The phosphorylation of protein I, a specific neuronal protein, has been found to be regulated both by cyclic AMP (cAMP) and by calcium, in intact as well as in lysed synaptosome preparations from rat brain. In order to determine the phosphorylation site(s) of protein I that were regulated by cAMP and calcium, protein I was purified after it was phosphorylated under various conditions. This purified protein I was then subjected either to peptide mapping after limited proteolysis in sodium dodecyl sulfate/polyacrylamide gels or to tryptic fingerprinting. 8-Br-cAMP selectively increased the phosphorylation of the same protein I peptide fragment in both intact and lysed synaptosomes. Depolarization-induced calcium influx into intact synaptosomes, or the addition of calcium to lysed synaptosomes, caused a stimulation of the phosphorylation not only of this peptide but also of other distinct peptides. Differential regulation by cAMP and calcium of the phosphorylation of multiple sites on the same neuronal protein may provide a molecular basis for interactions between these two second-messenger systems in certain nerve terminal functions.
