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• W2026007913 abstract "BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53Ser-15 following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53Ser-15 efficiently. Phosphorylation of p53Ser-15 is necessary for an IR-induced G1/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53Ser-15 phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G1/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G1/S cell cycle progression after DNA damage. BRCA1 is a major player in the DNA damage response. This is evident from its loss, which causes cells to become sensitive to a wide variety of DNA damaging agents. The major BRCA1 binding partner, BARD1, is also implicated in the DNA damage response, and recent reports indicate that BRCA1 and BARD1 co-operate in this pathway. In this report, we utilized small interfering RNA to deplete BRCA1 and BARD1 to demonstrate that the BRCA1-BARD1 complex is required for ATM/ATR (ataxia-telangiectasia-mutated/ATM and Rad3-related)-mediated phosphorylation of p53Ser-15 following IR- and UV radiation-induced DNA damage. In contrast, phosphorylation of a number of other ATM/ATR targets including H2AX, Chk2, Chk1, and c-jun does not depend on the presence of BRCA1-BARD1 complexes. Moreover, prior ATM/ATR-dependent phosphorylation of BRCA1 at Ser-1423 or Ser-1524 regulates the ability of ATM/ATR to phosphorylate p53Ser-15 efficiently. Phosphorylation of p53Ser-15 is necessary for an IR-induced G1/S arrest via transcriptional induction of the cyclin-dependent kinase inhibitor p21. Consistent with these data, repressing p53Ser-15 phosphorylation by BRCA1-BARD1 depletion compromises p21 induction and the G1/S checkpoint arrest in response to IR but not UV radia-tion. These findings suggest that BRCA1-BARD1 complexes act as an adaptor to mediate ATM/ATR-directed phosphorylation of p53, influencing G1/S cell cycle progression after DNA damage. Damage to genetic material, which occurs as a consequence of exposure to environmental genotoxins or the byproducts of oxidative metabolism, represents a ubiquitous and persistent threat to genomic integrity. In eukaryotes and prokaryotes alike, the deleterious effects of genotoxic stress are countered by a robust DNA damage response pathway that monitors the genome for the presence of abnormal DNA structures, including single-stranded DNA, DNA double strand breaks, and chemically modified DNA bases. The DNA damage response can be reduced to detection, signal transduction, and effector phases, which are analogous to the signaling paradigms of growth factors and their cognate receptors. In mammals, the ATM 1The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM and Rad3-related; A-T, ataxia-telangiectasia; BrdUrd, bromodeoxyuridine; IR, ionizing radiation; UVC, ultraviolet light wave-length band C; siRNA, small interfering RNA; Gy, gray (unit of measure); GFP, green fluorescent protein; YFP, yellow fluorescent protein; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. 1The abbreviations used are: ATM, ataxia-telangiectasia-mutated; ATR, ATM and Rad3-related; A-T, ataxia-telangiectasia; BrdUrd, bromodeoxyuridine; IR, ionizing radiation; UVC, ultraviolet light wave-length band C; siRNA, small interfering RNA; Gy, gray (unit of measure); GFP, green fluorescent protein; YFP, yellow fluorescent protein; FITC, fluorescein isothiocyanate; PBS, phosphate-buffered saline. (ataxia-telangiectasia-mutated) and ATR (ATM and Rad3-related) protein kinases function as critical regulators of the cellular DNA damage response (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Google Scholar, 2Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Google Scholar). ATM and ATR are Ser/Thr-Gln-directed protein kinases with overlapping substrate specificities that are activated in response to distinct, as well as partially overlapping, types of genotoxic stimuli (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Google Scholar, 2Khanna K.K. Jackson S.P. Nat. Genet. 