Abstract
Inactivation of the TNFAIP3 gene, encoding the A20 protein, is associated with critical inflammatory diseases including multiple sclerosis, rheumatoid arthritis and Crohn’s disease. However, the role of A20 in attenuating inflammatory signalling is unclear owing to paradoxical in vitro and in vivo findings. Here we utilize genetically engineered mice bearing mutations in the A20 ovarian tumour (OTU)-type deubiquitinase domain or in the zinc finger-4 (ZnF4) ubiquitin-binding motif to investigate these discrepancies. We find that phosphorylation of A20 promotes cleavage of Lys63-linked polyubiquitin chains by the OTU domain and enhances ZnF4-mediated substrate ubiquitination. Additionally, levels of linear ubiquitination dictate whether A20-deficient cells die in response to tumour necrosis factor. Mechanistically, linear ubiquitin chains preserve the architecture of the TNFR1 signalling complex by blocking A20-mediated disassembly of Lys63-linked polyubiquitin scaffolds. Collectively, our studies reveal molecular mechanisms whereby A20 deubiquitinase activity and ubiquitin binding, linear ubiquitination, and cellular kinases cooperate to regulate inflammation and cell death.
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Change history
13 January 2016
Nature 528, 370–375 (2015); doi: 10.1038/nature16165. In this Article, owing to a typesetter error the ‘received date’ was incorrectly shown as ‘5 November 2015’ instead of ‘5 November 2013’; this has been corrected in the online versions of the paper.
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Acknowledgements
The authors thank J. Ernst, N. Kayagaki, K. O’Rourke, S. Hymowitz, I. Bosanac, E. Varfolomeev, T. Goncharov, M. diAlmagro, Z. Zhang, C. Klijn, D. Bustos, A. Maltzman, K. Wickliffe, J. Heideker, P. Liu, A. Ashkenazi, T.-K. Chang, B. Brasher, and C. Schwerdtfeger for reagents and discussions, A. Ma for A20 null MEFs, and the Genentech Protein Expression Group, Sequencing Facility, Luminex Core Group, Animal Facility and Genotyping Laboratory, J. Z. Solorio and M. Roose-Girma and the Mouse Models Group for dedication and support.
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Contributions
I.E.W., C.L., F.M., M.T., E.D., W.P.L., C.C.G., M.B. and V.M.D. coordinated studies. I.E.W., K.N., S.K., D.S., J.Z., N.P., E.H., A.S., S.J., N.R., L.K., K.H., D.D., W.S., A.Z., S.M., J.D.V., E.D., K.Y. and G.K. designed and performed experiments. I.E.W., K.N., D.S., J.Z., N.P., E.H., A.S., S.J., N.R., L.K., K.H., D.D., W.S., F.M., C.Q., W.J.F., M.T., S.W., J.D.V., J.L., E.D., P.C., W.P.L., C.C.G., M.B., K.Y., G.K. and V.M.D. interpreted data. I.E.W. wrote the manuscript. I.E.W., K.N., N.P., S.M., E.H., R.J.N., C.Q., J.T. and S.W. prepared reagents.
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I.E.W., K.N., D.S., S.K., C.L., J.Z., N.P., E.H., A.S., S.J., N.R., L.K., R.J.N., D.D., W.S., S.M., A.Z., F.M., C.Q., J.T., W.J.F., M.T., S.W., J.D.V., J.L., E.D., P.C., W.P.L., M.B., K.Y., G.K. and V.M.D. are currently or have been employees of Genentech.
Extended data figures and tables
Extended Data Figure 1 A model for A20 OTU and A20 ZnF4 regulation of TNF- and LPS-activated signalling.
