Cell Cycle Regulation of Human CDC6 Protein

doi:10.1074/jbc.274.36.25927

Cell Cycle Regulation of Human CDC6 Protein

INTRACELLULAR LOCALIZATION, INTERACTION WITH THE HUMAN MCM COMPLEX, AND CDC2 KINASE-MEDIATED HYPERPHOSPHORYLATION*

From the ^‡Laboratories of Viral Oncology and ^¶Biochemistry, Research Institute, Aichi Cancer Center, Chikusa-ku, Nagoya 464-8681, Japan

Abstract

The binding of mammalian MCM complexes to chromatin is cell cycle-regulated and under CDC2 kinase negative control. Here, we investigated the properties of mammalian CDC6 protein, a candidate regulator of MCM. The levels of CDC6 were relatively constant during the HeLa cell cycle. In asynchronous cells, CDC6 was mainly detected in the nuclei with immunostaining, but some CDC6 was not extractable with nonionic detergent. In contrast to the chromatin-bound MCM, this fraction of CDC6 was resistant to DNase I treatment, suggesting that it binds to the detergent- and nuclease-resistant nuclear structure. In S phase cells, CDC6 became detectable in the cytoplasm with immunostaining; however, the level of the bound CDC6 was unchanged. In G₂/M phase cells, the level of the bound CDC6 was still maintained, which was hyperphosphorylated by CDC2 kinase. These data suggest that some CDC6 protein is associated with the specific nuclear structure throughout the cell cycle and that major binding sites on chromatin differ between MCM and CDC6. However, co-immunoprecipitation assays with chemical cross-linking indicated that a small part of the chromatin-bound MCM is present close to the bound CDC6.

Footnotes

↵* This work was supported in part by a Grant from the Ministry of Education, Science, Sports 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.
↵§ To whom correspondence should be addressed: Lab. of Viral Oncology, Research Inst., Aichi Cancer Center, Kanokoden 1-1, Chikusa-ku, Nagoya 464-8681, Japan. Tel./Fax: 81-52-764-2981; E-mail: mfujita@aichigw.aichi-cc.pref.aichi.jp.
↵2 Y. Tatsumi, T. Tsurimoto, K. Shirahige, H. Yoshikawa, and C. Obuse, submitted for publication.
Abbreviations:

ARS

autonomously replicating sequence

ORC

origin recognition complex

GST

glutathioneS-transferase

PAGE

polyacrylamide gel electrophoresis

DSP

dithiobis-succinimidylpropionate

hMCM

human MCM

hCDC6

human CDC6

hORC

human ORC
- Received April 1, 1999.
- Revision received June 23, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.