ARF repression of NF-κB activity is ATR and Chk1 dependent. (A, B) siRNAs targeting ATR and Chk1 abolish ARF-induced repression of Bcl-xL. PCR analysis of Bcl-xL expression was performed following treatment of NARF2-E6 cells with the indicated siRNAs. Cells were either treated with IPTG to induce ARF expression (A) or stimulated with UV-C (40 J/m²) (B). (C, D) Inhibition of ATR or Chk1 activity abolishes ARF-mediated repression of the Bcl-xL promoter. In all, 1.5 μg of the Bcl-xL or Bcl-xL ΔκB luciferase reporter plasmids was transfected into NARF2-E6 cells. Either (C) 1 μg of kdATR or kdChk1 expression plasmids were cotransfected as indicated or (D) cells were treated with 2 mM caffeine or 1 μM Gö6976 as indicated at the time of ARF induction. In this and subsequent luciferase assays, results are expressed as fold activation or repression, relative to levels seen in the relevant untreated cell controls. The absence of error bars for some data points, in this and subsequent figures, results from them being below the resolution of the graph. All data points in this and subsequent figures result from at least three independent experiments. (E, F) Chk1 is required for ARF-mediated repression of the RelA transcriptional activation domain. The Gal4 E1B luciferase reporter plasmid (1 μg) was transfected into NARF2-E6 cells together with expression plasmids encoding either Gal4 alone (0.75 ng) or Gal4 RelA(TAD) (0.75 ng). Where indicated, cells were also cotransfected with 1 μg of kinase dead dominant-negative Chk1 expression plasmid (E) or treated with 1 μM Gö6976 at the time of ARF induction (D).

ARF-induced sensitivity to TNF-induced cell death requires ATR and Chk1. NARF2 E6 cells were transfected once with control, ATR and Chk1 siRNAs (oligos A and B combined). At 48 h after siRNA transfection, cells were treated with IPTG to induce ARF. After a further 24 h, cells were treated with TNF (30 ng/ml) and, 24 h later, percentage cell death was determined as described previously (Rocha et al, 2003).

Phosphorylation of RelA at Thr505. (A) ARF induces Thr505 phosphorylation. Whole-cell lysates were prepared from NARF2 cells either treated with IPTG to induce ARF for 24 h, stimulated with TNF (10 ng/ml for 30 min) or left untreated. Phosphorylated RelA was then immunoprecipitated by incubating 150 μg of lysate with purified phospho-RelA T505A antibody. The immunoprecipitate was resolved by SDS–PAGE before Western blotting with an anti-RelA antibody (sc-372 Santa Cruz). (B) UV light does not induce Thr505 phosphorylation. The experiment was performed as in (A), except that immunoprecipitations were performed with 100 μg of nuclear protein extract. Input lanes represent 10 μg of nuclear extract. As indicated, some cells were treated with UV-C (40 J/m²) and harvested after the times shown. (C) ARF-induced Thr505 phosphorylation is prevented by the Chk1 inhibitor Gö6976. The experiment was performed as in (A), except that that indicated cells were incubated with 1 μM Gö6976 at the same time as IPTG addition. (D) Schematic diagram showing species conservation of the RelA Thr505 region and similarity to the Chk1 consensus phosphorylation sequence. The human Thr505 residue is underlined. (E) The RelA Thr505 motif is phosphorylated by Chk1 in vitro. Purified GST, GST RelA TAD (amino acids 428–551) and GST RelA TAD T505A were incubated with recombinant purified GST-Chk1 and unlabelled ATP. Proteins were then resolved by SDS–PAGE and immunoblotted with the anti-phospho RelA Thr505 antibody (upper panel). Immunoblotting with anti-GST antibody demonstrated equivalent loading of GST-RelA (TAD) and GST-RelA TAD (T505A) proteins (lower panel). (F) ARF induces nuclear phospho-Thr505-modified RelA. NARF2 cells were plated onto coverslips and fixed at the indicated times after IPTG treatment. Cells were stained with mouse anti-p14^ARF and rabbit anti-P-T505 RelA antibodies. In this and subsequent experiments, cells were analysed and images were acquired using a DeltaVision microscope. (G) Induction of RelA Thr505 phosphorylation in Hs68 E2F1–ER cells. The experiment was performed as in (A), except that immunoprecipitations were performed with 300 μg of whole-cell lysates from Hs68 E2F1–ER cells. Input lanes represent 30 μg of extract. As indicated, cells were treated with Tamoxifen and 2 mM caffeine and harvested 24 h later.

