title: Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation.
abstract: TopBP1 and Claspin are adaptor proteins that facilitate phosphorylation of Chk1 by the ATR kinase in response to genotoxic stress. Despite their established requirement for Chk1 activation, the exact way in which TopBP1 and Claspin control Chk1 phosphorylation remains unclear. We show that TopBP1 tightly colocalizes with ATR in distinct nuclear subcompartments generated by DNA damage. Although depletion of TopBP1 by RNA interference (RNAi) strongly impaired phosphorylation of multiple ATR targets, including Chk1, Nbs1, Smc1, and H2AX, it did not interfere with ATR assembly at the sites of DNA damage. These findings challenge the current concept of ATR activation by recruitment to damaged DNA. In contrast, Claspin, like Chk1, remained distributed throughout the nucleus both before and after DNA damage. Consistently, the RNAi-mediated ablation of Claspin selectively abrogated ATR's ability to phosphorylate Chk1 but not other ATR targets. In addition, downregulation of Claspin mimicked Chk1 inactivation by inducing spontaneous DNA damage. Finally, we show that TopBP1 is required for the DNA damage-induced interaction between Claspin and Chk1. Together, these results suggest that while TopBP1 is a general regulator of ATR, Claspin operates downstream of TopBP1 to selectively regulate the Chk1-controlled branch of the genotoxic stress response.
authors.affiliation: Institute of Cancer Biology and Centre for Genotoxic Stress Research, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark.
authors.forename: Shizhou
authors.initials: S
authors.lastname: Liu
authors.forename: Simon
authors.initials: S
authors.lastname: Bekker-Jensen
authors.forename: Niels
authors.initials: N
authors.lastname: Mailand
authors.forename: Claudia
authors.initials: C
authors.lastname: Lukas
authors.forename: Jiri
authors.initials: J
authors.lastname: Bartek
authors.forename: Jiri
authors.initials: J
authors.lastname: Lukas
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D064007:Ataxia Telangiectasia Mutated Proteins; D002352:Carrier Proteins / Q000378:metabolism*; D018797:Cell Cycle Proteins / Q000378:metabolism*; D000071877:Checkpoint Kinase 1; D004249:DNA Damage / Q000235:genetics; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins; D015536:Down-Regulation / Q000235:genetics; D004789:Enzyme Activation; D006801:Humans; D008957:Models, Genetic; D009687:Nuclear Proteins; D010641:Phenotype; D010766:Phosphorylation; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D015398:Signal Transduction; D014407:Tumor Cells, Cultured
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; C110513:TOPBP1 protein, human; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases