Ataxia Telangiectasia Mutated (ATM) Kinase and ATM and Rad3 Related Kinase Mediate Phosphorylation of Brca1 at Distinct and Overlapping Sites

IN VIVO ASSESSMENT USING PHOSPHO-SPECIFIC ANTIBODIES*

Magtouf GateiDagger , Bin-Bing Zhou§, Karen HobsonDagger , Shaun ScottDagger , David YoungDagger , and Kum Kum KhannaDagger ||

From the Dagger  Queensland Institute of Medical Research, P.O. Royal Brisbane Hospital, Brisbane Qld 4029, Australia, the § Department of Oncology Research, Glaxo SmithKline Beecham, King of Prussia, Pennsylvania 19406, and the  Department of Pathology, University Of Queensland, Brisbane Qld 4029, Queensland, Australia

Received for publication, December 26, 2000, and in revised form, February 12, 2001


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Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1·Mre11·Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
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DISCUSSION
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Damage to DNA, which can occur from exogenous agents such as IR,1 UV radiation, and certain chemotherapeutic drugs or from endogenously generated reactive oxygen species, poses a great threat to genomic stability. Cells respond to DNA damage by activating a complex DNA damage-response pathway that includes cell cycle arrest, transcriptional and posttranscriptional regulation of genes associated with repair, and under some circumstances, the triggering of programmed cell death. Biochemically, the DNA damage-response pathway provides a mechanism for transducing a signal from a sensor that recognizes the damage, through a transduction cascade, to a series of downstream effector molecules, which implement the appropriate response. At or close to the top of this pathway in mammalian cells are the members of the phosphoinositide kinase-related kinase (PIKK) family, which includes ATM (ataxia telangiectasia mutated) and ATR (ATM and Rad3 related). These proteins play important roles in signaling the presence of DNA damage, activating cell cycle checkpoints, and repairing DNA (1). ATM is homozygously mutated in the germline of patients with the neurodegenerative and cancer predisposition syndrome, ataxia telangiectasia (AT). Cells derived from AT patients are hypersensitive to agents that cause double strand breaks in DNA, such as IR, but retain normal resistance to UV and other damaging agents. AT cells exhibit cell cycle defects at multiple checkpoints (G1/S, intra S, and G2/M) in response to IR. ATM-deficient mice are viable but show growth retardation and a cancer predisposition. In mammals, ATM is an upstream regulator of p53, c-Abl, Chk2 (hCds1), Brca1, and nibrin in response to IR (2, 3). Less is known about ATR because no ATR mutations have been detected in human diseases. However, the overexpression of the dominant negative ATR derivative in mammalian cells sensitizes them to a range of DNA-damaging agents (4). The homozygous inactivation of ATR in mice results in the early embryonic lethality associated with the loss of cellular proliferative potential and chromosomal fragmentation leading to mitotic catastrophe, a phenotype reminiscent of the phenotype of the Brca1-null embryos (5, 6). Recent studies have provided evidence that Brca1 suppresses cancer, at least in part, by participating in the signaling of DNA damage and DNA repair (7, 8). Brca1 is hyperphosphorylated after the exposure of cells to DNA-damaging agents (9), indicating that it is a target of protein kinases activated by DNA damage. We and others have recently provided evidence that ATM and ATM-dependent kinase Chk2 mediate the phosphorylation of Brca1 after IR (10-12). ATM is required only for the rapid and immediate phosphorylation of Brca1 after IR because Brca1 is still phosphorylated in ATM-null cells, albeit with delayed kinetics (11). Furthermore, the phosphorylation of Brca1 after UV and hydroxyurea is ATM-independent (13) and probably mediated by ATR because ATM and ATR have been shown to have overlapping substrate specificity in vitro (14). Despite the apparent overlap of function between ATM and ATR, it has been clearly shown that the immediate phosphorylation of p53 in response to IR is largely ATM-dependent, whereas ATR mediates the UV-induced rapid phosphorylation of p53. Recently, evidence has been presented that ATR regulates the phosphorylation of human Chk1 in response to UV treatment (15). Therefore, this study was designed to investigate the role of ATR in UV-induced modifications of Brca1. During the preparation of this manuscript, similar studies have suggested the role of ATR in Brca1 phosphorylation (13, 16). However, using phospho-specific antibodies, our results provided further details of BRCA1 phosphorylations at various sites by ATM and ATR. We found that ATM and ATR, in addition to their shared phosphorylation sites, also phosphorylate distinctively different sites in BRCA1 in vivo.