2001; 27: 247-254Google Scholar). Despite their structural similarity and overlapping substrate specificities, ATM and ATR are functionally nonredundant protein kinases, and this is most convincingly demonstrated by comparing their respective gene knock-out phenotypes. In humans, inactivating mutations in ATM result in the cancer predisposition/neurodegeneration syndrome ataxia-telangiectasia (A-T) (3Shiloh Y. Curr. Opin. Genet. Dev. 2001; 11: 71-77Google Scholar). Cells from A-T patients or ATM-nullizygous mice are exquisitely sensitive to ionizing radiation (IR) and other agents that induce double strand breaks and fail to activate the IR-induced G1/S or G2/M checkpoints. In addition, A-T cells exhibit radioresistant DNA synthesis, the failure to transiently down-regulate DNA replication in response to IR, which is indicative of an S-phase checkpoint defect. In contrast to ATM–/– mice, which are viable, ATR-deficient mice die early during embryogenesis, and conditional knock-out of ATR gene function in human cells leads to a loss of cellular viability (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Google Scholar). ATR mediates responses to a broad spectrum of genotoxic stimuli, including DNA replication inhibitors (e.g. hydroxyurea), UV radiation, IR, and agents such as cis-platinum that induce DNA interstrand cross-links. The checkpoint functions of ATM and ATR are mediated, in part, by a pair of checkpoint effector kinases termed Chk1 and Chk2/Cds1 (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Google Scholar). Although structurally distinct, Chk1 and Chk2 are functionally related kinases that phosphorylate an overlapping pool of cellular substrates (5O'Neill T. Giarratani L. Chen P. Iyer L. Lee C.H. Bobiak M. Kanai F. Zhou B.B. Chung J.H. Rathbun G.A. J. Biol. Chem. 2002; 277: 16102-16115Google Scholar, 6Bartek J. Falck J. Lukas J. Nat. Rev. Mol. Cell. Biol. 2001; 2: 877-886Google Scholar). Chk1 is phosphorylated on two Ser residues (Ser-317 and Ser-345) in an ATM-dependent manner following IR (7Gatei M. Sloper K. Sorensen C. Syljuasen R. Falck J. Hobson K. Savage K. Lukas J. Zhou B.B. Bartek J. Khanna K.K. J. Biol. Chem. 2003; 278: 14806-14811Google Scholar) and in an ATR-dependent manner following cellular exposure to hydroxyurea or UV light, two classic ATR stimuli. Chk2 is inducibly phosphorylated on multiple Ser/Thr residues, including a regulatory site at Thr-68, by ATM in response to double strand breaks. Substrates for Chk1 and Chk2 include members of the Cdc25 family of protein phosphatases, which are essential for S-phase and G2/M-phase cell cycle transitions (1Abraham R.T. Genes Dev. 2001; 15: 2177-2196Google Scholar). Like ATM and ATR, Chk1 and Chk2 are nonredundant protein kinases that regulate distinct and partially overlapping cellular responses to genotoxic stimuli (8Bartek J. Lukas J. Cancer Cell. 2003; 3: 421-429Google Scholar). Although essential for transmission of DNA damage signals, the ATM/ATR and Chk1/Chk2 kinases are not sufficient for checkpoint signaling to occur. Genetic studies using budding and fission yeast have identified gene products that participate in the initiation and propagation of checkpoint signals (4Zhou B.B. Elledge S.J. Nature. 