The ubiquitin ligase HERC3 attenuates NF-κB-dependent transcription independently of its enzymatic activity by delivering the RelA subunit for degradation

Abstract

Activation of NF-κB-dependent transcription represents an important hallmark of inflammation. While the acute inflammatory response is per se beneficial, it can become deleterious if its spatial and temporal profile is not tightly controlled. Classically, NF-κB activity is limited by cytoplasmic retention of the NF-κB dimer through binding to inhibitory IκB proteins. However, increasing evidence suggests that NF-κB activity can also be efficiently contained by direct ubiquitination of NF-κB subunits. Here, we identify the HECT-domain ubiquitin ligase HERC3 as novel negative regulator of NF-κB activity. We find that HERC3 restricts NF-κB nuclear import and DNA binding without affecting IκBα degradation. Instead HERC3 indirectly binds to the NF-κB RelA subunit after liberation from IκBα inhibitor leading to its ubiquitination and protein destabilization. Remarkably, the regulation of RelA activity by HERC3 is independent of its inherent ubiquitin ligase activity. Rather, we show that HERC3 and RelA are part of a multi-protein complex containing the proteasome as well as the ubiquitin-like protein ubiquilin-1 (UBQLN1). We present evidence that HERC3 and UBQLN1 provide a link between NF-κB RelA and the 26S proteasome, thereby facilitating RelA protein degradation. Our findings establish HERC3 as novel candidate regulating the inflammatory response initiated by NF-κB.

Figure 1.

HERC3 suppresses NF-κB transcriptional activity. (A) BAEC and RelA^−/− 3T3 were transfected with κB-luciferase reporter (200 ng) and CMV-β-galactosidase control (40 ng) plasmids together with RelA (20 ng) and increasing amounts of HERC3 (BAEC: 80, 160 and 320 ng; RelA^−/- 3T3: 35, 70 and 140 ng). Luciferase activities were measured 3 days after transfection and normalized to β-galactosidase activities. Bars represent mean + SEM (n = 9, derived from 3 independent experiments). RelA-induced reporter activity was set to 100 and activity of all other data sets was calculated relative to this value. (B) Assay was carried out in RelA^−/− 3T3, transfected as in (A) with different NF-κB decameric consensus binding sites and constant amounts of HERC3. Sites were derived from the human IL8, ICAM1, IκBα and MHC class I enhancer sequences. (C) BAEC were transfected with reporter plasmids, control vector or HERC3, and either left untreated or 2 days later stimulated with TNF for 16 h. Bars represent mean + SEM (n = 12, derived from four independent experiments). TNF-induced reporter activity without HERC3 co-expression was set to 100. (D) BAEC transiently transfected with control, RelA or RelA plus HERC3 were harvested, RNA was isolated and expression levels of endogenous Icam1, Vcam1, Sele and IκBα were evaluated by qRT-PCR. Expression levels of control-transfected cells were set to 1, and relative induction for all other experimental groups was calculated. Bars represent mean + SEM (n = 5, derived from 5 independent experiments). In all experiments appropriate protein expression was monitored by Western Blotting (see Supplementary Figure S1A–D). Data were considered significant at *P < 0.05. RLU, relative luciferase units.

Figure 2.

HERC3 reduces NF-κB DNA-binding by limiting its nuclear import. (A) BAEC were transfected with control vector or myc-HERC3, stimulated for 0, 0.5 or 1 h with TNF or TNF/LMB and processed for immunofluorescence. Nuclei were stained with DAPI. Bar represents 10 μm. The graph shows ratios of cytosolic and nuclear fluorescence obtained by automatic quantification of RelA compared to DAPI staining (n = 61–147 cells, derived from 2–3 experiments). Values N/C > 1 indicate predominantly nuclear RelA. (B) Subcellular localization of GFP-LacZ harboring a SV40-derived nuclear localization sequence (NLS) was tested in absence and presence of myc-HERC3 in BAEC. GFP-LacZ, not containing an active NLS, was used as control. DAPI staining served as nuclear reference. Size bar = 10 μm. Data from 18–33 cells for each condition, derived from two independent experiments, were quantified. Percent of nuclear GFP staining was: for GFP-LacZ –HERC3 0 ± 0; +HERC3 0.12 ± 0.02, and for GFP-SV40NLS-LacZ –HERC3 100 ± 0; +HERC3 100 ± 0. (C) RelA DNA-binding in absence and presence of HERC3 was observed by electrophoretic mobility shift assay (EMSA). Total cell extracts from transfected BAEC were incubated with double-stranded purified γ-³²P ATP-labeled Igκ light chain enhancer oligonucleotide. Protein–DNA complexes were separated on 5% Tris/glycine/EDTA-PAGE, bands were visualized by autoradiography and results from three experiments were quantified. Equal RelA protein expression with and without HERC3 was ensured by parallel observation of protein levels by Western Blotting (see Supplementary Figure S1E). (D) RelA binding was verified by competition with either non-labeled sense oligonucleotide (co Ig κB) or non-labeled scrambled oligonucleotide (co sc Ig κB), and by super-shift with RelA antibody. Experiments were performed in triplicates. The arrows in (C) and (D) indicate the specific NF-κB band. Values were considered significant at *P < 0.05. h, hours.

