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Volume 271, Number 47, Issue of November 22, 1996 pp. 30077-30082
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Threonine 1342 in Human Topoisomerase II Is Phosphorylated Throughout the Cell Cycle

(Received for publication, August 6, 1996)

Ryoji Ishida , Miwako Iwai ^¶ , Katherine L. Marsh ^** , Caroline A. Austin ^** , Takeo Yano , Masao Shibata , Naohito Nozaki ^§§ and Akira Hara ^¶

From the  Laboratory of Biochemistry, Aichi Cancer Center Research Institute, Chikusa-Ku, Nagoya 464, Japan; ^¶ Department of Biochemistry, Gifu Pharmaceutical University, Mitahora, Gifu 502, Japan; ^** Department of Biochemistry and Genetics, The Medical School, The University of Newcastle upon Tyne, Newcastle-upon-Tyne NE2 4HH, United Kingdom;  Medical and Biological Laboratories, Ina, Nagano 396, Japan; and ^§§ Department of Oral Biochemistry, Kanagawa Dental College, Yokosuka, Kanagawa 238, Japan

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 II 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 II immunoprecipitants. Furthermore, the antibody did not react with the human topoisomerase II 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 II through the cell cycle, HeLa cells were stained simultaneously for phosphorylated topoisomerase II and DNA and analyzed by flow cytometry. Cells in the G₂-M phase contained about double the PT1341-reacted topoisomerase II 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 II antibody. Thus, threonine 1342 in topoisomerase II is phosphorylated throughout the cell cycle.

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