PROTEIN KINASE C--IN VITRO STUDIES

本申请旨在继续支持一个项目， 表征蛋白激酶C（PKC）与其 辅因子，并确定其激活的机制。 PKC 是一种重要的细胞内调节蛋白， 肿瘤促进剂佛波醇酯的亲和受体。 活动 PKC的活性依赖于钙和磷脂。低钙 在高浓度下，活性也被佛波醇酯和/或 甘油二酯 PKC以钙依赖性方式与膜结合， 它也是一个新发现的钙反应类别的成员 仅在膜存在时结合钙的蛋白质。 PKC形式 两种膜结合状态，一种是可逆的和钙依赖的， 另一种具有整合膜蛋白的性质。 形成 后者由佛波醇酯刺激。 本研究建议， 继续阐明激活和相互作用的机制 PKC与这些辅因子的结合。 磷脂酰乙醇胺的作用， 细胞内主要的中性磷脂，对PKC及其各种 将研究属性。 初步结果显示， 磷脂酰胆碱，主要的中性 细胞表面的磷脂。 我们计划记录和解释 这种差异的基础是使用酶活性和 蛋白膜结合 其他目标包括确定 条件和磷脂，增强形成的整体 PKC的膜结合形式。 这种形式的蛋白质将通过 其辅因子非依赖性活性和其用磷脂洗脱 在EGTA存在下的凝胶过滤色谱上。 一个主要目标 将是确定佛波酯或 甘油二酯在其可逆的膜结合状态下激活PKC。 蛋白质-膜相互作用将通过荧光或光来研究 散射技术和活性将通过蛋白质 用放射性ATP磷酸化。 荧光和圆二色光谱 与磷脂相互作用诱导的PKC变化， 将测量佛波醇酯和钙以确定 与这些相互作用相关的蛋白质结构变化。 另一 已经分离出一组丰富的蛋白质，它们结合钙， 与PKC相似的方式。 第一个目标是 确定它们的身份，如果它们是膜联蛋白家族的成员， proteins.这些不同的蛋白质对膜特性的影响 将进行研究，以确定它们是否产生膜渗透性， 其可逆或不可逆的膜结合形式。 扩展 我们的目标是继续研究各种PKC激活剂的机制 和抑制剂，并比较这些不同的性质， PKC亚型。 这些结果应该有助于解开的基础， 佛波酯效应及某些第二类药物的作用机制 使者

This application is for continuation of support for a project designed to characterize the interaction of Protein Kinase C (PKC) with its cofactors, and to determine the mechanisms by which it is activated. PKC is an important intracellular regulatory protein that is also the high affinity receptor for the tumor promotors, phorbol esters. The activity of PKC is calcium and phospholipid- dependent. At low calcium concentrations, the activity is also stimulated by phorbol esters and/or diacylglycerol. PKC binds to membranes in a calcium-dependent manner and it is also a member of a newly identified category of calcium response proteins that bind calcium only in the presence of membranes. PKC forms two membrane-bound states, one is reversible and calcium-dependent while the other has properties of an integral membrane protein. Formation of the latter is stimulated by phorbol esters. This study proposes to continue the elucidation of the mechanism of activation and interaction of PKC with these cofactors. The effect of phosphatidyl- ethanolamine, the major intracellular neutral phospholipid, on PKC and its various properties will be studied. Preliminary results show a great preference for this phospholipid over phosphatidyl-choline, the major neutral phospholipid on the cell surface. We plan to document and explain the basis for this difference using assays for both enzyme activity and protein-membrane binding. Other objectives include determination of the conditions and phospholipids that enhance formation of the integral membrane-bound form of PKC. This form of the protein will be detected by its cofactor- independent activity and by its elution with phospholipids on gel filtration chromatography in the presence of EGTA. A major goal will be to determine the mechanism by which Phorbol esters or diacylglycerol activate PKC in its reversible membrane-bound state. Protein-membrane interaction will be studied by fluorescence or light scattering techniques and activity will be measured by protein phosphorylation with radioactive ATP. The fluorescence and CD spectral changes in PKC that are induced by interaction with phospholipids, phorbol esters and calcium will be measured to determine the extent of protein structural change associated with these interactions. Another abundant group of proteins has been isolated that bind calcium and membranes in a manner similar to PKC. The first objective will be to determine their identity and if they are members of the annexin family of proteins. The effects of these various proteins on membrane properties will be studied to determine if they produce membrane permeability in either their reversible or irreversible membrane-bound forms. Extended goals are to continue to examine the mechanisms of various PKC activators and inhibitors, and to compare these various properties for the different isoforms of PKC. These results should help unravel the basis for the phorbol ester effect and the mechanism of action of some second messengers.