Figure 3.

HERC3 has no effect on IκBα degradation. (A) IκBα turnover was monitored in HEK293T cells transfected with empty vector, myc-HERC3 or flag-dnIKK2 after stimulation with 10 ng/ml TNF for 0, 20 or 60 min. Where indicated cells were pre-treated with 50 μM MG132. Presence of transfected proteins was verified by immunoblotting with myc- or flag-specific antibodies. (B) HEK293T cells were transfected with HA-IκBα together with either empty vector or myc-HERC3. Cells were treated with 10 ng/ml TNF for 20 min and where indicated pre-treated for 3 h with 50 μM MG132. IκBα was pulled down from cell lysates with HA-coupled agarose and its ubiquitination was assessed by detection with an anti-ubiquitin antibody. All experiments were carried out in triplicates. HC, heavy chain; IB, immunoblot; IP, immunoprecipitation; min, minutes.

Figure 4.

HERC3 interacts with RelA and mediates its ubiquitination. (A) RelA was precipitated with RelA antibody from total HEK293T cell lysates transfected with myc-RelA and myc-HERC3. RelA and HERC3 co-precipitation was assessed by Western Blotting with anti-myc antibody. (B) HEK293T cells were transfected with HA-RelA and myc-HERC3. HERC3 was pulled down from cell lysates with myc-specific antibody and RelA was detected in the precipitate with anti-RelA antibody. (C) HEK293T were transfected with myc-RelA, his-ubiquitin and myc-HERC3. Ubiquitinated RelA was detected by pull down of his-ubiquitin under denaturing conditions, followed by Western Blotting with RelA-specific antibody. (D) BAEC were transfected with his-ubiquitin and myc-HERC3. After 24 h cells were treated with TNF for 30 min and assay was performed as described in (C). All experiments were carried out in triplicates. IB, immunoblot; IP, immunoprecipitation.

Figure 5.

HERC3 mediates RelA K48 ubiquitination and protein destabilization. (A) His-ubiquitin, myc-RelA, with or without myc-HERC3 were transiently introduced into HEK293T cells. RelA was precipitated from whole cell lysates with RelA-specific antibody and the nature of RelA ubiquitination was examined by Western Blotting with anti-ubiquitin antibodies recognizing total, K48 or K63 ubiquitin, respectively. (B) HEK293T cells were transfected with myc-RelA with or without myc-HERC3. Twenty four hours after transfection, cells were pulse-labeled with [³⁵S]-methionine for 1 h and chased for 0, 2, 4 and 8 h. RelA was precipitated from total cell extracts with myc-affinity agarose, and protein levels were detected by autoradiography. (C) RelA was precipitated from transfected BAEC total cell extracts after pulse-labeling with [³⁵S]-methionine for 1 h and chasing for 0 and 8 h. Protein levels in precipitates were assessed by separation on SDS-PAGE and autoradiography. Average percent protein remaining before and after chase is indicated. All experiments were carried out at least in triplicates. h, hours; IB, immunoblot; IP, immunoprecipitation.

Figure 6.

