ACTIVATION OF PROTEIN-KINASE-C BY ARACHIDONIC-ACID SELECTIVELY ENHANCES THE PHOSPHORYLATION OF GAP-43 IN NERVE-TERMINAL MEMBRANES

Arachidonic acid (AA), a cis-unsaturated fatty acid that activates cer tain subspecies of protein kinase C (PKC), has been proposed to act as a retrograde messenger in modifying the efficacy of synapses during l ong-term potentiation (LTP). One prominent PKC substrate of the nerve terminal membrane, GAP-43 (F1, B-50, neuromodulin), shows an increase in phosphorylation that correlates with the persistence of LTP. The pr esent study investigated whether AA might exert its effects on presyna ptic endings by modulating the phosphorylation of GAP-43 and other mem brane-bound proteins. Using synaptosomal membranes from the rat cerebr ocortex, in which in vivo relationships between protein kinases and th eir native substrates are likely to be preserved, we found that in the absence of Ca2+, AA exerted a modest effect on the phosphorylation of GAP-43 and several other proteins; however, when AA was applied in co njunction with Ca2+, GAP-43 showed a particularly striking response: a t Ca2+ levels likely to exist at the nerve terminal membrane during sy naptic activity (10(-7) to 10(-5) m), AA (50 muM) increased the sensit ivity of GAP-43 phosphorylation to Ca2+ by an order of magnitude, and increased its maximal level of phosphorylation by 50%. At resting Ca2 levels, AA potentiated the stimulation in GAP-43 phosphorylation prod uced by 4beta-phorbol 12,13-dibutyrate, a diacylglycerol (DAG) analog. The stimulatory effect of AA and its synergistic interaction with Ca2 + were found to be mediated by PKC, since they were blocked by a speci fic peptide inhibitor of PKC, [Ala25]PKC(19-31), but were unaffected b y an inhibitor of protein phosphatase activity or by scavengers of fre e radicals. Since GAP-43 has been implicated in the development and pl asticity of synaptic relationships, the synergistic effects of AA and the intracellular signals Ca2+ and DAG on the phosphorylation of GAP-4 3 may serve as an AND gate to modify presynaptic function and/or struc ture in response to coincident pre- and postsynaptic activity.