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• W2023961264 abstract "Article1 June 2003free access A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein Nicolas Foray Nicolas Foray Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK RSRM Team, ID17, Synchrotron Radiation and Medical Research, BP 220, 38043 Grenoble, France Search for more papers by this author Didier Marot Didier Marot Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Anastasia Gabriel Anastasia Gabriel Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Voahangy Randrianarison Voahangy Randrianarison Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Antony M. Carr Antony M. Carr Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK Search for more papers by this author Michel Perricaudet Michel Perricaudet Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Alan Ashworth Alan Ashworth Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Penny Jeggo Corresponding Author Penny Jeggo Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK Search for more papers by this author Nicolas Foray Nicolas Foray Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK RSRM Team, ID17, Synchrotron Radiation and Medical Research, BP 220, 38043 Grenoble, France Search for more papers by this author Didier Marot Didier Marot Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Anastasia Gabriel Anastasia Gabriel Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Voahangy Randrianarison Voahangy Randrianarison Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Antony M. Carr Antony M. Carr Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK Search for more papers by this author Michel Perricaudet Michel Perricaudet Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France Search for more papers by this author Alan Ashworth Alan Ashworth Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK Search for more papers by this author Penny Jeggo Corresponding Author Penny Jeggo Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK Search for more papers by this author Author Information Nicolas Foray1,2, Didier Marot3, Anastasia Gabriel4, Voahangy Randrianarison3, Antony M. Carr1, Michel Perricaudet3, Alan Ashworth4 and Penny Jeggo 1 1Genome Damage and Stability Centre, University of Sussex, Falmer, Brighton, BN1 9RQ UK 2RSRM Team, ID17, Synchrotron Radiation and Medical Research, BP 220, 38043 Grenoble, France 3Vectorologie et Transfert de Gènes, CNRS UMR 1582, Institut Gustave-Roussy, 94805 Villejuif, France 4Breakthrough Breast Cancer Research Centre, Institute of Cancer Research, Fulham Road, London, SW3 6JB UK *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:2860-2871https://doi.org/10.1093/emboj/cdg274 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info BRCA1 is a central component of the DNA damage response mechanism and defects in BRCA1 confer sensitivity to a broad range of DNA damaging agents. BRCA1 is required for homologous recombination and DNA damage-induced S and G2/M phase arrest. We show here that BRCA1 is required for ATM- and ATR-dependent phosphorylation of p53, c-Jun, Nbs1 and Chk2 following exposure to ionizing or ultraviolet radiation, respectively, and is also required for ATM phosphorylation of CtIP. In contrast, DNA damage-induced phosphorylation of the histone variant H2AX is independent of BRCA1. We also show that the presence of BRCA1 is dispensable for DNA damage-induced phosphorylation of Rad9, Hus1 and Rad17, and for the relocalization of Rad9 and Hus1. We propose that BRCA1 facilitates the ability of ATM and ATR to phosphorylate downstream substrates that directly influence cell cycle checkpoint arrest and apoptosis, but that BRCA1 is dispensable for the phosphorylation of DNA-associated ATM and ATR substrates. Introduction DNA damage induced by ionizing (IR) and ultraviolet (UV) irradiation or caused by abnormal structures, such as stalled replication forks, triggers a complex cascade of phosphorylation events that ultimately serve to influence or effect DNA repair, cell cycle delay and apoptosis with the overall purpose of maintaining genome stability. Two members