DIFFERENTIAL PROTEIN-KINASE-C LIGAND REGULATION DETECTED IN-VIVO BY APHENOTYPIC YEAST ASSAY

The molecular dissection of protein kinase C (PKC) action has been bas ed in part on time-consuming functional assays such as the mouse skin model for testing the tumor promoter activity of phorbol esters and re lated PKC activators. To help overcome the limitations imposed by the complexity of such assays, we developed the yeast Saccharomyces cerevi siae as an alternative, rapid, and simple experimental system. This mo del has a specific phenotype, an increase in the cell doubling time, t hat is proportional to the level of enzymatic activity of expressed ma mmalian PKC isoforms. We used this phenotype to assay and compare the regulation of native bovine PKC alpha and mutants in the conserved reg ulatory region C1 in vivo by various activators: two diterpenes, the p horbol ester phorbol-12-myristate-13-acetate (PMA) and mezerein, and t he indole alkaloid indolactam V. We found that PMA activated PKC mutan ts lacking either Cys-rich, zinc finger-like repeat of the conserved r egion C1 to comparably reduced levels, whereas indolactam V activated native PKC alpha but none of the mutants at normal doses. in contrast, mezerein activated native PKC alpha and a mutant lacking the second C ys repeat equally well but mutants lacking the first Cys repeat of C1 at a greatly reduced level. These differential responses were supporte d by the observed in vitro PKC catalytic activities. Therefore, PMA re gulates PKC alpha activity comparably well via either Cys repeat, wher eas mezerein regulation predominantly occurs via the first Cys repeat of C1. Indolactam V activation was less potent, it was greatly reduced in the absence of either Cys repeat, and displayed no preference. We introduce this phenotypic assay as a rapid and general screen for the PKC-activating or possibly inhibitory potential of drug candidates and to identify the PKC regulatory sites involved in these interactions. (C) 1995 Wiley-Liss, Inc.