From the
Department of Oncology, Mayo Clinic and Foundation, Rochester, Minnesota 55905 and the ¶Division of Oncology, Columbia University, New York, New York 10032
Received for publication, March 14, 2003 , and in revised form, April 15, 2003.
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ABSTRACT |
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INTRODUCTION |
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Despite this recent progress made toward 53BP1 function little is known about the initial activation of 53BP1. Recruitment of 53BP1 to -H2AX foci seems to be a crucial step. H2AX-deficient cells lack normal 53BP1 foci formation and, like 53BP1-deficent cells, manifest a G2-M checkpoint defect after exposure to low doses of ionizing radiation (12). Moreover, H2AX/ mice show a radiation sensitivity similar to 53BP1/ mice (14). In this study we mapped the region required for 53BP1 foci formation in response to DNA damage. We show that a region upstream of the BRCT motifs is sufficient for 53BP1 foci formation and that this region interacts directly with phosphorylated H2AX. Using H2AX-deficient cells retransfected with either wild-type H2AX or an H2AX phosphomutant we confirm that phosphorylation of H2AX at Ser-140 is required for 53BP1 accumulation at DNA break areas. In contrast, radiation-induced phosphorylation of 53BP1 by ATM is not essential for the recruitment of 53BP1 to foci and occurs independently.
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EXPERIMENTAL PROCEDURES |
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AntibodiesAnti-S6P, anti-S25P/29P, and anti-S784P specific antibodies were generated by coupling synthetic 53BP1 peptides (S6P, CDPTG(P)SQLD; S25P/29P, CIED(P)SQPE(P)SQVLEDD; S784P, CSD(P)SQSWEDI where (P)S represents phosphoserine) to KLH using Imject maleimide-activated mcKLH (Pierce) prior to immunizing rabbits (Cocalico Biological). The antibodies were affinity-purified on agarose columns coupled with the non-phosphorylated or phosphorylated peptide (SulfoLink Coupling Gel, Pierce). Anti-53BP1 and anti--H2AX antibodies were generated as described previously (3). Monoclonal antibody HA11 specific for HA was purchased from BabCO Berkeley Antibody Co. Anti-ATM antibody Ab3 was purchased from Oncogene Research Products.
Immunofluorescence Staining, Immunoblots, and ImmunoprecipitationCells grown on coverslips were fixed with 3% paraformaldehyde 1 h after exposure to 0 or 1 Gy of IR. After permeabilization with 0.5% Triton X-100, cells were blocked with 5% goat serum and incubated successively with the primary and secondary antibodies, each for 25 min at 37 °C. In case of DNase or RNase treatment, cells were irradiated, permeabilized with 0.5% Triton X-100 for 3 min, and incubated with either DNase I (10 units/ml) or RNase A (50 µg/ml) in phosphate-buffered saline plus calcium and magnesium for 30 min at 37 °C prior to fixation with 3% paraformaldehyde. Immunoprecipitation and immunoblot assays were done as described previously (3).
ATM Kinase AssaysATM was precipitated from K562 cells, and aliquots of the ATM-protein A-Sepharose immunocomplex were resuspended in 25 µl of kinase buffer (10 mM Hepes (pH 7.4), 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM dithiothreitol, 10 nM ATP). ATM kinase reactions were carried out at 30 °C for 20 min with 10 µCi of [-32P]ATP and 1 µg of 53BP1 GST fusion proteins.
GST Pull-down AssaysGST pull-down experiments were performed by incubating 3 µg of various GST-labeled 53BP1 fragments with C-terminal H2AX peptide that was either phosphorylated or unphosphorylated at Ser-140 (CKATQA(P)SQEY) and had been immobilized on SulfoLink Coupling Gel (Pierce). Bound GST proteins were isolated by incubating the mixture for 1 h at 4 °C in 200 µl of NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris (pH 8), 0.5% Nonidet P-40), washing five times with NETN, eluting the proteins with 2x Laemmli buffer, separating them by SDS-PAGE, and immunoblotting with horseradish peroxidase-conjugated anti-GST (B-14, Santa Cruz Biotechnology).
Generation of 53BP1-deficient Embryonic CellsA 53BP1-deficient embryonic cell line was derived from 53BP1/ blastocysts using a standard procedure. The generation of 53BP1-deficient mice is described in Ref. 13.
