Evidence for the Identity of Nuclear and Cytoplasmic Adenosine-3':5'-Monophosphate-Dependent Protein Kinase from Porcine Ovaries and Nuclear Translocation of the Cytoplasmic Enzyme

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Abstract
Protein phosphokinase [EC 2.7.1.37] activity from a 0.5 M NaCl extract of purified porcine ovary nuclei was resolved by Sephadex G-200 gel filtration into 3 forms of protein kinase, protein kinase I and III, both independent of cyclic[c]AMP, and cAMP-dependent protein kinase II. cAMP-binding activity was associated with protein kinase II but not with protein kinases I and III. Protein kinases I, II and III exhibited different cyclic nucleotide dependency and substrate specificity. Protein kinase II was inhibited by a heat-stable protein from rabbit skeletal muscle, whereas protein kinases I and III were not inhibited. According to previously established criteria nuclear protein kinase II can be classified as cAMP-dependent protein kinase consisting of regulatory and catalytic subunits. Nuclear protein kinases I and III are cAMP-independent enzymes. Evidence for the identity of nuclear cAMP-dependent protein kinase II with cytosol (105,000 .times. g supernatant fraction) cAMP-dependent protein kinase was obtained in several ways. Nuclear and cytosol cAMP-dependent protein kinases exhibited identical elution characteristics on DEAE-cellulose and Sephadex G-200 indicating that both kinases are of similar molecular size and possess similar ionic charge. Both kinases exhibited an identical Km for ATP of 8 .mu.M, showed similar substrate specificity and revealed similar antigenic properties. cAMP-dependent protein kinase II was also identified in nuclei isolated in nonaqueous media, eliminating the possibility that the cAMP-dependent protein kinase activity identified in nuclei isolated in aqueous media may have arisen as the result of cytoplasmic contamination. After incubation of neonatal porcine ovaries which lack nuclear cAMP-dependent protein kinase with 0.1 .mu.M 8-p-chlorophenylthio cAMP, considerable cAMP-dependent protein kinase II activity was identified in nuclei isolated in nonaqueous media. The nuclear cAMP-dependent protein kinase II is apparently related to or identical with the ovary cytoplasmic cAMP-dependent protein kinase, supporting the concept that nuclear cAMP-dependent protein kinase is of cytoplasmic origin.