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The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation.

Zemirli N, Pourcelot M, Dogan N, Vazquez A, Arnoult D - Cell Commun. Signal (2014)

Bottom Line: From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process.However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex.These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR_S 1014, Hôpital Paul Brousse, Villejuif, 94800, France. naima.zemirli@inserm.fr.

ABSTRACT

Background: The nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.

Findings: We performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor-α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.

Conclusions: These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

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Related in: MedlinePlus

Participation of RNF121 in TNFR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 46 transmembrane E3 ubiquitin ligases (2 siRNAs/target) in HEK293T cells. Cells were stimulated with TNFα (10 ng/ml) for 6 hrs and fold activation compared to non-specific (NS) siRNA-treated cells was calculated. Red and green histograms indicate siRNA against RNF121 and TRAF2, respectively. TRAF2 was used as a positive control. (B) Cell extracts from HEK293T cells transfected as in (A) were analyzed by immunoblot as indicated. (C) HEK293T cells transfected with a control non-specific (NS) siRNA or with siRNAs against RNF121 (RNF121 a or b), were also transfected 48 hrs later with an NF-κB reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with TNFα (10 ng/ml) for 6 hrs and then were analyzed by luciferase assay. The results were normalized against Renilla luciferase activity [analysis of variance (ANOVA)]. ns: not significant. RLU, Relative Light Units. Inset: Immunoblotting analysis of the knockdown of RNF121 by the specific siRNAs. (D) NF-κB reporter luciferase assay in HEK293T cells transfected with increasing concentrations (200 or 500 ng) of a Myc-tagged plasmid coding for RNF121 and left unstimulated (left panel) or stimulated with TNFα (1 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (E) NF-κB reporter luciferase assay in HEK293T cells transfected with 200 ng of a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A and left unstimulated (left panel) or stimulated with TNFα (10 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (F) HEK293T cells were transfected with a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A. 24 hrs later, cell extracts were analyzed by immunoblotting. (Ub)n indicates poly-ubiquitylated species.
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Fig1: Participation of RNF121 in TNFR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 46 transmembrane E3 ubiquitin ligases (2 siRNAs/target) in HEK293T cells. Cells were stimulated with TNFα (10 ng/ml) for 6 hrs and fold activation compared to non-specific (NS) siRNA-treated cells was calculated. Red and green histograms indicate siRNA against RNF121 and TRAF2, respectively. TRAF2 was used as a positive control. (B) Cell extracts from HEK293T cells transfected as in (A) were analyzed by immunoblot as indicated. (C) HEK293T cells transfected with a control non-specific (NS) siRNA or with siRNAs against RNF121 (RNF121 a or b), were also transfected 48 hrs later with an NF-κB reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with TNFα (10 ng/ml) for 6 hrs and then were analyzed by luciferase assay. The results were normalized against Renilla luciferase activity [analysis of variance (ANOVA)]. ns: not significant. RLU, Relative Light Units. Inset: Immunoblotting analysis of the knockdown of RNF121 by the specific siRNAs. (D) NF-κB reporter luciferase assay in HEK293T cells transfected with increasing concentrations (200 or 500 ng) of a Myc-tagged plasmid coding for RNF121 and left unstimulated (left panel) or stimulated with TNFα (1 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (E) NF-κB reporter luciferase assay in HEK293T cells transfected with 200 ng of a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A and left unstimulated (left panel) or stimulated with TNFα (10 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (F) HEK293T cells were transfected with a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A. 24 hrs later, cell extracts were analyzed by immunoblotting. (Ub)n indicates poly-ubiquitylated species.

