Functional interactions between BRCA1 and the checkpoint kinase ATR during genotoxic stress
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Published on: November 5, 2012
R S Tibbetts1, D Cortez, K M Brumbaugh
1Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Abstract:
The BRCA1 gene encodes a tumor suppressor that is mutated in 50% of familial breast cancers. The BRCA1 protein has been implicated in the DNA damage response, as DNA damage induces the phosphorylation of BRCA1 and causes its recruitment into nuclear foci that contain DNA repair proteins. The ataxia-telangiectasia-mutated (ATM) gene product controls overall BRCA1 phosphorylation in response to gamma-irradiation (IR). In this study, we show that BRCA1 phosphorylation is only partially ATM dependent in response to IR and ATM independent in response to treatment with UV light, or the DNA replication inhibitors hydroxyurea (HU) and aphidicolin (APH). We provide evidence that the kinase responsible for this phosphorylation is the ATM-related kinase, ATR. ATR phosphorylates BRCA1 on six Ser/Thr residues, including Ser 1423, in vitro. Increased expression of ATR enhanced the phosphorylation of BRCA1 on Ser 1423 following cellular exposure to HU or UV light, whereas doxycycline-induced expression of a kinase-inactive ATR mutant protein inhibited HU- or UV light-induced Ser 1423 phosphorylation in GM847 fibroblasts, and partially suppressed the phosphorylation of this site in response to IR. Thus, ATR, like ATM, controls BRCA1 phosphorylation in vivo. Although ATR isolated from DNA-damaged cells does not show enhanced kinase activity in vitro, we found that ATR responds to DNA damage and replication blocks by forming distinct nuclear foci at the sites of stalled replication forks. Furthermore, ATR nuclear foci overlap with the nuclear foci formed by BRCA1. The dramatic relocalization of ATR in response to DNA damage points to a possible mechanism for its ability to enhance the phosphorylation of substrates in response to DNA damage. Together, these results demonstrate that ATR and BRCA1 are components of the same genotoxic stress-responsive pathway, and that ATR directly phosphorylates BRCA1 in response to damaged DNA or stalled DNA replication.
Insights
The ATM and Rad3-related (ATR) kinase directly phosphorylates the BRCA1 tumor suppressor protein in response to DNA damage and replication stress, revealing a shared genotoxic stress pathway.
Area of Science:
- Molecular Biology
- Cellular Biology
- Genetics
Background:
- The BRCA1 gene is a tumor suppressor frequently mutated in familial breast cancers.
- BRCA1 protein is crucial for DNA damage response, with phosphorylation and recruitment to nuclear foci upon DNA damage.
- The ataxia-telangiectasia-mutated (ATM) kinase regulates BRCA1 phosphorylation after gamma-irradiation (IR).
Purpose of the Study:
- To investigate the kinases responsible for BRCA1 phosphorylation beyond ATM.
- To elucidate the role of ATR in BRCA1 phosphorylation and DNA damage response.
- To determine if ATR and BRCA1 function within the same genotoxic stress pathway.
Main Methods:
- Assessed BRCA1 phosphorylation in response to IR, UV light, hydroxyurea (HU), and aphidicolin (APH).
- Investigated the role of ATR using increased ATR expression and a kinase-inactive ATR mutant.
- Examined ATR and BRCA1 localization in nuclear foci following DNA damage.
Main Results:
- BRCA1 phosphorylation is only partially ATM-dependent upon IR and ATM-independent upon UV, HU, or APH treatment.
- ATR directly phosphorylates BRCA1 on multiple residues, including Ser 1423, in vitro and in vivo.
- ATR forms distinct nuclear foci at stalled replication forks and these foci overlap with BRCA1 foci.
Conclusions:
- ATR, similar to ATM, controls BRCA1 phosphorylation in response to genotoxic stress.
- ATR directly phosphorylates BRCA1, indicating they are part of the same DNA damage response pathway.
- ATR relocalization to sites of DNA damage suggests a mechanism for regulating substrate phosphorylation.
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