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RESULTS |
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To determine the minimal region required for the recruitment of 53BP1 to damage-induced foci, we generated various HA-tagged 53BP1 deletion mutants and examined their distribution in transiently transfected U2OS cells (Fig. 1, B and C, and data not shown). A 3xNLS motif fused to the N terminus of 53BP1 ensured nuclear expression of the various constructs. Truncation of the 53BP1 N terminus (11052) or the BRCT domains (
17001972) did not affect 53BP1 foci formation as assessed by IF 1 h after exposure to 1 Gy of IR (Fig. 1C). However, increasing C-terminal deletions (
13051972 and
10521972) or deletion of a region upstream of the tandem BRCT motifs (
10521305) abolished 53BP1 foci formation (Fig. 2, B and C, and data not shown). Moreover a 53BP1 construct expressing residues 10521639 including the tudor domain was found to be sufficient for 53BP1 foci formation (Fig. 1, B and C) suggesting that the domain required for foci formation is contained within this region.
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53BP1 Focus Localization Region Interacts Directly with -H2AXH2AX-deficient cells show greatly reduced 53BP1 foci formation implying that H2AX is involved in the recruitment of 53BP1 to radiation-induced foci (14). H2AX becomes phosphorylated at a conserved C-terminal SQ site upon exposure of cells to ionizing radiation (18). Phosphorylation of H2AX at Ser-140 is impaired in ATM-deficient cells suggesting that this site is dominantly phosphorylated by ATM (12). To analyze whether phosphorylation of H2AX at Ser-140 is required for 53BP1 redistribution we transiently expressed wild-type H2AX or a S140A phosphomutant in H2AX-deficient ES cells and assessed 53BP1 foci formation. As shown in Fig. 2A, expression of wild-type H2AX reconstituted 53BP1 foci formation in response to IR. In marked contrast, expression of the H2AX S140A phosphomutant was insufficient to induce 53BP1 accumulation at the sites of DNA strand breaks.
We had shown earlier that phosphorylated H2AX co-immunoprecipitates with 53BP1 upon exposure of cells to DNA damage (3). To determine whether the region required for 53BP1 focus localization interacts directly with -H2AX, we used an in vitro pull-down assay. Six different 53BP1 GST fragments spanning the entire 53BP1 protein were incubated with immobilized C-terminal H2AX peptide that was either phosphorylated or non-phosphorylated at Ser-140. Only 53BP1 fragment 9561354, which overlaps with the mapped focus localization region, showed strong interaction with the phosphorylated H2AX peptide (Fig. 2B). As a control, no binding was detected to the non-phosphorylated peptide bearing identical sequence (Fig. 2B).
Since H2AX directs 53BP1 accumulation in response to DNA damage, we asked whether H2AX is also required for the kinetochore localization of 53BP1 in mitotic cells (19). As shown in Fig. 2C, 53BP1 can be readily detected at the kinetochores in H2AX-deficient mitotic cells suggesting that the kinetochore localization of 53BP1 is not mediated by phospho-H2AX.
Phosphorylation of 53BP1 Is Not Required for Foci FormationWe had previously demonstrated that 53BP1 becomes hyperphosphorylated in response to IR, and three regions at the N terminus of 53BP1 were found to be phosphorylated by ATM in vitro (3). To map the phosphorylation sites we designed a series of GST fusion peptides containing one or two ATM binding motifs (SQ or TQ sites). ATM kinase assays using these purified GST fusion proteins as substrates, and ATM kinase immunoprecipitated from either K562 lysates (containing wild-type ATM) or ATM-deficient GM03189D lysates revealed peptides aa 112, aa 1837, and aa 778791 as putative ATM substrates (Fig. 3A). To examine whether the respective SQ sites become phosphorylated in vivo, we raised polyclonal antibodies against phosphorylated Ser-6 (anti-S6P), phosphorylated Ser-25 and Ser-29 (anti-S25P/29P), and phosphorylated Ser-784 (anti-S784P). All affinity-purified antisera recognized 53BP1 in irradiated cells but not in untreated controls when assessed by immunofluorescence analysis (data not shown). In addition, anti-53BP1 S25P/29P antibodies detected 53BP1 from irradiated ATM wild-type but not ATM-deficient cells by Western blot analyses (Fig. 3B). Pretreatment with -phosphatase abolished the antibody binding further validating that anti-53BP1 S25P/29P specifically recognizes the phosphorylated form of 53BP1 (Fig. 3B).
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To test whether phosphorylation of 53BP1 is required for the recruitment of 53BP1 to sites of DNA lesions, we generated a phosphorylation-deficient mutant (53BP1 4SA) by mutating the mapped ATM target sites (Ser-6, Ser-25/Ser-29, and Ser-784) to alanines. 53BP1/ embryonic cells transfected with this phosphomutant showed normal 53BP1 foci formation in response to IR (Fig. 3C), indicating that ATM-dependent phosphorylation of 53BP1 is not required for recruitment to or retention of 53BP1 at DNA break sites.
