CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C（PKC）是磷脂依赖性激酶家族，已知其在肿瘤促进和进展中的作用，尽管单个PKC的作用尚未确定。 PKC异构体在体内介导不同的生物学效应，尽管它们在体外具有相似的性质。 体外相似性和体内选择性之间的差异可以通过多种PKC相互作用蛋白来解释，这些蛋白将PKC引导至上游激活剂、下游底物靶点并整合与其他途径的信号传导。识别PKC底物并确定PKC如何修饰其功能是理解PKC在生理过程和肿瘤促进中的作用的关键。为了实现这一目标，我们根据它们直接结合PKC的能力克隆了一组PKC底物，并将它们命名为STICK，即与C-激酶相互作用的底物。在上一个资助期间，我们绘制了磷酸化位点，证明了STIKs是同工酶选择性的体内底物，并开发了一组磷酸化状态选择性抗体STIKs，可用作内源性PKC活性的报告。在正常细胞中的研究表明，STIKs主要定位于皮质骨架，因此被定位为快速响应调节细胞骨架重塑的粘附信号。因此，我们发现2种STIKs，α-内收蛋白和STIK 72的PKC磷酸化与粘附、扩散和迁移刺激的细胞骨架重塑偶联。在进行性转化的REF 52细胞中PKC、STIKs及其磷酸化的比较揭示了可能有助于细胞转化的几种PKC信号传导缺陷，包括：同工酶表达改变、内源性活性增加、一些底物磷酸化增加和其他底物表达改变。 我们的总体假设是，蛋白激酶C结合和磷酸化的一组细胞骨架蛋白，磷酸化功能改变其在粘附，扩散和迁移的作用。转化细胞中增加的和/或不受调节的活性改变了这些性质，从而有助于肿瘤细胞转化。

Protein kinase C (PKC) is a family of phospholipid-dependent kinases that are known for their role in tumor promotion and progression, although the roles of individual PKCs have not been defined. PKC isoforms mediate distinct biological effects in vivo although they have similar properties in vitro. The discrepancy between in vitro similarity and in vivo selectivity can be explained by a variety of PKC interacting proteins that direct PKCs to upstream activators, downstream substrate targets and integrate signaling with other pathways. Identifying PKC substrates and determining how PKC modifies their functions is key to understanding the role of PKCs in physiological processes and tumor promotion. To achieve this goal, we cloned a panel of PKC substrates according to their ability to directly bind PKC and named them STICKs for Substrates That interact with C-Kinase. During the previous funding period, we mapped phosphorylation sites, demonstrated that STICKs are isozyme-selective in vivo substrates and developed a panel of phosphorylation state selective antibodies to STICKs that can be used as reporters of endogenous PKC activities. Studies in normal cells revealed that STICKs are mainly localized to the cortical skeleton, and thus are positioned to rapidly respond to adhesion signals that regulate cytoskeletal remodeling. Accordingly, we found that PKC phosphorylations of 2 STICKs, alpha- adducin and STICK72, are coupled to cytoskeletal remodeling stimulated by adhesion, spreading and migration. Comparisons of PKCs, STICKs and their phosphorylation in progressively transformed REF52 cells revealed several PKC signaling defects that could contribute to cell transformation including: altered isozyme expression, increased endogenous activity, increased phosphorylation of some substrates and altered expression of other substrates. Our overall hypothesis is that PKCs bind to and phosphorylate a group of cytoskeletal proteins, and that phosphorylation functionally modifies their roles in adhesion, spreading and migration. Increased and/or unregulated activity in transformed cells alters these properties and thereby contributes to tumor cell transformation.