Tumor suppressor p53 binding protein 1 (53BP1) is involved in DNA damage-signaling pathways

Abstract

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 damage-signaling pathways. Thus, we investigated the potential role of 53BP1 in DNA damage-signaling 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 (gamma-H2AX), which has been previously demonstrated to localize at sites of DNA strand breaks. 53BP1 foci formation is not restricted to gamma-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 gamma-radiation compared with cells expressing wild-type ATM. Second, wortmannin treatment strongly inhibits gamma-radiation-induced 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 damage-signaling pathways in mammalian cells.

Figure 3

53BP1 foci formation is reduced in cells lacking ATM. (A) ATM-deficient cell lines (FT169A, GM03189D, and GM05849C) and wild-type ATM cell lines (YZ5, GM02184D, and GM00637H) were irradiated with 1 Gy and immunostained with anti-53BP1 mAb BP13 at different time points before and after irradiation as indicated. FT169A cells and YZ5 cells are isogenic, whereas the other lines are unrelated. (B) The numbers of 53BP1 foci in at least 75 FT169A (ATM−) and YZ5 (ATM+) cells were counted for each time point. Data represent mean ± SD of three independent experiments. The difference in foci number between ATM+ and ATM− cells 10 or 20 min after irradiation was significant at P < 0.001 using a Student's t test. (C) Wortmannin inhibits γ-radiation–induced 53BP1 foci formation. HeLa cells were pretreated for 30 min with 0, 50, or 200 μM wortmannin before exposure to 1 Gy of γ-radiation. After recovery for 1 h, the control or irradiated cells were immunostained with anti-53BP1 antibodies.

Figure 3

53BP1 foci formation is reduced in cells lacking ATM. (A) ATM-deficient cell lines (FT169A, GM03189D, and GM05849C) and wild-type ATM cell lines (YZ5, GM02184D, and GM00637H) were irradiated with 1 Gy and immunostained with anti-53BP1 mAb BP13 at different time points before and after irradiation as indicated. FT169A cells and YZ5 cells are isogenic, whereas the other lines are unrelated. (B) The numbers of 53BP1 foci in at least 75 FT169A (ATM−) and YZ5 (ATM+) cells were counted for each time point. Data represent mean ± SD of three independent experiments. The difference in foci number between ATM+ and ATM− cells 10 or 20 min after irradiation was significant at P < 0.001 using a Student's t test. (C) Wortmannin inhibits γ-radiation–induced 53BP1 foci formation. HeLa cells were pretreated for 30 min with 0, 50, or 200 μM wortmannin before exposure to 1 Gy of γ-radiation. After recovery for 1 h, the control or irradiated cells were immunostained with anti-53BP1 antibodies.

Figure 3

53BP1 foci formation is reduced in cells lacking ATM. (A) ATM-deficient cell lines (FT169A, GM03189D, and GM05849C) and wild-type ATM cell lines (YZ5, GM02184D, and GM00637H) were irradiated with 1 Gy and immunostained with anti-53BP1 mAb BP13 at different time points before and after irradiation as indicated. FT169A cells and YZ5 cells are isogenic, whereas the other lines are unrelated. (B) The numbers of 53BP1 foci in at least 75 FT169A (ATM−) and YZ5 (ATM+) cells were counted for each time point. Data represent mean ± SD of three independent experiments. The difference in foci number between ATM+ and ATM− cells 10 or 20 min after irradiation was significant at P < 0.001 using a Student's t test. (C) Wortmannin inhibits γ-radiation–induced 53BP1 foci formation. HeLa cells were pretreated for 30 min with 0, 50, or 200 μM wortmannin before exposure to 1 Gy of γ-radiation. After recovery for 1 h, the control or irradiated cells were immunostained with anti-53BP1 antibodies.

Figure 1

53BP1 forms nuclear foci in response to DNA damage. HeLa cells were exposed to γ-irradiation (1 and 10 Gy) or a 50 J/m² UV pulse 1 h before immunostaining with anti-53BP1 mAb BP13. Alternatively, cells were treated for 1 h with the following drugs: 2 μg/ml 4-nitroquinoline 1-oxide (4NQO), 1 mM hydroxyurea (HU), 1 μM camptothecin (CPT), 40 μg/ml etoposide (VP16), 0.01% methylmethanesulfonate (MMS), 40 μM cisplatin (CDDP), 1 μM 7-hydroxystaurosporine (UCN-01), and 1 μM paclitaxel (Taxol).

Figure 2

(A) 53BP1 colocalizes with γ-H2AX in response to γ-radiation. WI38 cells were coimmunostained with anti-53BP1 antibody BP13 and affinity-purified anti–γ-H2AX serum before (0 min) and at the indicated time points after exposure to 1 Gy (10–160 min). (B) Coimmunoprecipitation of 53BP1 and γ-H2AX after DNA damage. HBL100 cells were exposed to 0 or 20 of Gy γ-radiation 1 h before lysis in NETN buffer (150 mM NaCl, 1 mM EDTA, 20 mM Tris, pH 8, 0.5% NP-40) including 0.3 M NaCl. Immunoprecipitation experiments were performed using anti-γH2AX or anti-53BP1 antibodies. A fivefold higher amount of cell lysate was used for anti–γ-H2AX immunoprecipitation than that used for anti-53BP1 immunoprecipitation. The samples were separated on 4–15% SDS-PAGE and Western blotting was performed using either anti–γ-H2AX or anti-53BP1 antibodies as indicated.

Figure 4

γ-Radiation–induced hyperphosphorylation of 53BP1 requires ATM. (A) γ-Radiation induces hyperphosphorylation of 53BP1. K562 cells were exposed to 0 or 20 Gy of γ-radiation and immunoprecipitated using polyclonal anti-53BP1 antibody. Immunoprecipitates were incubated for 1 h at 30°C with 800U λ-phosphatase (λPPase) in 100 μl incubation buffer or only incubation buffer. The samples were separated on a 3–8% gel and immunoblotted with anti-53BP1. (B) Wortmannin inhibits 53BP1 hyperphosphorylation. K562 cells were pretreated with 50 μM wortmannin for 30 min before exposure to 1 Gy of radiation. Whole cell lysates prepared from treated and control samples were separated on a 3–8% gradient gel (30 μg protein per lane) and immunoblotted with anti-53BP1 antibodies. (C) ATM is required for the γ-radiation–induced hyperphosphorylation of 53BP1. ATM-deficient GM03189D cells or ATM wild-type GM02184D cells were treated as described in the legend to B, and 30 μg lysates were separated on a 3–8% gel before immunoblotting with anti-53BP1 antibodies. (D) All three ATM-deficient cell lines tested (FT169A, GM03189D, and GM05849C) show no hyperphosphorylation 1 h after 20 Gy. (E) 53BP1 is a substrate of ATM in vitro. Six GST fusion proteins containing overlapping 53BP1 fragments were used as substrates in an ATM in vitro kinase assay. GST protein alone and GST fusion protein containing 13 residues surrounding the serine-15 of the p53 coding sequence were used, respectively, as negative and positive controls.