Binding of [26-3H]bryostatin 1 and analogs to calcium-dependent and calcium-independent protein kinase C isozymes.
In this study we explored the pattern of protein kinase C (PKC) isozyme selectivity of the bryostatins, a unique class of PKC activators that induce only a subset of the typical phorbol ester responses and antagonize those phorbol ester-mediated responses that they themselves fail to induce. The binding properties of individual recombinant PKC isozymes that had been expressed in insect cells, isolated, and reconstituted in Triton X-100/phosphatidylserine mixed micelles were determined. [3H]Bryostatin 1 showed lower affinity for PKC-beta 1 and -gamma, compared with PKC-alpha, -delta, -epsilon, and -eta. This pattern contrasts with that observed for other PKC ligands. These latter assays were conducted with isozymes reconstituted in phosphatidylserine, conditions that unfortunately do not permit quantitation of bryostatin 1 binding under equilibrium conditions. Using delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10, we could distinguish the respective roles of ligand and lipid in the pattern of selectivity. When isozymes were reconstituted in phosphatidylserine vesicles, delta 19,20-bryostatin 10 and delta 19,20-isobryostatin 10 showed similar affinities for PKC-alpha and -gamma, similarly to the phorbol esters. However, in the mixed micellar system, PKC-gamma showed a significantly lower binding affinity, as had been observed for bryostatin 1. These results suggest that the unique pattern of biological responses to the bryostatins does not represent a unique pattern of isotype recognition. Furthermore, the lipid environment of PKC plays an important role in determining the binding selectivity for individual isozymes.
Kazanietz
M G
MG
Molecular Mechanisms of Tumor Promotion Section, National Cancer Institute, Bethesda, Maryland 20892.
Lewin
N E
NE
Gao
F
F
Pettit
G R
GR
Blumberg
P M
PM
D000818:Animals; D001665:Binding Sites; D054713:Bryostatins; D002118:Calcium / Q000378:metabolism*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002478:Cells, Cultured; D007313:Insecta; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D007783:Lactones / Q000737:chemistry / Q000378:metabolism*; D018942:Macrolides; D008823:Micelles; D015394:Molecular Structure; D011493:Protein Kinase C / Q000235:genetics / Q000378:metabolism*; D014316:Tritium
D054713:Bryostatins; D007527:Isoenzymes; D007783:Lactones; D018942:Macrolides; D008823:Micelles; D014316:Tritium; C046785:bryostatin 1; D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium