Correspondence to: Junjie Chen, Guggenheim 1306, Division of Oncology Research, Mayo Clinic, 200 First Street, S.W., Rochester, MN 55905. Tel:(507) 538-1545 Fax:(507) 284-3906 E-mail:chen.junjie{at}mayo.edu.
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Abstract |
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The tumor suppressor p53 binding protein 1 (53BP1) binds to the DNA-binding domain of p53 and enhances p53-mediated transcriptional activation. 53BP1 contains two breast cancer susceptibility gene 1 COOH terminus (BRCT) motifs, which are present in several proteins involved in DNA repair and/or DNA damagesignaling pathways. Thus, we investigated the potential role of 53BP1 in DNA damagesignaling pathways. Here, we report that 53BP1 becomes hyperphosphorylated and forms discrete nuclear foci in response to DNA damage. These foci colocalize at all time points with phosphorylated H2AX (-H2AX), which has been previously demonstrated to localize at sites of DNA strand breaks. 53BP1 foci formation is not restricted to
-radiation but is also detected in response to UV radiation as well as hydroxyurea, camptothecin, etoposide, and methylmethanesulfonate treatment. Several observations suggest that 53BP1 is regulated by ataxia telangiectasia mutated (ATM) after DNA damage. First, ATM-deficient cells show no 53BP1 hyperphosphorylation and reduced 53BP1 foci formation in response to
-radiation compared with cells expressing wild-type ATM. Second, wortmannin treatment strongly inhibits
-radiationinduced hyperphosphorylation and foci formation of 53BP1. Third, 53BP1 is readily phosphorylated by ATM in vitro. Taken together, these results suggest that 53BP1 is an ATM substrate that is involved early in the DNA damagesignaling pathways in mammalian cells.
Key Words:
53BP1, DNA damage, nuclear foci, -H2AX, ATM
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Introduction |
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Cells have evolved various sophisticated pathways to sense and overcome DNA damage as a mechanism to preserve the integrity of the genome. Environmental attacks like radiation or toxins, as well as spontaneous DNA lesions, trigger checkpoint activation and consequent cell cycle arrest and/or apoptosis. One key protein that coordinates DNA repair with cell cycle progression and apoptosis is the tumor suppressor protein p53. P53 is activated and posttranslationally modified in response to DNA damage (
P53 interacts with p53 binding protein 1 (53BP1). 53BP1 has been identified in a yeast two hybrid screen as a protein that interacts with the central DNAbinding domain of p53 (
The presence of BRCT domains in 53BP1 and the reported interaction with p53 prompted us to investigate whether 53BP1 is involved in DNA damageresponse pathways. Here we report that 53BP1 becomes hyperphosphorylated and rapidly relocates to the sites of DNA strand breaks in response to ionizing radiation. 53BP1 foci formation is reduced in ATM-deficient cells and can be inhibited by wortmannin in ATM wild-type cells. Moreover, radiation-induced hyperphosphorylation of 53BP1 is absent in cells treated with wortmannin, as well as in ATM-deficient cells. Taken together, these results strongly suggest that 53BP1 participates in DNA damagesignaling pathways and is regulated by ATM after -radiation.
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Materials and Methods |
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Cell Culture and Treatments with DNA-damaging Agents
Cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C with 5% CO2. FT169A and YZ5 cells were provided by Dr. Y. Shilon (Tel Aviv University, Ramat Aviv, Israel). Cells grown on coverslips were irradiated in a JL Shepherd 137Cs radiation source at a rate of 1 Gy/min for doses of 15 Gy or 10 Gy/min for a dose of 10 Gy. UV light was delivered in a single pulse (50 J/m2) using a Stratalinker UV source (Stratagene). Before UV irradiation, the culture medium was removed and the medium was replaced immediately after irradiation. All cells were returned to the incubator for recovery and harvested at the indicated times. Genotoxic agents and other drugs were used at the indicated concentrations. After a 1-h exposure, the cells were harvested for immunostaining.
Immunoprecipitation, Immunoblotting, and Immunostaining
Immunoprecipitation, immunoblotting, and immunostaining were performed as described previously (
ATM Kinase Assay
ATM was immunoprecipitated from K562 cells using anti-ATM antibody Ab3 (Oncogene Research Products). Aliquots of the ATMprotein A Sepharose immunocomplexes were resuspended in 25 µl kinase buffer (10 mM Hepes, pH 7.4, 50 mM NaCl, 10 mM MgCl2, 10 mM MnCl2, 1 mM DTT, 10 nM ATP) and incubated for 20 min at 30°C with 10 µCi of [32]P-ATP and 1 µg of various affinity-purified GST fusion proteins containing different fragments of 53BP1.