ATR is required for ARF-induced p53 activation. (A) ARF induces the association of ATR with p53. ATR was immunoprecipitated with 200 μg of whole-cell lysate prepared from NARF2 cells treated as indicated. The immunoprecipitated complex was resolved by SDS–PAGE and immunoblotted with an anti-p53 and anti ATR antibodies. (B) ARF-induced p53 transcriptional activity requires ATR. In all, 1.5 μg of the p53-dependent PG13 luciferase and p21^WAF1 luciferase reporter plasmids were transfected into NARF2 cells. Where indicated, cells were cotransfected with 1 μg kdATR expression. (C, D) Caffeine treatment abolishes ARF induction of p53. Western blot analysis of whole-cell lysates showing the induction of ARF, p53 and p21 following treatment of NARF2 cells with IPTG or Hs68 E2F1–ER cells with Tamoxifen in the presence or absence of 2 mM caffeine. (E, F) ATR siRNA treatment inhibits ARF induction of p53 activity. Western blot analysis of whole-cell lysates from NARF2 (E) or Hs68 E2F1–ER (F) cells showing the induction of ARF, p53 and p21. Cells were transfected with either ATR or control siRNAs. (G) ATM siRNA treatment does not abolish ARF induction of p53 in NARF2 cells. Western blot analysis of NARF2 cell whole-cell lysates showing the induction of ARF, p53 and phospho-serine 15-modified p53 following treatment with either ATM or control siRNAs. In the top panel, PCR analysis of ATM RNA levels is shown following treatment with ATM siRNAs. Inhibition of ATM protein expression by ATM siRNA is shown in Supplementary Figure 1G. (H) ATM siRNA inhibits p53 activation following DNA damage. Western blot analysis of whole-cell lysates showing the induction of p53 and phospho-serine 15-modified p53 following treatment with 2 μM doxorubicin for 4 h in NARF2 cells treated with either ATM or control siRNAs.

ARF-induced inhibition of cell proliferation requires p53 and ATR. NARF2 cells were transfected with the indicated siRNA oligonucleotides and, 48 h later, ARF was induced with IPTG. Cell proliferation was measured every 24 h, as indicated, by alamarBlue assay. Results are obtained from four separate experiments performed in triplicate.

ARF induces ATR activity. (A) ARF induces the phosphorylation of ATM/ATR substrates. Whole-cell lysates were prepared from NARF2 cells either treated with IPTG to induce ARF for 24 h, stimulated with UV-C (40 J/m²) or left untreated. The lysate (20 μg) was then resolved by SDS–PAGE and Western blotted with the anti-phospho-SQ/TQ antibody. (B) Induction of ATR activity in Hs68 E2F1–ER cells by endogenous ARF. Western blot analysis of whole-cell lysates using the anti-phospho-SQ/TQ antibody following treatment of Hs68 E2F1–ER cells with Tamoxifen for 24 h. Cells were transfected twice with either ATR (A, B combined), ARF (A, B combined) or control siRNAs as indicated. ARF siRNA validation is shown in Supplementary Figure 1H.

ARF induces phosphorylation of Chk1. Whole-cell lysates were prepared from NARF2 cells either treated with IPTG to induce ARF for the indicated times, stimulated with UV-C (40 J/m²) or left untreated.

