CORRELATION BETWEEN PROTEIN-KINASE-C BINDING-PROTEINS AND SUBSTRATES IN REF52 CELLS

We have used a blot overlay assay to identify phosphatidylserine-depen dent interactions between protein kinase C (PKC) and PKC binding prote ins. The purpose of the present studies was to compare the properties of PKC binding proteins and PKC substrates detected by in vivo and in vitro phosphorylation assays. The major binding proteins and substrate s in REF52 cells shared similar properties including enrichment by cal modulin-Sepharose chromatography, binding to phosphatidylserine, and r esistance to heat denaturation. In addition, several of the major bind ing proteins and substrates were coordinately down modulated in SV40-t ransformed REF52 cells. The major PKC substrate, MARCKS, was also dete cted as a PKC binding protein. These results emphasize that the phosph atidylserine-dependent interactions between PKC and several substrates are of sufficient affinity to be detected in a blot overlay. Down mod ulation of binding proteins/substrates in transformed cells may reflec t either decreased expression or increased basal phosphorylation of th e target proteins and is likely to be involved in maintenance of the t ransformed phenotype.