Preferential phosphorylation of basic non-histone proteins by nuclear protein kinase NII from rat liver

Rat liver nuclear protein kinase NII, which is independent of cyclic nucleotides, phosphorylated both the acidic protein casein and the basic histone preparation, soluble in 0.25 M HCl, which was extracted from the cell nuclei. When the isolated nuclei were incubated with [32P]ATP in the presence of protein kinase NII, more than 95% of the 32P-labelled proteins was recovered in the 0.25 M HCl extract. In order to analyze the substrate proteins of protein kinase NII, a histone preparation free from the endogenous protein kinase activities was used as substrate. The results demonstrated that histones were not phosphorylated, except for a faint 32P incorporation into the H3 fraction. Instead, multiple non-histone proteins were highly phosphorylated, which were present in a minor quantity in the histone preparation. The molecular weights of the main phosphorylated proteins were 72000, 68000, 56000, 47000, 46000, 43000, 38000 and 32000. Isoelectric focusing demonstrated the basic nature of the phosphorylated proteins, and as much as 72% of 32P radioactivity was distributed in the pH region higher than 7.0. Furthermore, many proteins phosphorylated in the isolated nuclei with added protein kinase NII were also found to be basic non-histone proteins. These results indicate that protein kinase NII preferentially phosphorylates in vitro a set of nuclear basic non-histone proteins.