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. 2005 Aug;25(15):6592–6602. doi: 10.1128/MCB.25.15.6592-6602.2005

FIG. 5.

FIG. 5.

Akt phosphorylation of Ser-1834 enhances the transcriptional activity of p300 by increasing its promoter recruitment, histone acetylation, and enhanceosome formation on the ICAM-1 promoter. (A) The p300 S1834A mutant inhibited TNF-α- or TSA-induced ICAM-1 promoter activity. A549 cells were cotransfected with pIC339 and the Flag-tagged wt p300 or p300 S1834A mutant, or the empty vector, and then treated with 10 ng/ml of TNF-α or 1 μM TSA for 6 h. Luciferase activity was measured. (Inset) Immunoblots of extracts from each sample to detect the level of Flag-tagged wt p300 or S1834A mutant using anti-Flag antibody. (B) The p300 S1834A mutant inhibited ICAM-1 promoter activity induced by the constitutive-active mutant of p110 or Akt. Cells were cotransfected with myc-p110*, HA-myr-Akt, or empty vector and the Flag-tagged wt or S1834A mutant of p300. After 24 h of transfection, luciferase activity was measured. (Inset) Immunoblots of extracts from each sample to detect the level of myc-p110*, HA-myr-Akt, or Flag-p300 using anti-myc, anti-HA, or anti-Flag antibody, respectively. (C) Phosphorylation of p300 at Ser-1834 is critical for NF-κB-, CREB-, and AP-1-mediated transcriptions. Cells were cotransfected with the Flag-tagged wt or the S1834A or S1834E mutant of p300 and NF-κB-, CRE-, or AP-1-luciferase reporters, and then luciferase activity was measured. (D) Mutation of Ser-1834 to Ala abolished the recruitment of p300, acetylation of nucleosomal histones, and RNA polymerase II to the ICAM-1 promoter. ChIP assays were performed in cells transfected with the Flag-tagged wt or S1834A mutant of p300 or the empty vector. Cells were pretreated with 50 μM Ly294002 for 30 min, followed by TNF-α stimulation for 60 min, and then chromatin was immunoprecipitated with anti-Flag, anti-acetylated histone H3, or anti-RNA ploymerase II (Pol II) antibody or without antibody (control). The precipitated ICAM-1 promoter region (−24 to −346) was amplified by PCR; 1% of the chromatin was assayed to verify equal loading (Input). All results were expressed as the mean plus standard error of the mean from three independent experiments.