of the phosphatidylinositol 3-kinase-related kinase (PIKK) family, ATM and ATR, play a central role in damage recognition and the initial phosphorylation events (for reviews, see Tibbetts et al., 2000; Zhou and Elledge, 2000; Khanna et al., 2001; Rouse and Jackson, 2002). These kinases are activated by different forms of DNA damage: ATM responds to DNA double-strand breaks (DSBs), whereas ATR functions following exposure to other forms of DNA damage such as bulky lesions or stalled replication forks (O'Connell et al., 2000; Khanna et al., 2001; Shiloh, 2001). Mutation of the ATM gene causes ataxia–telangiectasia (A-T), an autosomal recessive disorder associated with clinical radiosensitivity and cancer predisposition (Savitsky et al., 1995). Cell lines derived from A-T patients display severe radiosensitivity and defects in G1/S, S and G2/M phase checkpoint arrest following exposure to IR (Lavin and Khanna, 1999; Khanna et al., 2001). ATM phosphorylates multiple substrates including RPA, BRCA1, Nbs1, CtIP, Chk1, Chk2, c-Jun, p53, MdM2, H2AX, Rad9 and Rad17 (for reviews, see Lavin and Khanna, 1999; Khanna et al., 2001; Shiloh, 2001). Importantly, ATM is not activated in response to UV irradiation and A-T cell lines are neither UV sensitive nor impaired in UV-induced DNA damage checkpoints (Khanna et al., 2001). In contrast to ATM, ATR is an essential gene required for cell proliferation. When deleted in mice, it causes early embryonic lethality (Brown and Baltimore, 2000; de Klein et al., 2000; Cortez et al., 2001). Since ATR null cell lines are not available, the study of ATR function has relied on either the overexpression of a dominant-negative construct or cre-lox-mediated gene loss. These studies demonstrate that ATR-dependent phosphorylation events occur after exposure to various forms of DNA damage and replication blocks, but not following exposure to IR (Tibbetts et al., 2000; Zhou and Elledge, 2000; Cortez et al., 2001). The yeast homologues of ATM are known as Tel1 in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. ATR is the sequence and functional homologue of S.pombe Rad3 and S.cerevisiae Mec1 (Carr, 2000). In both yeasts, Tel1 plays only a minor role in signalling the presence of DSBs, while Mec1- or Rad3-dependent pathways respond to all forms of DNA damage. Thus, S.cerevisiae Mec1 or S.pombe Rad3 mutants show sensitivity to IR, UV and hydroxyurea (HU) (Paciotti et al., 2001). Notwithstanding the difference between yeasts and mammalian cells, studies with the lower organisms have been highly informative in investigations of signalling responses to DNA damage and have directed studies with mammalian cells. Using information from the yeasts as a model system, recent work has shown that ATR functions in cooperation with ATR-interacting protein (ATRIP) to bind to the sites of DNA damage (Cortez et al., 2001). Furthermore, Rad17 (an RFC-like protein) binds chromatin independently at damage sites and forms a complex with the PCNA-like proteins Rad1, Rad9 and Hus1 (Rauen et al., 2000; Zou et al., 2002). ATR-dependent phosphorylation of H2AX, and most probably ATRIP (based on analogy with yeast), is not dependent on Hus1, whereas phosphorylation of the downstream ATR substrate Chk1 is dependent on this protein (Cortez et al., 2001; Ward and Chen, 2001; Zou et al., 2002). ATR-dependent substrates include Chk1, Chk2, p53 and BRCA1 (Tibbetts et al., 1999, 2000; Xu et al., 2002; Zhao and Piwnica-Worms, 2002). Of these substrates, p53 is required to effect p21-dependent G1/S checkpoint arrest. Chk1 and Chk2 kinases, which are regulated by their phosphorylation status, phosphorylate various targets including Cdc25A (which is degraded in response to DNA damage) and p53. Ultimately, the phosphorylation of cell cycle regulators prevents progression through G1/S and G2/M via the regulation of Cdk activities (Zeng et al., 1998; Mailand et al., 2000). Heterozygous germ-line mutations in BRCA1 are responsible for a subset of hereditary breast cancers, indicating that BRCA1 encodes a tumour suppressor gene. Cell lines impaired in BRCA1 display hypersensitivity to a range