Adenosine 3':5'-Monophosphate-dependent Protein Kinase and the Stimulation of Ovarian Nuclear Ribonucleic Acid Polymerase Activities

Abstract

Abstract The cyclic adenosine 3':5'-monophosphate-dependent (cyclic AMP-dependent) protein kinase of calf ovary cytosol has been partially purified and characterized. This preparation activated and possibly phosphorylates ovarian nuclear RNA polymerases Ia, Ib, and II. Cyclic AMP-dependent protein kinase of calf ovary cytosol has been dissociated into a cyclic AMP-binding protein subunit and into a cyclic AMP-independent protein kinase subunit. Isolation of the cyclic AMP-binding protein and of the cyclic AMP-independent protein kinase resulted in a 110-fold purification of both sub-units. The cyclic AMP-independent protein kinase used histone F1, protamine, phosvitin, casein, and Escherichia coli RNA polymerase as substrates and appeared to exhibit little substrate specificity. The apparent Km value of the cyclic AMP-independent protein kinase for ATP was determined as 1.8 x 10-5 m. The cyclic AMP-binding protein was determined by Sephadex gel filtration to have a molecular weight of about 135,000. The molecular weight of the cyclic AMP-independent protein kinase was estimated to be in the range of about 375,000. Dissociation of the cyclic AMP-independent protein kinase by sodium dodecyl sulfate resulted in the formation of three subunits exhibiting molecular weights of 26,000, 64,000, and 90,000, respectively. The cyclic AMP-dependent protein kinase in the presence of 5 x 10-9 m cyclic AMP, and the cyclic AMP-independent protein kinase stimulated the incorporation of 32P from [γ-32P] ATP into fractions containing nuclear RNA polymerases Ia, Ib, and II. Chromatography on DEAE-cellulose indicated a coincidence of the phosphorylated proteins with the three polymerase activities but no proof of the incorporating of 32P into the RNA polymerases themselves has yet been provided. The fraction containing RNA polymerase II activity was the preferred substrate as judged by the significantly higher incorporation of 32P. The cyclic AMP-dependent phosphorylation of the RNA polymerase preparations was associated with an increase of the RNA-synthesizing activity of all three enzyme preparations. The activity of the polymerase II was increased 9-fold after phosphorylation. Only a 2.5- to 3-fold stimulation of RNA synthesis by the phosphorylated polymerases Ia and Ib was observed. These results suggest that cyclic AMP and protein kinase play an important role in the hormonal control of ovarian nuclear RNA synthesis through regulation and perhaps phosphorylative modification of the ovarian nuclear RNA polymerases.