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. 2009 Mar 4;28(5):513-22.
doi: 10.1038/emboj.2008.285. Epub 2009 Jan 8.

The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling

Noula Shembade  1 , Kislay Parvatiyar, Nicole S Harhaj, Edward W Harhaj
Affiliations

The ubiquitin-editing enzyme A20 requires RNF11 to downregulate NF-kappaB signalling

Noula Shembade et al. EMBO J. .

Abstract

The RING domain protein RNF11 is overexpressed in breast cancers and promotes tumour growth factor-beta (TGF-beta) signalling. RNF11 has been proposed to regulate TGF-beta signalling by interacting with HECT- and SCF-type E3 ligases; however, the role of RNF11 in other signalling pathways is poorly understood. Here, we demonstrate a novel function of RNF11 as a negative regulator of NF-kappaB and jun N-terminal kinase (JNK) signalling pathways. Knockdown of RNF11 with siRNA resulted in persistent tumour necrosis factor (TNF)- and lipopolysaccharide (LPS)-mediated NF-kappaB and JNK signalling. RNF11 interacted with the NF-kappaB inhibitor A20 and its regulatory protein TAX1BP1 in a stimulus-dependent manner. RNF11 negatively regulated RIP1 and TRAF6 ubiquitination upon stimulation with TNF and LPS, respectively. Furthermore, RNF11 was required for A20 to interact with and inactivate RIP1 to inhibit TNF-mediated NF-kappaB activation. Our studies reveal that RNF11, together with TAX1BP1 and Itch, is an essential component of an A20 ubiquitin-editing protein complex that ensures transient activation of inflammatory signalling pathways.

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Figures

Figure 1
Figure 1
Dynamic interactions between RNF11 and the A20 ubiquitin-editing complex. (A) 293T cells were transfected with plasmids encoding Flag-tagged TAX1BP1, A20 and STAT1 in the absence or presence of Myc–RNF11. After 36 h, lysates were immunoprecipitated with anti-Myc followed by immunoblotting with anti-Flag. Lysates were examined for the expression of ectopic proteins by immunoblotting with anti-Flag and Myc. IgH, immunoglobulin heavy chain. (B) MEFs were untreated or treated with TNF (10 ng/ml) for 30 min. Cells were lysed in RIPA buffer and immunoprecipitated with anti-RNF11 or isotype control immunoglobulin (control Ig) and immunoblotted with anti-TAX1BP1, A20, RIP1 and RNF11. (C) BMDMs or (D) MEFs were untreated or treated with TNF (10 ng/ml) for the indicated times. Lysates were immunoprecipitated with anti-RNF11 or control Ig and immunoblotted with anti-TAX1BP1, A20 and RIP1.
Figure 2
Figure 2
RNF11 specifically inhibits NF-κB activation. (A) THP-1 cells were transfected on day 1 with no siRNA (mock), control scrambled siRNA or RNF11 siRNA. On day 2, the same cells were transfected with pRL-tk internal control Renilla luciferase plasmid, κB-TATA Luc and Myc–RNF11 as indicated. After an additional 2 days, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way analysis of variance (ANOVA), followed by the Tukey–Kramer test for multiple comparisons (*P<0.001 compared with control TNF-treated samples). (B) THP-1 cells were transfected as described in (A) but NFAT-Luc was included in lieu of NF-κB Luc. After an additional 2 days, cells were treated with PMA (10 ng/ml) and ionomycin (2 mM) for 8 h for dual luciferase assays. The lysates were also subjected to immunoblotting to examine the expression of RNF11 and β-actin. Error bars indicate s.e.m. of triplicate samples.
Figure 3
Figure 3
RNF11 is essential for the termination of NF-κB signalling. (A) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK and RNF11. (B) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or A20 siRNAs. At 2 days after the second transfection, cells were treated with LPS (1 μg/ml) for the indicated times. Cells were lysed and immunoblotted with anti-IκBα, pIκBα, JNK, pJNK, A20, RNF11 and β-actin. (C) THP-1 cells were transfected with control scrambled or RNF11 siRNA as described in (A). At 2 days after the second transfection, cells were stimulated with TNF (10 ng/ml) for various times and RNA was subjected to real-time PCR for IκBα and A20 expression. This experiment was repeated twice with similar results. (D) THP-1 cells were transfected with siRNAs as described in (A). Supernatants were subjected to an IL-6 ELISA. Error bars indicate s.e.m. of triplicate samples. Statistical analysis was performed by one-way ANOVA, followed by the Tukey–Kramer test for multiple comparisons. *P<0.01 (compared with control IL-1-treated samples); **P<0.001 (compared with control TNF-treated samples).
Figure 4
Figure 4
RNF11 negatively regulates the ubiquitination of RIP1 and TRAF6. (A) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for the indicated times. IPs were performed with anti-RIP1 or isotype control (control Ig) and immunoblotted with anti-Ub or RIP1. Lysates were examined for RNF11 expression by immunoblotting with anti-RNF11. Molecular sizes are shown to the left of the Ub blot panel. (B) THP-1 cells were transfected with siRNAs as described in (A). Cells were treated with LPS (1 μg/ml) for the indicated times. IPs were performed with anti-TRAF6 or isotype control (control Ig) and immunoblotted with anti-Ub or TRAF6. Molecular sizes are shown to the left of the Ub blot panel. Lysates were examined for RNF11 expression by immunoblotting with anti-RNF11. NS, nonspecific band; IgH, immunoglobulin heavy chain.
Figure 5
Figure 5
RNF11 is essential for A20 to interact with and inactivate RIP1. (A) THP-1 cells were transfected on consecutive days with control scrambled, RNF11 or TAX1BP1 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 30 min. IPs were performed with anti-RIP1 or isotype control (cont. Ig) followed by immunoblotting with anti-A20 or RIP1. Lysates were immunoblotted with anti-RNF11, TAX1BP1 and β-actin. (B) THP-1 cells were transfected on consecutive days with control scrambled or RNF11 siRNAs. At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 30 min where indicated. IPs were performed with anti-Itch or isotype control (cont. Ig) followed by immunoblotting with anti-RIP1 or Itch. Lysates were immunoblotted with anti-RNF11 and β-actin. (C) Itch+/+ and Itch−/− MEFs were treated with TNF (10 ng/ml) for 30 min as indicated. Cells were lysed and immunoprecipitated with anti-RNF11 or isotype control (cont. Ig) followed by immunoblotting with anti-RIP1 or RNF11. (D) Tax1bp1+/− and Tax1bp1−/− MEFs were treated and subjected to co-IPs as in (C). (E) MEFs were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with 0.25, 0.5 or 1 μg of Flag A20 (wedges in lanes 3–5 and 6–8). After 2 days, the cells were lysed and endogenous TRAF6 was immunoprecipitated with anti-TRAF6 followed by immunoblotting with anti-Ub. Molecular sizes are shown to the left of the Ub blot panel. The expression of ectopic A20 was examined by immunoblotting with anti-Flag. The efficiency of RNF11 knockdown was determined by immunoblotting with anti-RNF11. (F) 293T cells were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with pRL-tk, κB-TATA-Luc and 0, 0.25, 0.5 or 1 μg of Flag–A20 or Flag–A20 C103A as indicated (wedges). At 2 days after the second transfection, cells were treated with TNF (10 ng/ml) for 8 h and lysates were subjected to dual luciferase assays. Error bars indicate s.e.m. of triplicate samples. (G) MEFs were transfected with control scrambled or RNF11 siRNAs. The following day, the cells were transfected with 1 μg of empty vector (lanes 1–4) or Flag A20 (lanes 5–12). At 2 days after the second transfection, cells were treated with CHX for the indicated times. Cells were lysed and the stability of endogenous RIP1 was examined by immunoblotting with anti-RIP1. Lysates were also immunoblotted with anti-Flag, RNF11 and β-actin.
Figure 6
Figure 6
RNF11 requires the RING domain and PPXY motif to interact with the A20 ubiquitin-editing complex and to terminate NF-κB signalling. (A) Schematic diagram of RNF11 domain organization and mutants used in this figure. The PPXY motif is located between amino acids 37 and 40. The RING finger domain is located between amino acids 99 and 140. The RNF11 C99A mutant has a non-functional RING domain. The RNF11 Y40A mutant has a disrupted PPXY motif. (B) THP-1 cells were transfected with a control scrambled or a single duplex RNF11 siRNA. The following day, the cells were transfected with siRNA-resistant forms of Flag–RNF11 (WTR), Flag–RNF11 C99A (C99AR) and Flag–RNF11 Y40A (Y40AR). After two more days, cells were treated with TNF (10 ng/ml) for the indicated times and lysates were immunoblotted with anti-pIκBα, IκBα, pJNK, JNK, Flag, RNF11 and β-actin. (C) THP-1 cells were transfected with siRNA and siRNA-resistant forms of RNF11 as described in (B). At 2 days after DNA transfections, cells were treated with TNF for 30 min where indicated. Lysates were subjected to immunoprecipitations with anti-RNF11 followed by immunoblotting with A20, TAX1BP1 and RIP1. The lysates were also used for immunoblotting using anti-IκBα, Flag, RNF11 and β-actin.
See this image and copyright information in PMC

