Comparison of protein kinase C functional assays to clarify mechanisms of inhibitor action.

The effects of inhibitors of protein kinase C on the activities of the intact enzyme, the proteolytically-generated catalytic domain, and [3H]phorbol 12,13-dibutyrate (PDBu) binding were compared in an effort to evaluate this approach for clarifying mechanisms of inhibitor action. Staurosporine, H-7 [1-(5-isoquinolinylsulfonyl)-2-methylpiperazine], and quercetin inhibited the catalytic fragment with similar potencies as for the intact enzyme while having little or no effect on binding, consistent with reports that they are competitive with ATP. Adriamycin, trifluoperazine, and tamoxifen, suggested to disrupt hydrophobic interactions between the regulatory domain of protein kinase C and phospholipid, were all most effective on the intact enzyme. They appear to possess a mixed mechanism, however, inhibiting activity of the catalytic domain with approximately 3-fold lower potencies. Gossypol inhibited intact enzyme, catalytic fragment, and PDBu binding with similar potencies. In light of multiple apparent sites of action for such protein kinase C inhibitors, comparison of their activities on the individual functional domains of the kinase may provide a useful complement to studies with the intact enzyme.