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

Abstract

THE important role of Ca²⁺ in the physiology of the nervous system is well documented^1,2. However, the biochemical mechanisms underlying certain of its physiological effects, such as stimulus–secretion coupling^3,4 and synthesis of cate-cholamines^5,6, 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 brain^7,8. 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 phosphodiesterase^12–14. A detergent-solubilised preparation of brain adenylate cyclase can also be activated by this calcium-binding protein¹⁵. Calcium stimulates protein phosphorylation in both intact^16,17 and lysed^18,19 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.