of DNA damaging agents including IR and UV irradiation (Venkitaraman, 2002). They also display a failure to effect both S and G2/M checkpoint arrest after DNA damage (Xu et al., 2001). BRCA1 is phosphorylated by ATM and ATR following IR and UV irradiation, respectively (Tibbetts et al., 2000; Gatei et al., 2001). While evidence suggests that BRCA1 functions in both ATM- and ATR-dependent signalling pathways, its precise role remains unclear. BRCA1 has an N-terminal ring finger motif and a C-terminal tandem BRCT domain which is thought to mediate protein–protein interactions (Brzovic et al., 2001; Williams et al., 2001). BRCA1 has been purified as part of a large protein complex known as BASC (BRCA1-associated genome surveillance complex), which contains a wide range of DNA repair and replication proteins (Wang et al., 2000). Further interactions of BRCA1 with ATM, ATR, Rad51, Rad50–Mre11–Nbs1, BLM, p53, Chk1, Chk2 and FANCD2 have been identified by co-immunoprecipitation analysis, supporting the notion that BRCA1 functions in DNA repair and/or cell cycle checkpoint arrest (Scully et al., 1997; Zhong et al., 1999; Lee et al., 2000; Tibbetts et al., 2000; Wang et al., 2000; Garcia-Higuera et al., 2001; Yarden et al., 2002). Additional interactions with hSNF/SW1, BACH1, CtIP and COBRA1 suggest a possible additional role in the maintenance of chromatin topology (Deng and Brodie, 2000; Cantor et al., 2001; Ye et al., 2001). Crb2, a S.pombe protein that shares sequence similarity with BRCA1, is phosphorylated in a Rad3-dependent manner after exposure to IR and is required for Rad3-dependent phosphorylation of Chk1 (Esashi and Yanagida, 1999). Furthermore, S.cerevisiae ScRad9p, the homologue of Crb2, is required for the phosphorylation of SpChk2 (Brondello et al., 1999). Based on these yeast studies, and considering the fact that many of the proteins that bind to BRCA1 are ATM or ATR substrates, we investigated whether BRCA1 is required for the phosphorylation of ATM and ATR substrates after IR and UV treatment, respectively. In an intriguing similarity with the yeast models, our data indicate that BRCA1 is required for downstream ATM- and ATR-dependent phosphorylation events including the phosphorylation of p53, c-Jun, Nbs1, CtIP and Chk2. In contrast, BRCA1 is not required for activation of ATM kinase activity or for phosphorylation of the DNA-associated substrates H2AX, Rad9, Hus1 or Rad17. Our results suggest a model in which BRCA1 functions as a scaffold for the two PIKKs, ATM and ATR, to pass on their phosphorylation to downstream components required for apoptosis and checkpoint activation. Results BRCA1 is required for multiple IR-induced phosphorylation events To determine whether BRCA1 is required for ATM-dependent phosphorylation events, we examined a BRCA1-mutated cell line, HCC1937, and derivatives obtained following infection with adenovirus alone (HCCAdco) or with adenovirus containing wild-type BRCA1 (HCCAdB1) for their ability to phosphorylate p53, c-Jun, Nbs1, CtIP and Chk2 following exposure to IR. The HCC1937 and HCCAdco cells express very low levels of a mutant BRCA1 protein. The HCCAdB1 line expresses similar levels of BRCA1 to the tumour cell line 293T, which is ∼5-fold higher than the levels seen in the transformed 1BRneo line (HeLa cells also show similar levels of BRCA1 to 293T cells) (Figure 1A). The HCCAdB1 cells are substantially corrected for the radiosensitive phenotype of HCC1937 cells observed using a clonogenic survival assay (Figure 1B) and the analysis of radiation-induced micronuclei, consistent with previous findings (Foray et al., 1999) (Figure 1C). Untreated HCC1937 cells also show enhanced levels of micronuclei relative to either 1BR3neo or 293T cells, consistent with their reported genomic instability, and this also appears to be reduced in HCCAdB1 cells (Figure 1C). To ascertain the requirement for BRCA1 in ATM-dependent phosphorylation events, we used phosphospecific antibodies to examine the phosphorylation status of p53 (anti-p53Ser15), c-Jun (anti-c-junSer63), Nbs1 (anti-Nbs1Ser343) and Chk2 (anti-Chk2thr68), and standard immunoblotting