Analysis of M Phase-specific Phosphorylation of DNA Topoisomerase II*
- From the ‡ Mitsubishi Kasei Institute of Life Sciences, Machida, Tokyo 194 and the
- ¶ Department of Molecular Cell Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba Aramaki, Aoba-ku, Sendai, Miyagi 980-77, Japan
- § To whom correspondence should be addressed. Tel.: 81-427-24-6261; Fax: 81-427-24-6317.
Abstract
In mammalian cells, two isoforms of DNA topoisomerase II (topo II), topo IIα and topo IIβ, are phosphorylated. The phosphorylation of topo IIβ changes its apparent molecular mass determined by SDS-polyacrylamide gel electrophoresis from 180 to 190 kDa in mitotic cells, whereas topo IIα 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 IIβ were removed within 4 h after release from mitotic block. On the other hand, 35% of topo IIα 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 IIβ was extracted at much lower concentrations of NaCl than topo IIα.
Footnotes
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↵* This work is supported in part by Grants-in-Aid for Scientific Research on Priority Areas from the Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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↵1 The abbreviations used are:
- topo II
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topoisomerase II
- log phase cell
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logarithmically growing cell
- PAGE
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polyacrylamide gel electrophoresis
- PAP
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potato acid phosphatase
- TBS
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Tris-buffered saline.
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- Received November 13, 1995.
- Revision received April 3, 1996.
- © 1996 by The American Society for Biochemistry and Molecular Biology, Inc.
