Stimulation of brain membrane protein phosphorylation by calcium and an endogenous heat-stable protein

regulation by calcium of calcium-dependent protein kinase activity may be mediated physiologically by the calcium-binding protein postulated to regulate cyclic nucleotide phosphodiesterase and adenylate cyclase.

Abstract

THE important role of Ca2+ in the physiology of the nervous system is well documented1,2. However, the biochemical mechanisms underlying certain of its physiological effects, such as stimulus–secretion coupling3,4 and synthesis of cate-cholamines5,6, have not yet been elucidated. Calcium has been implicated in several biochemical reactions of potential importance to synaptic function. Thus, calcium and a heat-stable calcium-binding protein activate the cyclic nucleotide phosphodiesterase from mammalian brain7,8. The calcium-dependent regulator (CDR) from porcine brain9, bovine heart10 and bovine brain11 has been purified and characterised. CDR seems to be a calcium receptor as it binds calcium strongly and specifically. There is evidence that a CDR·Ca2+ complex is the true activator of cyclic nucleotide phosphodiesterase12–14. A detergent-solubilised preparation of brain adenylate cyclase can also be activated by this calcium-binding protein15. Calcium stimulates protein phosphorylation in both intact16,17 and lysed18,19 synaptosomes and this cyclic nucleotide-independent mechanism may mediate or modulate some of the intracellular effects of Ca2+ on the function of presynaptic nerve terminals. We report here that calcium-dependent phosphorylation of synaptosomal membrane fractions from rat cerebral cortex requires an endogenous protein factor present in the synaptosomal cytoplasm. Calcium stimulated phosphorylation is lost on purification of synaptic membranes and can be effectively recovered by reconstitution with either the synaptosomal cytoplasm or with a purified preparation of CDR. Thus, regulation by calcium of calcium-dependent protein kinase activity may be mediated physiologically by the calcium-binding protein postulated to regulate cyclic nucleotide phosphodiesterase and adenylate cyclase.