HUMAN TOPOISOMERASE II-ALPHA IS PHOSPHORYLATED IN A CELL-CYCLE PHASE-DEPENDENT MANNER BY A PROLINE-DIRECTED KINASE

Topoisomerase II is essential for chromosome condensation and segregat ion at mitosis in eukaryotic cells, but the mechanism of its regulatio n is not clearly understood. We have investigated whether or not the a lpha isozyme of human topoisomerase II is phosphorylated in a cell-cyc le phase-dependent manner. Two-dimensional tryptic phosphopeptide mapp ing revealed that several sites on HeLa topoisomerase II alpha protein were phosphorylated predominantly or exclusively during the G2 and hi phases. To identify the protein kinases involved in this cell-cycle p hase-specific phosphorylation, oligohistidine-tagged recombinant domai ns 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 domai n of the topoisomerase II alpha protein by the universal mitotic contr oller, p34(cdc2), generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M-phase-specific phosphorylation sites ob served in vivo. The same phosphopeptides were obtained following phosp horylation of the C-terminal domain in vitro by the mitogen-activated protein kinase. Site-directed mutagenesis studies identified five of t hese sites of phosphorylation, each of which comprised a serine-prolin e motif. Our data implicate one or more proline-directed kinases in th e cell-cycle-dependent regulation of topoisomerase II alpha enzyme act ivity in human cells.