CYTOSKELETAL ASSOCIATION OF PROTEIN KINASE C

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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.