a, Left complex. Upon TNF binding TNFR1 forms a trimer, thereby promoting recruitment of the adaptor protein TRADD and the RIP1 kinase (RIPK1). TRADD recruits TRAF2, TRAF5 and the ubiquitin ligases cIAP1 and cIAP2. The cIAP proteins promote K63-linked ubiquitination of signalling proteins including RIPK1, cIAP1/2 (autoubiquitination), and possibly TNFR1. K63 ubiquitination of cIAP1/2 subsequently recruits the LUBAC complex that promotes linear polyubiquitination of signalling proteins including RIPK1 and TNFR1. K63 ubiquitin chains on RIPK1 promote recruitment of the TAK1/TAB2/3 complex, whereas linear ubiquitin chains on RIPK1 promote IκK kinase complex recruitment via NEMO. Kinase complex recruitment promotes their subsequent activation and propagation of downstream JNK, p38 (via MKK3 and MKK4) and NFκB signalling pathways. We propose that A20 is recruited to the active TNFR1 signalling complex via ZnF7 binding to linear ubiquitin chains. The A20 OTU domain catalytic C103 is essential for attenuating TNF-activated signalling by removing K63 polyubiquitin chains from RIPK1 and other proteins including TNFR1, thereby promoting the dissociation of the active signalling complex. The A20 ZnF4 motif, that depends on C609/C612 for structural integrity and Y599/F600 for ubiquitin binding, is likely to collaborate with other proteins (not shown) to further downregulate TNF signalling by directing K48 polyubiquitination and subsequent degradation of proximal complex proteins, including RIPK1 and TNFR1. Right complex. LPS binding activates TLR4 and promotes the assembly of proximal signalling complexes via the adaptors TRIF and TRAM (not shown) or Mal and MyD88. Recruitment and activation of the proximal kinases IRAK4 and IRAK1, the ubiquitin ligase Pellino (not shown), and the LUBAC complex promote K63 and linear polyubiquitination of signalling proteins. As with TNFR1 signalling, this scaffolding-type ubiquitination promotes recruitment of TAK1/TAB2/3 and IκK kinase complexes, their subsequent activation, and propagation of downstream JNK, p38 and NFκB signalling pathways. A20 is probably recruited to the LPS-activated signalling complex via ZnF7 binding to linear ubiquitin chains. The A20 OTU domain catalytic C103 is essential for attenuating LPS-activated signalling by removing K63 polyubiquitin chains from TRAF6, and possibly other proximal signalling proteins. Although the structural integrity and the ubiquitin-binding function of A20 ZnF4 is dispensable for proper attenuation of TLR4 signalling, A20 ZnF4 could have a redundant function with another protein. b, In A20 OTU(C103A) cells removal of K63 ubiquitin chains on proximal signalling components is compromised, thus proteins are hyperubiquitinated with K63-linked chains. With sufficient linear ubiquitination, the infrastructure of the signalling complex is sustained, caspase recruitment to TNFR1 is prohibited, and pro-survival signalling is enhanced. c, In A20 OUT(C103A) cells with deficient linear ubiquitination, removal of K63 ubiquitin chains is still compromised; however, decreased linear chains favours enhanced association of hyperubiquitinated RIPK1 with FADD and caspase 8, the proximal components of the pro-death complex. Enhanced caspase 8 recruitment and activation in turn activates downstream effector caspases (such as caspase 3 and 7), culminating in cell death.
Extended Data Figure 2 Engineering and genotyping of A20 OTU mutant and A20 ZnF4 mutant knock-in mice.
a, Schematic diagrams of the A20 protein indicating the locations of the knock-in point mutations for each engineered mouse strain. The gene and protein names are also indicated, with abbreviated protein names indicated in parentheses. b, Tnfaip3Otu/Otu knock-in allele encoding A20 OTU(C103A) and representative genotyping data (right panel). c, Tnfaip3z4Cys/z4Cys knock-in allele encoding A20 ZnF4(C609A,C612A) and representative genotyping data (right panel). d, Tnfaip3z4Ub/z4Ub knock-in allele encoding A20 ZnF4(Y599A,F600A) and representative genotyping data (right panel). b–d, Correctly targeted ES cell clones were identified by long-range PCR followed by sequencing (data not shown). LoxP sites are illustrated as yellow arrows, frt sites as red arrows. Modified exons 3 and 7 are indicated in blue. For gel source data, see Supplementary Fig. 6.
