PROTEIN KINASE C--IN VITRO STUDIES

蛋白激酶C（PKC）是一种关键的调节酶，被认为是 参与许多细胞过程，如细胞生长， 分化，激素释放，血小板活化，以及许多 其他事件。 底物蛋白的磷酸化通常 需要Ca+2和磷脂以及第二信使， 甘油二酯（DAG）。 生物活性佛波醇酯 代替DAG。 尽管目前有大量关于这方面的研究， 蛋白质，很少有人知道的物理实体，产生 激酶活性以及各种激活剂如何发挥其影响。 我们已经发现，Ca+2和磷脂的需求， PKC大大超过PKC-膜结合所需的那些， 辅因子（磷脂、Ca+2和/或DAG）的需求 PKC的激活依赖于底物的选择。 在 为了解释这种行为，我们观察到， 底物与磷脂的相互作用是关键的 蛋白激酶C的作用方面和所有良好的体外底物不仅 与磷脂结合但引起磷脂聚集 囊泡 这些意见说明，需要评估所有 PKC与其底物和辅因子的不同相互作用。 本研究将探讨PKC与其他蛋白的相互作用， 各种状态的组分（例如磷脂囊泡和 单分子层），并将决定如何事件和他们的更多 详细的方面对于激酶活性的开发是重要的。 将尝试鉴定非聚集测定系统。 PKC的金属离子结合特性将通过几种方法进行检查。 包括直接结合测量的技术， 荧光法 PKC对膜的选择性 结构特征（例如成分和表面曲率）将 进行研究，以确定参与PKC-膜的因素 结合和产生不可解离的膜结合PKC。 磷酸化对蛋白质-膜组装的影响将 使用两种不同的蛋白质，组蛋白和髓鞘碱性蛋白进行评估。 蛋白 Ca ~（2+）、DAG或佛波酯对细胞凋亡的影响 蛋白激酶C-膜组装动力学的研究 和解离将使用荧光和CD进行研究 方法. 将所有相互作用与PKC的外观进行比较 活性，以帮助确定这些方面的酶底物， 膜-辅因子复合物，这是至关重要的发展 激酶活性 定义体外性质将提高我们的 了解这种酶的体内靶点是如何 鉴定

Protein kinase C (PKC) is a key regulatory enzyme believed to be involved in many cellular processes such as cell growth and differentation, hormone release, platelet activation, and many other events. Phosphorylation of substrate proteins usually requires Ca+2 and phospholipid as well as the second messenger, diacylglycerol (DAG). Biologically active phorbol esters substitute for DAG. Despite abundant current studies on this protein, little is known about the physical entity that generates kinase activity and how various activators exert their influence. We have found that the Ca+2 and the phospholipid requirements of PKC greatly exceed those needed for PKC-membrane binding and that the cofactor (phospholipid, Ca+2, and/or DAG) requirements of PKC activation are dependent on the choice of substrate. In searching for an explanation for this behavior, we observed the interaction of substrates with the phospholipid was a critical aspect of PKC action and all good in vito substrates not only bound to phospholipid but caused aggregation of phospholipid vesicles. These observations illustrate the need to evaluate all of the different interactions of PKC, its substrates and cofactors. This study will investigate the interactions of PKC and other components in various states (e.g. phospholipid vesicles and monolayers) and will determine how the events and their more detailed aspects are important to develoment of kinase activity. Identification of nonaggregated assay systems will be attempted. Metal ion binding properties of PKC will be examined by several techniques including direct binding measurements and fluorescence methods. Selectivity of PKC for membrane structural features (e.g. composition and surface curvature) will be studied to determine the factors involved in PKC-membrane binding and in generating nondissociable membrane-bound PKC. The effect of phosphorylation on protein-membrane assembly will be assessed using two different proteins, histones and myelin basic protein. The effect of Ca+2, DAG or phorbol esters on the conformation and on the dynamics of PKC-membrane assembly and dissociation will be investigated using fluorescence and CD methods. All interactions will be compared to appearance of PKC activity to help define those aspects of the enzyme-substrate- membrane-cofactor complex that are critical to development of kinase activity. Defining the in vitro properties will improve our understanding of how in vivo targets for this enzyme are identified.