The intrinsic substrate specificity of a cyclic nucleotide-independent protein (histone) kinase

Abstract

Phosphorylation and dephosphorylation of proteins play a pivotal role in the regulation of intracellular processes (reviewed in [l-3]). Beside several well-characterized protein kinases a number of protein phosphorylating enzymes have been found; however, their natural substrates and regulatory properties are not known. A possible way to identify these enzymes is the determination of their intrinsic substrate specificity; i.e., the most important residues of the amino acid sequence that they recognize in their substrate. A cyclic nucleotide-independent histone kinase (designated as HK II) was described in 1451. We have also reported a type of cyclic nucleotide-independent histone kinase found in the cytoplasm and nucleus of human tonsillar lymphocytes [6,7] and in the extract of bovine thymus [8]. This enzyme phosphorylates the Ser-32 residue of calf thymus H2b histone and a synthetic peptide containing the amino acid sequence of H2b from Gly-26-Lys-34, but it does not act on Ser-36 which is phosphorylated preferentially by the catalytic subunit of the cyclic AMP-dependent protein kinase 19-l 11. Separation of the cyclic nucleotide-independent histone kinase from the catalytic subunit of the cyclic AMP-dependent protein kinase was carried out by DEAE-cellulose chromatography in the presence of cyclic AMP, as in 181. The histone kinase fraction obtained by this procedure from bovine thymus was dialyzed against 0.005 M potassium phosphate (pH 7.5) and it was applied onto a hydroxylapatite (Bio-Gel HTP) column. The column was washed by 5 vol. of each of 0.01 M, 0.07 M, 0.085 M, 0.1 M and 0.4 M potassium phosphate (pH 7.5). The histone kinase was eluted by the last volumes of 0.07 M and by the first volume of 0.085 M potassium phosphate. The specific activity of the enzyme preparation obtained was lo-12 nmol phosphate transferred mg protein -’ . min -‘, as measured in the presence of 1 mg histone Hi/ml and 2 x lo-’ M ATP. This preparation yielded a single (Mr 55 000) histone kinase peak on Sephadex G-100 gel-chromatography.