Protein Kinase C Regulation by Diacylglycerols: Structure-Function Relationships and Mechanism
A mixed micellar assay for protein kinase C was developed to investigate the specificity and stoichiometry of activation by phospholipids, diacylglycerols, and diacylglycerol analogues. Triton X-100 micelles containing 8 mol% phosphatidylserine (PS) and 2.5 mol% sn-1,2-dioleoylglycerol (diC18:1) activated rat brain protein kinase C in the presence of Ca2+ to the same degree as sonicated PS/diC18:1. Protein kinase C activity was almost totally dependent on diC18:1 in the mixed micellar assay. At 8 mol% PS, diC18:1 stimulated maximally at 1 mol%. At 2.5 mol% diC18:1, PS did not activate until 3 mol% and then did so cooperatively with maximal stimulation occurring at 6–8 mol%. Molecular sieve chromatography demonstrated that monomeric protein kinase C interacts with Triton X-100 micelles in a PS and Ca2+ dependent manner. Interpretations follow: 1) a single molecule of diC18:1 activates monomeric protein kinase C; 2) a phospholipid bilayer is not required; 3) four or more molecules of PS are required. In addition, several diacylglycerol analogues were synthesized to determine the exact structural features required for activation. The data suggest that both carbonyls of the oxygen esters and the 3-hydroxyl are required. A model of protein kinase C activation by PS and diacylglycerol was formulated.
KeywordsProtein Kinase Mixed Micelle Epidermal Growth Factor Binding Stimulate Protein Kinase Molecular Sieve Chromatography
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