Research ArticleImmunology

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

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Science Signaling  23 Aug 2016:
Vol. 9, Issue 442, pp. ra85
DOI: 10.1126/scisignal.aab2820
  • 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 are 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 intravenously 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 intravenously 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 intravenously 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 messenger RNA (mRNA) abundance relative to that in untreated liver samples. (E) WT (n = 8) and S534A (n = 10) mice were injected intravenously 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 grays (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 14 mice per genotype. *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 intravenously 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 intravenously with LPS (1 μg/kg) and then were sacrificed 8 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 intravenously with LPS (20 mg/kg). (D) Survival was monitored for 96 hours. (E and F) Serum concentrations of TNF-α (E) and interleukin-1β (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; nd, not detected; ns, not significant.

  • 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 intravenously 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. AU, arbitrary units. (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 glyceraldehyde-3-phosphate dehydrogenase (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 (Pos ctrl) (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. Data are means ± SD of six mice per genotype.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/442/ra85/DC1

    Fig. S1. Phenotypic characterization of S534A knock-in mice.

    Fig. S2. Determination of NF-κB activation in MEFs, spleens, and livers from wild-type and S534A mice.

    Fig. S3. Analysis of gene expression in the livers of wild-type and S534A mice injected with low-dose LPS.

    Fig. S4. Expression of NF-κB–dependent genes in the spleens of wild-type and S534A mice at early and late time points after injection with low-dose LPS.

    Fig. S5. Analysis of p65 protein abundance and NF-κB target gene expression in macrophages from wild-type and S534A mice.

    Fig. S6. IKK phosphorylates p65 at multiple sites.

    Table S1. Complete blood counts in wild-type and S534A mice.

    Table S2. Hepatic gene expression profiles in untreated wild-type and S534A mice.

    Table S3. Hepatic gene expression profiles in wild-type and S534A mice treated with high-dose LPS.

    Table S4. Hepatic gene expression profiles in wild-type and S534A mice treated with low-dose LPS.

  • Supplementary Materials for:

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

    Jean-Philippe Pradère, Céline Hernandez, Christiane Koppe, Richard A. Friedman, Tom Luedde, Robert F. Schwabe*

    *Corresponding author. Email: rfs2102{at}cumc.columbia.edu

    This PDF file includes:

    • Fig. S1. Phenotypic characterization of S534A knock-in mice.
    • Fig. S2. Determination of NF-κB activation in MEFs, spleens, and livers from wildtype and S534A mice.
    • Fig. S3. Analysis of gene expression in the livers of wild-type and S534A mice injected with low-dose LPS.
    • Fig. S4. Expression of NF-κB–dependent genes in the spleens of wild-type and S534A mice at early and late time points after injection with low-dose LPS.
    • Fig. S5. Analysis of p65 protein abundance and NF-κB target gene expression in macrophages from wild-type and S534A mice.
    • Fig. S6. IKK phosphorylates p65 at multiple sites.
    • Table S1. Complete blood counts in wild-type and S534A mice.

    [Download PDF]

    Technical Details

    Format: Adobe Acrobat PDF

    Size: 1.1 MB

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). Hepatic gene expression profiles in untreated wild-type and S534A mice.
    • Table S3 (Microsoft Excel format). Hepatic gene expression profiles in wild-type and S534A mice treated with high-dose LPS.
    • Table S4 (Microsoft Excel format). Hepatic gene expression profiles in wild-type and S534A mice treated with low-dose LPS.

    [Download Tables S2 to S4]


    Citation: J.-P. Pradère, C. Hernandez, C. Koppe, R. A. Friedman, T. Luedde, R. F. Schwabe, Negative regulation of NF-κB p65 activity by serine 536 phosphorylation. Sci. Signal. 9, ra85 (2016).

    © 2016 American Association for the Advancement of Science

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