Extended Data Figure 3 Analysis of TNF-challenged A20 wild-type, A20 OTU(C103A), A20 ZnF4(C609A,C612A) and A20 ZnF4(Y599A,F600A) mice.
a, Body temperatures of mice in response to 300 μg TNF per kg body weight treatment. Error bars are indicated for each data point and represent the mean ± standard deviation of 3 or 4 mice per genotype. b, A heat map representing profiles of serum cytokines in 12 different genotype/TNF treatment groups. Mice (n = 3 or 4 per group) were treated for the indicated time with 300 μg TNF per kg body weight; mean values per group are represented in the heat map. Each row represents one cytokine, whose values were standardized to z-scores with a mean of zero and a standard deviation of 1, and colour-coded according to the colour key. Variances of selected serum cytokines from A20 wild-type, A20 OTU(C103A) (OTU), or A20 ZnF4(C609A,C612A) (ZnF4 Cys) mice in response to TNF stimulation were evaluated using the Student’s t-test: IL6 WT versus ZnF4 Cys 2 h P = 0.009, 4 h P = 0.030; IL6 WT versus OTU 2 h P = 0.023, 4 h P = 0.035; Cxcl1 WT versus ZnF4 Cys 2 h P = 0.011, 4 h P = 0.017; Cxcl1 WT versus OTU 2 h P = 0.047, 4 h P = 0.043; Csf3 WT versus ZnF4 Cys 4 h P = 0.017, Csf3 WT versus OTU 4 h P = 0.042; Ccl11 WT versus ZnF4 Cys 4 h P = 0.0003, Ccl11 WT versus OTU 4 h P = 0.036. c, Body temperatures of ZnF4 mutant mice in response to 300 μg TNF per kg body weight treatment. Error bars are indicated for each data point and represent the mean ± standard deviation of 4 mice per genotype. d, A heat map representing profiles of serum cytokines as in Extended Data Figure 3b, but the indicated mice (n = 4 per group) were treated for four hours with 300 μg TNF per kg body weight or PBS vehicle control. Variances of selected serum cytokines from A20 ZnF4(C609A,C612A) (ZnF4 Cys), A20 ZnF4(Y599A,F600A) (ZnF4 Ub) or the respective wild-type control mice in response to TNF stimulation were evaluated using the Student’s t-test: IL6 WT versus ZnF4 Cys P = 0.000057; IL6 WT versus ZnF4 Ub P = 0.014; Cxcl1 WT versus ZnF4 Cys P = 0.016; Cxcl1 WT versus ZnF4 Ub P = 0.012; Csf3 WT versus ZnF4 Cys P = 0.024, Csf3 WT versus ZnF4 Ub P = 0.0061; Ccl11 WT versus ZnF4 Cys P = 0.005, Ccl11 WT versus ZnF4 Ub P = 0.001. e, Analysis of myelin oligodendrocyte glycoprotein-induced experimental autoimmune encephalomyelitis (MOG-EAE) studies in A20 WT, A20 OTU(C103A), and A20 ZnF4(C609A,C612A) mice. Top panel, MOG-EAE disease scores over time (mean ± s.e.m.) for A20 WT (n = 15) and A20 OTU(C103A) (n = 14). A20 OTU(C103A) average daily clinical scores (ADCS) P = 0.012, Dunnett’s test versus A20 WT. Bottom panel, EAE disease scores over time (mean ± s.e.m.) for A20 WT (n = 13) and A20 ZnF4(C609A,C612A) (n = 12). A20 ZnF4(C609A,C612A) ADCS P = 0.046, Dunnett’s test versus A20 WT. f, Lower power (upper panel A) and higher power (lower panel B) microscopic images of a representative lumbar spinal cord section derived from a A20 ZnF4(C609A,C612A) mouse with a grade 3 EAE clinical score at study termination (day 30). The section is stained with haematoxylin, eosin and Luxol fast blue. A, Foci of myelinopathy and gliosis (arrows). Gr, grey matter; Wh, white matter; scale bar, 200 μm. B, Focally severe myelinopathy and gliosis (double arrows) extending from the meninges close to the grey matter (delineated by white dashed line). Scale bar, 50 μm. Data represent at least two biological replicates.