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Constructs and Recombinant Proteins-- Epstein-Barr virus-transformed lymphoblastoid cell lines generated from normal controls and AT patients have been described previously (17). The generation and characterization of GM847 cells that express wild-type (wt) or kinase-dead (kd) ATR under the control of the bacterial tet operator have been described previously (4). Lymphoblastoid cell lines were cultured in RPMI 1640 medium supplemented with 10% fetal calf serum at 37 °C in a humidified atmosphere of 5% CO2. The SV40-transformed GM847 cells were grown in Dulbecco's modified Eagle's medium containing 10% fetal calf serum. Following transfection, cells were grown in complete medium containing G418 (Life Technologies, Inc.). The expression of wt and kd ATR was induced by incubating the cells in culture medium containing 1 mM doxycycline (Sigma) for 48 h. Cells were grown to 80% confluence and then treated with either IR or UV light. The indicated cells were irradiated (6 Gy) with a 137Cs gamma -ray source or exposed to UV-C (80 J/m2).

Antisera and Plasmid Constructs-- Phospho-specific antibodies against ATM phosphorylation sites in BRCA1 (anti-Ser-1423, anti-Ser-1457, and anti-Ser-1387) have been described previously (11). Anti-phospho-Ser-1524 rabbit antiserum was generated in this study by immunizing with keyhole limpet hemocyanin-conjugated phosphopeptide (QNRNYP-Phospho-Ser(P)-QEELIK). Bleeds were assayed by enzyme-linked immunosorbent assay, and the serum was collected on days corresponding to the peak antibody response and then affinity-purified against the same peptide. The affinity-purified polyclonal antibodies recognized the phosphorylated form of a Brca1 peptide but not the unphosphorylated form employing an enzyme-linked immunosorbent assay. Anti-BRCA1 monoclonal antibodies (Ab-1) and anti-ATR polyclonal antibodies were from Oncogene Science, Inc. (Manhasset, NY). Anti-FLAG was purchased from Sigma. A series of glutathione S-transferase (GST)-BRCA1 fusion proteins was generated and purified as described previously (11).

Immunoblotting, Co-immunoprecipitation, and Kinase Assays-- Cellular extracts were prepared by resuspending the cells in lysis buffer (50 mM Tris-HCL, pH 7.4, 150 mM NaCl, 2 mM EGTA, 2 mM EDTA, 25 mM NaF, 25 mM beta -glycerophosphate, 0.1 mM sodium orthovandate, 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. 40 µg of protein/sample was then analyzed by SDS-polyacrylamide gel electrophoresis and immunoblotting with the appropriate antibodies. For co-immunoprecipitations, protein samples were precleared with protein A beads for 1 h at 4 C. The supernatants were then incubated with the required antibody for 2 h. The immune complexes were collected with protein A- and G-Sepharose beads. The complexes were washed twice with lysis buffer and then fractionated by SDS-polyacrylamide gel electrophoresis for immunoblot analysis. For kinase assays, cells were lysed in TGN buffer (50 mM Tris-HCl, pH 7.5, 50 mM beta -glycerophosphate, 150 mM NaCl, 10% glycerol, 1% Tween 20, 1 mM NaF, 1 mM sodium orthovandate, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin, 10 µg/ml aprotinin, 5 µg/ml leupeptin, and 1 mM dithiothreitol) as described previously (18). The immunoprecipitation was carried out as described above using either anti-ATR or anti-FLAG antibodies. The immunoprecipitates were washed twice with TGN buffer, once with 100 mM Tris-HCl (pH 7.5) and 0.5 M LiCl, and twice with kinase buffer (10 mM Hepes, pH 7.5, 50 mM beta -glycerophosphate, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 5 µM ATP, and 1 mM dithiothreitol). Kinase reactions were prepared by resuspending washed beads in 30 µl of kinase buffer containing 10 µCi of [gamma -32P]ATP and 1 µg of GST-BRCA1 fusion protein. Immune complex reactions were incubated at 30 °C for 30 min and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography.

