TOPO-II PHOSPHORYLATION AND CELL DIFFERENTIATION

TOPO-II 磷酸化和细胞分化

• 批准号: 2103251
• 负责人: ANDREAS I. CONSTANTINOU
• 金额: $ 9.64万
• 依托单位: UNIVERSITY OF ILLINOIS AT CHICAGO
• 依托单位国家: 美国
• 财政年份: 1993
• 资助国家: 美国
• 起止时间: 1993-04-01 至 1998-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2103251
• 关键词: DNA topoisomerases; cell cycle; cell differentiation; cell free system; diacylglycerols; enzyme activity; enzyme inhibitors; phorbols; phosphorylation; protein kinase; tissue /cell culture

The long term research objectives of this proposal are: a) to precisely define how the activity of DNA topoisomerase II (topo II) is regulated by phosphorylation in cultured cells, and b) to identify the specific phosphorylation sites which influence cell growth and differentiation. Preliminary data strongly suggest that topo II is phosphorylated at multiple sites. Increased topo II phosphorylation is associated with elevated enzymatic activity in cell-free systems; however, in phorbol ester-treated HL-60 cells it is associated with reduced activity. Based on these observations and other phosphorylation/activity relationships, we form the hypothesis that phosphorylation of certain topo II sites renders this enzyme less active, while the phosphorylation of other sites renders it more active. To scrutinize this hypothesis, and to define the biological significance of topo II phosphorylation in cultured cells, we propose to: a) map the multiple phosphorylation sites of topo II; b) identify the specific topo II sites modulated by protein kinase activators and inhibitors, define their impact on the catalytic (strand passing) activity of the enzyme; and c) identify specific topo II determinants which, when phosphorylated, influence the ability of the enzyme to ligate DNA in the presence of topo II-reactive antitumor agents. At the end of this project, it is anticipated that the regulation of cell growth and differentiation will be further clarified and the interactions of topo II and protein kinases will be documented.

本提案的长期研究目标是：a) 精确地 定义 DNA 拓扑异构酶 II (topo II) 的活性如何受到调节 培养细胞中的磷酸化，以及 b) 鉴定特定的 影响细胞生长和分化的磷酸化位点。 初步数据强烈表明拓扑 II 在 多个站点。 topo II 磷酸化增加与 无细胞系统中酶活性升高；然而，在佛波醇 酯处理的 HL-60 细胞与活性降低有关。 基于 根据这些观察结果和其他磷酸化/活性关系， 我们假设某些拓扑 II 位点被磷酸化 使该酶活性降低，而其他位点的磷酸化 使其更加活跃。 为了检验这一假设并定义 培养细胞中拓扑 II 磷酸化的生物学意义，我们 建议： a) 绘制拓扑 II 的多个磷酸化位点图； b) 识别由蛋白激酶调节的特定拓扑 II 位点 活化剂和抑制剂，定义它们对催化（链 通过）酶的活性； c) 识别特定的拓扑 II 当磷酸化时，影响能力的决定因素 在拓扑 II 反应性抗肿瘤药物存在的情况下连接 DNA 的酶 代理。 在该项目结束时，预计 细胞生长和分化的调控将进一步阐明 拓扑 II 和蛋白激酶的相互作用将被记录。