PHOSPHORYLATION OF HISTONES ON N-AMINO ACIDS

这项研究工作的长期目标是剖析 负责组蛋白和其他DNA磷酸化的系统- N-氨基酸组氨酸、赖氨酸和精氨酸上的结合蛋白， 哺乳动物细胞的细胞核[N-磷酸化]。 组蛋白是 大量的共价修饰事件。 正如已经说明的那样， 发现许多细胞分裂周期基因， 导致复制过程的停滞--编码组蛋白激酶， 这些共价修饰形成复合物的重要组分 负责建立、维护和调制的机器 染色质结构和功能。 本机器或其 病原体和其他因素的破坏，例如在 致癌转化，对健康的严重后果， 个人参与。 但是，为了详细了解 这一机制的运作，必须确定和 研究其各个组成部分。 因此，我们的目的是a） 鉴定和表征进行N- B）以确定它们如何与磷酸化、N-激酶和N-磷酸酶结合， c）确定场地和功能后果 哺乳动物细胞核染色质蛋白的N-磷酸化。 本研究建议概述了这一努力的开始阶段。 其 中心目标有三个。 一是差异化 这些组蛋白N-磷酸化事件是动态的， 那些是静态的。 这将通过检查 细胞核组蛋白N-磷酸化与核N-激酶活性的关系 3 T3细胞在使染色质发生结构变化的情况下 --细胞分裂过程中基因组的复制和基因突变 转录发生积极转录染色质不同于 那些在大量染色质中的。 第二个目标是分离和研究一种 N-激酶，步行者256大鼠的“生长相关”组蛋白H4激酶 癌 将检查其物理和催化性能， 通过克隆其cDNA确定其氨基酸序列。 这 序列将与丝氨酸/苏氨酸和酪氨酸的那些序列进行比较。 以确定是否N-和O-激酶 家庭在结构上是相互关联的。 虽然已经检测到N-激酶 在许多真核细胞的细胞核中，尚不清楚是否存在 这些酶的活性被N-磷酸酶的活性抵消。 因此，本提案的第三个目标是确定 哺乳动物细胞核中是否存在N-磷酸酶活性。

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.