Because the calmodulin in postsynaptic densities (PSDs) activates a cyclic nucleotide phosphodiesterase, we decided to explore the possibility that the PSD also contains a calmodulin-activatable protein kinase activity. As seen by autoradiographic analysis of Coomassie Blue-stained SDS polyacrylamide gels, many proteins in a native PSD preparation were phosphorylated in the presence of [γ-32P]ATP and Mg2+ alone. Addition of Ca2+ alone to the native PSD preparation had little or no effect on phosphorylation. However, upon addition of exogenous calmodulin there was a general increase in background phosphorylation with a statistically significant increase in the phosphorylation of two protein regions: 51,000 and 62,000 M(r). 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+. In addition, when a calmodulin-deficient PSD preparation was prepared, sonicated, and incubated with [γ-32P]ATP, Mg2+ and Ca2+, one could not induce a Ca2+-stimulation of protein kinase activity unless exogenous calmodulin was added back to the system, indicating a reconstitution of calmodulin into the PSD. We have also attempted to identify the two major phosphorylated proteins. Based on SDS polyacrylamide gel electrophoresis, it appears that the major 51,000 M(r) PSD protein is the one that is phosphorylated and not the 51,000 M(r) component of brain intermediate filaments, which is a known PSD contaminant. In addition, papain digestion of the 51,000 M(r) protein revealed multiple phosphorylation sites different from those phosphorylated by the Mg2+-dependent kinase(s). Finally, although the calmodulin-activatable protein kinase may phosphorylate proteins I(a) and I(b), the cyclic AMP-dependent protein kinase, which definitely does phosphorylate proteins I(a) and I(b) and is present in the PSD, does not phosphorylate the 51,000 and 62,000 M(r) proteins, because specific inhibition of this kanse has no effect on the levels of the phosphorylation of these latter two proteins.
|Original language||English (US)|
|Number of pages||9|
|Journal||Journal of Cell Biology|
|State||Published - 1981|
ASJC Scopus subject areas
- Cell Biology