Wells N J, Hickson I D
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, England.
Eur J Biochem. 1995 Jul 15;231(2):491-7. doi: 10.1111/j.1432-1033.1995.tb20723.x.
Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood. We have investigated whether or not the alpha isozyme of human topoisomerase II is phosphorylated in a cell-cycle phase-dependent manner. Two-dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and M phases. To identify the protein kinases involved in this cell-cycle phase-specific phosphorylation, oligohistidine-tagged recombinant domains of the topoisomerase II alpha protein were expressed in Escherichia coli, purified by affinity chromatography and phosphorylated in vitro by different protein kinases. Phosphorylation of the C-terminal domain of the topoisomerase II alpha protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites observed in vivo. The same phosphopeptides were obtained following phosphorylation of the C-terminal domain in vitro by the mitogen-activated protein kinase. Site-directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine-proline motif. Our data implicate one or more proline-directed kinases in the cell-cycle-dependent regulation of topoisomerase II alpha enzyme activity in human cells.
拓扑异构酶II对于真核细胞有丝分裂时的染色体浓缩和分离至关重要，但其调节机制尚不清楚。我们研究了人类拓扑异构酶II的α同工酶是否以细胞周期阶段依赖性方式被磷酸化。二维胰蛋白酶磷酸肽图谱显示，HeLa细胞拓扑异构酶IIα蛋白上的几个位点主要或仅在G2期和M期被磷酸化。为了鉴定参与这种细胞周期阶段特异性磷酸化的蛋白激酶，拓扑异构酶IIα蛋白的寡组氨酸标签重组结构域在大肠杆菌中表达，通过亲和层析纯化，并在体外被不同的蛋白激酶磷酸化。通用有丝分裂调控因子p34cdc2对拓扑异构酶IIα蛋白的C末端结构域进行磷酸化，产生多个胰蛋白酶磷酸肽，其中许多与体内观察到的G2/M期特异性磷酸化位点相对应。有丝分裂原激活的蛋白激酶在体外对C末端结构域进行磷酸化后也得到了相同的磷酸肽。定点诱变研究确定了其中五个磷酸化位点，每个位点都包含一个丝氨酸-脯氨酸基序。我们的数据表明，一种或多种脯氨酸定向激酶参与了人类细胞中拓扑异构酶IIα酶活性的细胞周期依赖性调节。