to examine CtIP since the phosphorylated protein has an altered mobility on SDS–PAGE. Following exposure to IR (20 Gy), the level of phosphorylated product was increased in control cells (1BRneo and 293T) for all the proteins examined. Phosphoprotein was normally evident 30 min post-irradiation and increased over a 4 h post-irradiation period (Figure 2A). However, phosphorylation of each protein was either decreased or absent in the A-T cell line AT5BIVA and in HCC1937 (BRCA1-mutated) cells. To demonstrate that these were BRCA1-dependent phosphorylation events, we examined the phosphorylation status of each protein in HCCAdco cells and in HCC1937 cells expressing ectopic BRCA1 (HCCAdB1), and compared this with the level of phosphoprotein in 293T cells which express similar BRCA1 protein levels. The phosphorylation of each substrate occurs to similar levels and with similar kinetics in 1BRneo, 293T and HCCAdB1 cells, demonstrating that the levels of BRCA1 do not appreciably affect this aspect of the response to DNA damage (Figure 2A). It has previously been reported that p53Ser15 phosphorylation is observed at later times after irradiation in A-T-defective cell lines and that this represents an ATR-dependent event (Siliciano et al., 1997). Consistent with these observations, phosphorylation of p53Ser15 was observed in AT5BIVA cells 8 h post-irradiation (Figure 2B). In striking contrast, no phosphorylation was observed in HCC1937 cells even up to 12 h post-irradiation (Figure 2B; data not shown), indicating that BRCA1 might also be required for the delayed ATR-dependent p53 phosphorylation after IR. Figure 1.Adenovirus-infected HCC1937 cells express BRCA1 and are complemented for their radiosensitive phenotype. (A) Expression of BRCA1 protein in adenovirus-infected HCC1937 cells. Nuclear extracts from the indicated cell lines were subjected to immunoblotting using anti-BRCA1 antibody. The same extracts were also examined using anti-ATM antibody as a loading control and to verify ATM expression in BRCA1-defective cells. 1BRneo and AT5BIVA are transformed fibroblasts derived from a normal and an A-T patient, respectively. 293T is a tumour cell line. HCCAdco and HCCAdB1 are HCC1937 cells infected with empty adenovirus vector or adenovirus expressing full-length BRCA1 protein, respectively. (B) Clonogenic survival after exposure of the indicated cell lines to 2 Gy irradiation. The results shown were obtained from a single survival analysis carried out on the corrected cell lines. (C) Radiosensitivity monitored by the formation of micronuclei in the indicated cell lines. Either unirradiated cells (empty boxes) or cells irradiated with 6 Gy γ-rays (filled boxes) were scored for micronuclei. The results represent the mean of three experiments and the error bars represent the standard deviation of the mean between these experiments. Each experiment was carried out after a separate infection with adenovirus. Download figure Download PowerPoint Figure 2.Phosphorylation of p53, c-Jun, Nbs1, CtIP and Chk2 is impaired in HCC1937 cells following exposure to IR. (A) Phosphorylation of the indicated substrates was examined in the absence of irradiation and at 0.5, 1 and 4 h after exposure to 20 Gy γ-rays. Phosphospecific antibodies against p53, c-Jun, Nbs1 and Chk2 were employed. Below these samples are the non-phosphospecific antibodies used as expression controls. CtIP was examined by immunoblotting. Phosphorylation was also examined in HCC1937 cells expressing BRCA1 following adenovirus infection (HCCAdB1) and HCC1937 cells infected with empty adenovirus (HCCAdco). Phosphorylation was compared with 293T cells which express similar levels of BRCA1 to HCCAdB1 cells. The majority of immunoblots have been analysed from three independently prepared cellular extracts. (B) Phosphorylation of p53 at 8 h post-irradiation. At this later time point, marked phosphorylation was observed in AT5BIVA but not in HCC1937 cells. Similar results were also obtained 12 h post-irradiation (data not shown). Download figure Download PowerPoint To confirm these observations, we also employed immunofluorescence