QUANTITATION OF THE INTERACTION OF PROTEIN-KINASE-C WITH DIACYLGLYCEROL AND PHOSPHOINOSITIDES BY TIME-RESOLVED DETECTION OF RESONANCE ENERGY-TRANSFER

Quantitative studies of the binding of protein kinase C (PKC) to lipid cofactors were performed by monitoring resonance energy transfer with time-resolved fluorescence techniques. For that purpose, diacylglycer ol (DG), phosphatidylinositol 4,5-biphosphate (PIP2), phosphatidylinos itol 4-phosphate (PIP), phosphatidylinositol (PI), phosphatidylcholine (PC), and phosphatidylserine (PS) were labeled with a pyrenyl decanoy l moiety at the sn-2 position of the lipid glycerol. These labeled lip ids proved excellent energy acceptors of light-excited tryptophan resi dues in PKC. The quenching efficiency of the tryptophan fluorescence w as determined as function of lipid probe concentration in mixed micell es consisting of poly(oxyethylene)-9-lauryl ether, PS, and various mol e fractions of probe lipid. The experimental conditions and method of data analysis allowed the estimation of binding constants of single or multiple pyrene lipids to PKC. The affinity of PKC for inositide lipi ds increases in the order PI < PIP < PIP2. The affinity of PKC for PIP and PIP2 is higher than that for DG. Determination of PKC activity in the presence of labeled lipids and PS showed that only PIP2 and DG ac tivate PKC. Double-labeling experiments suggest that PIP2 and DG are n ot able to bind simultaneously to PKC, indicating a reciprocal binding relationship of both cofactors. The results support the notion that, besides DG, PIP2 can be a primary activator of PKC.