Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication
Phosphorylation of MCM4 at sites inactivating DNA helicase activity of the MCM4-MCM6-MCM7 complex during Epstein-Barr virus productive replication
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DOI：
10.1128/jvi.00678-06
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发表时间：
2006-10-01
影响因子：
5.4
通讯作者：
Tsurumi, Tatsuya
中科院分区：
文献类型：
--
作者：
Kudoh, Ayumi;Daikoku, Tohru;Tsurumi, Tatsuya
EB病毒（EBV）裂解性复制的诱导阻断染色体DNA复制，尽管具有高细胞周期蛋白依赖性激酶（CDK）活性的S期样细胞环境。我们在这里报告，磷酸化形式的MCM 4，MCM复合物的亚基染色体DNA复制所必需的，增加裂解复制的进展，Thr-19和Thr-110的CDK 2/CDK 1的目标，其磷酸化灭活MCM 4-MCM 6-MCM 7（MCM 4 -6-7）复合物相关的DNA解旋酶。EBV编码的蛋白激酶（EBV-PK）在HeLa细胞中的表达引起MCM 4上这些位点的磷酸化，导致细胞生长停滞。在体外，证实了MCM 4 -6-7六聚体的MCM 4位点被EBV-PK磷酸化，与CDK 2/细胞周期蛋白A相同的解旋酶活性丧失。在MCM 4的N-末端6个Ser和Thr残基中引入突变降低了CDK 2/细胞周期蛋白A的抑制作用，而EBV-PK抑制了野生型和突变型MCM 4 -6-7六聚体的解旋酶活性，这可能是因为EBV-PK可以磷酸化MCM 6和MCM 4的除N-末端残基之外的另一个位点。因此，在裂解性复制过程中，CDK和EBV-PK的冗余作用导致MCM复合物磷酸化，这可能提供了一种通过MCM 4 -6-7复合物灭活DNA解旋来阻断感染细胞中染色体DNA复制的机制。
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.