2000; 408: 433-439Google Scholar, 9O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Google Scholar). A critical element of the DNA damage-signaling network is the DNA replication factor C-related protein, Rad17. Studies in yeasts and human cells indicated that Rad17 interacts genetically and biochemically with a heterotrimeric complex of proteins consisting of Rad9, Rad1, and Hus1 (Rad9 complex). The Rad9 complex is structurally, and presumably functionally, related to the PCNA sliding clamp, leading to the hypothesis that hRad17-replication factor C functions as a clamp loader for the Rad9 complex at or near sites of DNA damage (10Thelen M.P. Venclovas C. Fidelis K. Cell. 1999; 96: 769-770Google Scholar). Recently, Rad17 has been shown to be required for recruitment of Rad9 complex on damaged DNA and for ATR-dependent phosphorylation of Chk1 after IR (11Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Google Scholar). Another protein with a putative adaptor/scaffolding function is the tumor suppressor, BRCA1. BRCA1 is a phosphoprotein that is mutated in ∼50% of familial breast and ovarian cancers (12Miki Y. Swensen J. Shattuck-Eidens D. Futreal P.A. Harshman K. Tavtigian S. Liu Q. Cochran C. Bennett L.M. Ding W. Bell R. Rosenthal J. Hussey C. Tran T. McClure M. Frye C. Hattier T. Phelps R. Haugen-Strano A. Katcher H. Yakumo K. Gholami Z. Shaffer D. Stone S. Bayer S. Wray C. Bogden R. Dayananth P. Ward J. Tonin P. Narod S. Bristow P.K. Norris F.H. Helvering L. Morrison P. Rosteck P. Lai M. Barrett J.C. Lewis C. Neuhausen S. Cannon-Albright L. Goldgar D. Wiseman R. Kamb A. Skolnick M.H. Science. 1994; 266: 66-71Google Scholar). BRCA1 is phosphorylated by ATM and ATR on multiple Ser/Thr residues in response to genotoxic stimuli (13Cortez D. Wang Y. Qin J. Elledge S.J. Science. 1999; 286: 1162-1166Google Scholar, 14Gatei M. Zhou B.B. Hobson K. Scott S. Young D. Khanna K.K. J. Biol. Chem. 2001; 276: 17276-17280Google Scholar), as well as by Chk2 (15Lee J.S. Collins K.M. Brown A.L. Lee C.H. Chung J.H. Nature. 2000; 404: 201-204Google Scholar). BRCA1 contains a BRCT motif that is found in a variety of DNA repair and checkpoint proteins (16Callebaut I. Mornon J.P. FEBS Lett. 1997; 400: 25-30Google Scholar), including budding yeast ScRad9p. ScRad9p mediates interaction between the ATM/ATR ortholog ScMec1p and its downstream target, the Chk2 ortholog ScRad53p (17Gilbert C.S. Green C.M. Lowndes N.F. Mol. Cell. 2001; 8: 129-136Google Scholar). The adaptor function of Rad9 is dependent upon prior phosphorylation by ScMec1p. BRCA1 is an ATM/ATR target (14Gatei M. Zhou B.B. Hobson K. Scott S. Young D. Khanna K.K. J. Biol. Chem. 2001; 276: 17276-17280Google Scholar). Therefore, based on this analogy, it is conceivable that ATM/ATR-dependent phosphorylation of BRCA1 generates binding sites for BRCA1-associated factors, which are ATM/ATR substrates. Consistent with this theory, BRCA1 in cells exists as a part of a complex known as BASC (BRCA1-associated genome surveillance complex), which contains many of the known ATM targets including NBS1, BLM, and SMC1 (18Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Google Scholar). BRCA1 is almost always found complexed with BARD1 in vivo. Based on these studies, we investigated whether BRCA1-BARD1 complexes are required for phosphorylation of ATM/ATR targets after exposure of cells to IR and UV radiation. The present study demonstrates that BRCA1-BARD1 dimers are required for efficient phosphorylation of p53 after exposure of cells to IR and UV