Protein kinase C isozymes in progressively transformed rat embryo fibroblasts

The role of individual protein kinase C (PKC) isoforms in progressive transformation of a rat embryo fibroblast cell line (REF52 cells) has been evaluated. Normal (REF A) cells were transfected with SV40 to produce the partially transformed REF B cell line. REF B cells are morphologically transformed but have only limited capacity for growth in soft agar. Message and protein levels for PKC-alpha and PKC-delta were similar in REF A and B cells, indicating that expression of SV40 large T did not directly influence the amounts of PKCs. However, PKC-alpha localization was influenced. PKC-alpha was associated with focal contacts of REF A but not REF B cells, indicating that changes in location rather than content are an early event in REF52 cell transformation. Clones of REF B cells were selected for growth in soft agar (REF C cells). Levels of PKC-delta, but not PKC-alpha or epsilon, were increased in several of these clones, suggesting that increased PKC-delta content may facilitate anchorage-independent growth. In other studies, we have determined that PKCs interact with their binding proteins/substrates through their regulatory domains (RD; L. Liao et al., Biochemistry, 33: 1229-1233, 1994). These results predict that RDs could potentially inhibit PKC signaling by competing with endogenous wild-type PKCs for binding protein/substrate interactions. Overexpression of the RD of PKC-delta inhibited growth in soft agar of one representative REF C clone, whereas overexpression of the RD of PKC-alpha promoted growth in soft agar. These results suggest that RD expression may be a useful approach for dominant negative PKC inhibitors with potential isozyme specificity.