Ca2+-calmodulin-dependent phosphorylation of soluble and nuclear proteins in the rat ovary

Studies were undertaken to determine if calmodulin-regulated Ca2+-dependent protein kinase system(s) exist in the prepubertal rat ovary. Phosphorylation studies were performed with ovarian cytosol, calmodulin-depleted cytosol, and nuclear extracts. In vitro phosphorylation of endogenous substrate proteins was accomplished by incubation of tissue fractions with [gamma-32P]ATP followed by electrophoretic separation and autoradiographic demonstration of phosphorylated proteins. Calmodulin-dependent phosphorylations of a cytosol protein, mol wt, 95,000, and of three nuclear proteins in the mol wt range of 50,000-60,000, were established by demonstrating: Ca2+ requirement; inhibition by the phenothiazine derivative chlorpromazine; and dependence upon addition of exogenous calmodulin to the calmodulin-depleted cytosol, or to the nuclear extract. These findings demonstrate the presence of rat ovarian cytosol and nuclear Ca2+-calmodulin-dependent protein kinase activities capable of recognizing endogenous substrate proteins.