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Interaction of the double-strand break repair kinase DNA-PK and estrogen receptor-alpha - PubMed

  • ️Fri Jan 01 2010

Interaction of the double-strand break repair kinase DNA-PK and estrogen receptor-alpha

Senad Medunjanin et al. Mol Biol Cell. 2010.

Abstract

Estrogens are suggested to play a role in the development and progression of proliferative diseases such as breast cancer. Like other steroid hormone receptors, the estrogen receptor-alpha (ERalpha) is a substrate of protein kinases, and phosphorylation has profound effects on its function and activity. Given the importance of DNA-dependent protein kinase (DNA-PK) for DNA repair, cell cycle progression, and survival, we hypothesized that it modulates ERalpha signaling. Here we show that, upon estrogen stimulation, DNA-PK forms a complex with ERalpha in a breast cancer cell line (MELN). DNA-PK phosphorylates ERalpha at Ser-118. Phosphorylation resulted in stabilization of ERalpha protein as inhibition of DNA-PK resulted in its proteasomal degradation. Activation of DNA-PK by double-strand breaks or its inhibition by siRNA technology demonstrated that estrogen-induced ERalpha activation and cell cycle progression is, at least, partially dependent on DNA-PK.

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Figures

Figure 1.
Figure 1.

KU70 interacts with ERα. (A) MELN cells were grown in phenol red–free medium supplemented with 10% charcoal-dextran–stripped FBS for 3 d. Lysates from 100 nM E2-stimulated cells were immunoprecipitated (IP) with anti-ERα followed by immunoblotting (IB) with anti-ERα and anti-Ku70 (top). Lysates were immunoprecipitated with anti-Ku70 followed by immunoblotting with anti-Ku70 and anti-ERα (bottom). CL, cell lysate. (B) Lysates from E2-treated MELN cells were treated with 400 μg/ml ethidium bromide and further immunoprecipitated with anti-ERα followed by immunoblotting with anti-Ku70, anti-Ku80, and anti-DNA-PKcs. CL, cell lysate. (C) COS-7 cells were transfected with pcDNA3.1ERα2xFlag and stimulated with 100 nM E2. Immunostaining of the ERα was performed with anti-Flag M2 antibody. Scale bar, 20 μm. (D) In vitro kinase assay using recombinant human ERα 1 μg or p53 1 μg as substrate for DNA-PK (0.05 μg). (E) DNA-PK, was incubated with 1 μg recombinant human ERα and 10 μCi of [γ-32P]ATP at 30°C for different time points.

Figure 2.
Figure 2.

Ku70 interacts with the B domain of ERα. (A) ERα deletion mutants generated by PCR. (B) COS-7 cells were cotransfected with Flag-tagged wild-type ERα or ERα deletion mutants (ΔF, ΔA, ΔAB, ΔABC, and ΔABCD). Forty-eight hours after transfection, cells were lysed, and Ku70 was immunoprecipitated with anti-Ku70. The lysates (top) and immunoprecipitates (IP; bottom) were immunoblotted (IB) with anti-Flag M2 antibody. (C) In vitro kinase assay using wild-type and mutant GST-ERα-(76-176) fusion proteins (1 μg) as substrates for DNA-PK (top, pERαSer118; bottom, Coomassie staining). (D) MELN cells were incubated for 10 min with 10 mM LiCl, 5 μM NU7026, or 10 μM PD98059 and thereafter with 100 nM E2 for 20 min. ERα phosphorylation was detected by immunoblotting. Detection of β-actin was used as a loading control.

Figure 3.
Figure 3.

Silencing DNA-PK decreases ERα protein and influences the proliferative response to E2. (A) MELN cells were transfected either with GL3 control siRNA (siGL3) or with siRNA targeting DNA-PK subunits (siKu70 and siDNA-PKcs). After 24-h pretreatment with siRNA, cells were treated or not with 10 nM E2 for 48 h, and lysates were analyzed by immunoblotting (IB) with specific antibodies as indicated. (B) MELN cells were transfected either with GL3 control siRNA (siGL3-C) or with siRNA targeting DNA-PK subunits (Ku70 and DNA-PKcs siRNA) and stimulated with 10 nM E2. ERE-dependent gene expression was quantified by measuring luciferase activity. Fold induction is the ratio of stimulated to unstimulated cells. (C) MELN cells were transfected either with GL3 control siRNA (siGL3) or with siRNA targeting DNA-PK subunits (siKu70 and siDNA-PKcs) and stimulated with 10 nM E2. ERα-dependent proliferation was quantified using a proliferation assay (Roche, Mannheim, Germany). Fold induction is the ratio of stimulated to unstimulated cells. (D) The effect of 10 nM E2 and DNA-PK inhibitor NU7026 5 μM on phosphorylation of the ERα at Ser118 and proliferation marker proteins. Detection of β-actin on the same membrane was used as a loading control. (E) MELN cells were grown in phenol red–free medium supplemented with 10% charcoal-dextran–stripped FBS for 3 d. After pretreatment with 5 μM NU7026 and 100 nM E2 for 20 h, cells were treated with IR (3Gy) and incubated for another 6 h. The lysates were assayed for specific protein expression. Detection of β-actin was used as loading control.

Figure 4.
Figure 4.

DNA-PK prevents ERα ubiquitination. (A) The total RNA from the GL3 or DNA-PKcs knockdown cells was analyzed for expression of ERα and DNA-PKcs by semiquantitative RT-PCR. (B) To detect ubiquitinated forms of endogenous ERα from MELN cells, the cells were treated with 5 μM NU7026 for 24 h, after treatment with 100 nM E2 for 6 h. The lysates were immunoprecipitated with antibody against ERα. Ubiquitination from the immunoprecipitates was examined by ubiquitin antibody. (C) COS-7 cells were transfected with ERα-WT and ERαSer118A-mutant. Forty-eight hours after transfection, the cells were treated with 5 μM MG132 for 24 h and 100 nM E2 for 6 h before cell lysis. Anti-ERα immunoprecipitates were resolved by SDS-PAGE and immunoblotted with the indicated antibodies. Detection of ERα on the same membrane was used as a loading control.

Figure 5.
Figure 5.

Ku70 occupancy of estrogen-responsive gene promoters. (A) EMSAs with nuclear extracts from E2-treated (100 nM) MELN cells. Extracts were incubated with biotin-labeled double-stranded oligonucleotide probes containing ERE for 20 min. Antibodies for ERα, Ku70, and nonspecific (NS-AB) were incubated for 30 min. Competition reactions were performed with unlabeled competitor (comp.) at a 200-fold molar excess. (B) Cells were grown in phenol red–free medium supplemented with 10% charcoal-dextran–stripped FBS for 3 d. After treatment with 100 nM E2 for 1 h, cells were cross-linked with 1%formaldehyde and monitored by ChIP assays. Soluble chromatin from control and E2-treated MELN cells was immunoprecipitated with either anti-ERα, anti-Ku70, anti RNA-pol II, or a control IgG. The final DNA extractions were amplified by PCR with pairs of primers covering the indicated EREs (ERE) or control regions (CR) of the pS2 and cathepsin D promoters. (C) In ReChIP experiments, soluble chromatin from the indicated cells was immunoprecipitated with anti-Ku70. The immune complexes were eluted by incubation with 10 mM DTT for 30 min at 37°C. After centrifugation, the supernatant was diluted 30 times with ReChIP buffer followed by reprecipitation with anti-ERα and then detection of the indicated EREs or CRs in the pS2 promoter.

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