Recruitment of ATR and BRCA1 to ARF-containing nucleolar complexes. (A, B) Immunofluorescence analysis of ARF, ATR and BRCA1 following ARF induction. NARF2 cells were plated onto coverslips and fixed at the indicated times after IPTG treatment. Cells were stained with rabbit anti-p14^ARF, goat anti-ATR and mouse anti-BRCA1 antibodies as indicated. In (B), selected data were further analysed and colocalisation of the indicated proteins was determined by examination of merged and enlarged images. Since these are digital images, colour designation is arbitrary in this and all subsequent images. (C) ATR coimmunoprecipitates with ARF. ARF was immunoprecipitated from 150 μg of whole-cell lysate prepared from NARF2 cells. The immunoprecipitated complex was resolved by SDS–PAGE and immunoblotted with anti-ARF or ATR antibodies. (D) E2F-induced ARF colocalises with ATR. Immunofluorescence analysis of ARF and ATR in Hs68 E2F-ER cells. Cells were plated onto coverslips and fixed 24 h after Tamoxifen treatment. Cells were stained with rabbit anti-p14^ARF and goat anti-ATR antibodies. An enlarged section of the merged ATR/ARFs image is also shown.

ARF-induced p53 activation requires BRCA1. Western blot analysis of NARF2 whole-cell lysates showing the induction of ARF, p53, phospho-serine 15 p53 and p21. Cells were doubly transfected with either ATR (oligos A and B combined), BRCA1 or control siRNAs as indicated. BRCA1 siRNA validation is shown in Supplementary Figure 1I.

Analysis of Chk1 localisation and ATR association after ARF induction. (A) ARF does not recruit Chk1 to nucleoli. NARF2 cells were fixed 24 h after ARF induction and stained with goat anti-ATR, rabbit anti-ARF and sheep anti-Chk1. Enlarged sections from merged Chk1/ATR images are shown. (B) ATR coimmunoprecipitates with Chk1 following ARF induction. ATR was immunoprecipitated from 150 μg of whole-cell lysate prepared from NARF2 cells with or without ARF induction. The immunoprecipitated complex was resolved by SDS–PAGE and immunoblotted with anti-Chk1 and anti-ATR antibodies.

Serine 15-phosphorylated p53 localises to ARF and ATR-containing nucleolar complexes. (A) Immunofluorescence analysis of p53 following ARF induction. NARF2 cells were stained for p53 with mouse anti-p53 DO1 antibody. (B) Imaging of serine 15-phosphorylated p53 following UV-C treatment. Immunofluorescence analysis of serine 15-phosphorylated p53 in U-2 OS cells 4 h after treatment with UV-C (40 J/m²). Cells were stained with mouse 16G8 monoclonal anti-phospho serine 15 p53 antibody. (C) Serine 15-phosphorylated p53 associates with ARF and ATR in the nucleolus. Immunofluorescence analysis of ARF, ATR and serine 15-phosphorylated p53 following induction of ARF in NARF2 cells. Cells were stained with rabbit anti-p14^ARF, goat anti-ATR and mouse 16G8 monoclonal anti-phospho-serine 15 p53 antibodies. Additional images can be seen in Supplementary data.

Schematic model depicting engagement of the ATR/Chk1 pathway by ARF. Data presented in this report demonstrate that ARF engages ATR through two pathways distinct to those following DNA damage. In the nucleoplasm, Chk1 is activated by ATR and is required for Thr505 phosphorylation of the RelA NF-κB subunit. This inhibits RelA transactivation resulting in decreased Bcl-xL levels, sensitising cells to apoptotic stimuli. At the nucleoli, ARF induces the assembly of an ATR/BRCA1 complex that appears to be required for activation of p53 at serine 15. It is probable that nucleoplasmic ATR/BRCA1 will also contribute to p53 activation. In addition, both pathways are likely to regulate many other cellular processes and make a wider contribution to ARF-mediated tumour suppression.