Monoclonal antibodies to protein kinase Cγ

Abstract

Four monoclonal antibodies (mAbs), derived from high‐responder Biozzi mice immunized with purified protein kinase C, were selected by ELISA and further characterized by immunoblot: 3G12, 5A2, 36G9 are of isotype γ₁, K and 15G4 is of isotype γ_2b,K. Competition analysis between 15G4 and the three other mAbs showed that 15G4 and 3G12 are directed against either the same or overlapping epitope(s). All four mAbs are specific of the bovine γ isoform of protein kinase C and cross‐react with protein kinase Cγ from a variety of animal species. Immunoblot analysis of protein kinase C tryptic fragments revealed that the mAbs recognize the regulatory domain and not the catalytic domain. Two of the mAbs, 36G9 and 5A2, inhibit protein kinase Cγ cofactor‐dependent activity (80% and 50% respectively). Consistent with the epitope mapping, none of mAbs inhibit the cofactor‐independent catalytic activity of protein kinase Cγ. Competition analysis between these mAbs and phosphatidylserine, 12‐O‐tetradecanoylphorbol 13‐acetate and Ca²⁺ showed that 36G9 and 5A2 block cofactor binding to protein kinase Cγ. These four mAbs thus interact with at least three distinct epitopes in the regulatory domain of protein kinase Cγ.