radiation, respectively. Furthermore, ATM and ATR-dependent phosphorylation of BRCA1 is necessary for its adaptor function. Consequently, we show that the BRCA1-BARD1 complex is required for a G1/S arrest following IR but not UV radiation. These findings highlight the role of the BRCA1-BARD1 complex as an important mediator in the DNA damage response, affecting checkpoint function. Cell Culture and Transfection—293T is a human embryonic kidney carcinoma cell line that has been transformed with the SV40 large T-antigen. U2OS is a human osteosarcoma cell line, and MCF-7 is a human breast carcinoma cell line. HeLa is a human cervical carcinoma cell line. HCC1937 is a human breast carcinoma cell line harboring a pathogenic hemizygous mutation in BRCA1 (19Tomlinson G.E. Chen T.T. Stastny V.A. Virmani A.K. Spillman M.A. Tonk V. Blum J.L. Schneider N.R. Wistuba I.I. Shay J.W. Minna J.D. Gazdar A.F. Cancer Res. 1998; 58 (I. I.): 3237-3242Google Scholar). These cells have been stably transfected with pcDNA vector, pcDNA-BRCA1wt, or pcDNA-BRCA1S1423A/S1524A to generate three cell lines as described previously (20Zhang J. Willers H. Feng Z. Ghosh J.C. Kim S. Weaver D.T. Chung J.H. Powell S.N. Xia F. Mol. Cell. Biol. 2004; 24: 708-718Google Scholar). C3ABR is a human lymphoblastoid cell line. All cells were maintained in RPMI 1640 medium supplemented with 10% fetal calf serum and grown at 37 °C in a humidified 5% CO2 atmosphere. Cells were seeded onto 15-cm2 dishes and transfected at 50–60% confluence with 15 μg of plasmid DNA by electroporation for immunoblotting. For immunofluorescence studies, a portion of the cells was taken from the above samples and cytospun onto slides prior to lysis. Alternatively, cells were seeded onto coverslips and transfected at 50–60% confluence with 1.5 μg of plasmid DNA with LipofectAMINE (Invitrogen) according to the manufacturer's instructions. For flow cytometry analysis, cells were seeded onto 6-cm2 dishes and transfected at 50–60% confluence with 5 μg of plasmid DNA with LipofectAMINE according to the manufacturer's instructions. Cells were analyzed 72 h post-transfection. When required, cells were either irradiated with a 137Cs γ-ray source or exposed to UVC. Plasmid Construction—The BRCA1 small interfering RNA (siRNA) construct was generated by annealing the following complementary oligonucleotides and inserted into the pSuper vector as a BglII/HindIII fragment: forward, 5′-G ATC CCC GAA AGT ACG AGA TTT AGT CTT CAA GAG AGA CTA AAT CTC GTA CTT TCT TTT TGG AAA-3′, and reverse, 5′-AG CTT TTC CAA AAA GAA AGT ACG AGA TTT AGT CTC TCT TGA AGA CTA AAT CTC GTA CTT TCG GG-3′. The BARD1 siRNA construct was generated in a similar manner with the following oligonucleotides: forward, 5′-G ATC CCC CAT TCT GAG AGA GCC TGT GTT CAA GAG ACA CAG GCT CTC TCA GAA TGT TTT TGG AAA-3′, and reverse, 5′-AG CTT TTC CAA AAA CAT TCT GAG AGA GCC TGT GTC TCT TGA ACA CAG GCT CTC TCA GAA TGG GG-3′. The oligonucleotides used to generate the GFP siRNA construct were as follows: forward, 5′-GAT CCC GCT GGA GTA CAA CTA CAA CTT CAA GAG AGT TGT AGT TGT ACT CCA GCT TTT GGA AA-3′, and reverse, 5′-TT TCC AAA AGC TGG AGT ACA ACT ACA ACT CTC TTG AAG TTG TAG TTG TAG TTG TAC TCC AGC GGG ATC-3′. The nucleotides in boldface indicate the regions of BRCA1, BARD1, and GFP that were targeted for silencing by siRNA. Full-length wild-type YFP-tagged BRCA1 has been described previously (21Rodriguez J.A. Henderson B.R. J. Biol. Chem. 2000; 275: 38589-38596Google Scholar). The QuikChange site-directed mutagenesis kit (Stratagene) was used according to the manufacturer's instructions to introduce