Human Topoisomerase IIα is Phosphorylated in a Cell‐Cycle Phase‐Dependent Manner by a Proline‐Directed Kinase

Abstract

Topoisomerase I1 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 a isozyme of human topoisomerase I1 is phosphorylated in a cell-cycle phase-dependent manner. Twodimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase IIa 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 IIa 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 IIa protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2Af-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 IIa enzyme activity in human cells.Keywords. Topoisomerase 11; mitosis ; ~3 4 "~'~; phosphorylation ; cell cycle.Topoisomerases are ubiquitous enzymes with essential roles in DNA metabolism. The type-I1 enzymes, such as topoisomerase IV in bacteria and topoisomerase I1 in eukaryotic cells, are required both for chromosome segregation and for faithful partitioning of plasmids into daughter cells (DiNardo et al., 1984;Holm et al., 1985; Uemura and Yanagida, 1986;Uemura et al., 1987;Rose et al., 1990; Kato et al., 1990;Adams et al., 1992). In addition to this essential function during cell division, topoisomerase I1 enzymes play numerous other roles in DNA metabolism in eukaryotic cells, including mitotic chromosome condensation, relief of torsional stress and suppression of genomic instability (reviewed by Osheroff et al., 1991;Wang and Lynch, 1993;Watt and Hickson, 1994). Evidence has been presented that topoisomerase I1 performs a structural role in eukaryotic cell nuclei, anchoring topologically constrained domains of chromatin to the nuclear matrix or scaffold (reviewed by Roberge and Gasser, 1992).In contrast to lower eukaryotes, mammalian cells express two closely related, but genetically distinct, isozymes of topoisomerase I1 (Tsai-Pflugfelder et al., 1988;Drake et al., 1989;Chung et al., 1989;Tan et al., 1992;Jenkins et al., 1992;Austin et al., 1993), designated a (170-kDa form) and p (180-kDa form differential expression during the cell cycle and following oncogenic transformation (Woessner et al., 1990(Woessner et al., , 1991 that at least some functional specialization of these isozymes has evolved in ...

Ataxia telangiectasia mutated‐dependent regulation of topoisomerase II alpha expression and sensitivity to topoisomerase II inhibitor

“…Topoisomerase II alpha is phosphorylated on multiple Ser and Thr residues, most of which are located in the C‐terminal domain . Several potential ATM‐dependent or ‐regulated phosphorylation sites (Ser1424, 1466, 1522, and 1524) were reported .…”

“…Topoisomerase II alpha is phosphorylated on multiple Ser and Thr residues, most of which are located in the C-terminal domain. (34)(35)(36)(37)(38)(39) Several potential ATM-dependent or -regulated phosphorylation sites (Ser1424, 1466, 1522, and 1524) were reported. (40) Our findings showed phosphorylation of a new target site, Ser1512, induced by DNA damage in an ATMdependent manner.…”

Epstein-Barr Virus BGLF4 Kinase Induces Disassembly of the Nuclear Lamina To Facilitate Virion Production

“…The supercoiling and knotting activities of condensin and Topo II␣ are activated by cellular Cdc2 kinase-induced phosphorylation (28,56). To explore whether BGLF4 can phosphorylate condensin and Topo II␣ directly, recombinant BGLF4 kinase and the kinase-dead mutant K102I were expressed in a recombinant baculovirus system.…”

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“…TopoIIα undergoes a myriad of phosphorylations during mitosis, including those catalysed by CDC2 and protein kinase C [6,7]. TopoIIα is in fact the major mitotic chromosomal protein recognized by MPM-2 [8].…”