Inhibition of NF-kappaB signaling by A20 through disruption of ubiquitin enzyme complexes

Abstract

A20 negatively regulates inflammation by inhibiting the nuclear factor kappaB (NF-kappaB) transcription factor in the tumor necrosis factor-receptor (TNFR) and Toll-like receptor (TLR) pathways. A20 contains deubiquitinase and E3 ligase domains and thus has been proposed to function as a ubiquitin-editing enzyme downstream of TNFR1 by inactivating ubiquitinated RIP1. However, it remains unclear how A20 terminates NF-kappaB signaling downstream of TLRs. We have shown that A20 inhibited the E3 ligase activities of TRAF6, TRAF2, and cIAP1 by antagonizing interactions with the E2 ubiquitin conjugating enzymes Ubc13 and UbcH5c. A20, together with the regulatory molecule TAX1BP1, interacted with Ubc13 and UbcH5c and triggered their ubiquitination and proteasome-dependent degradation. These findings suggest mechanism of A20 action in the inhibition of inflammatory signaling pathways.

Fig. 1

Disruption of interactions between E2 and E3 enzymes in the TNFR and TLR4/IL-1R pathways by A20 and TAX1BP1. (A) Kinetics of TRAF6, Ubc13, UbcH5c, Itch, RNF11, A20, and TAX1BP1 interactions in control and A20-deficient MEFs. A20^+/+ and A20^−/− MEFs were stimulated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with TRAF6 antibody and detected by immunoblotting with antibodies to A20, Ubc13, UbcH5c, Itch, RNF11, TAX1BP1, or TRAF6. Lysates were subjected to immunoblotting with antibodies to IκBα, A20, TAX1BP1, Ubc13, UbcH5c, RNF11, Itch, and β-actin. (B) Specificity of TRAF6, Ubc13, A20, UbcH5c, and TAX1BP1 interactions. A20^+/+ MEFs were stimulated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with TRAF6 or control rabbit antibody [Cont. IgG (immunoglobulin G)] and detected by immunoblotting with antibodies to A20, Ubc13, UbcH5c, TAX1BP1, or TRAF6. Lysates were subjected to immunoblotting with antibodies to IκBα and β-actin. (C) Kinetics of TRAF2, Ubc13, Itch, RNF11, A20, and TAX1BP1 interactions in control and A20-deficient MEFs. A20^+/+ and A20^−/− MEFs were stimulated with TNF-α, and proteins from lysates were immunoprecipitated with TRAF2 antibody followed by immunoblotting with antibodies to A20, Ubc13, Itch, RNF11, TAX1BP1, and TRAF2. Lysates were subjected to immunoblotting with antibodies to IκBα, TAX1BP1, A20, Ubc13, RNF11, Itch, and β-actin. (D and E) Interaction of TAX1BP1 with Ubc13. A20^+/+ and A20^−/− MEFs were stimulated with TNF-α (D) or LPS (E) for 30 and 60 min, and proteins from lysates were immunoprecipitated with antibody to TAX1BP1, followed by immunoblotting with antibodies to Ubc13, UbcH5c, or TAX1BP1. Lysates were subjected to immunoblotting with antibodies to IκBα, A20, TAX1BP1, Ubc13, and β-actin. The results shown are representative of three independent experiments. IP, immunoprecipitation; IB, immunoblot.

Fig. 2

Negative regulation of TRAF2 and TRAF6 polyubiquitination by A20. (A) Kinetics of TRAF6 ubiquitination. A20^+/+ and A20^−/− MEFs were stimulated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with antibody to TRAF6, eluted with 1% SDS, diluted in lysis buffer, reimmunoprecipitated with TRAF6 antibody followed by immunoblotting with K63-specific antibody to Ubi or antibody to TRAF6. Lysates were subjected to immunoblotting with antibodies to IκBα or β-actin. (B) Kinetics of TRAF2 ubiquitination. A20^+/+ and A20^−/− MEFs were stimulated with TNF-α for the indicated times. Proteins from lysates were immunoprecipitated with antibody to TRAF2, eluted with 1% SDS, diluted in lysis buffer, and reimmunoprecipitated with TRAF2 antibody, followed by immunoblotting with antibodies to Ubi or TRAF2. Lysates were subjected to immunoblotting with antibodies to IκBα and β-actin. (C) Stability of ubiquitinated TRAF6. A20^+/+ and A20^−/− MEFs were pretreated with cycloheximide, followed by IL-1 stimulation for the indicated times. Proteins from lysates were immunoprecipitated with antibody to TRAF6 as described (A), followed by immunoblotting with antibodies to Ubi, Ubc13, or TRAF6. Lysates were subjected to immunoblotting with antibodies to TRAF6, IκBα, and β-actin. The results shown are representative of three independent experiments.

