Department of Pharmacology, Case Comprehensive Cancer Center, Cleveland, Ohio 44106, USA.
J Biol Chem. 2012 Jul 20;287(30):25501-9. doi: 10.1074/jbc.M112.350397. Epub 2012 Jun 11.
Chk1 plays a key role in regulating the replication checkpoint and DNA damage response. Recent evidence suggests that mammalian Chk1 regulates both the nuclear and cytoplasmic checkpoint events. However, mechanisms regulating cellular mobilization of Chk1 were not well understood. Here, we report the identification of regions of human Chk1 that regulate its protein cellular localization and checkpoint function. We demonstrate that the two highly conserved motifs (CM1 and CM2) at the C terminus of Chk1 function as a nuclear export signal and nuclear localization signal, respectively. Mutating five highly conserved residues within these two motifs of Chk1 resulted in its accumulation mainly in the cytoplasm. These cytoplasmic Chk1 mutants were less stable and exhibited significantly reduced phosphorylation by DNA damage treatment, yet they retained, at least partially, checkpoint function. Using an adenovirus-mediated gene targeting technique, we attempted to create an HCT116 cell line in which endogenous Chk1 is mutated so that it is expressed exclusively in the cytoplasm. However, we failed to obtain homozygous mutant cell lines. We found that even the heterozygous mutant cell lines showed cell survival defects accompanied by spontaneous cell death. Together, these results reveal novel regulatory mechanisms that couple protein cellular localization with the checkpoint response and cell viability of Chk1.
Chk1 在调节复制检验点和 DNA 损伤反应方面起着关键作用。最近的证据表明，哺乳动物 Chk1 调节核和细胞质检验点事件。然而，调节细胞内 Chk1 动员的机制还不太清楚。在这里，我们报告了鉴定人类 Chk1 调节其蛋白细胞定位和检验点功能的区域。我们证明 Chk1 羧基末端的两个高度保守的模体（CM1 和 CM2）分别作为核输出信号和核定位信号起作用。改变这两个模体中五个高度保守的残基导致 Chk1 主要在细胞质中积累。这些细胞质 Chk1 突变体不太稳定，并且在用 DNA 损伤处理后其磷酸化显著减少，但它们至少部分保留了检验点功能。使用腺病毒介导的基因靶向技术，我们试图创建一种 HCT116 细胞系，其中内源性 Chk1 发生突变，使其仅在细胞质中表达。然而，我们未能获得纯合子突变细胞系。我们发现，即使是杂合子突变细胞系也表现出细胞存活缺陷，并伴有自发细胞死亡。总之，这些结果揭示了将蛋白细胞定位与检验点反应和 Chk1 细胞活力联系起来的新的调节机制。
