Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin
Phosphorylation of MCM4 by Cdc7 kinase facilitates its interaction with Cdc45 on the chromatin
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DOI：
10.1074/jbc.m608935200
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发表时间：
2006-12-22
影响因子：
4.8
通讯作者：
Arai, Ken-ichi
中科院分区：
文献类型：
--
作者：
Masai, Hisao;Taniyama, Chika;Arai, Ken-ichi
Cdc 7激酶在DNA复制中起着重要作用，是一种从酵母到人类都很保守的激酶。然而，它刺激DNA复制起始的机制在很大程度上仍不清楚。我们已经分析了磷酸化的MCM亚基在细胞周期中，通过检查迁移率的变化在SDS-PAGE。染色质上的MCM 4在S期经历特异性磷酸化。Cdc 7磷酸化MCM复合物中的MCM 4以及MCM 4 N-末端多肽。磷酸化氨基酸特异性抗体的实验表明，S期特异性迁移率的变化是由于在特定的N-末端（S/T）（S/T）的MCM 4蛋白的P残基的磷酸化。这些特异性磷酸化事件在Cdc 7缺陷的小鼠ES细胞中没有观察到，或者在用Cdc 7特异性siRNA处理的细胞中减少，表明它们由Cdc 7激酶介导。MCM 4的N端磷酸化刺激Cdc 45与染色质的结合，这表明它可能是Cdc 7激活复制起点的重要磷酸化事件。MCM 4的N-末端非保守的150个氨基酸的缺失导致生长抑制，并且添加携带推定的Cdc 7靶序列的氨基酸部分恢复生长。此外，MCM 4 N-末端缺失与丙氨酸取代的组合以及分别缺失MCM 2和MCM 6的N-末端区段（其含有丝氨酸/苏氨酸簇并且也可能是Cdc 7的靶标）导致明显的非存活表型。这些结果是一致的概念，N-末端磷酸化的MCM 2，MCM 4，和MCM 6可能发挥功能冗余，但在启动DNA复制的重要作用。
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.