Cell cycle regulation of human CDC6 protein. Intracellular localization, interaction with the human mcm complex, and CDC2 kinase-mediated hyperphosphorylation.
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 G(2)/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.
Fujita
M
M
Laboratories of Viral Oncology, Research Institute, Aichi Cancer Center, Chikusa-ku, Nagoya 464-8681, Japan. mfujita@aichigw.aichi-cc.pref.aichi.jp
Yamada
C
C
Goto
H
H
Yokoyama
N
N
Kuzushima
K
K
Inagaki
M
M
Tsurumi
T
T
D002453:Cell Cycle; D018797:Cell Cycle Proteins / Q000378:metabolism*; D002843:Chromatin / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D006367:HeLa Cells; D006801:Humans; D064168:Minichromosome Maintenance Complex Component 7; D009687:Nuclear Proteins / Q000378:metabolism*; D010766:Phosphorylation; D011233:Precipitin Tests; D029701:Saccharomyces cerevisiae Proteins
C066503:CDC6 protein, S cerevisiae; D018797:Cell Cycle Proteins; D002843:Chromatin; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D029701:Saccharomyces cerevisiae Proteins; C101624:MCM7 protein, human; D064168:Minichromosome Maintenance Complex Component 7