Mentions: As we recently reported an accumulation of the ubiquitylated transmitters leading to NF-κB activation to the endomembrane fraction [8], we set up a siRNA screen with two oligoribonucleotides against each of the 46 membrane spanning E3 ubiquitin ligases [9] (Additional file 1) to uncover new regulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. Indeed, engagement of TNFR promotes a rapid NF-κB activation through the recruitment to the receptor of the adaptor protein TRADD together with the E3 ubiquitin ligases c-IAPs and TRAF2, which are responsible for catalyzing the polyubiquitination of the kinase RIP1 that acts as a specific ubiquitylated transmitter for this pathway [10]. The impact of the knock down of the 46 membrane spanning E3 ubiquitin ligases was assessed in a gene reported assay and silencing of the key regulator of TNFR-mediated NF-κB TRAF2 was used as a control (Figure 1A, B; see Additional file 2 for detailed Methods description). Among the top hits was RNF121 (Figure 1A). RNF121 is part of a chromosomal band (11q13) that may contain a high penetrance gene for breast cancer [11]. We then used two additional siRNA sequences against RNF121 and confirmed that TNFR-mediated NF-κB activation was decreased, further validating the results from our initial screen (Figure 1C). Interestingly, the enforced expression of RNF121 activated NF-κB and NF-κB activation following TNFR stimulation was potentiated in a dose dependent-manner (Figure 1D). RNF121 specifically triggered NF-κB activation because it did not stimulate the expression of IFNβ-, ISRE-, NFAT-, AP1- or p53-dependent reporter genes (data not shown). The ability of RNF121 to activate NF-κB was dependent on the catalytic activity of its RING domain, because the RNF121C226-229A mutant gave significantly lower levels of NF-κB activation (Figure 1E). Based on the available RING finger protein structure [12,13], the replacement of the cys-226 and 229 residues with an alanine was predicted to prevent Zn2+ coordination, thereby impeding the overall function of the RING domain as assessed by the level of auto-ubiquitination (Figure 1F).Figure 1


The E3 ubiquitin ligase RNF121 is a positive regulator of NF-κB activation.

Zemirli N, Pourcelot M, Dogan N, Vazquez A, Arnoult D - Cell Commun. Signal (2014)

Participation of RNF121 in TNFR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 46 transmembrane E3 ubiquitin ligases (2 siRNAs/target) in HEK293T cells. Cells were stimulated with TNFα (10 ng/ml) for 6 hrs and fold activation compared to non-specific (NS) siRNA-treated cells was calculated. Red and green histograms indicate siRNA against RNF121 and TRAF2, respectively. TRAF2 was used as a positive control. (B) Cell extracts from HEK293T cells transfected as in (A) were analyzed by immunoblot as indicated. (C) HEK293T cells transfected with a control non-specific (NS) siRNA or with siRNAs against RNF121 (RNF121 a or b), were also transfected 48 hrs later with an NF-κB reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with TNFα (10 ng/ml) for 6 hrs and then were analyzed by luciferase assay. The results were normalized against Renilla luciferase activity [analysis of variance (ANOVA)]. ns: not significant. RLU, Relative Light Units. Inset: Immunoblotting analysis of the knockdown of RNF121 by the specific siRNAs. (D) NF-κB reporter luciferase assay in HEK293T cells transfected with increasing concentrations (200 or 500 ng) of a Myc-tagged plasmid coding for RNF121 and left unstimulated (left panel) or stimulated with TNFα (1 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (E) NF-κB reporter luciferase assay in HEK293T cells transfected with 200 ng of a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A and left unstimulated (left panel) or stimulated with TNFα (10 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (F) HEK293T cells were transfected with a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A. 24 hrs later, cell extracts were analyzed by immunoblotting. (Ub)n indicates poly-ubiquitylated species.
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Fig1: Participation of RNF121 in TNFR-mediated NF-κB activation. (A) NF-κB reporter luciferase assay screen of a siRNA library targeting 46 transmembrane E3 ubiquitin ligases (2 siRNAs/target) in HEK293T cells. Cells were stimulated with TNFα (10 ng/ml) for 6 hrs and fold activation compared to non-specific (NS) siRNA-treated cells was calculated. Red and green histograms indicate siRNA against RNF121 and TRAF2, respectively. TRAF2 was used as a positive control. (B) Cell extracts from HEK293T cells transfected as in (A) were analyzed by immunoblot as indicated. (C) HEK293T cells transfected with a control non-specific (NS) siRNA or with siRNAs against RNF121 (RNF121 a or b), were also transfected 48 hrs later with an NF-κB reporter. 24 hrs later, the cells were either left unstimulated or were stimulated with TNFα (10 ng/ml) for 6 hrs and then were analyzed by luciferase assay. The results were normalized against Renilla luciferase activity [analysis of variance (ANOVA)]. ns: not significant. RLU, Relative Light Units. Inset: Immunoblotting analysis of the knockdown of RNF121 by the specific siRNAs. (D) NF-κB reporter luciferase assay in HEK293T cells transfected with increasing concentrations (200 or 500 ng) of a Myc-tagged plasmid coding for RNF121 and left unstimulated (left panel) or stimulated with TNFα (1 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (E) NF-κB reporter luciferase assay in HEK293T cells transfected with 200 ng of a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A and left unstimulated (left panel) or stimulated with TNFα (10 ng/ml) for 6 hrs (right panel) [analysis of variance (Student’s t-tests)]. (F) HEK293T cells were transfected with a Myc-tagged plasmid coding for RNF121 or for the mutant RNF121C226-229A. 24 hrs later, cell extracts were analyzed by immunoblotting. (Ub)n indicates poly-ubiquitylated species.
Mentions: As we recently reported an accumulation of the ubiquitylated transmitters leading to NF-κB activation to the endomembrane fraction [8], we set up a siRNA screen with two oligoribonucleotides against each of the 46 membrane spanning E3 ubiquitin ligases [9] (Additional file 1) to uncover new regulators of NF-κB activation, using tumor necrosis factor–α (TNF-α) receptor (TNFR) stimulation as a model. Indeed, engagement of TNFR promotes a rapid NF-κB activation through the recruitment to the receptor of the adaptor protein TRADD together with the E3 ubiquitin ligases c-IAPs and TRAF2, which are responsible for catalyzing the polyubiquitination of the kinase RIP1 that acts as a specific ubiquitylated transmitter for this pathway [10]. The impact of the knock down of the 46 membrane spanning E3 ubiquitin ligases was assessed in a gene reported assay and silencing of the key regulator of TNFR-mediated NF-κB TRAF2 was used as a control (Figure 1A, B; see Additional file 2 for detailed Methods description). Among the top hits was RNF121 (Figure 1A). RNF121 is part of a chromosomal band (11q13) that may contain a high penetrance gene for breast cancer [11]. We then used two additional siRNA sequences against RNF121 and confirmed that TNFR-mediated NF-κB activation was decreased, further validating the results from our initial screen (Figure 1C). Interestingly, the enforced expression of RNF121 activated NF-κB and NF-κB activation following TNFR stimulation was potentiated in a dose dependent-manner (Figure 1D). RNF121 specifically triggered NF-κB activation because it did not stimulate the expression of IFNβ-, ISRE-, NFAT-, AP1- or p53-dependent reporter genes (data not shown). The ability of RNF121 to activate NF-κB was dependent on the catalytic activity of its RING domain, because the RNF121C226-229A mutant gave significantly lower levels of NF-κB activation (Figure 1E). Based on the available RING finger protein structure [12,13], the replacement of the cys-226 and 229 residues with an alanine was predicted to prevent Zn2+ coordination, thereby impeding the overall function of the RING domain as assessed by the level of auto-ubiquitination (Figure 1F).Figure 1