Extended Data Figure 4 A20 proteins from wild-type, A20 OTU(C103A) cells, A20 ZnF4(C609A,C612A) cells, and A20 ZnF4(Y599A,F600A) cells are efficiently recruited to TNFR1 and regulate downstream signalling.
a, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 OTU(C103A) MEFs. b, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 ZnF4(C609A,C612A) MEFs. c, Immunoblot analysis Flag–TNF-engaged immunocomplexes and the corresponding whole-cell lysates in wild-type and A20 ZnF4(Y599A,F600A) MEFs. d, Immunoblot analysis Flag–TNF-treated whole-cell lysates in wild-type and in A20 OTU(C103A) MEFs. Asterisk, background band; arrow, phospho-MKK4. UnRx, untreated. e, Immunoblot analysis Flag–TNF-treated whole-cell lysates in wild-type and in A20 ZnF4(C609A,C612A) MEFs. Asterisk, background band; arrow, phospho-MKK4. UnRx, untreated. f, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 OTU(C103A) MEFs. Immunoblot analysis of whole-cell lysates from TNF-treated wild-type, A20 OTU(C103A), and A20 null MEFs following TNF pre-treatment to induce A20 expression, as well as TNF-treated A20 wild-type and A20 OTU(C103A) primary BMDMs, and TNF-treated A20 wild-type and A20 OTU(C103A) immortalized BMDMs all showed similar trends (not shown). g, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4 C609,612A E1A transformed MEFs. UnRx, untreated. Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4 C609,612A MEFs following TNF pre-treatment to induce A20 expression, and analysis of whole-cell lysates from TNF-treated A20 wild-type and A20 ZnF4 C609,612A primary BMDMs all showed similar trends (not shown). h, Immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 ZnF4(Y599A,F600A) primary MEFs. For gel source data, see Supplementary Figs 6, 7. Data represent two to four biological replicates.
Extended Data Figure 5 Additional analysis of ubiquitination status analysis of TNFR1 and associated proteins.
a, A summary table of selected proteins identified in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis (left columns) and a summary of the ubiquitination sites identified on the indicated proteins from TNF-treated A20 wild-type MEFs with the PTMscan approach using anti-K-ε-GG antibodies and LC-MS/MS (right column). b, Analysis of TNF-engaged TNFR1 in untreated or Flag–TNF-treated E1A-transformed A20 wild-type or A20 OTU(C103A) MEFs, or in A20 null primary MEFs. Anti-Flag immunocomplexes were purified using Flag peptide elution and elutions were blotted for TNFR1. Immunoblots of the corresponding whole-cell lysates are indicated below. c, Lysates corresponding with Fig. 1c. d, Murine TNFR1(K256R) attenuates TNFR1 ubiquitination and downstream signalling. Murine wild-type or TNFR1(K256R) was transfected in human 293T cells and cells were treated with Flag–TNF as indicated. Equal inputs of lysates were immunoprecipitated with anti-Flag, dissociated and re-immunoprecipitated with anti-linear ubiquitin antibody, and blotted for murine TNFR1, or lysates were blotted with the indicated antibodies. e, Analysis of TNFR1-associated RIPK1 K63 ubiquitination (Ub) status and TNFR1 immunoprecipitates in Flag–TNF-treated wild-type and A20 OTU(C103A) MEFs. f, Comparison of activated TNFR1 ubiquitination status in E1A transformed A20 wild-type and OTU(C103A) MEFs. Treated cells were lysed in buffer containing 6 M urea and immunoprecipitated with the indicated antibodies under denaturing conditions. g, Summary table of RIPK1 ubiquitination sites identified from TNF-treated A20 wild-type and OTU(C103A) MEFs with the PTMscan approach using anti-K-ε-GG antibodies and LC-MS/MS. Peptides were quantified with area under curve (AUC) and summarized to site level. The equivalent human RIPK1 residues are also indicated. The average ratio of endogenous RIPK1 ubiquitination sites in A20 OTU(C103A):wild-type A20 is 1.7. Additional TNFR1 mass spectrometry data are shown in Supplementary Information a–c. For gel source data, see Supplementary Figs 7, 8. Data represent two to four biological replicates.
Extended Data Figure 6 In vitro deubiquitination assays.
