Protein kinase C activation in mixed micelles. Mechanistic implications of phospholipid, diacylglycerol, and calcium interdependencies

The phospholipid, sn-1,2-diacylglycerol, and calcium dependencies of rat brain protein kinase C were investigated with a mixed micellar assay (Hannun, Y., Loomis, C., and Bell, R.M. (1985) J. Biol. Chem. 260, 10039-10043). Protein kinase C activity was independent of the number of Triton X-100, phosphatidylserine (PS) and sn-1,2-dioleoylglycerol (diC(18:1)) mixed micelles. Activation was strongly dependent on the mole per cent of PS and diC(18:1). Activity of protein kinase C was dependent on PS, diC(18:1), and calcium in mixed micelles prepared from detergents other than Triton X-100. This is consistent with the micelle providing an inert surface into which the lipid cofactors partition. Molecular sieve chromatography provided direct evidence for the homogeneity of Triton X-100, PS, and diC(18:1) mixed micelles. Mixing studies and surface dilution studies indicated that PS and diC(18:1) rapidly equilibrate among the mixed micelles. At saturating calcium, the diC(18:1) dependence was strongly dependent on the mole per cent PS present. At 10 mol % PS, 0.25 mol % diC(18:1) gave maximal activity whereas 6 mol % PS and 6 mol % diC(18:1) did not give maximal activity. diC(18:1) dependencies were hyperbolic at all PS levels tested. The data support the conclusion that a single molecule of diC(18:1)/micelle is sufficient to activate monomeric protein kinase C. The mole per cent PS required for maximal activation was reduced markedly as the mole per cent diC(18:1) increased. Under all conditions tested, the PS dependence of protein kinase C activation lagged until greater than 3 mol % PS was present. Then activation occurred in a cooperative manner with Hill numbers near 4. These data indicate that 4 or more molecules of PS are required to activate monomeric protein kinase C. PS was the most effective of all phospholipids tested in the mixed micelle assay. diC(18:1) was found to modulate the amount of calcium required for maximal activity. As the level of Ca²⁺ increased, the mole per cent PS required reached a limiting value of 3 mol %. A number of sn-1,2-diacylglycerols containing short chain fatty acids activated protein kinase C in a saturable manner in mixed micelles. The data are discussed in relation to a model for protein kinase activation.