HERC3 affects RelA ubiquitination independently of its catalytic domains as part of a larger molecular weight complex. (A) Schematic representation of HERC3 constructs used in (B and C). (B) Transcriptional activity of RelA was examined in presence of different HERC3 constructs as described in Figure 1A. Bars represent mean + SEM (n = 9–18, derived from 3–6 independent experiments). Values were significant at *P < 0.05. (C) HEK293T cells were transfected with his-ubiquitin, myc-RelA and indicated myc-HERC3 wild type, mutant or truncation constructs. Ubiquitinated RelA was pulled down with Ni-agarose beads bound to his-tagged ubiquitin and detected in immunoblotting with a RelA-specific antibody. (D) HEK293T cells were transfected with flag-RelA alone, flag-RelA and myc-HERC3 or flag-HERC3 alone. Lysates were subjected to precipitation with flag-agarose beads, followed by flag-peptide elution. Eluates were loaded on 3–12% Native Bis-Tris Gels and separated by Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE). (E) HEK293T cell transfection, lysate precipitation and elution were performed as outlined in (D). Eluates were loaded on a Sephacryl S-300 HiPrep 16/60 column for size exclusion chromatography (SEC). Thyroglobulin, aldolase and ovalbumin with calculated relative molecular masses (Mr) of 669, 158 and 44, respectively, served as molecular weight indication. All experiments were carried out in triplicates. aa, amino acids; CA, cysteine to alanine; ev, empty vector; FR, fraction; IB, immunoblot; IP, immunoprecipitation; Mr, relative molecular mass; RLU, relative luciferase units; V₀, column void volume; wt, wild type.

Figure 7.

HERC3/RelA associate with the ubiquitin-like protein UBQLN1 and the 26S proteasome. (A) List of ubiquitination/degradation-associated proteins found by MS/MS analysis to bind to HERC3/RelA. (B) HEK293T cells transfected with indicated constructs were lysed and immunoprecipitation was performed with myc-specific antibody to pull down HERC3. RelA, PSMC2 and PSMA4 co-association was detected with specific antibodies recognizing these proteins. (C) HEK293T cells were transfected with control, myc-RelA, myc-HERC3 or myc-RelA/myc-HERC3 together. The endogenous proteasomal subunit PSMD4 was precipitated with PSMD4 antibody and association of RelA and HERC3 was tested in Western Blotting with respective antibodies. (D) HA-RelA, flag-tagged UBQLN1 and myc-HERC3 were introduced into HEK293T cells. Association of HERC3 and RelA, as well as the proteasomal subunit PSMC2 with UBQLN1 was tested by immunoprecipitation of UBQLN1 with flag-beads, followed by Western Blot detection. All experiments were performed 3 times. The asterisks mark the heavy chain detected by Western Blotting after immunoprecipitation. IB, immunoblot; IP, immunoprecipitation.

Figure 8.

HERC3 and UBQLN1 conjointly affect NF-κB activity by linking RelA to the proteasome and promoting its degradation. (A) UBQLN1 expression was knocked down by siRNA transfection into HEK293T cells. At the same time myc-HERC3 and HA-RelA were introduced and interaction between these two proteins was studied by co-immunoprecipitation approach. Association of endogenous proteasomal subunits PSMC2 and PSMA4 was also tested. The asterisks point out the antibody heavy chains detected by Western Blotting after pull down. (B) HEK293T were transfected with siRNA targeting UBQLN1, myc-HERC3 and myc-RelA. Binding of proteins to RelA was examined by pull down with anti-RelA antibody and detection of HERC3 and PSMC2 by Western Blotting. (C) Stability of RelA protein in presence of HERC3 and absence of UBQLN1 was determined in HEK293T by metabolic labeling as described in Figure 5B. Results from 4 independent experiments were quantified. Average percent of protein before and after chase is noted on top of the respective graph columns. (D) Endogenous RelA protein stability was assessed in HUVEC exposed to siRNA targeting HERC3 and/or UBQLN1. Non-targeting siRNA was used as control. HUVEC were incubated in [³⁵S]-methionine-containing growth medium for 2 h on day 3 after siRNA exposure. Pulse medium was removed and cells were left in culture for another 7 h in normal growth medium substituted with 10 ng/ml TNF. HUVEC were harvested and RelA was precipitated from total cell extracts with RelA-specific antibody. Data from four experiments were plotted on a graph. Average percent protein remaining before and after chase is indicated. (E) HUVEC transfected with non-targeting control or Herc3/Ubqln1-targeting siRNAs were stimulated with 10 ng/ml TNF for 0, 0.5 and 6 h. Protein levels of endogenous RelA, HERC3, UBQLN1 and β-actin were determined. Shown blots are representative of 2 independent experiments. (F) mRNA expression of three NF-κB-regulated genes Icam1, Vcam1 and Sele was examined in siRNA-treated HUVEC. Three days after siRNA exposure NF-κB-dependent transcription was stimulated with TNF for 6 and 16 h. RNA was isolated and gene expression was assessed by qRT-PCR. HERC3 and UBQLN1 knock down efficiency was tested in un-stimulated cells and plotted in the first two panels. Data were normalized to each group and represent mean +SEM from 5–9 independent experiments. Values were considered significant at *P < 0.05. IB, immunoblot; IP, immunoprecipitation; si, small interfering RNA.