Immunofluoresence Studies-- Cells were synchronized by a double thymidine block at the G1/S transition and then released into thymidine-free medium for a synchronous passage through S phase. 2 h after release, cells were either left untreated or irradiated at 10 Gy, and 4 h after exposure, cells were fixed in methanol:acetone (1:1) at -20 °C, washed in phosphate-buffered saline, and blocked with a blocking solution (3% bovine serum albumin, 0.1% Triton in phosphate-buffered saline). For double labeling with rabbit (anti-ATR) and mouse (anti-Brca1) antibodies, cells were incubated with a mixture of antibodies for 2 h at 4 °C. Cells were then washed three times in phosphate-buffered saline and incubated with a mixture of fluorescein-labeled anti-rabbit IgG (Zymed Laboratories Inc.) and indocarbocyanine-labeled anti-mouse IgG (Zymed Laboratories Inc.) for 30 min. After extensive washing, samples were mounted onto glass slides with a drop of anti-fade mounting reagent (Vectashield, Vector Laboratories). Samples were analyzed with a confocal laser scanning microscope (Bio-Rad). Images were digitally captured and overlaid using the RGB function of Adobe Photoshop.

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INTRODUCTION
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Ultraviolet or gamma  Irradiation and Brca1 Phosphorylation-- We previously raised anti-phospho-specific antibodies against three of the ATM phosphorylation sites in Brca1 (Ser-1387, Ser-1423, and Ser-1457). In this report, polyclonal rabbit antibodies against an additional ATM phosphorylation site in Brca1 (Ser-1524) were produced, affinity-purified, and termed alpha -1524. In the present study, we monitored the phosphorylation of Brca1 in intact cells by using the phospho-specific antibodies. Control and AT cells were exposed to IR (6 Gy) or UV radiation (80 J/m2), and the cells were harvested for Western blot analysis 30 min after damage. The Brca1 protein level and phosphorylation state were then analyzed by Western blotting by using anti-Brca1 (Ab-1, Oncogene Science) or phospho-specific antibodies (alpha -1387, alpha -1423, alpha -1457, and alpha -1524) against ATM phosphorylation sites in Brca1. In agreement with our earlier study (11), Brca1 phosphorylation after IR but not after UV was ATM-dependent (Fig. 1). The alpha -1524 and alpha -1423 antibodies detected an increased level of phosphorylation of Brca1 after UV and IR. The IR-induced phosphorylation was clearly ATM-dependent because ATM-deficient cells (L3) failed to show increased phosphorylation at these sites; however, UV-C-induced phosphorylation was ATM-independent. Immunoblotting with alpha -1457 also revealed dramatically increased levels of phosphorylated Brca1 in response to UV but not in response to IR (Fig. 1). Again, this UV-C-induced increase was ATM-independent. However, previously (11) we have shown that alpha -1457 did detect phosphorylated Brca1 after IR when Brca1 was immunoprecipitated from cell lysate (1 mg) with anti-Brca1 antibody (Ab-1) followed by blotting with alpha -1457. These results suggest that only a small fraction of Brca1 is phosphorylated at this site after IR, which is below the threshold level of detection after straight Western blot. However, a significant proportion of Brca1 is phosphorylated at Ser-1457 after UV. By contrast the alpha -1387 antibodies detected an increase in Brca1 phosphorylation after IR but not after UV radiation.


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Fig. 1.   ATM is required for IR-induced phosphorylation of Brca1 but is not required to phosphorylate Brca1 following UV treatment. Synchronously growing cultures from the control lymphoblastoid cell line (C3ABR) and ATM-deficient cell line (L3) were exposed to either IR (6 Gy) or UV-C (80 J/m2) and harvested at 30 min after treatment. The proteins were analyzed by Western blotting (WB) with anti-BRCA1 (detects total pool of Brca1: phosphorylated and unphosphorylated) and anti-phospho-specific Brca1 antibodies against ATM phosphorylation sites in Brca1 (alpha -1524, alpha -1457, alpha -1423, and alpha -1387).