to examine the IR- and UV-induced phosphorylation of three proteins for which phosphospecific antibodies were utilized (p53, c-Jun, Nbs1). We did not observe significant phosphorylation of c-Jun, p53 and Nbs1 by immunofluorescence in HCC1937 cells (data not shown). We conclude that both ATM and BRCA1 are required for the IR-induced phosphorylation of these DNA damage response substrates. BRCA1 is also required for multiple UV-induced phosphorylation events Most of the substrates phosphorylated by ATM in response to IR are phosphorylated by ATR following exposure to UV. Therefore we investigated whether these ATR-dependent phosphorylation events are also BRCA1 dependent. 1BRneo control cells showed phosphorylation of all these substrates, except CtIP, following exposure to UV (Figure 3). Previous studies have also reported that CtIP is not phosphorylated following exposure to UV (Li et al., 2000). As expected, these ATR-dependent phosphorylation events were evident in AT5BIVA cells. With the exception of p53 and Chk2, none of the substrates was phosphorylated following exposure of HCC1937 or HCC1937Adco cells to UV, whereas phosphorylation was observed in control and HCC1937AdB1 cells (Figure 3A). Although phosphorylated p53 was substantially delayed in HCC1937 cells, marked phosphorylation was evident at 4 h post-irradiation. Similarly, phosphorylation of Chk2 was observed after longer times (4 h) in some experiments in HCC1937 cells (see Discussion). Immunofluorescence using the phosphospecific antibodies against p53Ser15, c-JunSer63 and Nbs1Ser343 was also performed at 4 h post-irradiation, and using all three antibodies phosphoprotein was detected in 1BRneo and AT5BIVA but not in HCC1937 cells (data not shown). Figure 3.Phosphorylation of p53, c-Jun, Nbs1 and Chk2 is impaired in HCC1937 cells following exposure to UV. Phosphorylation of the indicated substrates was examined in the absence of irradiation and at 0.5, 1 and 4 h after exposure to UV (20 J/m2). Phosphorylation was also examined in HCC1937AdB1 and HCCAdco cells, and compared with that observed in 293T cells. Details of the antibodies used are given in Figure 2. CtIP is not phosphorylated after exposure to UV irradiation. Download figure Download PowerPoint Taken together, our results show that whilst phosphorylation of a range of damage response substrates is ATM or ATR dependent after exposure to IR or UV, respectively, all the phosphorylation events examined require BRCA1. Activation of ATM kinase is not dependent upon BRCA1 A possible explanation for these findings is that BRCA1 is required for the activation of ATM kinase. We examined ATM activation in vivo using immunoprecipitation and kinase assays with 1BRneo, AT5BIVA, HCCAdco1 and HCCAdB1 cells (Figure 4A). Following immunoprecipitation with anti-ATM antibodies, a GST–p531–40 fusion peptide was used as a phosphorylation substrate. Phosphorylation was monitored using the anti-p53Ser15 antibody, a technique that gives a low background in unirradiated cells (Girard et al., 2002). Activation of ATM kinase activity is seen clearly in 1BRneo and HCC1937 cells whether or not they express BRCA1 protein. In contrast, no phosphorylation is seen using AT5BIVA cells (Figure 4A). We conclude that IR-induced activation of ATM does not require BRCA1. Figure 4.HCC1937 cells activate ATM normally and phosphorylate H2AX, Rad17, Rad9 and Hus1 after exposure to IR and UV irradiation. (A) ATM was immunoprecipitated from the indicated cell lines without exposure to IR and 4 h after exposure to 20 Gy. α-ATM indicates the level of immunoprecipitated ATM in each sample. The immunoprecipitated material was examined for ATM kinase activity using a p53 peptide. Phosphorylation of p53 was determined using the anti-p53Ser15 antibody. ATM kinase activity is activated normally independently of BRCA1 expression. (B) The phosphorylation of H2AX phosphospecific anti-p-H2AXSer139 antibody was examined 1 h after exposure of HCC1937 cells to IR (20 Gy) or UV (20 J/m2). Results are only shown for HCC1937 cells, but similar results were observed with 1BRneo and 293T cells. Results