Threonine 1342 in human topoisomerase IIalpha is phosphorylated throughout the cell cycle

To investigate the relationship between the modulation of topoisomerase II activity and its phosphorylation state during the cell cycle, a monoclonal antibody against C-terminal peptide (residues 1335-1350) of topoisomerase IIalpha containing a consensus sequence of casein kinase II, TDDE and its phosphorylated threonine were prepared. In an enzyme-linked immunosorbent assay, the antibody, named PT1342, recognized the immunogenic phosphopeptide but not the non-phosphorylated form of the peptide. The PT1342 antibody reacted only with a 170-kDa protein from HeLa cells and recognized anti-topoisomerase IIalpha immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase IIalpha mutated at codon 1342 from threonine to alanine, showing that PT1342 was directed against the phosphorylated threonine 1342. To examine the level of phosphorylation of threonine 1342 of topoisomerase IIalpha through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase IIalpha and DNA and analyzed by flow cytometry. Cells in the G2-M phase contained about double the PT1341-reacted topoisomerase IIalpha than did cells in G1 or S phases. The antibody stained the nuclei in interphase and mitotic chromosomes and its periphery, as seen with anti-topoisomerase IIalpha antibody. Thus, threonine 1342 in topoisomerase IIalpha is phosphorylated throughout the cell cycle.