Research ArticleBiochemistry

The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100

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Science Signaling  27 Sep 2016:
Vol. 9, Issue 447, pp. ra96
DOI: 10.1126/scisignal.aad9413
  • Fig. 1 RelB inhibits p100 degradation.

    (A) Top: WT MEFs, Relb−/− MEFs, and Relb−/− MEFs reconstituted with WT relb [relb WT transgenic (Tg)] were stimulated with α-LTβR for the indicated times. Samples were then analyzed by Western blotting [immunoblotting (IB)] with antibodies against the indicated proteins to monitor the processing of p100 to generate p52. β-Actin was used as a loading control. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (B) Top: WT MEFs were left untreated, whereas Relb−/− MEFs were treated with 10 μM MG132 for the indicated times before the samples were analyzed by Western blotting with an antibody against p100. β-Actin served as a loading control. Bottom: Densitometric analysis of the intensity of the p100 band, normalized to that of β-actin, for each sample. Data are means ± SEM of three experiments. (C) RelB blocks p100 processing in human embryonic kidney (HEK) 293T cells. Top: HEK 293T cells were transiently transfected with the indicated combinations of plasmids encoding Flag-p100, green fluorescent protein (GFP)–RelB, and hemagglutinin (HA)–NIK. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated targets. β-Actin served as a loading control. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (D) RelA plays no role in p100 degradation. Top: HEK 293T cells were transiently transfected with the indicated combinations of plasmids encoding Flag-RelA, GFP-RelB, Flag-p100, and HA-NIK. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated targets. β-Actin served as a loading control. Western blots are representative of four experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of four independent experiments.

  • Fig. 2 RelB competes with NIK and IKK1 for binding to p100.

    (A) Top: HEK 293T cells transiently transfected to express the indicated combinations of constructs were subjected to immunoprecipitation (IP) of Flag-p100 with anti-Flag M2 agarose beads. Samples were then analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. Bottom: Densitometric analysis of the ratio of p52 abundance to p100 abundance for the indicated samples. Data are means ± SEM of three independent experiments. (B) Top: HEK 293T cells were transiently transfected with plasmids encoding Flag-p100 and HA-NIK and then subjected to immunoprecipitation with anti-Flag M2 agarose beads. Another set of HEK 293T cells was transfected with two different amounts of plasmids encoding GFP-RelB or GFP-RelA, and lysates of these cells were incubated with beads bound to the Flag-p100:NIK complex for 30 min. Cell lysates (Input) and immunoprecipitated samples were analyzed by Western blotting with antibodies against the indicated proteins. Western blots are representative of three experiments. Bottom: Densitometric analysis of the abundance of NIK normalized to that of p100 for the indicated samples. Data are means ± SEM of three independent experiments. (C) WT MEFs, Relb−/− MEFs, and Relb−/− MEFs reconstituted with WT relb were treated with α-LTβR for the indicated times. Cell lysates were analyzed by Western blotting to assess NIK abundance. β-Actin served as a loading control. Western blots are representative of three experiments. (D) HEK 293T cells were transfected with the indicated combinations of plasmids encoding full-length HA-tagged NIK, an HA-tagged N-terminal truncated NIK NIKΔN324, Flag-p100, and GFP-RelB. Forty-eight hours later, the cells were lysed, and cell lysates were analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. (E) HEK 293T cells were left untreated or were treated with the indicated concentrations of the IKK inhibitor XII before being transfected with plasmids encoding Flag-p100 and HA-NIK. Twenty-four hours later, the cells were analyzed by Western blotting with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments. (F) RelB blocks the IKK1-stimulated processing of p100 in HEK 293T cells. HEK 293T cells were transiently transfected with the indicated combination of plasmids encoding Flag-p100, HA-IKK1, and GFP-RelB. Forty-eight hours later, the cells were lysed and analyzed by Western blotting with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments. (G) A model depicting competition between the NIK:IKK1 complex and RelB for binding to p100 and its processing. The NIK:IKK1 complex induces the processing of p100, whereas RelB displaces the NIK:IKK1 complex that is bound to p100 to prevent its processing. The model also depicts the reduction in the abundance of free NIK (p100-unbound) by IKK1.

  • Fig. 3 RelB favors p100-kappaBsome formation.

