Ishida R, Takashima R, Koujin T, Shibata M, Nozaki N, Seto M, Mori H, Haraguchi T, Hiraoka Y
Laboratory of Chemotherapy, Aichi Cancer Center Research Institute, Nagoya, Japan.
Cell Struct Funct. 2001 Aug;26(4):215-26. doi: 10.1247/csf.26.215.
It is known that topoisomerase IIalpha is phosphorylated by several kinases. To elucidate the role of phosphorylation of topoisomerase IIalpha in the cell cycle, we have examined the cell cycle behavior of phosphorylated topoisomerase IIalpha in HeLa cells using antibodies against several phospho-oligopeptides of this enzyme. Here we demonstrate that serine1212 in topoisomerase IIalpha is phosphorylated only in the mitotic phase. Using an antibody against an oligopeptide containing phosphoserine-1212 in topoisomerase IIalpha (PS1212), subcellular localization of topoisomerase IIalpha phosphorylated at serine1212 was examined by indirect immunofluorescence staining, and compared with that of overall topoisomerase IIalpha. Serine1212-phosphorylated topoisomerase IIalpha was localized specifically on mitotic chromosomes, but not on interphase chromosomes; this result contrasts with overall topoisomerase IIalpha which was observed on chomosomes in both interphase and mitosis. Serine1212-phosphorylated topoisomerase lIalpha first appeared on chromosome arms in prophase, became concentrated on the centromeres in metaphase, and disappeared in early telophase. In addition, ICRF-193, a catalytic inhibitor of topoisomerase II, prevented accumulation of serine1212-phosphorylated topoisomerase IIalpha at the centromeres. These results indicate that serine1212 of topoisomerase IIalpha is phosphorylated specifically during mitosis, and suggest that the serine1212-phosphorylated topoisomerase IIalpha acts on resolving topological constraint progressively from the chromosome arm to the centromere during metaphase chromosome condensation.
已知拓扑异构酶IIα可被多种激酶磷酸化。为阐明拓扑异构酶IIα磷酸化在细胞周期中的作用，我们使用针对该酶几种磷酸化寡肽的抗体，研究了HeLa细胞中磷酸化拓扑异构酶IIα的细胞周期行为。在此我们证明，拓扑异构酶IIα中的丝氨酸1212仅在有丝分裂期被磷酸化。使用针对拓扑异构酶IIα中含磷酸丝氨酸-1212的寡肽（PS1212）的抗体，通过间接免疫荧光染色检测了丝氨酸1212磷酸化的拓扑异构酶IIα的亚细胞定位，并与整体拓扑异构酶IIα的定位进行了比较。丝氨酸1212磷酸化的拓扑异构酶IIα特异性定位于有丝分裂染色体上，而不是间期染色体上；这一结果与在间期和有丝分裂期染色体上均观察到的整体拓扑异构酶IIα形成对比。丝氨酸1212磷酸化的拓扑异构酶IIα首先在前期出现在染色体臂上，在中期集中于着丝粒，并在末期早期消失。此外，拓扑异构酶II的催化抑制剂ICRF-193可阻止丝氨酸1212磷酸化的拓扑异构酶IIα在着丝粒处积累。这些结果表明，拓扑异构酶IIα的丝氨酸1212在有丝分裂期间被特异性磷酸化，并表明丝氨酸1212磷酸化的拓扑异构酶IIα在中期染色体浓缩过程中，从染色体臂到着丝粒逐步作用于解决拓扑约束。
