CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

蛋白激酶C （PKC）是一个磷脂依赖性激酶家族，以其在肿瘤促进和进展中的作用而闻名，尽管个体PKC的作用尚未明确。PKC异构体在体内介导不同的生物效应，尽管它们在体外具有相似的特性。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引导到上游激活剂、下游底物靶点，并与其他途径整合信号。鉴定PKC底物并确定PKC如何改变其功能是了解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PKC直接结合PKC的能力克隆了一组PKC底物，并将其命名为与c -激酶相互作用的底物stick。在之前的资助期内，我们绘制了磷酸化位点，证明了stick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，这些抗体可以作为内源性PKC活性的报告者。对正常细胞的研究表明，STICKs主要定位于皮质骨架，因此定位于快速响应调节细胞骨架重塑的粘附信号。因此，我们发现PKC的2个stick （α -内缩蛋白和STICK72）磷酸化与粘附、扩散和迁移刺激的细胞骨架重塑相关。在逐渐转化的REF52细胞中，PKC、STICKs及其磷酸化的比较揭示了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，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PKCs结合并磷酸化一组细胞骨架蛋白，磷酸化在功能上改变了它们在粘附、扩散和迁移中的作用。转化细胞中活性的增加和/或不受调节改变了这些特性，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.