Tibbetts R S, Cortez D, Brumbaugh K M, Scully R, Livingston D, Elledge S J, Abraham R T
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Genes Dev. 2000 Dec 1;14(23):2989-3002. doi: 10.1101/gad.851000.
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.
BRCA1基因编码一种肿瘤抑制因子，在50%的家族性乳腺癌中发生突变。BRCA1蛋白与DNA损伤反应有关，因为DNA损伤会诱导BRCA1磷酸化，并使其募集到含有DNA修复蛋白的核灶中。共济失调毛细血管扩张症突变（ATM）基因产物在γ射线照射（IR）时控制BRCA1的整体磷酸化。在本研究中，我们发现BRCA1磷酸化在响应IR时仅部分依赖ATM，而在响应紫外线、DNA复制抑制剂羟基脲（HU）和阿非迪霉素（APH）处理时不依赖ATM。我们提供的证据表明，负责这种磷酸化的激酶是ATM相关激酶ATR。ATR在体外使BRCA1的六个丝氨酸/苏氨酸残基磷酸化，包括丝氨酸1423。ATR表达增加会增强细胞暴露于HU或紫外线后BRCA1丝氨酸1423的磷酸化，而强力霉素诱导表达的激酶失活ATR突变蛋白会抑制GM847成纤维细胞中HU或紫外线诱导的丝氨酸1423磷酸化，并部分抑制响应IR时该位点的磷酸化。因此，ATR与ATM一样，在体内控制BRCA1磷酸化。虽然从DNA损伤细胞中分离出的ATR在体外未显示出增强的激酶活性，但我们发现ATR通过在停滞的复制叉位点形成不同的核灶来响应DNA损伤和复制阻滞。此外，ATR核灶与BRCA1形成的核灶重叠。ATR响应DNA损伤时的显著重新定位表明了其增强底物对DNA损伤磷酸化能力的一种可能机制。总之，这些结果表明ATR和BRCA1是同一基因毒性应激反应途径的组成部分，并且ATR在响应受损DNA或停滞的DNA复制时直接使BRCA1磷酸化。
