Functional PKC isoforms in platelets

血小板中的功能性 PKC 亚型

• 批准号: 8281478
• 负责人: Satya P. Kunapuli
• 金额: $ 43.23万
• 依托单位: TEMPLE UNIV OF THE COMMONWEALTH
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-05-01 至 2014-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8281478
• 关键词: 1,2-diacylglycerol; Agonist; Biochemical; Biological; Blood Platelets; Calcium; Cell physiology; Cells; Collagen Receptors; Coupled; Cytoplasmic Granules; Data; Diglycerides; Event; Fibrinogen Receptors; G-Protein-Coupled Receptors; GTP-Binding Proteins; Genetic; Hemostatic function; Injury; Knockout Mice; Laboratories; Lead; Link; Mediating; Modeling; Molecular; Molecular Genetics; Pathway interactions; Phospholipase C; Phosphorylation; Physiological; Platelet Activation; Play; Poly(ADP-ribose) Polymerases; Protein Isoforms; Protein Kinase; Protein Kinase C; Protein Tyrosine Kinase; Protein-Serine-Threonine Kinases; Proteomics; Receptor Activation; Receptor Protein-Tyrosine Kinases; Regulation; Research; Role; Signal Pathway; Signal Transduction; Signaling Molecule; Site-Directed Mutagenesis; Stream; Surface; Testing; Therapeutic; Thrombin; Thrombin Receptor; Thrombosis; Thromboxane A2; Tyrosine; Tyrosine Phosphorylation; Work; base; calcium-dependent protein kinase; new therapeutic target; novel; protein kinase C kinase; receptor; response

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.

蛋白激酶是细胞功能的关键调节剂。本实验室和其他已建立的实验室的工作 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异构体的身份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中起负调节作用- 介导致密颗粒释放，而它促进凝血酶受体下游的分泌。与此类似， 血小板新型PKC（nPKC）亚型酪氨酸磷酸化的生理意义需要 进一步说明。我们的总体假设是，不同的nPKC亚型在血小板活化中发挥不同的作用。 功能和差异酪氨酸磷酸化PKCd亚型触发不同的信号级联反应， to diverse多样functional功能responses反应.我们将使用互补细胞生物学， 药理学、生物化学和分子遗传学方法。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活化和分泌中的功能作用。我们将测试 假设“血栓素A2和凝血酶激活调节致密颗粒特异性PKC亚型 然而，ADP不能激活这些同种型”。为了支持这个想法，我们最近证明了 不被ADP激活的PKCd同种型在致密颗粒释放中起重要作用。目标二是 描绘了血小板中PKCd对致密颗粒释放的差异调节的分子基础。我们 假设在GPVI和PARS下游PKCd的差异调节由于其差异而发生， 与SHIP 1的联系初步研究显示SHIP 1与PKCd选择性相关， 下游的GPVI，但不是PAR，支持这一假设。目的3是研究分子生物学 血小板中PKCd和SHIP 1的差异相互作用机制。我们假设酪氨酸 磷酸化PKCd触发不同的信号级联。PKC亚型具有几个酪氨酸残基， 可以被磷酸化。我们假设G蛋白偶联的下游不同的信号通路 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，这些 差异磷酸化修饰了这些同种型的功能含义。我们的初步研究 表明G蛋白偶联PAR和酪氨酸激酶连接的胶原蛋白受体GPVI 分别磷酸化Y-311和Y-155残基。我们建议测试这些磷酸酪氨酸的作用， 通过分子细胞生物学方法研究了SHIP 1与其他氨基酸残基的相互作用。最后，我们将确定 通过生物化学和生物化学方法与差异磷酸化PKCd相关的其他信号分子 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， 血栓的治疗。