Bottom Line: From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process.However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex.These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

View Article: PubMed Central - PubMed

Affiliation: INSERM, UMR_S 1014, Hôpital Paul Brousse, Villejuif, 94800, France. naima.zemirli@inserm.fr.

ABSTRACT

Background: The nuclear factor κB (NF-κB) family members regulate several biological processes as cell proliferation and differentiation, inflammation, immunity and tumor progression. Ubiquitination plays a key role in NF-κB activation and the ubiquitylated transmitters of the NF-κB signaling cascade accumulate in close proximity to endomembranes.

Findings: We performed an unbiased siRNA library screen targeting the 46 E3 ubiquitin ligases bearing transmembrane domains to uncover new modulators of NF-κB activation, using tumor necrosis factor-α (TNF-α) receptor (TNFR) stimulation as a model. We report here the identification of a new Golgi Apparatus-resident protein, RNF121, as an enhancer of NF-κB promoter activity through the catalytic function of its RING domain. From a molecular standpoint, while knocking down RNF121 did not alter RIP1 ubiquitination and IKK activation, the proteasomal degradation of IκBα was impaired suggesting that this E3 ubiquitin ligase regulates this process. However, RNF121 did not directly ubiquitinate IκBα While they were found in the same complex. Finally, we discovered that RNF121 acts as a broad regulator of NF-κB signaling since its silencing also dampens NF-κB activation following stimulation of Toll-Like Receptors (TLRs), Nod-Like Receptors (NLRs), RIG-I-Like Receptors (RLRs) or after DNA damages.

Conclusions: These results unveil an unexpected role of Golgi Apparatus and reveal RNF121 as a new player involved in the signaling leading to NF-κB activation.

Show MeSH
Related in: MedlinePlus