Ca2+-controlled competitive diacylglycerol binding of protein kinase C isoenzymes in living cells.

Abstract

The cellular decoding of receptor-induced signaling is based in part on the spatiotemporal activation pattern of PKC isoforms. Because classical and novel PKC isoforms contain diacylglycerol (DAG)-binding C1 domains, they may compete for DAG binding. We reasoned that a Ca2+-induced membrane association of classical PKCs may accelerate the DAG binding and thereby prevent translocation of novel PKCs. Simultaneous imaging of fluorescent PKC fusion proteins revealed that during receptor stimulation, PKCα accumulated in the plasma membrane with a diffusion-limited kinetic, whereas translocation of PKCɛ was delayed and attenuated. In BAPTA-loaded cells, however, a selective translocation of PKCɛ, but not of coexpressed PKCα, was evident. A membrane-permeable DAG analogue displayed a higher binding affinity for PKCɛ than for PKCα. Subsequent photolysis of caged Ca2+ immediately recruited PKCα to the membrane, and DAG-bound PKCɛ was displaced. At low expression levels of PKCɛ, PKCα concentration dependently prevented the PKCɛ translocation with half-maximal effects at equimolar coexpression. Furthermore, translocation of endogenous PKCs in vascular smooth muscle cells corroborated the model that a competition between PKC isoforms for DAG binding occurs at native expression levels. We conclude that Ca2+-controlled competitive DAG binding contributes to the selective recruitment of PKC isoforms after receptor activation.