ROLE AND REGULATION OF PLATELET PROTEIN KINASE C

血小板蛋白激酶 C 的作用和调节

• 批准号: 3362435
• 负责人: YUSUF AWNI HANNUN
• 金额: $ 16.06万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-07-01 至 1995-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3362435
• 关键词: biological signal transduction; enzyme induction /repression; enzyme mechanism; enzyme substrate; high performance liquid chromatography; human subject; immunocytochemistry; intracellular transport; isozymes; monoclonal antibody; phorbols; platelet activation; platelets; protein kinase C; protein purification

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.

蛋白激酶 C (PKC) 已成为信号中的关键酶 转导、细胞调节、细胞分化和肿瘤促进。 现在已知该酶作为密切相关的亚型家族而存在 其个体机制和功能尚未明确。在人类 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 （关于血小板活化）。我们已经鉴定出 PKC 的四种同工酶 血小板。长期目标是确定 PKC 在血小板中的作用 功能。该提案旨在研究不同 PKC 的假设 同工酶受到差异性调节以转导选择性功能。 我们的实验室在生化方面拥有丰富的经验 PKC 的表征以及在血小板生物学研究中具有独特的意义 适合评估这些问题。该提案的具体目标是， 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（使用 FPLC）并在体外研究它们 监管（使用我们开发的混合胶束方法）； 2） 确定静息和静息状态下 PKC 同工酶的细胞内定位 分别激活血小板（通过监测酶活性、佛波醇 结合和蛋白质印迹）和； 3）研究原位（生理） 血小板 PKC 同工酶的调节及其选择性 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化背景 用于确定 PKC 同工酶的机制和生理调节。 这些知识对于提高我们对事物的认识具有重要意义。 血小板在止血和病理生理学中的作用 动脉血管疾病。这种生化知识的提高可能会导致 更有效、更合理的抗血小板药物研发。知识 从血小板模型中获得的结果应该被证明对于 研究人员研究 PKC 同工酶在各种疾病中的调节和作用 其他细胞系统。