Selective regulation of protein kinase C isoenzymes by oleic acid in human platelets

Cis-unsaturated fatty acids activate soluble protein kinase C (PKC) in vitro and in intact platelets. The following studies were conducted to determine the effects of oleate on individual isoenzymes of PKC in human platelets. Human platelets were found to contain predominantly PKC α, β_I, β_II, and δ with minor immunoreactivity for PKC ε, ζ, and η. In intact platelets sodium oleate caused a time-dependent redistribution of PKC α, β_II, and δ from cytosol to membrane fractions with little effects on PKC β_I. On the other hand, PMA and thrombin induced translocation of all four isoenzymes of PKC. In vitro, oleate partially activated (50% of V_max) purified calcium-dependent PKC (α, β_I, and β_II) with an EC₅₀ of 50 μM whereas it fully activated (100% of V_max) purified calcium-independent PKC (predominantly δ) with an EC₅₀ of 5 μM. The selective effects of oleate on PKC isoenzymes were investigated in platelet cytosol which contains endogenous PKC and its physiologic substrates. Under these conditions, oleate potently activated calcium-independent PKC causing the phosphorylation of the 40-kDa substrate. Activation of calcium-dependent isoforms occurred only at higher concentrations of oleate. Thus, oleate activates multiple isoenzymes of PKC with predominant effects on calcium-independent PKC.