TOPO-II PHOSPHORYLATION AND CELL DIFFERENTIATION

这项建议的长期研究目标是： 定义DNA拓扑异构酶II（topo II）的活性是如何通过 B）鉴定培养细胞中的特异性磷酸化， 磷酸化位点影响细胞生长和分化。 初步数据强烈表明，topo II是磷酸化的， 多个站点。 拓扑异构酶II磷酸化的增加与 在无细胞系统中酶活性升高;然而，在佛波醇中 酯处理的HL-60细胞，其与活性降低有关。 基于 基于这些观察结果和其它磷酸化/活性关系， 我们形成了这样一种假设，即某些topo II位点的磷酸化 使这种酶活性降低，而其他位点的磷酸化 使其更加活跃。 仔细检查这个假设，并定义 topo II磷酸化在培养细胞中的生物学意义，我们 建议：a）绘制拓扑异构酶II的多个磷酸化位点; B） 确定蛋白激酶调节的特异性topo II位点 活化剂和抑制剂，定义其对催化（链） 通过）酶的活性;和c）鉴定特异性拓扑异构酶II 决定因素，当磷酸化，影响的能力， 在topo II反应性抗肿瘤剂存在下连接DNA的酶 剂. 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 并记录拓扑异构酶II和蛋白激酶的相互作用。

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.