alanine point mutations into the YFP-BRCA1 construct to disrupt the DNA damage-induced phosphorylation sites: Ser-1387, Ser-1423, and Ser-1524. To mutate residue serine 1387 to alanine, the following primers were used: forward, 5′-TCA GGG CTA TCC GCT CAG AGT GAC ATT-3′, and reverse, 5′-AAT GTC ACT CTG AGC GGA TAG CCC TGA-3′. To mutate residue serine 1423 to alanine, the following primers were used: forward, 5′-GAA CAG CAT GGG GCC CAG CCT TCT AAC-3′, and reverse, 5′-GTT AGA AGG CTG GGC CCC ATG CTG TTC-3′. To mutate residue serine 1524 to alanine, the following primers were used: forward, 5′-GA AAC TAC CCA GCT CAA GAG GAG CTC-3′, and reverse, 5′-GAG CTC CTC TTG AGC TGG GTA GTT TC-3′. The codons in boldface indicate the amino acids mutated to alanine. All plasmid mutations were confirmed by DNA sequencing. Immunoblotting—Cellular extracts were prepared by resuspending the cells in lysis buffer B (50 mm Tris-HCl, pH 7.4, 150 mm NaCl, 2 mm EGTA, 2 mm EDTA, 25 mm NaF, 25 mm β-glycerophosphate, 0.1 mm sodium orthovanadate, 0.1 mm phenylmethylsulfonyl fluoride, 5 μg/m1 leupeptin, 1 μg/ml aprotinin, 0.2% Triton X-100, and 0.3% Nonidet P-40) and incubating on ice for 30 min. Supernatants were collected following centrifugation at 14,000 × g for 15 min. The protein sample was then analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with the appropriate antibodies. The following primary antibodies were used: ATMSer-1981 (Rockland Diagnostic), ATM (2C-1, Genetex), p21 (Oncogene Research), Chk1Ser-317 (7Gatei M. Sloper K. Sorensen C. Syljuasen R. Falck J. Hobson K. Savage K. Lukas J. Zhou B.B. Bartek J. Khanna K.K. J. Biol. Chem. 2003; 278: 14806-14811Google Scholar), Chk1 (Santa Cruz Biotechnology), Chk2Thr-68 (Cell Signaling), Chk2 (Santa Cruz), p53Ser-15 (Cell Signaling), p53 (Novo Castra), Nbs1 (Novus), c-junSer-63 (Santa Cruz), Cdc25A (Santa Cruz), BRCA1 (Ab-1; Oncogene Research), BRCA1Ser-1387, BRCA1Ser-1423, and BRCA1Ser-1524 (14Gatei M. Zhou B.B. Hobson K. Scott S. Young D. Khanna K.K. J. Biol. Chem. 2001; 276: 17276-17280Google Scholar), BARD1 (provided by Dr. Richard Baer), and γ-tubulin (T5192; Sigma). Antibody bound to the above proteins was detected by incubation with the horseradish peroxidase-conjugated secondary antibody (Sigma). Blotted proteins were visualized using the ECL detection system (Amersham Biosciences). Markers (Bio-Rad) were used as molecular size standards. Immunofluorescence Microscopy—Cells were fixed in 4% paraformaldehyde/PBS for 15 min at room temperature, washed three times in PBS, and then permeabilized in 0.2% Triton X-100/PBS. Cells were then washed three times in PBS and blocked in 3% bovine serum albumin/PBS for 45 min before the required primary antibody was applied. BRCA1 was detected with a monoclonal antibody Ab-1 (Oncogene Research) followed by incubation with fluorescein-labeled antimouse IgG (Zymed Laboratories Inc.. The phosphospecific antibodies p53Ser-15 (Cell Signaling) and H2AXSer-139 (Upstate Biotechnology) were used to detect the phosphorylation status of p53 and H2AX, respectively. Antibody bound to p53Ser-15 or H2AXSer-139 was detected with fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Molecular Probes). Cell nuclei were counterstained with the chromosome dye Hoechst 33285 (Sigma). After extensive washing, samples were mounted onto glass slides with a drop of Vectashield anti-fade mounting reagent (Vector Laboratories). Samples were analyzed with an epifluorescence microscope (Olympus). Images were captured digitally and processed with Adobe Photoshop. Bromodeoxyuridine (BrdUrd) Incorporation Assay—Cells