Role of ATR in Damage-induced Phosphorylation of Brca1-- Both ATM and ATR are members of the PIKK family and contribute to the phosphorylation and accumulation of p53 after cellular exposure to gamma  and UV radiation, respectively (19, 20). No ATR mutations have so far been documented in human disease; therefore, human cell lines that carry mutant ATR are not available. Furthermore, a homozygous knockout of ATR in mice is embryonic lethal (5, 6). Therefore, for this study we have used cells transfected with FLAG-tagged kd ATR, which has previously been shown to exert a dominant negative effect (4). Cells were induced with 1 mM doxycycline for 48 h to allow the expression of transfected cDNA prior to harvesting. Western blotting of cell lysates using anti-FLAG antibody revealed the presence of ectopic ATR protein induced with doxycycline (Fig. 2A). This protein was not detected in untransfected GM847 cells. Immunoblotting of cell lysates with the anti-ATR antibody, which recognizes both endogenous and ectopic ATR, revealed an approximately 2-3-fold increase in ATR protein expression after the cells were induced with doxycycline (Fig. 2A). The extracts from uninduced and induced cells transfected with kd ATR, with and without prior exposure to IR (6 Gy) and UV (80 J/m2), were analyzed for damage-induced modifications of Brca1 by Western blotting with alpha -Brca1 (Ab-1) and various anti-phospho-specific antibodies (alpha -1524, alpha -1457, alpha -1423, and alpha -1387) against Brca1. The results, shown in Fig. 2B, clearly demonstrate that the immediate UV-induced mobility shift of Brca1 is compromised in cells overexpressing kd ATR protein. This is reflected as the markedly reduced phosphorylation of Brca1 on serine residues 1524, 1423, and 1457 in these cells in response to UV (but not IR), suggesting thereby that rapid UV-induced modifications of Brca1 are dependent upon the presence of functional ATR kinase.


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Fig. 2.   Rapid UV-induced phosphorylation of Brca1 is dependent upon the presence of functional ATR kinase. A, inducible expression of ATR wt and kd in GM847 (GM) fibroblasts. Immunoblotting with anti-FLAG or anti-ATR antibodies was carried out in the presence (+) or absence (-) of 1 mM doxycycline (dox) for 48 h prior to harvesting the cells. WB, Western blot. B, ATR is required for the UV-induced phosphorylation of Brca1. wt or kd ATR-transfected cells were induced with 1 mM doxycycline (dox) for 48 h. After induction, the cells were treated with either IR (6 Gy) or UV-C (80 J/m2) and harvested 30 min following damage. Proteins were fractionated by SDS-polyacrylamide gel electrophoresis and immunoblotted with anti-Brca1 and anti-phospho-specific Brca1 antibodies. GM, GM847. WB, Western blot.

ATR Phosphorylates Brca1 Directly-- Recently it has been shown that Brca1 exists as a part of a complex designated Brca1-associated genome surveillance complex that includes putative DNA damage sensors such as ATM (21). Therefore, we were interested in determining whether ATR exists in complex with Brca1. To study this association, cell lysates were co-immunoprecipitated with either anti-Brca1 or anti-ATR antibodies. The immunoblot analysis of the ATR immunoprecipitates with anti-Brca1 antibody demonstrated an interaction between the two proteins in control and AT cell lines, indicating that the interaction between ATR and Brca1 is ATM-independent (Fig. 3A). Similar results were obtained when Brca1 immunoprecipitates were blotted with the ATR antibody. The specificity of this association was confirmed by immunoprecipitation with nonspecific immunoglobulin, which failed to show an ATR or Brca1 band after blotting with the respective antibodies (results not shown). We next sought to examine whether ATR and Brca1 co-localize in cells with or without the prior treatment of cells with DNA damage. Prior to DNA damage, the majority of cells stained with anti-ATR antibodies indicated a nuclear diffuse pattern of staining. However, about 15% of the cells exhibit a punctate pattern (foci) of staining in the nucleus (data not shown). The synchronization of cells in S phase by a double thymidine block caused a dramatic increase in the proportion of cells exhibiting ATR foci (Fig. 3B). In synchronized cells, ATR foci overlapped Brca1 foci. The staining pattern of ATR and Brca1 also overlapped extensively at 4 h after the treatment of cells with IR and UV (Fig. 3B), indicating that the damage-induced modification of Brca1 by ATR might contribute to changes in Brca1 subnuclear localization after DNA damage. To investigate this possibility, we sought to examine whether the phosphorylated Brca1 is a part of the Brca1 foci induced after DNA damage by using a phospho-specific antibody against Ser-1524. Most of the undamaged cells (>85%) stained negative for the alpha -Ser-1524 antibody; however, by 4 h after the treatment of cells with IR or UV, >80% of cells exhibited the presence of phosphorylated Brca1 as nuclear foci (Fig. 3B). Most of the foci observed after staining cells with the anti-Brca1 antibody (which detects both phosphorylated and nonphosphorylated Brca1) co-localized with the foci observed with the phospho-specific antibody, indicating that the phosphorylation of Brca1 at Ser-1524 may be essential for regulating the localization of Brca1.