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Results |
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53BP1 Forms Nuclear Foci in Response to Various Types of DNA Damage
Several proteins, including BRCA1 and Mre11/Rad50/Nbs1, form DNA damageregulated, subnuclear foci in the cell. To determine whether 53BP1 participates in DNA damagesignaling pathways, we examined 53BP1 localization after various types of DNA damage using several anti-53BP1 polyclonal and monoclonal antibodies generated for this study. All antibodies specifically recognize endogenous, as well as HA-tagged, full-length 53BP1 as examined by Western blotting, immunoprecipitation, and immunostaining (data not shown). As shown in Fig 1, 53BP1 is diffusely localized in the nuclei of normal cells, but relocates to discrete subnuclear foci structures in response to ionizing radiation (e.g., 1 Gy). These 53BP1 foci can be detected as early as 5 min after irradiation (data not shown). Higher doses of radiation (e.g., 10 Gy) lead to more but smaller 53BP1 foci (Fig 1). The number of foci reaches a peak at 30 min after radiation. Thereafter, the foci number slowly decreases, whereas the foci size increases (data not shown).
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Foci formation is also observed in response to other DNA-damaging events. UV radiation induced the formation of numerous small foci, similar to that induced by 4NQO (a UV-mimetic agent) and hydroxyurea (Fig 1). Treatment with the DNA topoisomerase I poison camptothecin or the topoisomerase II poison etoposide (VP16), which cause DNA single strand and double strand breaks, respectively, also resulted in the formation of 53BP1 foci. Similar results were obtained with the alkylating agent methylmethanesulfonate. However, cisplatin, a DNA cross-linking agent, induced only a few 53BP1 foci during the first hour after drug application, whereas the protein kinase inhibitor UCN-01 and the antimitotic agent paclitaxel (Taxol; Bristol-Meyers Squibb Co.) did not induce 53BP1 foci formation. Thus, different types of DNA damage trigger the recruitment of 53BP1 into discrete nuclear foci.
53BP1 Colocalizes with -H2AX in Response to DNA Damage
The time course of 53BP1 foci formation and disappearance is very similar to that recently described for phosphorylated H2AX (-H2AX) appears within 13 min as discrete nuclear foci on sites of DNA double strand breaks (
-H2AX (
-H2AX at the various time points analyzed. The number of 53BP1 foci was identical to that of
-H2AX throughout the course of the experiment. In addition, coimmunoprecipitation analysis revealed that 53BP1 and
-H2AX biochemically interact after
-radiation (Fig 2 B). Small amounts of 53BP1 were detected in
-H2AX immunoprecipitates prepared from irradiated HBL100 cells. In unirradiated cells, H2AX was not phosphorylated and anti
-H2AX antibodies did not immunoprecipitate any phosphorylated H2AX. Similarly, 53BP1 was also not present in anti
-H2AX immunoprecipitates prepared from unirradiated cells. These results demonstrate that 53BP1 colocalizes and interacts with
-H2AX at the sites of DNA strand breaks after
-radiation.
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ATM Is Involved in 53BP1 Foci Formation
Several phosphatidylinositol 3kinase (PI3K)-related kinases, including DNA-dependent protein kinase (DNA-PK), ATM, and ATM-related kinase (ATR), participate in DNA damageresponsive pathways (
We first examined 53BP1 foci formation in the presence or absence of DNA-PK using two derivatives of the human glioma cell line MO59 (-radiation (data not shown). However, comparison was hampered by the high number of 53BP1 foci in unirradiated MO59K and MO59J cells and subtle differences might be overlooked.
We then examined whether the 53BP1 response to ionizing radiation is affected in cells lacking ATM. Immortalized ATM-deficient fibroblasts (FT169A) were compared with their isogenic derivative cells, YZ5, that have been reconstituted with wild-type ATM cDNA (
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To further corroborate the role of ATM in 53BP1 foci formation, we pretreated HeLa cells for 30 min with wortmannin before exposure to 1 Gy of irradiation. Wortmannin is a potent inhibitor of the PI3K-related kinases, including ATM and DNA-PK (-radiation. At an even higher dose (200 µM), wortmannin completely blocked 53BP1 foci formation. These results suggest that the kinase activities of ATM or other PI3K-related kinases are required for 53BP1 foci formation.
ATM Is Required for DNA Damage-induced Hyperphosphorylation of 53BP1
Many proteins involved in DNA damageresponse and/or DNA repair are phosphorylated upon DNA damage. To examine whether 53BP1 becomes phosphorylated in response to -radiation, K562 cells were irradiated (20 Gy) and harvested 1 h later. After immunoprecipitation using anti-53BP1 antisera, the samples were incubated for 1 h at 30°C in the presence or absence of
protein phosphatase and separated on a 38% gradient SDS gel. Phosphatase treatment of unirradiated K562 cells revealed a faster migrating form of 53BP1 (Fig 4 A). This indicates that 53BP1 is modified by phosphorylation in normal undamaged cells. Upon
-radiation, 53BP1 showed an even slower mobility that was reversed by phosphatase treatment (Fig 4 A). These results suggest that 53BP1 is phosphorylated in undamaged cells and becomes hyperphosphorylated after
-radiation.
