Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin

被引:157
作者
Masai, Hisao
Taniyama, Chika
Ogino, Keiko
Matsui, Etsuko
Kakusho, Naoko
Matsumoto, Seiji
Kim, Jung-Min
Ishii, Ai
Tanaka, Taku
Kobayashi, Toshiko
Tamai, Katsuyuki
Ohtani, Kiyoshi
Arai, Ken-ichi
机构
[1] Tokyo Metropolitan Inst Med Sci, Genome Dynam Project, Bunkyo Ku, Tokyo 1138613, Japan
[2] Ginkgo Biomed Res Inst, Tokyo 1080071, Japan
[3] Med & Biol Labs Co Ltd, Nagano 3960002, Japan
[4] Tokyo Med & Dent Univ, Human Gene Sci Ctr, Tokyo 1138510, Japan
[5] Univ Tokyo, Adv Sci & Technol Res Ctr, Tokyo 1538904, Japan
关键词
D O I
10.1074/jbc.M608935200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Cdc7 kinase, conserved from yeasts to human, plays important roles in DNA replication. However, the mechanisms by which it stimulates initiation of DNA replication remain largely unclear. We have analyzed phosphorylation of MCM subunits during cell cycle by examining mobility shift on SDS-PAGE. MCM4 on the chromatin undergoes specific phosphorylation during S phase. Cdc7 phosphorylates MCM4 in the MCM complexes as well as the MCM4 N-terminal polypeptide. Experiments with phospho-amino acid-specific antibodies indicate that the S phase-specific mobility shift is due to the phosphorylation at specific N-terminal (S/T)( S/T) P residues of the MCM4 protein. These specific phosphorylation events are not observed in mouse ES cells deficient in Cdc7 or are reduced in the cells treated with siRNA specific to Cdc7, suggesting that they are mediated by Cdc7 kinase. The N-terminal phosphorylation of MCM4 stimulates association of Cdc45 with the chromatin, suggesting that it may be an important phosphorylation event by Cdc7 for activation of replication origins. Deletion of the N-terminal non-conserved 150 amino acids of MCM4 results in growth inhibition, and addition of amino acids carrying putative Cdc7 target sequences partially restores the growth. Furthermore, combination of MCM4 N-terminal deletion with alanine substitution and deletion of the N-terminal segments of MCM2 and MCM6, respectively, which contain clusters of serine/threonine and are also likely targets of Cdc7, led to an apparent nonviable phenotype. These results are consistent with the notion that the N-terminal phosphorylation of MCM2, MCM4, and MCM6 may play functionally redundant but essential roles in initiation of DNA replication.
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页码:39249 / 39261
页数:13
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