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Phosphorylation of MCM4 at Sites Inactivating DNA Helicase Activity of the MCM4-MCM6-MCM7 Complex during Epstein-Barr Virus Productive Replication

Ayumi Kudoh,¹ Tohru Daikoku,^1, Yukio Ishimi,² Yasushi Kawaguchi,³ Noriko Shirata,¹ Satoko Iwahori,¹ Hiroki Isomura,¹ and Tatsuya Tsurumi^1*

Division of Virology, Aichi Cancer Center Research Institute, 1-1, Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan,¹ Department of Materials and Biological Sciences, Faculty of Science, Ibaraki University, Mito 310-8512, Japan,² Institute of Medical Science, University of Tokyo, Shirokanedai, Minato-ku, Tokyo 108-8639, Japan³

Received 4 April 2006/ Accepted 25 July 2006

Induction of Epstein-Barr virus (EBV) lytic replication blocks chromosomal DNA replication notwithstanding an S-phase-like cellular environment with high cyclin-dependent kinase (CDK) activity. We report here that the phosphorylated form of MCM4, a subunit of the MCM complex essential for chromosomal DNA replication, increases with progression of lytic replication, Thr-19 and Thr-110 being CDK2/CDK1 targets whose phosphorylation inactivates MCM4-MCM6-MCM7 (MCM4-6-7) complex-associated DNA helicase. Expression of EBV-encoded protein kinase (EBV-PK) in HeLa cells caused phosphorylation of these sites on MCM4, leading to cell growth arrest. In vitro, the sites of MCM4 of the MCM4-6-7 hexamer were confirmed to be phosphorylated with EBV-PK, with the same loss of helicase activity as with CDK2/cyclin A. Introducing mutations in the N-terminal six Ser and Thr residues of MCM4 reduced the inhibition by CDK2/cyclin A, while EBV-PK inhibited the helicase activities of both wild-type and mutant MCM4-6-7 hexamers, probably since EBV-PK can phosphorylate MCM6 and another site(s) of MCM4 in addition to the N-terminal residues. Therefore, phosphorylation of the MCM complex by redundant actions of CDK and EBV-PK during lytic replication might provide one mechanism to block chromosomal DNA replication in the infected cells through inactivation of DNA unwinding by the MCM4-6-7 complex.

Present address: Department of Virology, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan.

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