Analysis of M phase-specific phosphorylation of DNA topoisomerase II.
In mammalian cells, two isoforms of DNA topoisomerase II (topo II), topo IIalpha and topo IIbeta, are phosphorylated. The phosphorylation of topo IIbeta changes its apparent molecular mass determined by SDS-polyacrylamide gel electrophoresis from 180 to 190 kDa in mitotic cells, whereas topo IIalpha affects it only slightly (Kimura, K., Nozaki, N., Saijo, M., Kikuchi, A., Ui, M., and Enomoto, T. (1994) J. Biol. Chem. 269, 24523-24526). Here we examined the stability of the protein and the phosphate moiety of each topo II isoform, as the cells progressed from M to G1 phase. While its protein moiety remained intact, 75% of the phosphates attached to topo IIbeta were removed within 4 h after release from mitotic block. On the other hand, 35% of topo IIalpha protein and 52% of the attached phosphates disappeared. We verified that M phase-specific phosphorylation had no particular effect on the catalytic activities of both topo II isoforms after extensive phosphatase digestion. We also examined the binding of two isoforms to the nucleus or chromosomes. In logarithmically growing cells, both isoforms were extracted from nuclei at the same concentrations of NaCl. From the mitotic chromosomes, topo IIbeta was extracted at much lower concentrations of NaCl than topo IIalpha.
Kimura
K
K
Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194.
Nozaki
N
N
Enomoto
T
T
Tanaka
M
M
Kikuchi
A
A
D000595:Amino Acid Sequence; D002384:Catalysis; D002877:Chromosomes, Human; D004250:DNA Topoisomerases, Type II / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D006367:HeLa Cells; D006801:Humans; D007399:Interphase; D007527:Isoenzymes / Q000378:metabolism*; D008938:Mitosis; D008969:Molecular Sequence Data; D010766:Phosphorylation
D007527:Isoenzymes; D004250:DNA Topoisomerases, Type II