TOPO-II PHOSPHORYLATION AND CELL DIFFERENTIATION

这项建议的长期研究目标是：a)准确地 定义DNA拓扑异构酶II(Topo II)的活性是如何通过 培养细胞中的磷酸化，以及b)识别特定的 影响细胞生长和分化的磷酸化位点。 初步数据有力地表明Topo II在 多个站点。Topo II磷酸化增加与 在无细胞体系中，酶活性升高；然而，在佛波醇中 酯处理的HL-60细胞，它与活性降低有关。基座 根据这些观察和其他磷酸化/活性关系， 我们假设某些Topo II位点的磷酸化 使这种酶的活性降低，而其他位点的磷酸化 使其更加活跃。仔细研究这一假设，并定义 Topo II磷酸化在培养细胞中的生物学意义 建议：a)绘制Topo II的多个磷酸化位点图；b) 确定蛋白激酶调控的Topo II特异性位点 激活剂和抑制剂，定义它们对催化链的影响 通过)酶的活性；以及c)鉴定特定的Topo II 决定因素，当磷酸化，影响能力的能力 在Topo II反应性抗肿瘤药物存在下连接DNA的酶 探员们。在本项目结束时，预计 对细胞生长和分化的调控将进一步阐明 Topo II和蛋白激酶的相互作用将被记录在案。

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.