    (A) Top: HEK 293T cells were transfected with the indicated combinations of plasmids encoding Flag-p100, GFP-RelB, HA-RelA, and Myc-cRel. Forty-eight hours later, cell extracts were fractionated by size-exclusion chromatography (Superose 6), and the fractions were analyzed by Western blotting (IB) with an antibody against p100. Boxes denote the fractions with maximal p100 elution. Western blots are representative of three experiments. Bottom: Densitometric analysis of the abundance of p100 for the indicated samples. Data are representative of three independent experiments. (B) Extracts of HEK 293T cells transfected with the indicated combinations of plasmids encoding Flag-p100, HA-RelA, and Myc-cRel were fractionated as described in (A) and analyzed by Western blotting with antibodies against the indicated proteins. Rectangles denote the fractions with maximal amounts of p100. (C) WT MEFs were left unstimulated, were stimulated with α-LTβR for 12 hours alone, or were washed after the stimulation and incubated for a further 12 hours. Cell extracts were then subjected to immunoprecipitation with anti-p100 antibody and Western blot (IB) analysis with antibodies against the indicated proteins. Western blots are representative of three experiments. (D) A model depicting how RelB protects p100 from processing during stimulation. This model is a continuation of the model shown in Fig. 2G. The p100:RelB complex further associates with other NF-κB subunits to form the kappaBsome. Together, the models shown here and in Fig. 2G depict the opposing events that occur during noncanonical signaling.

  • Fig. 4 RelB protects p100 from complete degradation.

    (A) HEK 293T cells were transiently transfected with the indicated combination of plasmids encoding Flag-p100, HA-tagged NIK-WT or NIK-inactive mutant, and GFP-RelB. Forty-eight hours later, the cells were left untreated or were treated with CHX for the indicated times before cell extracts were analyzed by Western blotting with antibodies against the indicated targets. Western blots are representative of three experiments. (B) Relb−/− MEFs reconstituted with either WT RelB or the RelB Y300A mutant were left unstimulated or were stimulated with α-LTβR for the indicated times. The processing of p100 was monitored by Western blot analysis with antibodies against the indicated proteins. β-Actin served as a loading control. Western blots are representative of three experiments.

  • Fig. 5 p100, NIK:IKK1, and RelB form a transitional complex.

    (A) WT MEFs were left untreated or were stimulated with α-LTβR for 8 hours. The cells were also treated with 10 μM MG132 for 1 hour before they were lysed and subjected to immunoprecipitation with antibodies against p100, NIK, or RelB or with immunoglobulin G (IgG) as a control. Lysates (Input) and immunoprecipitated samples were analyzed by Western blotting (IB) with antibodies against the indicated proteins. Western blots are representative of three experiments. (B) WT, Relb−/−, and p100−/− MEFs were left untreated or were stimulated with α-LTβR for 8 hours. Cell lysates were subjected to immunoprecipitation with antibody against p100, and lysates (Input) and immunoprecipitated samples were analyzed by Western blotting (IB) with antibodies against the indicated proteins. Western blots are representative of three experiments. (C) A model showing the generation of either p52 or kappaBsomes from p100 in a single transient complex. In the transient complex, p100, NIK:IKK1, and RelB remain associated in a competing manner. If RelB causes the displacement of the NIK:IKK1 complex before p100 is phosphorylated by these kinases, then kappaBsomes will be formed. On the other hand, if p100 is phosphorylated while RelB remains bound to p100 (perhaps in a different conformation), this will result in the generation of the p52:RelB heterodimer. This model is an extension of the models shown in Figs. 2G and 3D.

Supplementary Materials

  • www.sciencesignaling.org/cgi/content/full/9/447/ra96/DC1

    Fig. S1. Aberrant subcellular distributions of p52 and p100 in Relb−/− cells.

    Fig. S2. NIK and RelB compete for binding to p100.

    Fig. S3. RelB stimulates kappaBsome formation.

    Fig. S4. RelB protects p100 from complete degradation.

    Fig. S5. RelB, NIK:IKK1, and p100 form a multiprotein complex.

  • Supplementary Materials for:

    The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100

    Amanda J. Fusco, Anup Mazumder, Vivien Ya-Fan Wang, Zhihua Tao, Carl Ware, Gourisankar Ghosh*

    *Corresponding author. Email: gghosh{at}ucsd.edu

    This PDF file includes:

    • Fig. S1. Aberrant subcellular distributions of p52 and p100 in Relb−/− cells.
    • Fig. S2. NIK and RelB compete for binding to p100.
    • Fig. S3. RelB stimulates kappaBsome formation.
    • Fig. S4. RelB protects p100 from complete degradation.
    • Fig. S5. RelB, NIK:IKK1, and p100 form a multiprotein complex.

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    Citation: A. J. Fusco, A. Mazumder, V. Y.-F. Wang, Z. Tao, C. Ware, G. Ghosh, The NF-κB subunit RelB controls p100 processing by competing with the kinases NIK and IKK1 for binding to p100m. Sci. Signal. 9, ra96 (2016).

    © 2016 American Association for the Advancement of Science

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