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，在肿瘤的发生、发展中起重要作用，但其作用机制尚未明确。尽管PKC亚型在体外具有相似的性质，但它们在体内介导不同的生物学效应。体外相似性和体内选择性之间的差异可以用多种PKC相互作用蛋白来解释，这些蛋白将PKC导向上游激活子、下游底物靶标，并将信号与其他途径整合。确定PKC底物并确定PKC如何修饰它们的功能是理解PKC在生理过程和肿瘤促进中作用的关键。为了实现这一目标，我们根据PKC底物直接结合PKC的能力克隆了一组PKC底物，并将它们命名为与C-Kinase相互作用的底物Sticts。在之前的资助期间，我们绘制了磷酸化位点图，证明了Stics在体内具有同工酶选择性底物，并开发了一组针对Stick的磷酸化状态选择性抗体，可用作内源性PKC活性的报告。在正常细胞中的研究表明，棍子主要定位于皮质骨骼，因此可以对调节细胞骨架重塑的黏附信号做出快速反应。因此，我们发现α-内收蛋白和STICK72这两条蛋白激酶C的磷酸化与黏附、铺展和迁移刺激的细胞骨架重塑有关。对进行性转化的REF52细胞中的PKC、STUCH及其磷酸化的比较发现，几种PKC信号缺陷可能有助于细胞转化，包括：同工酶表达改变，内源性活性增加，某些底物的磷酸化增加，以及其他底物的表达改变。我们的总体假设是PKCs与一组细胞骨架蛋白结合并磷酸化，而磷酸化在功能上改变了它们在黏附、扩散和迁移中的作用。转化细胞中增加的和/或不受调节的活性改变了这些属性，从而促进了肿瘤细胞的转化。

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.