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. 2005 Sep;25(18):7966–7975. doi: 10.1128/MCB.25.18.7966-7975.2005

FIG. 4.

FIG. 4.

Phosphorylation of RelA at serine 536 facilitates the acetylation of RelA on lysine 310. (A) Kinase-deficient mutants of IKK1 and IKK2 inhibit the acetylation (Ac) of RelA at lysine 310. 293T cells were cotransfected with the indicated expression plasmid DNA encoding T7-RelA (0.5 μg), p300 (2 μg), IKK1(K47M) (0.5 μg), and IKK2(K47A) (0.5 μg). Acetylation of RelA was detected by immunoblotting (IB) of anti-T7 (α-T7) immunoprecipitates (IP) with anti-acetylated lysine 310 (α-Ac-K310) antibodies (top). Levels of T7-RelA present in each of the lysates are shown in the bottom panel. (B) Alanine or glutamic acid substitutions at serine 536 impair or enhance, respectively, the acetylation of RelA at lysine 310. 293T cells were transfected with wild-type (WT) RelA, S536A, or S536E expression vectors (0.5 μg) together with p300 (2 μg). Levels of acetylation on each protein were detected by immunoblotting of anti-T7 immunoprecipitates with anti-acetylated lysine 310 antibodies. Levels of RelA, S536A, and S536E are shown in the bottom panel. (C) Phosphorylation of RelA at serine 536 by IKK2 enhances the acetylation of RelA in vitro. Recombinant RelA (1 μg) was phosphorylated with recombinant IKK2 (0.5 μl) (P-RelA) in an in vitro kinase assay. Reactions were then incubated with p300 immunoprecipitates in an in vitro acetylation assay as described in the legend to Fig. 1A. Levels of acetylation of RelA were detected by immunoblotting with anti-acetylated lysine 310 antibodies. Levels of RelA in each reaction are shown in the bottom panel. (D) RelA-deficient MEFs were transfected with E-selectin-luciferase reporter plasmid DNA (0.5 μg) and the various S276A, S276E, S536A, and S536E mutants of RelA (0.1 μg). After 36 h, cells were treated with or without TNF-α (20 ng/ml) for 6 h. Luciferase activity was measured as described previously (4). Expression levels of RelA and various RelA mutants in each transfection are shown in the bottom panel. Results represent the averages of three independent experiments ± standard deviations.