Brain histone kinase: The structure, the substrate specificity and the mechanism of action

Abstract

Cyclic adenosine 3′,5′-monophosphate-dependent histone kinase (ATP protein phosphotransferase, EC 2.7.1.37) was obtained from pig brain. The enzyme was purified 1140 times. It was homogeneous on polyacrylamide gel with electrophoresis and gel filtration. The established molecular weight of the enzyme is 120,000. The histone kinase dissociates into catalytic and regulatory subunits (molecular weight 40,000 and 90,000, respectively). With chromatography on DEAE-cellulose in the presence of excess cyclic AMP the homogeneous catalytic subunit was obtained. Using polylysylsepharose, which contains immobilized 8-(γ-carboxypropylthio)-cyclic AMP with affinity chromatography the homogeneous regulatory subunit was isolated. The methods permit us to obtain also individual catalytic and regulatory subunits of the histone kinase from partially purified preparations of the enzyme. Subunits of the histone kinase were homogeneous with polyacrylamide gel electrophoresis in the presence of SDS. The histone kinase had a high specificity of action according to lysine-rich histones F 1, F 2a2 and F 2b. Arginine-rich histones and other known protein substrates of cyclic AMP-dependent protein kinases (casein, E. coli, RNA polymerase, etc.) were not phosphorylated by this enzyme. The kinetics of phosphorylation of histones F 1, F 2a2, F 2b was investigated and corresponding sites of phosphorylation were determined in primary structures of these histones: Ser-38 of histone F 1, Ser-19 of histone F 2a2, Ser-14 and Ser-36 of histone F 2b. Phosphorylated serine residues were localized in all cases in sequence X-Y-Ser, where X is always a lysine or arginine residue, Y — an acidic or neutral amino acid. Using various original ATP and cyclic AMP analogs containing reactive groups, the functional topography of the active sites of catalytic and regulatory subunits active sites was investigated. Specific regions of ATP binding in the active centre of the catalytic subunit were determined and the imidazole ring of histidine was suggested as the catalytic group participating in the phosphotransferase reaction. The existence of at least three parts in the regulatory centre of histone kinase was suggested in which binding of a negatively charged hydroxyl group of the cyclophosphate system, the nucleophilic locus for binding of 2′-hydroxyl group of the ribose ring and the proton-donor group, which may take part in the interaction with the exo-amino group of the adenine base occurs. On this basis, the contribution of separate groups of molecule of the cyclic nucleotide to the enzyme activation was studied. The investigation of the primary structure of different subfractions of F 1 histone, the chemical modification reaction of F 1 histone and PMR spectra of native and phosphorylated Ser-38 molecules of F 1 histone in the complex with DNA permitted us to suggest that during the F 1 histone interaction with DNA the role of two cationic loci (15–36) and (107–212) of the F 1 histone molecule is determined by the realization of typical electrostatic contacts. The ionic interaction of one of the cationic loci, probably the cationic part, (15–36) is destroyed by the incorporation of phosphate group on Ser-38 of F 1 histone molecule. The phosphorylation on Ser-38 F 1 histone leads to the disorganization of the secondary structure of the central segment and leads to its more free movement in the complex with the polyanion template. PMR data demonstrated that the phosphorylation on Ser-38 residue of F 1 histone led to the weakening of the histone-DNA binding and influenced the secondary structure and protein-protein interactions in the nucleoproteid complex. Using the cytochemical approach the influence of the histone kinase on physico-chemical properties of chromatin was investigated. The hepatocyte treatment by the histone kinase led to the increase of acridine orange binding. The increase of acridine orange binding to DNP was the same both at 2.5 hr after the operation and after the histone kinase treatment of hepatocytes without operation: i.e., the cell treatment by the histone kinase had the same effect as partial hepatectomy. Thus, the phosphorylation of lysine-rich histones by the exogenous histone kinase led to the appearance of activated parts of genome and the stimulation of the transcription process.