Comment in

  • RNF11, a new piece in the A20 puzzle.
    Jacque E, Ley SC. Jacque E, et al. EMBO J. 2009 Mar 4;28(5):455-6. doi: 10.1038/emboj.2009.18. EMBO J. 2009. PMID: 19262463 Free PMC article. No abstract available.

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References

    1. Anderson LR, Betarbet R, Gearing M, Gulcher J, Hicks AA, Stefansson K, Lah JJ, Levey AI (2007) PARK10 candidate RNF11 is expressed by vulnerable neurons and localizes to Lewy bodies in Parkinson disease brain. J Neuropathol Exp Neurol 66: 955–964 - PubMed
    1. Azmi P, Seth A (2005) RNF11 is a multifunctional modulator of growth factor receptor signalling and transcriptional regulation. Eur J Cancer 41: 2549–2560 - PubMed
    1. Bohgaki M, Tsukiyama T, Nakajima A, Maruyama S, Watanabe M, Koike T, Hatakeyama S (2008) Involvement of Ymer in suppression of NF-κB activation by regulated interaction with lysine-63-linked polyubiquitin chain. Biochim Biophys Acta 1783: 826–837 - PubMed
    1. Boone DL, Turer EE, Lee EG, Ahmad RC, Wheeler MT, Tsui C, Hurley P, Chien M, Chai S, Hitotsumatsu O, McNally E, Pickart C, Ma A (2004) The ubiquitin-modifying enzyme A20 is required for termination of Toll-like receptor responses. Nat Immunol 5: 1052–1060 - PubMed
    1. Burger A, Li H, Zhang XK, Pienkowska M, Venanzoni M, Vournakis J, Papas T, Seth A (1998) Breast cancer genome anatomy: correlation of morphological changes in breast carcinomas with expression of the novel gene product Di12. Oncogene 16: 327–333 - PubMed

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