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Fig. 3.   Direct interaction and phosphorylation of BRCA1 by ATR. A, in vivo association of ATR and Brca1. Total cell lysates were subjected to immunoprecipitation (IP) with either anti-ATR or anti-Brca1 antibody, and the immunoprecipitated proteins were analyzed by immunoblotting with anti-Brca1 and anti-ATR antibodies. WB, Western blot. B, co-localization of ATR and Brca1 in cells synchronized in S phase (top panel, middle panel). Cells were synchronized by double thymidine block and then released into the fresh medium and cultured for 2 h. The cells were then either left untreated (top) or treated with either IR (10 Gy) or UV-C (20 J/m2) (middle) in m2. 4 h after treatment, cells were fixed, permeabilized, and stained with anti-ATR polyclonal antibody (Ab-1, Oncogene Science) and anti-Brca1 monoclonal antibody (Ab-1, Oncogene Science). Foci demonstrating coincident labeling with both antibodies appear yellow in the merged picture. Bottom panel, Brca1 phosphorylated on Ser-1524 localizes to nuclear foci. Normal human fibroblasts were fixed and permeabilized at 4 h after exposure to 10 Gy. Cells were then immunostained with a phospho-specific anti-Brca1 (alpha -1524) antibody and anti-Brca1 monoclonal antibody (Ab-1) that detects both unphosphorylated and phosphorylated Brca1. Foci that stain with both antibodies appear yellow in the merged picture. C, ATR phosphorylates Brca1 directly. Asynchronously growing cells were either untreated or treated with UV-C (80 J/m2) and then harvested at 30 min. ATR was immunoprecipitated using anti-ATR antibodies and assayed with Brca1 fragments (B1F1-B1F7) as substrates. D, wt but not kd ATR phosphorylates a fragment of Brca1. Whole cell lysates from uninduced, induced (dox) plus IR-treated, or induced plus UV-treated cells were immunoprecipitated with anti-FLAG antibodies to precipitate ectopic ATR and assayed for the phosphorylation of Brca1 (B1F6). GM, GM847. WB, Western blot.

To test whether Brca1 is a direct target for the protein kinase activity of ATR, we used a series of recombinant GST-Brca1 fragments (B1F1-B1F7) covering the entire open reading frame (11). Kinase reactions were performed with purified immunoprecipitated ATR with or without prior exposure to UV. Immunoprecipitated ATR from control lymphoblastoid cell lines phosphorylated the region in Brca1, B1F6 (amino acids 1241-1530 (Fig. 3C)); the same region was previously shown to be phosphorylated by ATM (11). This activity was also observed against an additional Brca1 fusion protein not phosphorylated by ATM, B1F3 (amino acids 521-757 (Fig. 3C)). None of the other Brca1-GST fusion proteins were phosphorylated by ATR. Immunoprecipitated ATR from ATM-deficient cells showed a similar phosphorylation of fragments 3 and 6 (data not shown), indicating that the activation of ATR is independent of ATM expression in cells. Although ATR is activated in vivo after the UV-C treatment of cells (demonstrated in Fig. 2B), we were experimentally unable to show the increased activation of ATR after UV-induced DNA damage in vitro. Ectopically expressed FLAG-tagged wt ATR revealed a similar phosphorylation of Brca1 at these sites (B1F6 is shown in Fig. 3D), whereas kd ATR failed to show any activity against Brca1, thus providing a specific control for ATR kinase activity (Fig. 3D).

    DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
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The present results demonstrate that there is a qualitative difference in Brca1 phosphorylation between gamma  radiation and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after both IR and UV. However, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These different responses in the phosphorylation of Brca1 after UV and IR exposure correspond to the fact that the nature of DNA damage caused by these two treatments is distinct; IR produces single or double strand breaks followed by ligation or recombination repair, whereas UV produces cyclobutane dimers followed by excision repair. The cellular molecules that detect and repair these distinct DNA lesions are different, which could well explain why the Brca1 response is also different. For example, ATM-deficient cells are defective in the cellular responses to double strand break-inducing agents (such as IR and other radiomimetic agents) but have a normal response to UV (22). However, there is not an absolute requirement for ATM in the cellular response to double strand breaks because the stabilization and phosphorylation of p53 (19, 22) is delayed but not abrogated in ATM-deficient cells in response to IR. Recently, the ATM-related kinase ATR was shown to mediate this late phosphorylation of p53 on Ser-15 in response to IR and the early phosphorylation of p53 in response to UV (20). These distinctions in kinetics may be a consequence of dynamic changes in initiating damage during the cellular exposures to IR and may dictate the need for particular components of the damage response pathway at particular times. These experiments reveal that the primary genetic damages induced by IR may initially activate ATM and, subsequently, that ATR is required to recognize the late DNA strand breakage. Moreover, these experiments also suggest that UV radiation may up-regulate the catalytic activity of ATR efficiently rather than regulating ATM activity. Although the mechanism by which ATM and ATR kinases are selectively regulated by distinct DNA damage signals remains unclear at present, it seems reasonable to propose that distinct DNA damage detectors might act to identify different types of DNA damage and that these distinct detectors might signal to Brca1 in a different way. ATR may be used immediately after UV damage, and ATM may be used after IR to detect damage in the DNA and then add phosphate groups to Brca1.

We have shown here that ATM is involved in the immediate phosphorylation of Brca1 at multiple sites (Ser-1387, Ser-1423, and Ser-1457) after IR but not after UV, which is consistent with accumulating data that ATM is primarily involved in the signaling of double strand breaks in DNA. The finding that the overexpression of catalytically inactive ATR inhibits the UV-induced phosphorylation of Brca1 at Ser-1423, Ser-1457, and Ser-1524 strongly suggests that the phosphorylation of BRCA1 in response to UV requires the presence of functional ATR. The precise mechanism by which the ATM- and ATR-dependent phosphorylation of Brca1 regulate Brca1 function is unclear at present. It is possible that phosphorylation might lead to a change in Brca1 subnuclear localization, which changes in response to DNA damage. In this regard, we observed that the Brca1 pool in cells is phosphorylated on Ser-1524 in response to IR and UV, and this phosphorylated form localizes to nuclear foci within 2 h after DNA damage, suggesting that phosphorylation might regulate the localization of Brca1. Interestingly, ATR also forms nuclear foci in cells synchronized in S phase, and these foci co-localize with Brca1. This observation is consistent with the essential roles of ATR and BRCA1 during the normal cell cycle, possibly facilitating DNA replication and preventing mitotic entry during S phase. A majority of cells in asynchronous culture show ATR-containing nuclear foci when exposed to DNA damage (IR and UV). Because an increase in ATR kinase activity has not been observed after treatment with various DNA-damaging agents, it is possible that ATR is regulated by its location to its substrate, such as BRCA1, after DNA damage. These results are also consistent with an earlier report that demonstrated the co-localization of Brca1 and ATR onto asynapsed regions of meiosis I chromosomes (23) and could represent the primary response to early lesions, which might initiate meiotic recombination and synapsis. It is quite conceivable that the ATR·Brca1 complex might control homologous recombination and homology-directed repair in somatic cells. The fact that ATR and BRCA1 mutant mice share similar phenotypes (5) and that ATR controls BRCA1 phosphorylation suggests that ATR is a key regulator of BRCA1 function. Uncovering the biochemical details of signaling to Brca1 will be an important step toward understanding why the defects in this gene product predispose people to breast cancer.

    ACKNOWLEDGEMENTS

Special thanks are extended to Dr. S. Handeli for providing cells expressing wild-type and dominant negative ATR.

    FOOTNOTES

* This work was supported by the National Health and Medical Research Council (Australia), the Queensland Cancer Fund (Australia), and the Susan G. Komen Breast Cancer Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| Supported by a Senior Research Fellowship from the Sylvia and Charles Viertel Foundation. To whom correspondence should be addressed. Tel.: 61-7-33620338; Fax: 61-7-33620106; E-mail: kumkumK@qimr.edu.au.

Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.M011681200

    ABBREVIATIONS

The abbreviations used are: IR, ionizing radiation; ATM, ataxia telangiectasia mutated; ATR, ATM and Rad3 related; AT, ataxia telangiectasia; wt, wild type; kd, kinase dead; Gy, gray; UV-C, ultraviolet-C; GST, glutathione S-transferase; PIKK, phosphoinositide kinase-related kinase.

    REFERENCES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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