Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation

TY - JOUR

T1 - Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation

AU - Liu, Shizhou

AU - Bekker-Jensen, Simon

AU - Mailand, Niels

AU - Lukas, Claudia

AU - Bartek, Jiri

AU - Lukas, Jiri

PY - 2006

Y1 - 2006

N2 - 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.

AB - 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.

KW - Adaptor Proteins, Signal Transducing

KW - Carrier Proteins

KW - Cell Cycle Proteins

KW - DNA Damage

KW - DNA Replication

KW - DNA-Binding Proteins

KW - Down-Regulation

KW - Enzyme Activation

KW - Humans

KW - Models, Genetic

KW - Nuclear Proteins

KW - Phenotype

KW - Phosphorylation

KW - Protein Binding

KW - Protein Kinases

KW - Protein-Serine-Threonine Kinases

KW - RNA Interference

KW - Signal Transduction

KW - Tumor Cells, Cultured

U2 - 10.1128/MCB.00492-06

DO - 10.1128/MCB.00492-06

M3 - Journal article

C2 - 16880517

SN - 0270-7306

VL - 26

SP - 6056

EP - 6064

JO - Molecular and Cellular Biology

JF - Molecular and Cellular Biology

IS - 16

ER -