Nuclear protein kinases. Evidence for their heterogeneity, tissue specificity, substrate specificities, and differential responses to cyclic adenosine 3':5'-monophosphate.

Abstract

Abstract The non-histone chromatin phosphoprotein fraction isolated from beef liver nuclei contains endogenous protein kinase activity which catalyzes the phosphorylation of these proteins in vitro in the presence of [γ-32P]ATP and magnesium. By using phosphocellulose chromatography we have separated this protein kinase activity into 12 distinct enzyme fractions. Individual enzyme fractions can be resolved further by acrylamide gel electrophoresis into multiple components, thus demonstrating an extraordinary degree of heterogeneity among the chromatin-associated protein kinases. Protein kinase fractions exhibit different specificities for casein, histone, and non-histone proteins as substrates, as demonstrated by both acid-precipitable radioactivity and analysis of radioactivity patterns of 32P-labeled substrates by acrylamide gel electrophoresis. Further evidence to suggest that we are dealing with a number of different protein kinases is presented by the specific effects on substrate phosphorylation which we observe in the presence of cyclic adenosine 3' : 5'-monophosphate (cyclic AMP). This cyclic nucleotide exhibits either stimulatory or inhibitory effects on protein phosphorylation, depending on both the substrate and the protein kinase fraction employed. A comparison of one of the nuclear protein kinase fractions with cytoplasmic histone kinase illustrates that these are different enzymes. Although both enzymes are able to catalyze the phosphorylation of non-histone chromatin proteins and f1 histone, the pattern of labeling is found to be quite different when analyzed by acrylamide gel electrophoresis. Cyclic AMP stimulates the phosphorylation of non-histone protein by both of these enzymes. However, the phosphorylation of f1 histone is stimulated by cyclic AMP when histone kinase is used but is inhibited by cyclic AMP with this particular nuclear protein kinase fraction, again demonstrating that these are distinctly different enzymes. On the basis of our results, we conclude that the protein kinases which are associated with the non-histone chromatin phosphorylated proteins of the nucleus can be fractionated into a large number of enzyme activities which have distinct substrate specificities and responses to cyclic AMP. Preliminary data showing the existence of different protein kinase profiles in liver and kidney emphasize the tissue specificity of these enzymes and suggest that these kinases may play an important role in the tissue-specific regulation of RNA synthesis and chromatin function.