Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation
Chk1 phosphorylation at Ser286 and Ser301 occurs with both stalled DNA replication and damage checkpoint stimulation
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DOI：
10.1016/j.bbrc.2008.10.119
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发表时间：
2008-12-26
影响因子：
3.1
通讯作者：
Inagaki, Masaki
中科院分区：
文献类型：
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作者：
Ikegami, Yosuke;Goto, Hidemasa;Inagaki, Masaki
我们之前报道了Chk1在有丝分裂过程中被细胞周期蛋白依赖性激酶(CDK)1磷酸化，分别位于Ser286和Ser301[T.Shiromizu等人，基因细胞11(2006)477-485]。在这里，我们证明了Chk1-Ser-286和-Ser301的磷酸化也发生在羟基脲(HU)处理或紫外线(UV)照射的细胞中。然而，与有丝分裂不同的是，Chk1不仅在Ser286和Ser301处被磷酸化，而且在检查点响应中也在Ser317和Ser345处被磷酸化。用CDK抑制剂处理后，Ser286和Ser301的Chk1磷酸化水平降低，而Ser317和Ser345的Chk1磷酸化水平没有下降。FIT体外分析显示Ser286和Ser301油Chk1是CDK2的两个主要磷酸化位点。免疫沉淀分析进一步证明，在检查点反应过程中，Ser286/Ser301和Ser317/Ser345在同一Chk1分子中发生了磷酸化。此外，在Ser317和Ser345突变为Ala的Chk1(S317A/S345A)中观察到CDK2对Ser286/Ser301的磷酸化，在S286A/S301A中也观察到ATR对Ser317/Ser345的磷酸化。因此，Chk1在检查点反应中的磷酸化不仅受ATR的调节，还受CDK2的调节。(C)2008 Elsevier Inc.保留所有权利。
We previously reported Chk1 to be phosphorylated at Ser286 and Ser301 by cyclin-dependent kinase (Cdk) 1 during mitosis [T. Shiromizu et al., Genes Cells 11 (2006) 477-485]. Here, we demonstrated that Chk1-Ser-286 and -Ser301 phosphorylation also occurs in hydroxyurea (HU)-treated or ultraviolet (UV)irradiated cells. Unlike the mitosis case, however, Chk1 was phosphorylated not only at Ser286 and Ser301 but also at Ser317 and Ser345 in the checkpoint response. Treatment with Cdk inhibitors diminished Chk1 phosphorylation at Ser286 and Ser301 but not at Ser317 and Ser345 with the latter. fit vitro analyses revealed Ser286 and Ser301 oil Chk1 to serve as two major phosphorylation sites for Cdk2. Immunoprecipitation analyses further demonstrated that Ser286/Ser301 and Ser317/Ser345 phosphorylation occur in the same Chk1 molecule during the checkpoint response. In addition, Ser286/Ser301 phosphorylation by Cdk2 was observed in Chk1 Mutated to Ala at Ser317 and Ser345 (S317A/S345A), as well as Ser317/Ser345 phosphorylation by ATR was in S286A/S301A. Therefore, Chk1 phosphorylation in the checkpoint response is regulated not only by ATR but also by Cdk2. (C) 2008 Elsevier Inc. All rights reserved.