Additional data corresponding to Fig. 2. Normalized A20 WT or OTU(C103A) inputs purified from E. coli or mammalian HEK 293T cells are shown in Fig. 2a. a, A20-mediated cleavage efficacy of K63- or K48-linked tetraubiquitin conjugated to a HA-tagged RIPK1 peptide. b, Sequence of the HA epitope-tagged human RIPK1 peptide. The HA epitope tag is shown in blue, the human RIPK1 residues in black, and K377 is highlighted in red. c, Cleavage time course of linear tetraubiquitin by purified A20 WT or OTU(C103A) from E. coli or from mammalian HEK 293T cells. Input protein levels are shown in Fig. 2a. d, A schematic of the human A20 protein indicating where the phosphorylation sites are localized. Mass spectrometry PhosphoSite analysis (http://www.phosphosite.org/) of A20 derived from mammalian expression systems is shown in Supplementary Information d. e, Comparison of the cleavage efficacy of K63-linked tetraubiquitin with increasing doses of human wild-type A20 or phospho-site mutant A20 (4× phos mut). 4× phos mut: S381A, S480A, S565A, and T625A. Wild-type or phos mut A20 proteins were expressed in and purified from mammalian HEK 293T cells. f, Tandem mass spectrum for the S381-containing peptide from human A20 expressed in E. coli and phosphorylated with recombinant IκKβ. g, Cleavage efficacy of linear tetraubiquitin chains by increasing doses of E. coli-derived wild-type A20, IκKβ alone, or IκKβ-phosphorylated A20. For gel source data, see Supplementary Figs 8, 9. Data represent two to five biological replicates.
Extended Data Figure 7 Effects of linear ubiquitination in modulating TNFR1 signalling and cell viability.
a, HOIP RNAi decreases linear ubiquitination of TNFR1. Wild-type MEFs were transfected with control or HOIP RNAi oligonucleotides and treated for the indicated times with TNF. Treated cells were lysed in buffer containing 6 M urea and immunoprecipitated with the indicated antibodies under denaturing conditions. Immunoprecipitates and whole-cell lysates were blotted as indicated. b, Area under the curve (AUC) data corresponding to cell death data in Fig. 3a. All error bars are s.e.m. for technical triplicates. ***P < 0.001 determined by t-test. c, Murine TNFR1(K256R) enhances caspase activation. Human HEK 293T cells were treated with control or with human TNFR1 RNAi oligonucleotides and transfected with murine wild-type or with TNFR1(K256R) as indicated. Cells were treated with TNF as indicated and equal inputs of lysates were immunoblotted with the indicated antibodies. d, Murine TNFR1(K256R) does not modulate MAPK or NF-κB signalling. Human HEK 293T cells were treated with control or with human TNFR1 RNAi oligonucleotides and transfected with murine wild-type or with TNFR1(K256R) as indicated. Cells were treated with TNF as indicated and equal inputs of lysates were immunoblotted with the indicated antibodies. e, Evaluation of the specificity of anti-caspase 8 antibodies. E1A-transformed MEFs of the indicated genotype were lysed in buffer containing 6 M urea, quantified, and immunoblotted with the indicated antibodies as detailed in the Methods. f, A summary table of cell death proteins identified in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis (also see Supplementary Fig. 6a). g, Sequences of the caspase 8 peptides in anti-Flag immunocomplexes from untreated or in Flag–TNF-treated wild-type MEFs by LC-MS/MS analysis. h, Left panels, analysis of FADD immunoprecipitates and FADD-associated RIPK1 K63 ubiquitination (Ub) status in TNF-treated wild-type and A20 OTU(C103A) MEFs transfected with control- or HOIP RNAi oligonucleotides. Right panels, immunoblot analysis of whole-cell lysates from TNF-treated wild-type and A20 OTU(C103A) MEFs transfected with control- or HOIP RNAi oligonucleotides. HOIP knockdown was validated by RT–PCR analysis (data not shown). For gel source data, see Supplementary Figs 9, 10. Data represent two to three biological replicates.
Extended Data Figure 8 A20 OTU domain, but not the ZnF4 motif, downmodulates LPS signalling.
