2的激活和血栓素的产生。最后，我们已经确定了一个负调控作用的PKC？巨核细胞生成和血小板生成因此，我们将集中精力研究PKC调控血小板功能和巨核细胞生成的分子机制。 在这段时间里， 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酪氨酸通过与其他信号分子相互作用形成信号转导的节点，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相关受体的下游途径磷酸化PKC上不同的酪氨酸残基？ 这些差异磷酸化 修饰该同种型的功能作用。 我们假设PKC？Y155磷酸化通过与SHIP 1和林恩的结合调节致密颗粒的释放，而PKC？Y311磷酸化调节cPLA 2活化和血栓素生成。我们将探讨磷酸化的Y155和Y311对PKC的功能作用？在血小板中使用蛋白激酶C？具有特异性突变Y155 F或Y311 F的敲入小鼠。 我们推测磷酸化的Y155和Y311对PKC？ 启动不同的信号通路比其他酪氨酸残基的PKC？我们将通过生物化学/蛋白质组学方法来检验这一假设。2)我们假设PKC？在巨核细胞分化和血小板产生中起着独特的作用。 我们建议使用缺乏PKC的小鼠骨髓巨核细胞来验证这一假设。 以及来自Y155 F和Y311 F基因敲入小鼠。 本申请中提出的研究将提高我们对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.