IDENTIFICATION, ACTIVITY, AND STRUCTURAL STUDIES OF PEPTIDES INCORPORATING THE PHORBOL ESTER-BINDING DOMAIN OF PROTEIN-KINASE-C

The family of homologous enzymes known as protein kinase C (PKC) has b een the object of intense interest because of its crucial role in cell ular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecula r modeling, and synthetic studies of both natural and designed activat ors, information about the structure of PKC itself has been limited by its large size and requirement for phospholipid cofactors. Additional ly, difficulties in the purification of truncated mutants of PKC have thus far prevented their analysis by nuclear magnetic resonance (NMR) or x-ray crystallographic methods. We describe the identification, syn thesis, ligand-binding analysis, cofactor requirements,and preliminary NMR evaluation of two subdomains (peptides B and C) of the regulatory domain of PKC-gamma. Peptides B and C bind [H-3]phorbol 12,13-dibutyr ate with good affinity (K-d = 6.4 mu M and 414 nM, respectively) in th e presence of phosphatidylserine. In comparison, the binding affinity of [H-3]phorbol 12,13-dibutyrate for PKC was found to be 2.6 nM, Like PKC itself, these peptides also recognize other PKC activators, includ ing dioctanoylglycerol and teleocidin B-4, and exhibit an ability to d ifferentiate phorbol ester from its C-4 epimer. NMR studies of PKC sub domains are also described, indicating that both peptides B and C are well behaved in solution and do not exhibit any concentration-dependen t changes. Finally, these studies reveal that peptide B becomes confor mationally ordered only in the presence of phospholipid, suggesting th at the regulatory domain of PKC itself might be organized for activati on only when associated with the lipid bilayer, where its activator (d iacylglycerol) is encountered.