PHOSPHORYLATION OF HISTONES ON N-AMINO ACIDS

N-氨基酸组蛋白的磷酸化

• 批准号: 3468320
• 负责人: PETER J. KENNELLY
• 金额: $ 9.88万
• 依托单位: VIRGINIA POLYTECHNIC INST AND ST UNIV
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 1995-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3468320
• 关键词: 3T3 cells; DNA binding protein; affinity chromatography; aminoacid; cell cycle; cell differentiation; cell growth regulation; chromatin; complementary DNA; enzyme mechanism; enzyme structure; enzyme substrate; erythrocytes; genetic transcription; genome; histones; laboratory rat; messenger RNA; molecular cloning; neoplastic transformation; phosphatase inhibitor; phosphorylation; protein sequence; protein tyrosine kinase; serine; threonine

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.

这项研究的长期目标是剖析 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 哺乳动物细胞的核[N-磷酸化]。组蛋白就是 许多共价修饰事件。正如所示， 发现许多细胞分裂周期基因--它们的破坏 导致复制过程的停止--编码组蛋白激酶， 这些共价修饰形成了络合物的重要组成部分 负责建立、维护和调整 染色质的结构和功能。这台机器的缺陷或它的 病原体和其他因素的破坏，例如发生在 致癌转化，对人的健康有严重后果 涉案的个人。然而，为了了解详细的信息， 这一机器运行的机制，至关重要的是识别和 研究它的各个组成部分。因此，我们的意图是) 鉴定和表征执行N-的过程的酶 磷酸化，N-激酶和N-磷酸酶，b)以确定它们如何 是受控的，以及c)确定场地和功能后果 在哺乳动物细胞中核染色质蛋白的N-磷酸化。 这项研究提案概述了这一努力的开始阶段。它的 核心目标有三个。一是差异化 这些组蛋白N-磷酸化事件本质上是动态的，来自 那些是静态的。这将通过检查以下级别来实现 组蛋白中的N-磷酸与培养的核N-激酶活性 3T3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的爆发 主动转录染色质的转录不同于 大量染色质的。第二个目标是分离和研究一个 N-激酶--Walker 256大鼠的“生长相关”组蛋白H4激酶 癌症。它的物理和催化性能将被检测，并 通过克隆ITS基因进行氨基酸序列测定。这 序列将与丝氨酸/苏氨酸和酪氨酸的序列进行比较 [O-激酶]以确定N-和O-激酶 族在结构上是相关的。尽管已经检测到N-激酶 在一些真核生物的细胞核中，尚不清楚 这些酶的活性被N-磷酸酶所抵消。 因此，这项提案的第三个目标将是确定 哺乳动物细胞的细胞核中是否存在N-磷酸酶活性。