ROLE AND REGULATION OF PLATELET PROTEIN KINASE C

蛋白激酶C(PKC)是一种重要的信号转导途径 转导、细胞调节、细胞分化和肿瘤促进。 现已知，该酶以一组密切相关的同工酶家族形式存在。 其个体机制和功能尚未明确。在人类中 血小板，PKC与许多血小板功能有关 包括分泌和聚集以及负反馈机制 (关于血小板激活)。我们已经鉴定了四种PKC同工酶。 血小板。长期目标是确定PKC在血小板中的作用 功能。该建议旨在研究不同的PKC的假设 同工酶是不同的调节，以转导选择性功能。 我们的实验室，在生化方面有丰富的经验 PKC的表征和在血小板生物学研究中的独特之处 适合于评估这些问题。这项建议的具体目标是， 因此，旨在确定PKC的机制和意义 来自血小板的同工酶。这些问题将通过以下方式解决：1)纯化PKC 血小板同工酶(FPLC)及其体外研究 监管(使用我们开发的混合胶束方法)；2) 静息状态下PKC同工酶在细胞内的定位 分别激活的血小板(通过监测酶活性，佛波尔 结合和Western blotts)；3)研究原位(生理学) 血小板蛋白激酶C同工酶的调节及其选择性测定 底物磷酸化(使用我们拥有的体外模型 发达)。这些研究将提供必要的生物化学背景。 以确定PKC同工酶的作用机制和生理调节。 这样的认识对于提高我们对 血小板在止血中的作用及其在心脏病理生理中的作用 动脉血管疾病。这种改进的生物化学知识可能会导致 更有效、更合理的抗血小板药物开发。知识 从血小板模型中获得的信息应该被证明对 研究PKC同工酶在不同类型生物体内的调节和作用 其他细胞系统。

Protein kinase C (PKC) has emerged as a critical enzyme in signal transduction, cell regulation, cell differentiation, and tumor promotion. The enzyme is now known to exist as a family of closely-related isoforms whose individual mechanism and function has not been defined. In human platelets, PKC has been implicated in a number of platelet functions including secretion and aggregation as well as negative feedback mechanisms (on platelet activation). We have identified four isoenzymes of PKC in platelets. The long-term goals are to define the role of PKC in platelet function. This proposal aims at studying the hypothesis that different PKC isoenzymes are differentially regulated to transduce selective functions. Our laboratory, which has extensive experience in the biochemical characterization of PKC and in the study of platelet biology, is uniquely suited to evaluate these questions. The specific aims of this proposal are, therefore, directed at determining the mechanism and significance of PKC isoenzymes from platelets. These will be addressed by: 1) purifying PKC isoenzymes from platelets (using FPLC) and studying their in vitro regulation (using mixed micellar methodologies that we have developed); 2) determining the intracellular localization of PKC isoenzymes in resting and activated platelets, respectively, (by monitoring enzyme activity, phorbol binding and by Western blots) and; 3) studying the in situ (physiologic) regulation of platelet PKC isoenzymes and determining their selective substrate phosphorylations (using an in vitro model that we have developed). These studies will provide the necessary biochemical background for determining the mechanism and physiologic regulation of PKC isoenzymes. Such knowledge is of great significance for improving our understanding of the role of platelets in hemostasis and in the pathophysiology of arteriovascular disease. This improved biochemical knowledge may lead to more efficacious and rational antiplatelet drug development. Knowledge gained from the platelet model should prove to be of great usefulness for investigators studying the regulation and role of PKC isoenzymes in various other cell systems.