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Since 53BP1 is hyperphosphorylated after -radiation, we then examined whether wortmannin would affect radiation-induced 53BP1 phosphorylation. As illustrated in Fig 4 B, there was no detectable radiation-induced 53BP1 mobility shift in wortmannin (50 µM)-pretreated cells. In contrast, the radiation-induced 53BP1 mobility shift was readily detected in cells that had received no drug treatment before radiation. We next repeated the experiment using the ATM-deficient GM03189D and GM02184D cells expressing wild-type ATM. Again, in ATM wild-type cells,
-radiation induced a 53BP1 mobility shift in control, but not in wortmannin-pretreated samples (Fig 4 C). However, no radiation-induced 53BP1 mobility shift was observed in ATM-deficient cells, with or without wortmannin treatment (Fig 4C and Fig D). Taken together, these results strongly suggest that ATM is required for 53BP1 hyperphosphorylation after
-radiation.
53BP1 Is a Substrate of ATM In Vitro
S/TQ sites have been described to be the minimal essential recognition sites for ATM (
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Discussion |
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Here we report that 53BP1 participates in the early DNA damageresponse. Using several antibodies specifically recognizing 53BP1, we show that 53BP1 becomes hyperphosphorylated and forms nuclear foci after exposure to ionizing radiation. -Radiationinduced 53BP1 hyperphosphorylation and foci formation are reduced in ATM-deficient cells. Moreover, 53BP1 hyperphosphorylation, as well as foci formation, is inhibited by wortmannin, an inhibitor of the PI3K-related kinases including ATM, DNA-PK, and, to a lesser extent, ATR (
In favor of a functional link between ATM and 53BP1, we also demonstrate that NH2-terminal fragments of 53BP1 are effectively phosphorylated by ATM in vitro. Similarly, Xia and colleagues have recently shown that Xenopus 53BP1 and a NH2-terminal fragment of human 53BP1 can be phosphorylated by ATM in vitro and in vivo (-radiation.
53BP1 rapidly colocalizes with -H2AX in response to ionizing radiation. H2AX is a histone H2A variant that becomes phosphorylated and forms foci at sites of DNA strand breaks after DNA damage (
-H2AX. Moreover, 53BP1 and
-H2AX physically interact after ionizing radiation, suggesting that 53BP1 relocates to the sites of DNA double strand breaks in response to
-radiation. Similar to 53BP1,
-H2AX foci formation is inhibited by wortmannin treatment (
Upon relocalizing to the sites of DNA damage, 53BP1 could participate in chromosome remodeling that makes DNA lesions accessible to DNA repair proteins. Alternatively, 53BP1 could be involved in the recruitment of repair proteins like BRCA1 and Rad51 to these DNA lesions. Both of these proteins colocalize with 53BP1 several hours after exposure to ionizing radiation (Rappold, I., and J. Chen, unpublished observations). In addition, BRCA1 biochemically interacts with 53BP1 after -radiation (Rappold, I., and J. Chen, unpublished observations).
53BP1 contains two BRCT motifs at its COOH terminus. 53BP1 BRCT motifs are closely related with those of BRCA1 and Saccharomyces cerevisiae Rad9 (scRad9) protein. Insight into the potential role of scRad9 comes from studies of its association with scRad53. ScRad53 is the homologue of mammalian Chk2 or Schizosaccharomyces pombe Cds1. After DNA damage, scRad9 is phosphorylated and this phosphorylated scRad9 associates with the forkhead homologyassociated (FHA) domain of scRad53 (
In conclusion, our data demonstrate that 53BP1 participates early in DNA damagesignaling pathways and is regulated by ATM after -radiation. The exact role of 53BP1 in these pathways remains to be resolved. Given the importance of these DNA damagesignaling pathways in cancer prevention, it will be interesting to examine whether 53BP1 is mutated in tumors.
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Footnotes |
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1 Abbreviations used in this paper: ATM, ataxia telangiectasia mutated; BRCA1, breast cancer susceptibility gene 1; BRCT, BRCA1 COOH terminus; DNA-PK, DNA-dependent protein kinase; 53BP1, binding protein 1; GST, glutathione S-transferase; PI3K, phosphatidylinositol 3kinase.
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Acknowledgements |
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We thank Drs. Scott Kaufmann, Larry Karnitz, and Jann Sarkaria for stimulating conversations. We also thank the Mayo Clinic Flow Cytometry and Protein Core facilities for their assistance. Initial studies of this work were performed in the laboratory of Dr. David M. Livingston. We specially thank Dr. Livingston for help and encouragement during the course of this study.
This work was supported by the Mayo Foundation, Mayo Cancer Center, Division of Oncology Research, and an Eagles grant to J. Chen.
Submitted: 24 October 2000
Revised: 23 March 2001
Accepted: 26 March 2001
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References |
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