Function of calmodulin in postsynaptic densities. II. Presence of a calmodulin- activatable protein kinase activity

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Abstract
Because the calmodulin in postsynaptic densities (PSD) activates a cyclic nucleotide phosphodiesterase, the possibility that the PSD also contains calmodulin-activatable protein kinase activity was investigated. As seen by autoradiographic analysis of Coomassie Blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation from canine cerebral cortex were phosphorylated in the presence of [.gamma.-32P]ATP and Mg2+ alone. Addition of Ca2+ alone to the native PSD preparation had little or no effect on phosphorylation. Upon addition of exogenous calmodulin there was a general increase in background phosphorylation with statistically significant increase in the phosphorylation of 2 protein regions: 51,000 and 62,000 Mr [molecular ratio]. Similar results were also obtained in sonicated or freeze-thawed native PSD preparations by addition of Ca2+ alone without exogenous calmodulin, indicating that the calmodulin in the PSD can activate the kinase present under certain conditions. The calmodulin dependency of the reaction was further strengthened by the observed inhibition of the calmodulin-activatable phosphorylation, but not of the Mg2+-dependent activity, by the Ca2+ chelator, EGTA, which also removes the calmodulin from the structure, and by the binding to calmodulin of the antipsychotic drug chlorpromazine in the presence of Ca2+. When a calmodulin-deficient PSD preparation was prepared, sonicated and incubated with [.gamma.-32P]ATP, Mg2+ and Ca2+, a Ca2+-stimulation of protein kinase activity could not be induced unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 Mr PSD protein is the one that is phosphorylated and not the 51,000 Mr component of brain intermediate filaments, which is a known PSD contaminant. Papain digestion of the 51,000 Mr protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg2+-dependent kinase(s). Although the calmodulin-activatable protein kinase may phosphorylate proteins Ia and Ib, the cAMP-dependent protein kinase, which definitely does phosphorylate proteins Ia and Ib, and is present in the PSD, does not phosphorylate the 51,000 and 62,000 Mr proteins, because specific inhibition of this kinase has no effect on the levels of the phosphorylation of these latter 2 proteins.