Coupling cellular localization and function of checkpoint kinase 1 (Chk1) in checkpoints and cell viability.
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.
Department of Pharmacology, Case Comprehensive Cancer Center, Cleveland, Ohio 44106, USA.
Jingna
J
Wang
Xiangzi
X
Han
Xiujing
X
Feng
Zhenghe
Z
Wang
Youwei
Y
Zhang
D021581:Active Transport, Cell Nucleus / Q000502:physiology; D020816:Amino Acid Motifs; D059447:Cell Cycle Checkpoints / Q000502:physiology*; D002467:Cell Nucleus / Q000235:genetics / Q000378:metabolism*; D002470:Cell Survival / Q000502:physiology; D000071877:Checkpoint Kinase 1; D003593:Cytoplasm / Q000235:genetics / Q000378:metabolism*; D057809:HEK293 Cells; D006367:HeLa Cells; D006579:Heterozygote; D006801:Humans; D009154:Mutation; D049790:Nuclear Export Signals / Q000502:physiology*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
D049790:Nuclear Export Signals; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1