title: Characterization of ligand and substrate specificity for the calcium-dependent and calcium-independent protein kinase C isozymes.
abstract: Analysis of [3H]phorbol-12,13-dibutyrate (PDBu) binding was performed with protein kinase C (PKC)-alpha, -beta 1, -gamma, -delta, -epsilon, -eta, and -zeta produced in Sf9 insect cells using the baculovirus expression system. With the exception of PKC-zeta, all of the PKC isozymes bound [3H]PDBu with high affinity (Kd < 1 nM), either in the presence or in the absence of calcium. Scatchard analysis using 100% phosphatidylserine vesicles revealed slightly lower affinity for the calcium-independent isozymes (PKC-delta, -epsilon, and -eta) than for the calcium-dependent isozymes (PKC-alpha, -beta, and -gamma). Competition for [3H]PDBu binding by different classes of PKC activators showed that 12-deoxyphorbol esters, mezerein, and octahydromezerein likewise possessed lower affinity for the calcium-independent isozymes. The mezerein analog thymeleatoxin was the most marked example, being almost 20-fold less potent for binding to PKC-epsilon and -eta than to PKC-beta 1. In contrast, the indole alkaloids (-)-indolactam V and (-)-octylindolactam V and the postulated endogenous activator 1,2-diacylglycerol bound with similar affinities to all of the PKC isoforms, suggesting that different residues/configurations in the binding sites of the different PKC isozymes might be involved in interaction with the pharmacophore of the activators. The seven PKC isozymes also showed clearly different substrate specificities with exogenous peptide and protein substrates. The heterogeneous behavior of the different members of the PKC family with ligands and substrates may contribute to the heterogeneity of PKC-mediated pathways at the cellular level.
authors.affiliation: Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892.
authors.forename: M G
authors.initials: MG
authors.lastname: Kazanietz
authors.forename: L B
authors.initials: LB
authors.lastname: Areces
authors.forename: A
authors.initials: A
authors.lastname: Bahador
authors.forename: H
authors.initials: H
authors.lastname: Mischak
authors.forename: J
authors.initials: J
authors.lastname: Goodnight
authors.forename: J F
authors.initials: JF
authors.lastname: Mushinski
authors.forename: P M
authors.initials: PM
authors.lastname: Blumberg
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002118:Calcium / Q000378:metabolism*; D002460:Cell Line; D004789:Enzyme Activation; D007527:Isoenzymes / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D015240:Phorbol 12,13-Dibutyrate / Q000378:metabolism*; D011493:Protein Kinase C / Q000737:chemistry / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D007527:Isoenzymes; D011994:Recombinant Proteins; D015240:Phorbol 12,13-Dibutyrate; D011493:Protein Kinase C; D002118:Calcium