were mock irradiated or exposed to 6 Gy of IR or 50 J/m2 UVC. After 16 h, cells were pulsed by 20 mm BrdUrd for 30 min and then harvested by washing with PBS. Cells were fixed in 70% ethanol at –20 °C for at least 1 h. After fixation, the cells were washed with 1% fetal calf serum, PBS and then treated with 2 m HCl containing 0.5% Triton X-100 for 30 min at room temperature. The acid was neutralized by resuspending the cell pellet in 0.1 m sodium tetraborate (pH 8.5) and incubating at room temperature for 5 min. Following centrifugation, the cell pellet was resuspended in 100 μl of wash/stain buffer (1% fetal calf serum, 0.5% Tween 20, PBS) and then 10 μl of anti-BrdUrd-FITC antibody (BD Biosciences). Cells were incubated in the dark for 30 min and then washed twice in wash/stain buffer before being stained with 25 μg of propidium iodide/ml and 0.1 mg of RNase A/ml in PBS. Cellular fluorescence was measured by a BD Biosciences FACS Calibur flow cytometer. The data were analyzed using CellQuest software. BRCA1 and BARD1 Require Heterodimerization to Maintain Stability—BRCA1 predominantly is found complexed in vivo with its major binding partner, BARD1 (22Baer R. Ludwig T. Curr. Opin. Gen. Dev. 2002; 12: 86-91Google Scholar). These proteins co-localize in nuclear foci during S-phase (23Jin Y. Xu X.L. Yang M.-C.W. Wei F. Ayi T.-C. Bowcock A.M. Baer R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 12075-12080Google Scholar) and following exposure to DNA-damaging agents relocate to sites of DNA repair (24Celeste A. Fernandez-Capetillo O. Kruhlak M.J. Pilch D.R. Staudt D.W. Lee A. Bonner R.F. Bonner W.M. Nussenzweig A. Nat. Cell Biol. 2003; 5: 675-679Google Scholar). Furthermore, they co-fractionate in various DNA repair-associated nuclear complexes (25Chiba N. Parvin J.D. J. Biol. Chem. 2001; 276: 38549-38554Google Scholar), implicating the BRCA1-BARD1 heterodimer in the DNA damage response. Previous studies have demonstrated that the stability of BRCA1 is dependent on the expression of BARD1 and vice versa (26Joukov V. Chen J. Fox E.A. Green J.B. Livingston D.M. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 12078-12083Google Scholar, 27Hashizume R. Fukuda M. Maeda I. Nishikawa H. Oyake D. Yabuki Y. Ogata H. Ohta T. J. Biol. Chem. 2001; 276: 14537-14540Google Scholar). Therefore, we sought to confirm these data by using siRNA to specifically inhibit the expression of either BRCA1 or BARD1 in cells. 293T cells were transfected with siRNAs targeting BRCA1 and GFP. Immunoblotting results show that only BRCA1 siRNA specifically decreased BRCA1 expression with or without prior exposure of cells to IR (10 Gy) or UV (50 J/m2). In contrast, GFP siRNA had no apparent effect (Fig. 1A). Similar results were obtained by immunostaining (Fig. 2C). Consistent with published data, BARD1 levels were reduced in BRCA1 siRNA transfected cells compared with control transfected cells (Fig. 1A, GFP siRNA). The same phenomenon applied to BARD1 siRNA, i.e. 293T cells transfected with BARD1 siRNA not only specifically reduced BARD1 expression but also repressed BRCA1 expression, whereas GFP siRNA had no effect on either protein (Fig. 1B). These findings indicate that BRCA1-BARD1 complex formation is essential for mutual stability.Fig. 2BRCA1-BARD1 complexes mediate ATM/ATR-dependent phosphorylation of p53Ser-15 following DNA damage. A, cellular extracts were obtained from 293T cells transfected with siRNAs targeting either GFP or BRCA1 in the absence or presence of irradiation at 1 h after exposure to 10 Gy IR or 50 J/m2 UV light. The level of Cdc25A was analyzed by