Negative regulation of NF-κB p65 activity by serine 536 phosphorylation

Abstract

Nuclear factor κB (NF-κB) is a master regulator of inflammation and cell death. Whereas most of the activity of NF-κB is regulated through the inhibitor of κB (IκB) kinase (IKK)-dependent degradation of IκB, IKK also phosphorylates subunits of NF-κB. We investigated the contribution of the phosphorylation of the NF-κB subunit p65 at the IKK phosphorylation site serine 536 (Ser(536)) in humans, which is thought to be required for the activation and nuclear translocation of NF-κB. Through experiments with knock-in mice (S534A mice) expressing a mutant p65 with an alanine-to-serine substitution at position 534 (the murine homolog of human Ser(536)), we observed increased expression of NF-κB-dependent genes after injection of mice with the inflammatory stimulus lipopolysaccharide (LPS) or exposure to gamma irradiation, and the enhanced gene expression was most pronounced at late time points. Compared to wild-type mice, S534A mice displayed increased mortality after injection with LPS. Increased NF-κB signaling in the S534A mice was at least in part explained by the increased stability of the S534A p65 protein compared to that of the Ser(534)-phosphorylated wild-type protein. Together, our results suggest that Ser(534) phosphorylation of p65 in mice (and, by extension, Ser(536) phosphorylation of human p65) is not required for its nuclear translocation, but instead inhibits NF-κB signaling to prevent deleterious inflammation.

Fig. 1. S534A mice display normal body weight, IκBα degradation, MAPK activation, and p65 nuclear translocation

(A) Left: Generation of S534A knock-in mice by BAC recombineering. Right: Base pair substitutions were confirmed by sequencing. (B) Weight gain in wild-type (WT) and S534A mice was monitored at the indicated times. Data are means ± SD of six mice per genotype. (C) WT and S534A MEFs were left unstimulated or were stimulated with TNF-α (30 ng/ml) and 10 nM calyculin A for 15 min. The cells were then analyzed by Western blotting with antibodies specific for p65 phosphorylated at the indicated residues. The antibody against human pSer⁵³⁶-p65 detects mouse pSer⁵³⁴-p65. Actin was used as a loading control. Western blots are representative of three experiments. (D) WT and S534A MEFs were stimulated with TNF-α (30 ng/ml) for the indicated times before being analyzed by Western blotting with antibodies specific for the indicated targets. Data representative of three experiments. (E) WT and S534A MEFs treated with TNF-α (30 ng/ml) for the indicated times were analyzed by immunofluorescence microscopy to detect the nuclear translocation of p65 (red). F-actin was stained by phalloidin (green), whereas nuclei were detected with Hoechst (blue). Images are representative of three experiments.

Fig. 2. S534A mice show increased expression of NF-κB–dependent genes in specific settings

(A) WT (n=4) and S534A (n=4) mice were injected i.v. with LPS (20 mg/kg) and sacrificed at the indicated times. Liver extracts were then analyzed by Western blotting with antibodies specific for the indicated targets. Western blots are representative of four experiments. (B) WT and S534A mice were injected i.v. with LPS (1 µg/kg) for the indicated times. Liver extracts were then analyzed by Western blotting with antibodies specific for the indicated proteins. Western blots are representative of three experiments. (C and D) WT (n = 9) and S534A (n = 9) mice were injected i.v. with LPS (1 mg/kg) and sacrificed 4 hours later. (C) Liver tissue was subjected to microarray analysis as described in Materials and Methods, and the data are presented as a heatmap showing genes with greater than a 2 log-fold change in expression and FDR < 0.05 in comparison to untreated mice. Red indicates genes that were increased in expression; green indicates genes that were decreased in expression. (D) Liver tissue from the indicated mice was subjected to qPCR analysis of the expression of the indicated NF-κB–responsive genes. Data are means ± SD of nine mice per genotype and show the fold-increase in mRNA abundance relative to that in untreated liver samples (E) WT (n = 8) and S534A (n = 10) mice were injected i.v with TNF-α (5 µg/kg) and were sacrificed 4 hours later. Splenic RNA was extracted and subjected to qPCR analysis of the expression of the indicated NF-κB–dependent genes. Data are means ± SD of at least eight mice per genotype (F) WT (n = 14) and S534A (n = 14) mice received whole-body irradiation (12 Gy) and were sacrificed 4 hours later. Liver RNA was extracted and subjected to qPCR analysis of the expression of the indicated NF-κB–dependent genes. Data are means ± SD of fourteen mice per genotye. *P < 0.05, **P < 0.01.

Fig. 3. S534A mice display increased expression of NF-κB–dependent genes and mortality

(A and B) WT (n = 7) and S534A (n = 7) mice were injected i.v. with LPS (1 µg/kg) and sacrificed 4 hours later. (A) Liver tissue was then subjected to microarray analysis. The heatmap shows those genes that were differentially regulated in expression in the livers of LPS-treated S534A mice compared to those in the livers of LPS-treated WT mice (FDR <0.05). (B) Liver tissue from the indicated LPS-treated mice was subjected to qPCR analysis of the expression of the indicated NF-κB–dependent genes. Data are means ± SD of seven mice per genotype. (C) WT and S534A mice were injected i.v. with LPS (1 µg/kg) and then were sacrificed eight hours later. Liver RNA was extracted and subjected to qPCR analysis of the expression of the indicated NF-κB–dependent genes. Data are means ± SD of at least six mice per genotype. (D to F) WT (n = 13) and S534A mice (n = 14) were injected i.v. with LPS (20 mg/kg). (D) Survival was monitored for 96 hours. (E and F) Serum concentrations of TNF-α (E) and IL-1β (F) were determined by ELISA. Data are means ± SD of at least 12 mice per genotype and time point). *P < 0.05, **P < 0.01, ***P < 0.001.

Fig. 4. S534 phosphorylation affects DNA binding and gene expression by NF-κB at late time points through regulation of p65 stability

(A and B) WT and S534A mice were injected i.v. with LPS (20 mg/kg) and sacrificed after the indicated times. (A) Liver tissue was analyzed by immunohistochemistry to monitor the translocation of p65 (red) to the nucleus (blue). (B) The percentages of cells with p65-positive nuclei were quantified. Data are means ± SD of five mice per genotype and time point. (C) Top: HEK 293 cells overexpressing human M2-p65 or M2-S536A-p65 were treated with TNF-α and then subjected to pulse-chase analysis for the indicated times to determine the half-life of p65 protein. Bottom: Data are means ± SD of three independent experiments, each performed in triplicate. (D) Top: WT and S534A MEFs were treated with cycloheximide (30 µg/ml) and then were left unstimulated or were stimulated with IL-1β for the indicated times. Samples were analyzed by Western blotting with antibodies against the indicated proteins. Bottom: The relative abundance of p65 protein, normalized to that of GAPDH, was determined for the indicated times by densitometric analysis. Data are means ± SD of three experiments. (E) Left: NF-κB DNA binding activity was determined in nuclear extracts from the livers of LPS-treated WT and S534A mice (n = 6 mice per genotype and time point). A value of 100 represents the positive control (recombinant human p65). The dashed line indicates NF-κB binding activity at baseline. Right: The competitive oligonucleotide suppressed DNA binding by the positive control. *P < 0.05; n.s., not significant. Data are means ± SD of six mice per genotype.