CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C（PKC）是磷脂依赖性激酶家族， 在体外具有相似的特性。 由于大多数细胞表达一种以上的类型 蛋白激酶C，确定个人的作用蛋白激酶C在细胞过程中， 很困难我们的总体假设是，PKCs的靶向是 特异性亚细胞地址通过同工酶选择性相互作用与 结合蛋白针对性是一种有吸引力的限制机制， 底物可及性，从而提供同工酶选择性 功能协调发展的为了支持这一假设，我们鉴定并克隆了 基于表观高亲和力相互作用的几种PKC结合蛋白 体外进一步的研究建议，以确定这些约束力 蛋白质在体内作为PKC的细胞内受体起作用。我们有 确定结合蛋白也是底物，并将它们用作 报告系统，以确定单个PKC的活性增加是否 一般引起底物磷酸化， 特别的。尽管PKC的激活与肿瘤的发生有关， 促进/进展，个别PKC在这些过程中的作用 没有被定义。蛋白激酶C或结合蛋白表达的变化 蛋白质/底物会干扰PKC信号传导，并可能有助于 转型我们在进行性转化的REF 52细胞中的研究表明， 与不同生长功能相关的单个PKC。诱导型 活性和非活性（显性阴性）PKC同工酶的表达 结构将被用来增加或减少的活动， 单个PKC以确定它们在REF 52细胞转化中的作用。 转化还与两种独特的 序列结合蛋白。为了检测结合蛋白和/或 底物表达，这些序列的表达将 被重新引入转化细胞并影响PKC定位， 将监测底物磷酸化和生长。 这种组合 方法的沿着与独特的试剂和 逐步转化的细胞模型将扩展我们对细胞在肿瘤中的作用的认识， 单个PKC在细胞生长和转化中的作用

Protein kinase C (PKC) is a family of phospholipid-dependent kinases with similar properties in vitro. Since most cells express more than one type of PKC, identifying the roles of individual PKCs in cellular processes has been difficult. Our overall hypothesis is that PKCs are targeted to specific subcellular addresses by isozyme-selective interactions with binding proteins. Targeting is an attractive mechanism for restricting substrate accessibility and thereby providing for isozyme-selective functions. In support of this hypothesis we have identified and cloned several PKC binding proteins based on apparent high affinity interactions in vitro. Further studies are proposed to determine if these binding proteins function as intracellular receptors for PKCs in vivo. We have determined that binding proteins are also substrates and will use them as reporter systems to determine if increased activity of individual PKCs causes phosphorylation of substrates in general or select subgroups in particular. Although PKC activation has been associated with tumor promotion/progression the roles of individual PKCs in these processes have not been defined. Changes in expression either of PKCs or the binding proteins/substrates would perturb PKC signaling and could contribute to transformation. Our studies in progressively transformed REF52 cells have associated individual PKCs with different growth functions. Inducible expression of active and inactive (dominant negative) PKC isozyme constructs will be used to increase or decrease the activities of individual PKCs to determine their roles in REF52 cell transformation. Transformation is also associated with decreased expression of two unique sequence binding proteins. To test the role of binding protein andlor substrate expression in PKC signaling, expression of these sequences will be reintroduced in transformed cells and effects on PKC localization, substrate phosphorylation and growth will be monitored. This combination of approaches along with the availability of unique reagents and the progressively transformed cell model will extend our knowledge on the role of individual PKCs in cell growth and transformation.