Western blotting. The phosphorylation of the indicated residues within the ATM/ATR substrates p53, Chk2, Chk1, and c-jun was also examined using phosphospecific antibodies. Chk1 phosphorylation was also assessed in C3ABR lymphoblastoid cells with (+) and without (–) prior exposure to ionizing radiation (10 Gy). B, 293T cells were transfected with either GFP or BARD1 siRNAs. At 72 h post-transfection, cells were treated with 10 Gy IR or 50 J/m2 UV light and were then incubated for 1 h. Cellular extracts were obtained and immunoblotted for p53 and Chk2 phosphorylation using phosphospecific antibodies against the indicated residues. The expression of Cdc25A was also assessed by immunoblotting. C, 293T cells were transfected and exposed to DNA-damaging agents as described in A. At 72 h post-transfection cells were immunostained for BRCA1 levels using an anti-BRCA1 antibody and for the phosphorylation status of p53 using the p53Ser-15 phosphospecific antibody. Microscopy images demonstrate that p53Ser-15 phosphorylation is repressed in cells lacking BRCA1 expression. D, 293T cells were transfected and treated as described in A and then immunostained with a H2AX phosphospecific antibody (H2AXSer-139). In contrast to the effect of p53Ser-15 phosphorylation, DNA damage-induced phosphorylation of H2AXSer-139 was not affected by the loss of BRCA1 expression.View Large Image Figure ViewerDownload (PPT) ATM is the primary signal transducer in response to DNA double strand breaks caused by exposure to IR. A recent paper illustrated that ATM is activated by autophosphorylation on Ser-1981 in response to IR (28Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Google Scholar). Therefore, to confirm that BRCA1-BARD1 complexes do not disrupt ATM activity directly or indirectly, we immunoblotted GFP, BRCA1, and BARD1 siRNA cellular extracts with a Ser-1981 phosphospecific antibody as a marker of ATM activity in cells. As expected, ATMSer-1981 phosphorylation was markedly increased following exposure to IR, but not UV radiation, in control transfected cells (Fig. 1, GFP siRNA). Importantly, phosphorylation of ATMSer-1981 was not decreased by the depletion of BRCA1 and BARD1 in cells transfected with siRNAs targeting these proteins (see Fig. 1). In contrast, IR-induced phosphorylation of ATM at Ser-1981 was slightly increased in BRCA1 and BARD1 siRNA-transfected cells relative to control cells (GFP siRNA), indicating that BRCA1-BARD1 complexes may set a threshold for ATM autophosphorylation in response to IR. These findings indicate that BRCA1-BARD1 complexes are not required for the activation of ATM. BRCA1-BARD1 Complexes Are Required for ATM/ATR-dependent Phosphorylation of p53Ser-15—Previous work from our laboratory and those of others (14Gatei M. Zhou B.B. Hobson K. Scott S. Young D. Khanna K.K. J. Biol. Chem. 2001; 276: 17276-17280Google Scholar, 29Tibbetts R.S. Cortez D. Brumbaugh K.M. Scully R. Livingston D. Elledge S.J. Abraham R.T. Genes Dev. 2000; 14: 2989-3002Google Scholar) has shown that although ATM and ATR phosphorylate the same set of targets in vitro, functionally they are nonredundant kinases in vivo. Rapid IR-induced phosphorylation of downstream targets are catalyzed by ATM in vivo, whereas ATR mediates rapid UVC-induced phosphorylation events. BRCA1 has been shown to bind many ATM/ATR targets as a component of BASC (BRCA1-associated genome surveillance complex) (18Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Google Scholar). To address whether the BRCA1-BARD1 heterodimer is required for the phosphorylation of ATM/ATR targets, we used siRNA to