Identification, activity, and structural studies of peptides incorporating the phorbol ester-binding domain of protein kinase C.
AUTOR(ES)
Wender, P A
RESUMO
The family of homologous enzymes known as protein kinase C (PKC) has been the object of intense interest because of its crucial role in cellular signal transduction. Although considerable information about the activation of PKC has been gained through structure-activity, molecular modeling, and synthetic studies of both natural and designed activators, information about the structure of PKC itself has been limited by its large size and requirement for phospholipid cofactors. Additionally, 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, synthesis, 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 [3H]phorbol 12,13-dibutyrate with good affinity (Kd = 6.4 microM and 414 nM, respectively) in the presence of phosphatidylserine. In comparison, the binding affinity of [3H]phorbol 12,13-dibutyrate for PKC was found to be 2.6 nM. Like PKC itself, these peptides also recognize other PKC activators, including dioctanoylglycerol and teleocidin B-4, and exhibit an ability to differentiate phorbol ester from its C-4 epimer. NMR studies of PKC subdomains are also described, indicating that both peptides B and C are well behaved in solution and do not exhibit any concentration-dependent changes. Finally, these studies reveal that peptide B becomes conformationally ordered only in the presence of phospholipid, suggesting that the regulatory domain of PKC itself might be organized for activation only when associated with the lipid bilayer, where its activator (diacylglycerol) is encountered.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=42853Documentos Relacionados
- Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C.
- Phorbol diester receptor copurifies with protein kinase C.
- Phorbol ester binding to protein kinase C requires a cysteine-rich zinc-finger-like sequence.
- A protein kinase C cDNA without the regulatory domain is active after transfection in vivo in the absence of phorbol ester.
- Overexpression of protein kinase C beta 1 enhances phospholipase D activity and diacylglycerol formation in phorbol ester-stimulated rat fibroblasts.