Effect of alpha-protein kinase C neutralizing antibodies and the pseudosubstrate peptide on phosphorylation, migration, and growth of REF52 cells.

REF52 cells are a line of rat embryo fibroblasts that express alpha-, delta-, epsilon-, and zeta-protein kinase Cs (PKCs). In this report, we have used neutralizing antibodies to alpha-PKC to study the role of this specific PKC isozyme in REF52 cell functions. Effects of the more general PKC inhibitor, the pseudosubstrate peptide, were also studied. Previous work demonstrated that alpha-PKC is concentrated in focal contacts of REF52 cells (Jaken, S., Leach, K., and Klauck, T. J. Cell Biol., 109: 697-704, 1989). alpha-PKC redistributed to the leading lamellopodia of cells stimulated to migrate into an artificial wound, indicating that alpha-PKC activation may be coupled to migratory stimuli. The effects of the alpha-PKC neutralizing antibodies and the pseudosubstrate peptide on responses associated with focal contact functions, namely attachment, migration, and growth, were studied. The data demonstrate that the antibodies and the pseudosubstrate peptide were all efficient inhibitors of phorbol ester-stimulated phosphorylation. However, only the pseudosubstrate peptide efficiently inhibited the migration and growth responses necessary to repopulate an artificial wound. These results indicate that PKCs, but probably not alpha-PKC in particular, are important in these responses. However, because the neutralizing antibodies did not completely inhibit phorbol 12,13-dibutyrate-stimulated phosphorylation, a potential role for alpha-PKC in migration and growth cannot be excluded.