The long range goal of this research effort is to dissect the components of the system responsible for the phosphorylation of histones and other DNA- binding proteins on the N-amino acids histidine, lysine, and arginine in the nuclei of mammalian cells [N-phosphorylation]. Histones are the sites of numerous covalent modification events. As has been illustrated by the finding that many of the cell division cycle genes -- whose disruption results in arrest of the replicative process -- encode histone kinases, these covalent modifications form an important component of the complex machinery responsible for the establishment, maintenance, and modulation of chromatin structure and function. Defects in this machinery or its sabotage by pathogens and other factors, such as that which occurs during oncogenic transformation, have grave consequences for the health of the individual involved. However, in order to understand the detailed mechanism by which this machinery operates, it is essential to identify and study its individual components. Therefore, it is our intention to a) identify and characterize the enzymes that carry out the process of N- phosphorylation, the N-kinases and N-phosphatases, b) to determine how they are controlled, and c) to determine the sites and functional consequences of the N-phosphorylation of nuclear chromatin proteins in mammalian cells. This research proposal outlines the opening stages of this effort. Its central objectives are three in number. The first is to differentiate those histone N-phosphorylation events that are dynamic in nature from those that are static. This will be accomplished by examining the level of N-phosphate in histones and the activity of nuclear N-kinases in cultured 3T3 cells under circumstances that subject chromatin to structural changes -- the replication of the genome during cell division and the burst in gene transcription that takes place actively transcribing chromatin differ from those in bulk chromatin. The second objective is to isolate and study an N-kinase, the """"""""growth-associated"""""""" histone H4 kinase of Walker 256 rat carcinomas. Its physical and catalytic properties will be examined, and its amino acid sequence determined through the cloning of its cDNA. This sequence will be compared to those of the serine/threonine and tyrosine kinases [O-kinases] in order to determine whether the N- and O-kinase families are structurally related. Although N-kinases have been detected in the nuclei of a number of eukaryots, it is not known whether the activity of these enzymes is counterbalanced by that of N-phosphatases. Therefore, the third objective of this proposal will be to determine whether N-phosphatase activity exists in the nuclei of mammalian cells.
