Kondapi Anand K, Padmaja Gade, Satyanarayana N, Mukhopadyaya Robin, Reitz Marvin S
Department of Biochemistry, University of Hyderabad, Hyderabad 500 046, India.
Arch Biochem Biophys. 2005 Sep 1;441(1):41-55. doi: 10.1016/j.abb.2005.06.021.
Human topoisomerase II plays a crucial role in DNA replication and repair. It exists in two isoforms: topoisomerase II alpha (alpha) and topoisomerase II beta (beta). The alpha isoform is localized predominantly in the nucleus, while the beta isoform exhibits a reticular pattern of distribution both in the cytosol and in the nucleus. We show that both isoforms of topoisomerase II are phosphorylated in HIV infected cells and also by purified viral lysate. An analysis of the phosphorylation of topoisomerase II isoforms showed that extracts of HIV infected cells at 8 and 32 h. post-infection (p.i.) contain maximal phosphorylated topoisomerase II alpha, whereas infected cell extracts at 4 and 64 h p.i. contain maximum levels of phosphorylated topoisomerase II beta. In concurrent to phosphorylated topoisomerase II isoforms, we have also observed increased topoisomerase II alpha kinase activity after 8h p.i and topoisomerase beta kinase activity at 4 and 64 h p.i. These findings suggest that both topoisomerase II alpha and beta kinase activities play an important role in early as well as late stages of HIV-1 replication. Further analysis of purified virus showed that HIV-1 virion contained topoisomerase II isoform-specific kinase activities, which were partially isolated. One of the kinase activities of higher hydrophobicity can phosphorylate both topoisomerase II alpha and beta, while lower hydrophobic kinase could predominantly phosphorylate topoisomerase II alpha. The phosphorylation status was correlated with catalytic activity of the enzyme. Western blot analysis using phosphoamino-specific antibodies shows that both the kinase activities catalyze the phosphorylation at serine residues of topoisomerase II alpha and beta. The catalytic inhibitions by serine kinase inhibitors further suggest that the alpha and beta kinase activities associated with virus are distinctly different.
人类拓扑异构酶II在DNA复制和修复过程中发挥着关键作用。它以两种异构体形式存在：拓扑异构酶IIα（α）和拓扑异构酶IIβ（β）。α异构体主要定位于细胞核，而β异构体在细胞质和细胞核中均呈现网状分布模式。我们发现拓扑异构酶II的两种异构体在HIV感染的细胞中以及经纯化的病毒裂解液作用下均会发生磷酸化。对拓扑异构酶II异构体磷酸化情况的分析表明，感染后8小时和32小时的HIV感染细胞提取物中含有最大量的磷酸化拓扑异构酶IIα，而感染后4小时和64小时的感染细胞提取物中含有最高水平的磷酸化拓扑异构酶IIβ。与磷酸化的拓扑异构酶II异构体同时出现的是，我们还观察到感染后8小时拓扑异构酶IIα激酶活性增加，以及感染后4小时和64小时拓扑异构酶β激酶活性增加。这些发现表明拓扑异构酶IIα和β激酶活性在HIV-1复制的早期和晚期阶段均发挥重要作用。对纯化病毒的进一步分析表明，HIV-1病毒粒子含有拓扑异构酶II异构体特异性激酶活性，这些活性已被部分分离。其中一种疏水性较高的激酶活性可使拓扑异构酶IIα和β均发生磷酸化，而疏水性较低的激酶主要使拓扑异构酶IIα发生磷酸化。磷酸化状态与该酶的催化活性相关。使用磷酸氨基酸特异性抗体进行的蛋白质印迹分析表明，这两种激酶活性均催化拓扑异构酶IIα和β丝氨酸残基的磷酸化。丝氨酸激酶抑制剂的催化抑制作用进一步表明，与病毒相关的α和β激酶活性明显不同。
