[CANCER RESEARCH 60, 3299 –3304, June 15, 2000] Role for ATM in DNA Damage-induced Phosphorylation of BRCA1 1 Magtouf Gatei, Shaun P. Scott, Igor Filippovitch, Natasha Soronika, Martin F. Lavin, Barbara Weber, and Kum Kum Khanna 2 Queensland Institute of Medical Research [M. G., S. P. S., M. F. L., K. K. K.] and Departments of Surgery [M. F. L.] and Pathology [K. K. K.], The University of Queensland, P. O. Royal Brisbane Hospital, Brisbane 4029, Australia; Laboratory of Molecular Radiology Institute of Biophysics, Russian Federation Ministry of Health, Moscow, 123182 Russia [I. F., N. S.]; and Department of Medicine and Genetics and Cancer Center, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104 [B. W.] ABSTRACT The human genetic disorder ataxia-telangiectasia is characterized by immunodeficiency, progressive cerebellar ataxia, radiosensitivity, cell cy- cle checkpoint defects, and cancer predisposition. The gene product [atax- ia-telangiectasia mutation (ATM)] mutated in this syndrome is a compo- nent of the DNA damage detection pathway. Loss of ATM function in human and mouse cells causes defects in DNA repair and cell cycle checkpoint control and, not surprisingly, humans and mice with compro- mised ATM function are prone to cancers. An excess of breast cancer in the relatives of ataxia-telangiectasia patients has also been reported by epidemiological studies. Predisposition to breast and ovarian cancers is also observed in women with germline mutations in BRCA1, a tumor suppressor gene. BRCA1 is a nuclear protein with a cell cycle-regulated expression pattern and is hyperphosphorylated in response to DNA- damaging agents. Here we show that rapid ionizing radiation-induced in vivo phosphorylation of BRCA1 requires the presence of functional ATM protein. Furthermore, we show that ATM interacts with BRCA1, and this association is enhanced by radiation. We also demonstrate that BRCA1 is a substrate of ATM kinase in vitro and in vivo. Using phospho-specific antibodies against serines 1387, 1423, and 1457 of BRCA1, we demon- strate radiation-induced, ATM-dependent phosphorylation of BRCA1 at these sites. These findings show that BRCA1 is regulated by an ATM- dependent mechanism as a part of the cellular response to DNA damage. This interaction between ATM and BRCA1 argues in favor of the involve- ment of particular aspects of ATM function in breast cancer predisposi- tion. INTRODUCTION A-T 3 is a multisystem genetic disease characterized by progressive cerebellar degeneration, DNA damage sensitivity, cell cycle check- point defects, and cancer proneness, particularly lymphoid malignan- cies. Long before the gene was cloned, an association with breast cancer was suggested by Swift et al. (1, 2) who reported an excess of breast cancers in the relatives of A-T patients and this was supported by Easton’s analysis of several published studies (3). The gene mu- tated in this syndrome, ATM (for A-T mutated), encodes a protein containing a phosphatidylinositol-3-kinase-like domain (4). ATM is related through this domain to a family of proteins involved in cell cycle control and DNA damage recognition (5, 6). With the opportu- nity to screen breast cancer patients for ATM mutations, many studies have been initiated to determine whether A-T carriers have an in- creased susceptibility to breast cancer. These studies are providing increasing evidence that while protein-truncating mutations in ATM may not occur more frequently in breast cancer cases than in controls, allelic variants that lead to amino acid substitutions, or short in frame deletions/insertions, may be (reviewed in Ref. 7). However, this issue remains unresolved because there are still only a few published studies on the frequency of breast cancer in A-T patients and carriers with defined mutations (8). Both the phenotype observed in A-T and the homology of ATM protein to other known damage-sensitive checkpoint genes suggest a crucial role for ATM in biochemical pathways involved in the recog- nition, signaling, and repair of double strand breaks (5, 9). ATM is a proximal component in the pathways that signal cell cycle checkpoint arrest after DNA damage or incomplete replication (9). It seems likely that the ATM protein exerts multiple effects through its interaction with different effectors, phosphorylating one or more substrates in response to DNA damage to activate radiation signal transduction pathways and/or recruit proteins to sites of DNA repair (10). ATM has been shown to phosphorylate proteins such as p53, c-Abl, RPA, and PHAS-1 (11–15), and the kinase activity of ATM increases several- fold after exposure of cells to ionizing radiation. ATM is also required for efficient DNA double strand break repair, but the mechanism by which ATM regulates repair is largely unknown. Mutations in BRCA1 predispose carriers to breast and ovarian cancer (16). Recent studies have postulated a role for BRCA1 in the DNA damage response pathway at several different levels: (a) cell cycle checkpoint activation; (b) induction of apoptosis; and (c) DNA repair (17). BRCA1 is a nuclear protein with a cell cycle-regulated expression pattern and is hyperphosphorylated in response to DNA- damaging agents (18). BRCA1 localizes to discrete nuclear foci during the S phase of the cycle, and these foci also contain Rad51 (18), a protein involved in homologous recombinational processing of DNA. Importantly, after DNA damage both BRCA1 and Rad51 foci disperse and relocate to sites of DNA synthesis where DNA repair may occur. The most convincing evidence for involvement of BRCA1 in repair is based on the finding that mouse embryonic stem cells, nullizygous for BRCA1, are defective in their ability to carry out transcription coupled repair of DNA damage (19, 20). These cells are also hypersensitive to ionizing radiation and hydrogen peroxide. BRCA1 mutations in mice result in genetic instability, defective G 2 -M checkpoint control, and reduced homologous recombination (21, 22). Genetic and biochemical approaches suggest that ATM is essential for signaling the presence of DNA damage and activating cell cycle checkpoints. Here we describe a link between ATM and BRCA1 in a DNA damage response pathway and provide evidence that ATM is required for damage-induced phosphorylation of BRCA1. MATERIALS AND METHODS Lysate Preparations, Coimmunoprecipitation, and Western Blotting. Control and A-T lymphoblastoid cells were exposed to genotoxic agents (- or UV radiation) and, unless otherwise stated, harvested 1 h later. Cell extracts were prepared by lysis in universal immunoprecipitation buffer [50 mM Tris- HCl (pH 7.4), 150 mM NaCl, 2 mM EDTA, 25 mM sodium fluoride, 25 mM -glycerophosphate, 0.1 mM sodium orthovanadate, 0.1 mM PMSF, 5 g/ml leupeptin, 1 g/ml aprotinin, 0.2% Triton X-100, and 0.3% NP40]. Whole cell extract was loaded at 100 g/lane on 5% SDS-PAGE gels. To detect the BRCA1 mobility change, samples were transferred to nitrocellulose using Towbin’s buffer at 100 V for 1 h, and membranes were probed with anti- Received 1/3/00; accepted 4/19/00. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by the National Health and Medical Research Council of Australia and the Queensland Cancer Fund. 2 To whom requests for reprints should be addressed, at The Bancroft Centre, 300 Herston Road, Herston, Queensland 4029, Australia. Phone: 61-7-33620338; Fax: 61-7- 33620106; E-mail: kumkumK@qimr.edu.au. 3 The abbreviations used are: A-T, ataxia-telangiectasia; ATM kinase, ataxia-telangi- ectasia-mutated kinase; GST, glutathione-S-transferase; IR, ionizing radiation; aa, amino acid; LCL, lymphoblastoid cell line. 3299 Research. on November 28, 2021. © 2000 American Association for Cancer cancerres.aacrjournals.org Downloaded from