Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity
Phosphorylation of Mcm4 at specific sites by cyclin-dependent kinase leads to loss of Mcm4,6,7 helicase activity
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DOI：
10.1074/jbc.m104480200
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发表时间：
2001-09-14
影响因子：
4.8
通讯作者：
Komamura-Kohno, Y
中科院分区：
文献类型：
--
作者：
Ishimi, Y;Komamura-Kohno, Y
在真核生物DNA复制中起重要作用的Mcm蛋白在体内被磷酸化，周期蛋白依赖性蛋白激酶至少部分负责Mcm4的磷酸化。我们的研究小组报道了Mcm4,6,7复合体的DNA解旋酶活性在体外被Cdk2/cyclin A磷酸化后被抑制，Mcm4,6,7复合体可能参与DNA复制的起始。在这里，我们进一步研究了小鼠Mcm4、6,7复合物和周期蛋白依赖性激酶之间的相互作用，并确定了Mcm4磷酸化所需的位点。磷酸化总共需要6个丝氨酸和苏氨酸残基。Cdk2/cyclin A对Mcm4、6,7解旋酶活性的抑制作用通过在这些残基Mcm4中引入突变而得到很大程度的缓解。针对这些位点之一的抗磷苏氨酸抗体与从HeLa细胞制备的Mcm4在有丝分裂期发生反应，但不与G(1)和G(1)/S位点的抗体结合，表明该位点主要在有丝分裂期磷酸化。从有丝分裂期的HeLa细胞中纯化的Mcm4,6,7复合物与从其他时期的细胞中制备的复合物相比，显示出较低的DNA解旋酶活性。这些结果表明，Mcm4在特定位点的磷酸化导致Mcm4,6,7 DNA解旋酶活性的丧失。
Mcm proteins that play an essential role in eukaryotic DNA replication are phosphorylated in vivo, and cyclin-dependent protein kinase is at least in part responsible for the phosphorylation of Mcm4. Our group reported that the DNA helicase activity of Mcm4,6,7 complex, which may be involved in initiation of DNA replication, is inhibited following phosphorylation by Cdk2/cyclin A in vitro. Here, we further examined the interplay between mouse Mcm4,6,7 complex and cyclin-dependent kinases and determined the sites required for the phosphorylation of Mcm4. Six Ser and Thr residues, in all, were required for the phosphorylation. Inhibition of Mcm4,6,7 helicase activity by Cdk2/cyclin A was largely relieved by introducing mutations in these residues Mcm4. Anti-phosphothreonine antibodies raised against one of these sites reacted with Mcm4 prepared fro HeLa cells at mitotic phase but did not bind to those at G(1) and G(1)/S, suggesting that this site is mainly phosphorylated in the mitotic phase. Mcm4,6,7 complex purified from HeLa cells at the mitotic phase exhibited a low level of DNA helicase activity, compared with the complexes prepared from cells at other phases. These results suggest that phosphorylation of Mcm4 at specific sites leads to loss of Mcm4,6,7 DNA helicase activity.