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 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.
Wells
N J
NJ
Imperial Cancer Research Fund Laboratories, Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, England.
Hickson
I D
ID
D000595:Amino Acid Sequence; D000951:Antigens, Neoplasm; D001483:Base Sequence; D016203:CDC2 Protein Kinase / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D004250:DNA Topoisomerases, Type II / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins; D005434:Flow Cytometry; D016195:G2 Phase; D006367:HeLa Cells; D006801:Humans; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010449:Peptide Mapping; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011392:Proline / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism
D000951:Antigens, Neoplasm; D004268:DNA-Binding Proteins; D007527:Isoenzymes; D010446:Peptide Fragments; D010748:Phosphopeptides; D011994:Recombinant Proteins; D011392:Proline; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D016203:CDC2 Protein Kinase; D004250:DNA Topoisomerases, Type II