Fig. 3

The ubiquitination and degradation of Ubc13 is promoted by A20 and TAX1BP1. (A) Polyubiquitination of Ubc13 in cells stimulated with IL-1. Control, A20^−/−, or Tax1bp1^−/− MEFs were treated with IL-1 for the indicated times. Proteins from lysates were immunoprecipitated with antibody to Ubc13, eluted with 1% SDS, diluted in lysis buffer, and reimmunoprecipitated with Ubc13 antibody, followed by immunoblotting with antibodies to Ubi or Ubc13. Lysates were subjected to immunoblotting with antibodies to IκBα, Ubc13, and β-actin. (B) K48-linked polyubiquitination of Ubc13. Control MEFs were transiently transfected with plasmids encoding HA-Ubi, HA-Ubi-K63 only, or HA-Ubi-K48 only. After 36 hours, cells were stimulated for 4 hours with either TNF-α or LPS. Ubiquitination assays were performed as described in (A). Lysates were subjected to immunoblotting with antibody to HA. (C and D) Requirement of A20 for the degradation of Ubc13 and UbcH5c. BMDCs (C) or BMDMs (D) were transiently transfected with pSuper or pSuper A20 siRNA plasmids. After 36 hours, cells were stimulated with either IL-1 or TNF-α for the indicated times. Lysates were subjected to immunoblotting with antibodies to Ubc13, UbcH5c, UbcH10, A20, IκBα,or β-actin. (E and F) Proteasome-dependent degradation of Ubc13. Control MEFs were treated with TNF-α (E) or IL-1 (F) for the indicated times in the presence of MG132 or vehicle [dimethyl sulfoxide (DMSO)]. Lysates were subjected to immunoblotting with antibodies to Ubc13, UbcH5c, and β-actin. The results shown are representative of three independent experiments.

Fig. 4

Requirement of A20 zinc finger 4 for TAX1BP1 binding and degradation of Ubc13 and UbcH5c. (A) Schematic of A20 deletion mutants. (B) Requirement of A20 ZnF4 for the degradation of Ubc13. A20^−/− MEFs were transiently transfected with the indicated mutants. After 36 hours, cells were treated with TNF-α for the indicated times. Lysates were subjected to immunoblotting with antibodies to Ubc13, IκBα, β-actin, or Flag. (C) A20 DUB and ZnF4 mutants do not interact with Ubc13. A20^−/− MEFs were transiently transfected with Flag-A20, Flag-A20 (C103A), and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to A20 or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα, and β-actin. (D) A20 ZnF4 mutant does not interact with Ubc13. A20^−/− MEFs were transiently transfected with Flag-A20 and Flag-A20 ZnF4 mutant (C624A and C627A). After 36 hours, cells were stimulated with TNF-α for 60 min, and proteins from lysates were immunoprecipitated with Ubc13 antibody and detected by immunoblotting with antibodies to Flag or Ubc13. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (E) A20 DUB and ZnF4 mutants do not interact with UbcH5c. A20^−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with IL-1 for the indicated times, and proteins from lysates were immunoprecipitated with UbcH5c antibody and detected by immunoblotting with antibodies to A20 or UbcH5c. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. (F) Differential requirement for the A20 OTU domain and C-terminal zinc fingers in binding to TAX1BP1, Ubc13, and UbcH5c. A20^−/− MEFs were transiently transfected with empty vector, Flag-A20, Flag-A20 (547-775) or Flag-A20Δ547-699. After 36 hours, cells were treated with IL-1 for either 30 or 60 min. Proteins from lysates were immunoprecipitated with antibody to Flag, followed by immunoblotting with antibodies to TAX1BP1, UbcH5c, or Ubc13. Lysates were subjected to immunoblotting with antibodies to IκBα, Flag, and β-actin. (G) A20 ZnF4 mutant does not interact with TAX1BP1. A20^−/− MEFs were transiently transfected with plasmids as described in (C). After 36 hours, cells were stimulated with LPS for the indicated times, and proteins from lysates were immunoprecipitated with TAX1BP1 antibody and detected by immunoblotting with antibodies to A20 and TAX1BP1. Lysates were subjected to immunoblotting with antibodies to Flag, IκBα,and β-actin. The results shown are representative of three independent experiments.