are shown at low-power magnification to show the response of multiple cells and also in individual cells. (C) The kinetics of p-H2AX foci formation at varying times after exposure of 1BR3, 293T and HCC1937 to IR (20 Gy) or UV (20 J/m2). (D) The mobility shift observed after immunoblotting of Rad9 and Hus1 was examined 4 h after exposure of 1BRneo cells to 20 J/m2 with and without treatment with λ-phosphatase (λ-PPase). (E) The phosphorylation of Rad17, Rad9 and Hus1 was examined in 1BRneo, AT5BIVA and HCC1937 cells following exposure to IR (20 Gy) and UV (20 J/m2). Phosphorylation of Rad17 was examined using anti-p-Rad17 antibody and anti-Rad17 antibodies were used as a control for Rad17 expression. Phosphorylation of Rad9 and Hus1 was examined by mobility shift after immunoblotting. Phosphorylation was observed to a similar extent in 1BRneo, AT5BIVA and HCC1937 cells. (F) Rad17 was phosphorylated efficiently in HCCAdB1 and HCCAdco cells after UV and IR. Phosphorylation of Rad17 was examined in 293T cells and cells infected with empty adenovirus or adenovirus expressing BRCA1. (G) Formation of Rad9, Hus1 and Rad17 foci occurs normally in HCC1937 cells following exposure to UV. Thirty minutes after exposure to UV (20 J/m2), cells were examined by immunofluorescence for foci using anti-Rad9, anti-Hus1 and anti-Rad17 antibodies. Foci formed to a similar level in 1BRneo and HCC1937 cells. Additionally, the Hus1 and Rad17 foci were shown to overlap in merged images. Only a single cell has been shown to enhance visualization of the foci, but foci were observed in ∼40% of the cells 30 min after UV treatment (20 J/m2). A slightly higher level of foci was observed in untreated HCC1937 cells relative to untreated 1BRneo cells, which may reflect the elevated spontaneous instability reported in these cells (data not shown). Download figure Download PowerPoint IR- and UV-induced phosphorylation of H2AX is not dependent upon BRCA1 One of the earliest substrates phosphorylated after DNA damage is a variant form of the histone H2A, known as H2AX (Burma et al., 2001; Ward and Chen, 2001). H2AX phosphorylation (formation of p-H2AX foci) by ATR occurs in a Hus1-independent manner, in contrast to the phosphorylation of other ATR-dependent substrates (Ward and Chen, 2001) Therefore we investigated whether BRCA1 is required for phosphorylation of H2AX. We examined H2AX phosphorylation using phosphospecific anti-p-H2AXSer139 antibody by immunofluorescence following exposure to IR (20 Gy) or UV (20 J/m2) in 1BRneo, 293T and HCC1937 cells. Phosphorylated H2AX was clearly observed in HCC1937 cells at 1 h post-exposure (Figure 4B). Untreated HCC1937 cells appear to represent a heterogeneous population for H2AX foci, with a small percentage of cells showing numerous foci, potentially a consequence of the high genomic instability of these cells. This heterogeneity is evident in the lower-scale image (Figure 4B). Detailed foci after damage are also shown in Figure 4B. We also examined the kinetics of appearance of foci after DNA damage. Although HCC1937 cells had a higher level of p-H2AX foci after damage compared with 1BRneo and 293T cells, the kinetics of their appearance was similar in all three cell lines (Figure 4C). Thus, in contrast to the above substrates, the phosphorylation of H2AX is BRCA1 independent. BRCA1 is dispensable for damage-induced phosphorylation of Hus1, Rad9 and Rad17 Rad17 binds to chromatin in the absence of DNA damage and is required for the recruitment of the Rad1–Rad9–Hus1 complex to the sites of DNA damage and its loading onto chromatin (Zou et al., 2002). ATR and ATM phosphorylate Rad17 after damage, but the phosphorylation of Rad17 is not required for the loading of the Rad1–Rad9–Hus1 complex (Bao et al., 2001). However, the phosphorylation of Rad17 does require Hus1, suggesting that the Rad1–Rad9–Hus1 complex recruited by Rad17 facilitates ATR-dependent phosphorylation of Rad17 (Zou et al., 2002). Furthermore, Rad9 is also phosphorylated following DNA damage and appears to be required for checkpoint activation (Kostrub et al., 1998; Chen et al., 2001). Since BRCA1 is