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.

Protein kinase C isozyme-specific potentiation of expressed Cav 2.3 currents by acetyl-β-methylcholine and phorbol-12-myristate, 13-acetate

“…The faster generation of DAGs from phosphatidylinositol hydrolysis vs. phosphatidylcholine hydrolysis (Nishizuka, 1995) may predispose to activation of cPKCs instead of nPKCs. Evidence from other studies suggests that the more rapid kinetics of cPKC translocation is determined by the calcium transients (Oancea, 1998;Lenz et al, 2002;Gallegos et al, 2006). In summary, although phosphorylation at specific PKC sites in the I-II linker and C-terminus would seem to be sufficient for activation of Ca v 2.2 channels, in the Ca v 2.3 channels, these sites may be necessary but not sufficient.…”

Identification of a Novel A-kinase Anchoring Protein 18 Isoform and Evidence for Its Role in the Vasopressin-induced Aquaporin-2 Shuttle in Renal Principal Cells

“…FRET from CFP to YFP was determined by excitation of CFP (425 nm) and measurement of fluorescence emitted from YFP (535/26 nm). The maximum FRET capability of the system was defined by determining FRET from CFP to YFP in a fusion protein consisting of the two proteins (42). Background fluorescence from a region with no cells was subtracted from the data.…”

A Critical Intramolecular Interaction for Protein Kinase Cϵ Translocation

“…Schaefer et al (31,32) suggested that differences in translocation between classical and novel PKCs are due to differences in diffusion rates, and collision efficiencies with the membrane. Although diffusion and collision with the membrane are likely factors in the translocation rate, our data demonstrate that conformational changes in the enzyme also occur, leading to at least a two-step process.…”