Regulation of Topoisomerase II-Drug-DNA Ternary Complex

描述（由申请人提供）：本申请的总体目标是确定丝氨酸(S) 1106磷酸化在拓扑异构酶（topo） iα中的功能作用以及调节该位点磷酸化的上游机制。Topo i - α改变DNA拓扑结构并维持细胞完整性。这种酶以细胞周期依赖的方式在特定位点磷酸化。最近，我们在催化结构域中发现了一个磷酸化位点，Ser1106，它调节酶活性和药物敏感性。初步研究表明，Ser1106以Ca2+依赖的方式被CKI磷酸化。因此，我们的工作假设是CKI（和/或CKII）对S1106的ca2 +依赖性磷酸化调节topo i α的生物活性。为了验证这一假设，将采用体外和体内研究表达重组topo ialpha（野生型或突变型）和人类白血病HL-60模型系统的酿酒葡萄球菌（菌株BJ201或JN394）。Topo IIalkpha的功能将通过以下方法评估：a)十烷磺酸活性；b)体外依托泊苷（VP-16）稳定的DNA可切割复合物形成；c)体内对VP-16的敏感性。将评估：a) Ser1106对谷氨酸或天冬氨酸的突变，以及b)体内CKII磷酸化位点，单独或与S1106对丙氨酸的突变，对topo IIa功能的影响。Ser1106磷酸化topo IIa的细胞周期期依赖性表达和亚细胞定位将通过质谱/2D磷酸化肽定位和免疫荧光染色来确定。将产生Phospho-S1106特异性抗体，并用于同步G1、S、G2或M细胞的免疫印迹分析和免疫荧光染色。CKI和/或CKI在磷酸化Ser1106中的作用将在体外和体内进行测试。对于体外检测，将使用纯化或重组CKI或CKII，纯化野生型或突变型topo IIa或含有Ser1106的合成肽。在酵母和HL-60细胞中进行的体内研究将检查topo IIa磷酸化和以下功能：a) S1106附近激酶一致序列的改变，b)通过药理学（CKI/CKII抑制剂）或分子（CKI/CKII反义寡核苷酸或siRNA，或酵母CKI同源敲除）方法消耗CKI和/或CKII。这些激酶在对topo II毒物表现出不同敏感性的细胞中的表达和活性将被确定。从长远来看，拟议的研究应该提供关于topo ialpha磷酸化和上游事件（即调节磷酸化的特定激酶）的功能意义的重要信息。

DESCRIPTION (provided by applicant): The overall goal of this application is to determine the functional role of phosphorylation of serine (S) 1106 in topoisomerase (topo) IIalpha and the upstream mechanisms regulating phosphorylation of this site. Topo IIalpha alters DNA topology and maintains cellular integrity. This enzyme is phosphorylated at specific sites in a cell cycle dependent manner. Recently we identified a phosphorylation site, Ser1106, in the catalytic domain, which regulates enzymatic activity and drug sensitivity. Preliminary studies suggest that Ser1106 is phosphorylated by CKI in a Ca2+dependent manner. Thus, our working hypothesis is that Ca 2+dependent phosphorylation of S1106 by CKI (and/or CKII) regulates the biologic activity of topo IIalpha. To test this hypothesis, in vitro and in vivo studies in S. cerevesiae (strains BJ201 or JN394) expressing recombinant topo IIalpha (wild-type or mutant) and the human leukemia HL-60 model systems will be employed. Topo IIalkpha function will be assessed by: a) decatenation activity b) etoposide (VP-16)-stabilized DNA cleavable complex formation in vitro, and c) sensitivity to VP-16 in vivo. The effect of mutation of: a) Ser1106 to glutamic acid or aspartic acid, and b) in vivo CKII phosphorylation sites, alone or in combination with S1106 to alanine, on topo IIa function will be evaluated. Cell cycle phase dependent expression and subcellular localization of Ser1106 phosphorylated topo IIa will be determined by mass spectrometry/2D phosphopeptide mapping and immunofluorescence staining. Phospho-S1106 specific antibodies will be generated and used for immunoblot analyses of synchronized G1, S, or G2 or M cells and for immunofluorescence staining. A role for CKI and/or CKII in phosphorylating Ser1106 will be tested in vitro and in vivo. For in vitro assays purified or recombinant CKI or CKII and purified wild type or mutant topo IIa or synthetic peptides containing Ser1106 will be employed. In vivo studies, performed in yeast and HL-60 cells, will examine topo IIa phosphorylation and function following: a) alteration of kinase consensus sequences around S1106, and b) depletion of CKI and/or CKII by pharmacological (inhibitors of CKI/CKII) or molecular (CKI/CKII antisense oligonucleotides or siRNA, or yeast CKI homolog knock out) approaches. Expression and activity of these kinases in cells exhibiting differential sensitivity to topo II poisons will be determined. In the long term, the proposed studies should provide salient information on the functional significance of topo IIalpha phosphorylation and the upstream events, i.e. specific kinase(s), that modulate phosphorylation.