Calcium-regulated phosphorylation in synaptosomal cytosol: dependence on calmodulin.

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Abstract
Ca stimulated the phosphorylation of several specific synaptosomal cytosolic proteins in rats. The effects of Ca were both concentration and time dependent and were most apparent for proteins MW of 50,000, 55,000 and 60,000 MW. Exogenous Ca (1.0-100 .mu.M) enhanced the net incorporation of phosphate into protein by as much as 23-fold. In the absence of added Ca, the Ca chelator [ethylenebis(oxyethylenenitrilo)]tetraacetic acid did not lower the phosphorylation of any protein below control levels. The antipsychotic, fluphenazine (1.0-100 .mu.M), caused a concentration-dependent decrease in Ca-stimulated protein phosphorylation. When the heat-stable Ca-binding protein, calmodulin, was removed from synaptosomal cytosol by affinity chromatography on fluphenazine-Sepharose, Ca-stimulated protein phosphorylation was abolished. Responsiveness to Ca could be restored by the addition of calmodulin to the phosphorylation assay. Ca-dependent protein kinases are apparently of major importance in regulating the phosphorylation of specific cytosolic proteins in neuronal tissue. One of the 3 substrates under investigation is apparently specific to synaptosomal cytosol whereas the other 2 are present in both the cytosol and membrane fractions.