Phosphorylation of 53BP1 might occur at the break areas. To test this possibility, we transiently transfected 53BP1-deficient embryonic cells with the HA-tagged mutant that lacks part of the 53BP1 focus localization region (10521305) and remains a diffuse nuclear localization upon exposure of cells to IR. Co-immunostaining with anti-HA and anti-53BP1 S25P/29P antibodies revealed that ATM-dependent 53BP1 phosphorylation does not require 53BP1 foci formation (Fig. 3D). Immunoprecipitation assays confirmed that 53BP1
10521305 becomes readily phosphorylated at Ser-25/Ser-29 in response to IR (Fig. 3E). These findings are consistent with a recent report describing phosphorylation of 53BP1 Ser-25 in H2AX-deficient cells (12). Taken together, these results suggest that 53BP1 focus localization and ATM-dependent phosphorylation of 53BP1 are regulated independently.
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DISCUSSION |
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53BP1 had been speculated to be involved in the DNA damage response based on its C-terminal tandem BRCT domains. This motif was first detected in the BRCA1 C terminus and has been reported to bind directly to DNA breaks (20). Surprisingly the BRCT domains of 53BP1 were found to be dispensable for 53BP1 foci formation. Instead a region upstream of the BRCT motifs proved to be essential for 53BP1 accumulation at sites of DNA strand breaks. Our data suggest that the damage-induced phosphorylation of H2AX directs 53BP1 accumulation at sites of DNA strand breaks. First, H2AX-deficient cells reconstituted with a H2AX phosphomutant failed to induce or sustain 53BP1 foci formation. Second, a 53BP1 fragment (residues 9561354) contained within the 53BP1 focus localization region interacted strongly with phosphorylated H2AX in vitro. Third, 53BP1 co-immunoprecipitates with H2AX in a DNA damage-dependent manner (3). Thus, it is likely that the DNA damage-induced phosphorylation of H2AX at Ser-140 increases the interaction between H2AX and 53BP1 and leads to the accumulation of 53BP1 at the sites of DNA breaks.
Interestingly the focus localization region we mapped includes a region required for 53BP1 kinetochore localization in mitotic cells (residues 12201601) (19). Very recently, Morales and colleagues (21) showed that the kinetochore localization region is also essential for 53BP1 foci formation in response to DNA damage suggesting that both events might be regulated in a similar fashion. However, the kinetochore localization of 53BP1 is unlikely to involve DNA lesions (19). We have shown that 53BP1 kinetochore localization appears normal in H2AX-deficient cells, suggesting that kinetochore localization of 53BP1 is not mediated by phospho-H2AX. Further fine mapping studies will be necessary to clarify whether the same 53BP1 region initiates the recruitment of 53BP1 to DNA strand breaks or the kinetochore, respectively.
Phosphorylation by the ATM kinase plays a key role in the activation of various proteins involved in the DNA damage response (for example, see Ref. 22). A recent study by Bakkenist and Kastan (23) revealed that ATM forms an inactive oligomer in unirradiated cells. Upon radiation, ATM is rapidly autophosphorylated and dissociates from the complex thereby providing other substrates access to its kinase domain. Interestingly autophoshorylation and activation of ATM seem to occur at some distance to DNA break sites, and ATM then migrates in the nucleus to phosphorylate various substrates either at the break sites or elsewhere in the nucleus (23). This model is consistent with our finding that ATM-dependent phosphorylation of 53BP1 is not restricted to sites of DNA strand breaks and can occur within the entire nucleus. However, phosphorylation of 53BP1 alone is unlikely to trigger 53BP1 activation since deletion of the ATM target sites does not affect 53BP1 relocalization. Moreover the relocalization of 53BP1 appears to be required for efficient phosphorylation of ATM substrates at the sites of DNA breaks (data not shown). We therefore speculate that the rapid recruitment of 53BP1 to DNA break sites and the retention of 53BP1 at the sites of DNA breaks by binding to phospho-H2AX is one of the key steps in the activation of 53BP1 following DNA damage.
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FOOTNOTES |
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Supported by a postdoctoral fellowship from the Department of Defense breast cancer research program.
|| Recipient of a Department of Defense breast cancer career development award. To whom correspondence should be addressed. Tel.: 507-538-1545; Fax: 507-284-3906; E-mail: Chen.junjie{at}mayo.edu.
1 The abbreviations used are: IR, ionizing radiation; -H2AX, phosphorylated histone H2AX; ATM, ataxia-telangiectasia mutated; NLS, nuclear localization sequence; HA, hemagglutinin; ES, embryonic stem; KLH, keyhole limpet hemocyanin; Gy, gray(s); GST, glutathione S-transferase; aa, amino acid(s); IF, immunofluorescence; BRCT, BRCA1 C terminus.
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ACKNOWLEDGMENTS |
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REFERENCES |
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