Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition
Novel Positive Feedback Loop between Cdk1 and Chk1 in the Nucleus during G2/M Transition
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DOI：
10.1074/jbc.c109.051540
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发表时间：
2009-12-04
影响因子：
4.8
通讯作者：
Inagaki, Masaki
中科院分区：
文献类型：
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作者：
Enomoto, Masato;Goto, Hidemasa;Inagaki, Masaki
Chk 1是DNA损伤/复制检查点中的关键转换器之一，通过抑制Cdk 1活性来防止进入有丝分裂。然而，目前还不清楚这种抑制是如何在G（2）/M转换被取消。我们最近报道了Cdk 1在有丝分裂过程中对Chk 1的Ser（286）和Ser（301）进行磷酸化。在这里，我们表明，有丝分裂Chk 1磷酸化伴随着Chk 1易位从细胞核到细胞质的前期。这种易位先进的根据前期进展，并调节Crm-1依赖的核输出。外源性Chk 1的Ser（286）和Ser（301）突变为Ala（S286 A/S301 A）主要在前期细胞的细胞核中观察到，尽管在野生型Chk 1中几乎没有观察到这种核积累。S286 A/S301 A的诱导导致有丝分裂进入的延迟。使用免疫沉淀的细胞周期蛋白B-1-Cdk 1复合物的生化分析显示S286 A/S301 A表达阻断Cdk 1的充分活化。S286 A/S301 A的表达使Wee 1保持在较高水平，而Cdk 1诱导的细胞周期蛋白B-1和波形蛋白磷酸化保持在较低水平。激酶死亡的S286 A/S301 A也主要定位于细胞核中，但失去了延迟有丝分裂进入的能力。这些结果表明，Chk 1磷酸化的Cdk 1参与细胞质螯合的Chk 1活性，释放Cdk 1抑制细胞核中，促进有丝分裂进入。
Chk1, one of the critical transducers in DNA damage/replication checkpoints, prevents entry into mitosis through inhibition of Cdk1 activity. However, it has remained unclear how this inhibition is cancelled at the G(2)/M transition. We reported recently that Chk1 is phosphorylated at Ser(286) and Ser(301) by Cdk1 during mitosis. Here, we show that mitotic Chk1 phosphorylation is accompanied by Chk1 translocation from the nucleus to the cytoplasm in prophase. This translocation advanced in accordance with prophase progression and was regulated by Crm-1-dependent nuclear export. Exogenous Chk1 mutated at Ser(286) and Ser(301) to Ala (S286A/S301A) was observed mainly in the nuclei of prophase cells, although such nuclear accumulation was hardly observed in wild-type Chk1. Induction of S286A/S301A resulted in the delay of mitotic entry. Biochemical analyses using immunoprecipitated cyclin B-1-Cdk1 complexes revealed S286A/S301A expression to block the adequate activation of Cdk1. In support of this, S286A/S301A expression retained Wee1 at higher levels and Cdk1-induced phosphorylation of cyclin B-1 and vimentin at lower levels. A kinase-dead version of S286A/S301A also localized predominantly in the nucleus but lost the ability to delay mitotic entry. These results indicate that Chk1 phosphorylation by Cdk1 participates in cytoplasmic sequestration of Chk1 activity, which releases Cdk1 inhibition in the nucleus and promotes mitotic entry.