CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C(PKC)是一类磷脂依赖的蛋白激酶家族，具有 在体外具有相似的特性。由于大多数细胞表达一种以上类型 对于PKC，识别单个PKC在细胞过程中的角色具有 真的很难。我们的总体假设是PKC的目标是 同工酶选择性相互作用的特定亚细胞地址 结合蛋白。目标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。为了支持这一假说，我们已经确定并克隆了 几种基于表观高亲和力相互作用的PKC结合蛋白 在试管中。建议进行进一步的研究，以确定这些结合 在体内，蛋白质是PKCs的细胞内受体。我们有 确定结合蛋白也是底物，并将它们用作 用于确定单个PKC活性是否增加的报告系统 导致底物或特定亚群的一般磷酸化 很特别。尽管PKC激活与肿瘤有关 在这些进程中，个别公钥基础设施的作用有 未定义。PKC的表达或结合的变化 蛋白质/底物会干扰PKC信号转导，并可能有助于 转型。我们对渐进式转化的REF52细胞的研究 将具有不同生长功能的单个PKC联系起来。可感应的 活性和非活性(显性阴性)PKC同工酶的表达 构造将用于增加或减少 单个PKC确定它们在REF52细胞转化中的作用。 转化还与两个独特的基因表达减少有关 序列结合蛋白。来测试结合蛋白的作用。 底物表达在PKC信号转导中，这些序列的表达将 在转化细胞中重新导入，并对PKC定位的影响， 将监测底物的磷酸化和生长。这种组合 方法以及独特试剂的可用性和 逐步转化的细胞模型将扩展我们对该角色的知识 单个PKCs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.