Phosphorylation of CBP by IKKα Promotes Cell Growth by Switching the Binding Preference of CBP from p53 to NF-κB  Wei-Chien Huang, Tsai-Kai Ju, Mien-Chie Hung, Ching-Chow Chen  Molecular Cell  Volume 26, Issue 1, Pages 75-87 (April 2007) DOI: 10.1016/j.molcel.2007.02.019 Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 1 Nuclear IKKα Is Involved in the Competition between NF-κB and p53 for CBP (A) IKK inhibitors reverse the TNF-α-induced protein interactions of CBP with p65 and p53. Whole-cell extract of HeLa cells, treated as indicated, was subjected to immunoprecipitation (IP) with anti-CBP antibody and then immunoblotted with anti-CBP, anti-IKKα, anti-p65, and anti-p53 antibodies. (B) IKK inhibitors reverse the TNF-α-induced recruitment of CBP to NF-κB and p53 promoters. Whole-cell extract of HeLa cells, treated as indicated, was subjected to ChIP assays with anti-CBP antibody. (C) Knockout of ikkα reverses the TNF-α-induced protein interactions of CBP with p65 and p53. Whole-cell extract of MEF cells, treated as indicated, was immunoprecipitated with anti-CBP antibody and then immunoblotted with anti-CBP, anti-p65, and anti-p53 antibodies. (D–G) Overexpression of CBP dose-dependently enhances IKKα-induced NF-κB-responsive and ICAM-1 transcriptions and reverses IKKα-induced suppression of p53 and p21 transcriptional activities. Lysates of HeLa cells transfected with NF-κB-(D), pICAM-1-(E), p53-(F), or p21-(G) promoter-Luc and β-galactosidase and indicated constructs were subjected to luciferase assay. The relative luciferase activities were normalized with β-galactosidase activity. Data are shown as the mean value ± standard error of the mean of three independent experiments. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 2 IKKα Phosphorylates CBP at Ser-1382 and Ser-1386 In Vitro (A) Consensus motif of IKK phosphorylation sites. Sequences of p300, CBP, and other known substrates are aligned for comparison. The conserved sequence for phosphorylation by IKKs is highlighted in gray. (ψ, hydrophobic amino acid; X, any amino acid.) (B–D) IKKα phosphorylates CBP at Ser-1382 and Ser-1386 in vitro. HeLa cells were stimulated with TNF-α for 60 min (B and D) or the indicated times (C). Total cell lysates were prepared and immunoprecipitated with anti-IKKα or anti-IKKβ antibody or IgG and then subjected to in vitro phosphorylation assay with various forms of GST-CBP or GST-IκBα as substrates. Proteins were separated by electrophoresis on 10% SDS polyacrylamide gels, and phosphorylated GST-fusion proteins were visualized by autoradiography. The amount of GST-CBP was detected by Coomassie Brilliant Blue staining. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 3 IKKα Phosphorylates CBP at Ser-1382 and Ser-1386 In Vivo (A and B) Lysates of HeLa cell transfected with indicated IKK and CBP constructs after TNF-α treatment and labeled with [32P]orthophosphate were subjected to immunoprecipitation with anti-Flag antibody and autoradiography. (C and D) Lysates of HeLa cells with siRNA knockdown of IKKα or IKKβ (C) and MEF ikk knockout cells treated with or without TNF-α (D) were subjected to western blotting with the indicated antibodies. (E–H) Lysates of WT or ikkα−/− MEF cells treated with the inducers of canonical and alternative NF-κB pathways (E, G, and H) or with paclitaxel (F) were subjected to immunoprecipitation/western blot with the indicated antibodies. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 4 Ser-1382 and Ser-1386 Phosphorylation of CBP Enhances the Interaction of CBP with p65 and Decreases the Interaction of CBP with p53 (A and B) p65 or p53 was immunoprecipitated from whole-cell lysates of HeLa cells treated with or without TNF-α and then incubated with in vitro-translated Flag-CBP WT and its Ser-1382 or Ser-1386 mutant and subjected to SDS-polyacrylamide gel electrophoresis separation and autoradiography. (C) Phospho-CBP was immunoprecipitated by anti-phospho-Ser-1382/1386-CBP antibody from HeLa cells, and the remaining unphosphorylated CBP in the supernatant was further isolated by anti-CBP antibody. The immunocomplex was subjected to western blotting with anti-phospho-Ser-1382/1386-CBP, anti-CBP, anti-phospho-Ser-276 p65, anti-p65, and anti-p53 antibodies. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 5 Phosphorylation of CBP by IKKα Enhances NF-κB-Mediated but Decreases p53-Mediated Gene Transcription (A) Lysates of HeLa cells transfected with IKKα and Flag-CBP WT or its Ser-1382 or Ser-1386 mutant and then treated with TNF-α were subjected to ChIP assay with anti-Flag antibody. (B and C) Endogenous phosphorylated CBP was immunoprecipitated by anti-phospho-Ser-1382/1386 CBP antibody, and the remaining unphosphorylated CBP in the supernatant was immunoprecipitated by anti-CBP antibody. The immunoprecipitates were further divided into three parts for direct ChIP and second ChIP with anti-p65 (B) and anti-p53 (C) antibody. (D and E) Lysates of HeLa cells transfected with NF-κB-, pICAM-1- (D), p53-, or p21- (E) promoter-luciferase and β-galactosidase and indicated constructs were subjected to luciferase assay. The relative luciferase activities were normalized with β-galactosidase activity. Data are shown as the mean value ± standard error of the mean of three independent experiments. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 6 Phosphorylation of CBP by IKKα Enhances the Intrinsic Transcriptional and HAT Activities of CBP (A and B) Lysates of HeLa cells cotransfected with Gal-Luc, HA-IKKα, and Gal-CBP-Flag (A) or cotransfected with Gal-Luc and Gal-CBP-Flag and then treated with TNF-α (B) were subjected to luciferase activity assay. The relative activities were normalized with β-galactosidase activity. Data are shown as the mean value ± standard error of the mean of three independent experiments. (C–E) Lysates of ikk knockout cells (C) or HeLa cells transfected with Flag-CBP and/or HA-IKKα (D) or Flag-CBP followed by TNF-α stimulation (E) were subjected to HAT activity assay. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions

Figure 7 Phosphorylation of CBP by IKKα Correlates with the Activation of IKKα in Several Tumor Cell Lines and May Contribute to Tumor Growth (A and B) Lysates of various cell lines (A) and paired human normal and malignant lung tissues (B) were subjected to western blotting with anti-phospho-Ser-1382/1386 CBP, anti-CBP, anti-phospho-IKKα/β, anti-IKKα, anti-phospho-p53 (Ser-15), and anti-p53 antibodies. The difference of phospho-IKKα or phospho-CBP in normal and tumor tissue was examined by Wilcoxon Signed-Rank test. The correlation between phospho-IKKα and phospho-CBP expression levels was evaluated by Spearman rank correlation coefficients. (HTSMC, human tracheal smooth muscle cell). (C and D) HeLa cell stable transfectants overexpressing WT CBP (five clones) and SSAA mutants (eight clones) or SSEE mutants (eight clones) were treated as indicated. Cell growth was determined by MTT assay. Data are shown as the mean value ± standard error of the mean of three independent experiments. (E) A diagram depicts the involvement of IKKα-mediated CBP phosphorylation in the crosstalk between NF-κB and p53. Molecular Cell 2007 26, 75-87DOI: (10.1016/j.molcel.2007.02.019) Copyright © 2007 Elsevier Inc. Terms and Conditions