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.
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