specifically inhibit the expression of BRCA1 and BARD1 in cells. By immunoblotting we examined the phosphorylation status of p53 (anti-p53Ser-15), Chk2 (anti-Chk2Thr-68), and c-jun (anti-c-JunSer-63) in BRCA1, BARD1, and GFP siRNA-transfected cells before and after exposure to IR (10 Gy) and UV radiation (50 J/m2). In cells transfected with GFP siRNA, phosphorylation of p53 and c-jun increased following exposure to IR and UV radiation, whereas phosphorylation of Chk2 only increased after IR (Fig. 2A). Interestingly, the IR- and UV-induced increase in p53 phosphorylation was significantly less in BRCA1 and BARD1 siRNA-transfected cells after DNA damage (Fig. 2, A and B). Similar results were obtained by immunostaining (Fig. 2C). In contrast, Chk2 and c-jun phosphorylation was not affected by the loss of BRCA1 and BARD1 expression (Fig. 2, A and B). The effect of Chk1 phosphorylation (anti-Chk1Ser-317) was also assessed in GFP and BRCA1 siRNA-transfected cells in the absence and presence of DNA-damaging agents. As expected, Chk1Ser-317 phosphorylation increased following exposure to UV radiation in control transfected cells (Fig. 2A, GFP siRNA). Interestingly, Chk1Ser-317 phosphorylation was not affected in cells depleted of BRCA1 (Fig. 2A). It should be noted that publications from our laboratory and others (7Gatei M. Sloper K. Sorensen C. Syljuasen R. Falck J. Hobson K. Savage K. Lukas J. Zhou B.B. Bartek J. Khanna K.K. J. Biol. Chem. 2003; 278: 14806-14811Google Scholar, 40Sorensen C.S. Syljuasen R.G. Falk J. Schroeder T. Ronnstrand L. Khanna K.K. Zouh B.B. Bartek J. Lukas J. Cancer Cell. 2003; 3: 247-258Google Scholar) have shown that phosphorylation of Chk1Ser-317 is increased following IR in a number of lymphoblastoid cell lines in an ATM-dependent manner; however, we did not observe an increase in Chk1Ser-317 phosphorylation in 293T cells following IR (Fig. 2A). In contrast, as a positive control, we demonstrate that Chk1Ser-317 phosphorylation is markedly increased 1 h post-IR in C3ABR lymphoblastoid cells (Fig. 2A). A recent publication (30Okubo E. Lehman J.M. Friedrich T.D. J. Virol. 2003; 77: 1257-1267Google Scholar) has demonstrated that SV40 transformation can interfere with Chk1 phosphorylation, which would provide an explanation as to why we did not observe an increase in this phosphorylation event in 293T cells. Cdc25A is a downstream target of the Chk1 and Chk2 kinases. Both kinases can mediate the phosphorylation of Cdc25A, stimulating its degradation and resulting in activation of the S-phase checkpoint (31Xiao Z. Chen Z. Gunasekera A.H. Sowin T.J. Rosenberg S.H. Fesik S. Zhang H. J. Biol. Chem. 2003; 278: 21767-21773Google Scholar). Therefore, Cdc25A expression was analyzed in GFP, BRCA1, and BARD1 siRNA-transfected cells. As expected, Cdc25A expression decreased in GFP siRNA-transfected cells following DNA damage compared with untreated cells. Consistent with our findings on Chk1/Chk2 phosphorylation, Cdc25A degradation was not affected by the loss of BRCA1 and BARD1 expression (Fig. 2, A and B). We conclude that BRCA1-BARD1 dimers are required for ATM/ATR-dependent phosphorylation of p53Ser-15 in response to DNA damage but are dispensable for phosphorylation of various other ATM/ATR targets. BRCA1 Is Not Required for DNA Damage-induced H2AX Phosphorylation—One of the first proteins phosphorylated by ATM after DNA double strand breaks (32Burma S. Chen B.P. Murphy M. Kurimasa A. Chen D.J. J. Biol. Chem. 2001; 276: 42462-42467Google Scholar) or by ATR after replication st" @default.
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