Ca++/calmodulin-regulated protein phosphorylation in the Aplysia nervous system.

Protein substrates for an endogenous CA++/calmodulin-dependent protein kinase were characterized in the Aplysia nervous system. Ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid-washed membrane fractions from Aplysia ganglia contain an endogenous Ca++/calmodulin-dependent protein kinase which phosphorylates a number of membrane proteins. Such washed membrane preparations exhibit little or no adenosine 3':5'-cyclic phosphate (cAMP)-dependent protein kinase activity but do contain substrates for exogenously added catalytic subunit of cAMP-dependent protein kinase. Low concentrations of catalytic subunit rapidly stimulate the phosphorylation of a protein of Mr 70,000 and of a 52,000-dalton doublet, indicating that these proteins are major substrates for this enzyme. Phosphopeptide patterns obtained after limited proteolysis suggest that the 70,000-dalton protein and the 52,000-dalton doublet are similar in structure and that their phosphorylation is stimulated both by Ca++/calmodulin and by catalytic subunit. The 52,000-dalton doublet consists of two closely spaced bands: the phosphorylation of the upper band is stimulated by catalytic subunit, whereas the phosphorylation of the lower band is stimulated by the endogenous Ca++/calmodulin-dependent protein kinase. The results suggest that in Aplysia membranes a number of proteins can be phosphorylated by both Ca++/calmodulin-dependent protein kinase and catalytic subunit of cAMP-dependent protein kinase. This convergence of biochemical effects of Ca++ and cAMP may play a role in some of their physiological actions in molluscan neurons.