Synergistic activation by cis-fatty acid and diacylglycerol of protein kinase C and protein phosphorylation in hippocampal slices

Abstract

cis-Unsaturated fatty acid, which activates protein kinase C in vitro, stimulates protein phosphorylation in intact hippocampal slices. Two protein bands (44,000 and 47,000 mol. wt) are particularly sensitive to cis-fatty acid and are phosphorylated in a dose- and time-dependent manner. The cis-fatty acid-stimulated protein phosphorylation can be further potentiated with diacylglycerol or 12-O-tetradecanoylphorbol 13-acetate. Several lines of evidence indicate that the cis-fatty acid-stimulated phosphorylation of these proteins is mediated by protein kinase C. First, the cis-fatty acid effect is mimicked by other protein kinase C activators such as diacylglycerol. Second, the stimulation of the phosphorylation by these activators can be blocked by staurosporine, which potently inhibits protein kinase C. Third, a concomitant application of cis-fatty acid and diacylglycerol or 12-O-tetradecanoylphorbol 13-acetate enhances the 44,000 and 47,000 mol. wt phosphorylation in a synergistic manner, which is a novel activation mode for protein kinase C. Fourth, they can be phosphorylated by purified protein kinase C (type III: alpha). Moreover, the synergistic activation of purified protein kinase C by cis-fatty acid and diacylglycerol leads to a drastic increase in the phosphorylation of these two protein bands. Two-dimensional gel electrophoresis and immunoblot analysis revealed that they are both acidic proteins. The 47,000 mol. wt band consists of two protein components; one is found to be F1/growth-associated protein-43 (pI = 4.5), and the other 47,000 mol, wt protein has broad pI ranging from 4.6 to 4.9. The 44,000 mol. wt component is a major phosphoprotein with pI of 4.8-5.1. Our results strongly indicate that cis-fatty acid can act as a regulator of endogenous protein kinase C in concert with diacylglycerol, and stimulate protein phosphorylation of its substrates such as F1/growth-associated protein-43 in the hippocampus.