required for ATR- and ATM-dependent phosphorylation of downstream substrates, but not for the phosphorylation of chromatin-associated H2AX, we investigated whether BRCA1 is required for the phosphorylation of Rad17, Rad9 and Hus1 following exposure to UV and IR. Phosphospecific anti-Rad17Ser635 antibody was used to examine DNA damage-induced Rad17 phosphorylation. Rad9 and Hus1 phosphorylation was examined by a change in migration following immunoblotting and SDS–PAGE analysis. Since Hus1 phosphorylation has not been reported previously in human cells and the shift in migration for Rad9 is small, we verified that these represent phosphorylated proteins by showing that the band disappears after λ-phosphatase treatment (Figure 4D). Following exposure to IR, phosphorylation of Rad17, Rad9 and Hus1 was observed in control 1BRneo cells (Figure 4E). The phosphorylation of Rad17 and Hus1 was delayed in AT5BIVA cells, consistent with previous findings (for Rad17) and the notion that ATR is activated in a delayed fashion by IR (Siliciano et al., 1997; Zou et al., 2002). In contrast, the kinetics of Rad17 phosphorylation in HCC1937 cells was similar to that observed in 1BRneo control cells (Figure 4E). The mobility shift of the Rad9 protein was small, as observed previously, but is ATM dependent after IR, as expected (Chen et al., 2001) (Figure 4C). Rad9 phosphorylation is efficient in HCC1937 cells. Similar results were obtained following exposure to UV, although in this case phosphorylation was efficient in AT5BIVA cells, consistent with the fact that this phosphorylation event is ATR dependent (Figure 4E). Hus1 phosphorylation was less marked after UV treatment in all cell lines, possibly because Hus1 does not harbour a consensus ‘S–TQ motif’ normally required for ATM/ATR-directed phosphorylation. Thus, Hus1 phosphorylation may be mediated via other serine-containing sites that are numerous in the Hus1 sequence or via an indirect ATM- and ATR-dependent phosphorylation event. Nothwithstanding this, the phosphorylation of all three proteins occurred normally in HCC1937 cells following UV treatment, as well as after IR. Finally, Rad17 phosphorylation was shown to occur to similar extents in HCCAdB1 and HCCAdco cells following exposure to both IR and UV. Taken together, these results demonstrate that the phosphorylation of Rad17, Rad9 and Hus1, like the phosphorylation of H2AX, does not require BRCA1, whereas the phosphorylation of many other substrates (p53, c-Jun, Nbs1, CtIP and Chk2) is BRCA1 dependent. Following exposure to UV, Rad9 and Hus1 are relocalized to the sites of DNA damage in a Rad17-dependent but ATR-independent manner (Zou et al., 2002). We employed immunofluorescence to examine the relocalization of Rad9, Hus1 and Rad17 following UV treatment. After UV irradiation, these three proteins could be seen to relocalize to discrete foci in control and HCC1937 cells (Figure 4F). In merged images, the Hus1 and Rad17 foci were shown to overlap. Thus, we conclude that BRCA1 does not play a major role in either the relocalization or phosphorylation of Rad17 or its ability to recruit Rad9 and Hus1 to the sites of DNA damage. Brca1 mutant embryonic stem cells are also defective in the phosphorylation of a subset of ATM/ATR targets Since HCC1937 is a tumour cell line, we sought to verify our findings using an independent system and exploited an embryonic stem (ES) cell line (Brca1−/co) with one targeted mutant Brca1 allele and one conditional allele (A.Gabriel and A.Ashworth, unpublished data). Trans fection of Cre recombinase generates a cell line homozygous for a deletion of Brca1 exons 22–24, resulting in a truncated protein of 1739 amino acids compared with the wild-type protein of 1812 amino acids, which will be designated Brca1−/−. Wild-type and B" @default.
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• W2023961264 date "2003-06-01" @default.
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• W2023961264 title "A subset of ATM- and ATR-dependent phosphorylation events requires the BRCA1 protein" @default.
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