a, Kaplan–Meier survival curves of A20 WT (n = 15) and A20 OTU(C103A) (n = 15) mice in response to 20 mg LPS per kg body weight. Log rank P = 0.0002, Wilcoxon P < 0.0001. b, Upper panel, analysis of TRAF6 K63 ubiquitination (Ub) status in LPS-treated WT and A20 OTU(C103A) primary BMDMs. Lower panels, immunoblot analysis of whole-cell lysates from LPS-treated wild-type and A20 OTU(C103A) primary BMDMs. Asterisk, background band. Similar trends were seen in wild-type and A20 OTU(C103A) MEFs in response to acute LPS treatment and following LPS pre-treatment to induce A20 expression (not shown). c, Kaplan–Meier survival curves of A20 wild-type (n = 10), A20 ZnF4(C609A,C612A) (n = 10), and A20 ZnF4(Y599A,F600A) (n = 9) mice in response to 20 mg LPS per kg body weight. Log rank P = 0.1531, Wilcoxon P = 0.1398 for A20 ZnF4(C609A,C612A) versus WT; Log rank P = 0.4103, Wilcoxon P = 0.3373 for A20 ZnF4(Y599A,F600A) versus WT. d, A heat map representing profiles of serum cytokines in 12 different genotype/LPS treatment groups. Mice (n = 3 or 4 per group) were treated for the indicated time with 40 mg LPS per kg body weight LPS as indicated; mean values per group are represented in the heat map. Each row represents one cytokine, whose values were standardized to z-scores with a mean of zero and a standard deviation of 1, and colour-coded according to the colour key. Variances of selected serum cytokines from A20 WT, A20 OTU(C103A) (OTU), or A20 ZnF4(C609A,C612A) (ZnF4 Cys) mice in response to LPS stimulation were evaluated using the Student’s t-test: TNF WT versus OTU 2 h P = 0.044; IFNγ WT versus OTU 4 h P = 0.033; Ccl4 WT versus OTU 4 h P = 0.040. Profiles of selected serum cytokines from A20 WT (n = 5), ZnF4 Cys (n = 4), or OTU(C103A) (n = 5) mice in response to PBS or low dose (5 mg LPS per kg body weight) LPS and collected at 2 h or 6 h post-stimulation showed similarly significant variances between A20 WT and A20 OTU(C103A) (OTU) but not between A20 WT and A20 ZnF4(C609A,C612A) (ZnF4 Cys) (not shown). e, Upper panel, immunoblot analysis of whole-cell lysates from LPS-treated wild-type A20 and A20 ZnF4 C609,612A MEFs. Lower panel, analysis of TRAF6 K63 ubiquitination (Ub) status in the corresponding MEFs. Similar trends were seen in lysates from LPS-treated wild-type and A20 ZnF4 C609,612A MEFs following LPS pre-treatment to induce A20 expression (not shown). For gel source data, see Supplementary Fig. 10. Data represent two biological replicates.
Extended Data Figure 9 Characterization of A20 ZnF mutants and their cellular effects.
a, Summary of binding data of wild-type human A20 ZnF motifs and ZnF mutants to mono-ubiquitin, as measured by NMR, and to tri-ubiquitin chains, as measured by biolayer interferometry. Data are shown in Fig. 5b and Supplementary Information h, g. b, Analysis of TNF-engaged TNFR1 in untreated or TNF-treated E1A transformed A20 wild-type or A20 OTU(C103A) MEFs. Anti-TNF immunocomplexes were captured using anti-TNFR1 antibody-coupled beads and elutions were blotted for TNFR1. c, Analysis of TNFR1-associated RIPK1 K48 ubiquitination (Ub) status in TNF-treated wild-type and A20 ZnF4(C609A,C612A) MEFs. Immunoblot analysis of the corresponding whole-cell lysates are shown in the lower panels. d, Flag-wild-type A20 ubiquitinates recombinant murine TNFR1 with K48 chains. Flag–wild-type A20 or Flag–A20 ZnF4(C609A,C612A) proteins purified from HEK 293T lysates were added to in vitro reactions with recombinant murine TNFR1 and ubiquitin system enzymes. Reactions were immunoprecipitated in 4 M urea using an anti-K48 ubiquitin antibody, and immunoblotted, or reaction inputs were blotted as indicated. e, IκKβ-phosphorylated A20, but not IκKβ alone, promotes in vitro ubiquitination. For gel source data, see Supplementary Fig. 11. Data represent two to three biological replicates.
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Wertz, I., Newton, K., Seshasayee, D. et al. Phosphorylation and linear ubiquitin direct A20 inhibition of inflammation. Nature 528, 370–375 (2015). https://doi.org/10.1038/nature16165
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DOI: https://doi.org/10.1038/nature16165
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