IAP RING domain constructs interact with E2 ubiquitin-conjugating enzymes by yeast two-hybrid analysis. (A) Summary of positive interactions for each IAP RING domain DNA-binding fusion bait construct (c-IAP1 RING, c-IAP2 RING, ML-IAP RING, XIAP RING) with E2 activation domain fusion prey constructs. Interactions were scored according to growth on selective medium; + indicates growth present; − indicates growth absent. (B) Summary of interactions for UbcH5b and wild-type IAP RING domain prey constructs with wild-type IAP RING, IAP RING E2-binding surface mutants (c-IAP1 RING V573A, c-IAP2 RING V559A, ML-IAP RING V254A, XIAP RING I452A), and IAP RING dimerization mutants (c-IAP1 RING F616A, c-IAP2 RING F602A, ML-IAP RING F296A, XIAP RING 495A) bait constructs. WT IAP RING^* denotes that the respective wild-type IAP RING prey constructs were tested for each set of interactions (for instance, wild-type c-IAP1 RING prey and c-IAP1 RING F616A bait). (C) Sequence alignment of IAP RING domains, bold black type indicates amino acid residues with conserved identities, bold grey type indicates amino acid residues with conserved side chain properties, and asterisks mark the locations of E2-binding surface and dimerization residues mutated in the IAP RING bait constructs.

siRNA knockdown of the UbcH5 family increases the stability of c-IAP1 and the c-IAP1 substrate NIK. (A) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases c-IAP1 protein levels following CD40 treatment. HKB11-CD40 cells were transfected with control siRNA, or siRNA targeting the UbcH5 family (UbcH5a, UbcH5b, UbcH5c isoforms), Ube2S, tsg101, Ube2Q2, or Rad6B for 48 h. Anti-CD40 antibody was cross-linked with secondary antibody for 10 min, and then added to cells at a concentration of 0.5 μg/ml for 0, 5, or 30 min. Lysates were collected and subjected to western blot analysis using antibodies for detection of c-IAP1, UbcH5 (the antibody recognizes all three isoforms), tsg101, and actin, as a loading control. In parallel, RNA samples were collected and purified to monitor the efficiency of Ube2Q2 and Rad6B siRNA-knockdown by quantative RT–PCR analysis (Supplementary Table I). (B) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases c-IAP1 protein levels following TWEAK treatment. HT1080 cells were transfected with control siRNA, or siRNA targeting the UbcH5 family (UbcH5a, UbcH5b, UbcH5c isoforms), or Ube2S for 48 h and then treated with TWEAK (100 ng/ml) for indicated time points. Lysates were collected and subjected to western blot analysis using antibodies for detection of c-IAP1, UbcH5 (the antibody recognizes all three isoforms), Ube2S, and actin, as a loading control. (C) Knockdown of UbcH5 family inhibits gene induction by TWEAK. HT1080 cells were treated with TWEAK for 4 h (or 7 h for RelB), and RNA samples from treated and untreated cells were analysed by quantitative real-time PCR analysis. All values were normalized to an RPL19 RNA internal control. Columns represent mean from triplicate experiments and bars represent s.d. (D) siRNA-mediated knockdown of the UbcH5 family of E2 enzymes increases NIK protein stability in the presence of the c-IAP proteins. HEK293T cells were transfected with control siRNA, or siRNA targeting the UbcH5 family, Ube2S, tsg101, Ube2Q2, or Rad6B. At 24 h later, cells were transfected with myc-NIK1 plus FLAG-c-IAP1, FLAG-c-IAP2, or FLAG-vector. The following day, lysates were collected and analysed by western blot, and RNA was prepared for quantitative RT–PCR analysis (Supplementary Table II) as in panel (A).

TNF signalling stimulates K11-linked polyubiquitination of RIP1 by c-IAP1 and UbcH5. (A) TNFR1-bound RIP1 undergoes K11-linked polyubiquitination in response to TNFα. HeLa S3 cells were pretreated with 20 μM MG132 for 10 min, and then incubated with or without 100 ng/ml TNFα for 5 min. Lysates were collected and subjected to serial immunoprecipitations, first using anti-TNFR1 antibodies, and then after eluting under denaturing (6 M urea) conditions secondary immunoprecipitations were performed using linkage-specific anti-ubiquitin antibodies (anti-K11, anti-K48, anti-K63). Input lysates, left panel, were analysed by western blot with anti-RIP1, anti-IκBα, and anti-c-IAP1 antibodies. Serially immunoprecipitated material, right panel, was detected using anti-RIP1 antibody. (B) Time course of K11-linked polyubiquitination of RIP1 following TNFα stimulation. HeLa S3 cells were treated as in panel (A), except additional 20 and 45 min time points were included. Serial immunoprecipitations and western blot analysis were performed as in (A), using the indicated antibodies. (C) K11-linked polyubiquitination of RIP1 is dependent on c-IAP1. HeLa S3 cells were pretreated with or without 3 μM IAP antagonist (BV6) for 15 h, followed by treatment with 20 μM MG132 for 10 min, and then incubation with or without 100 ng/ml TNFα for 5 min. Serial immunoprecipitations and western blot analysis were performed as in (A), using the indicated antibodies. (D) K11-linked polyubiquitination of RIP1 involves the UbcH5 family of E2 enzymes. HeLa S3 cells were transfected with control siRNA, or siRNA targeting the UbcH5 family, Ube2S, or UbcH13. At 48 h later, cells were pretreated with 20 μM MG132 for 10 min, and then incubated with or without 100 ng/ml TNFα for 5 min. Lysates were prepared in lysis buffer containing 6 M urea, diluted and immunoprecipitated using the anti-K11-linkage-specific antibody. siRNA knockdown efficiency was analysed by western blot analysis of input lysates, right panel, using the following antibodies: anti-RIP1, anti-UbcH5, anti-UbcH13, anti-Ube2S, and anti-actin, as a loading control. Ubiquitinated endogenous RIP1, right panel, was detected in the immunoprecipitated material using an anti-RIP1 antibody.

Ube2S can promote ubiquitin chain extension in combination with c-IAP1 and UbcH5a. (A) Coomassie-stained gel containing equivalent amounts of in vitro c-IAP1 autoubiquitination reactions (rxn) performed in two steps with the first-step reaction conducted in the absence or the presence of UbcH5a as described in Materials and methods. The red boxes mark the boundaries of gel regions that were excised and subjected to in-gel trypsin digestion, followed by Ubiquitin-AQUA analysis. (B) Western blot using anti-ubiquitin antibody (P4D1) of autoubiquitination reactions. (C) Quantification of the amount of total ubiquitin in each corresponding gel region for each sample. A full-colour version of this figure is available at The EMBO Journal Online.

c-IAP1 promotes K11 polyubiquitin linkage formation on RIP1. (A) Ectopic expression of c-IAP1 and RIP1 promotes K11-linked polyubiquitination in 293T cells. HEK293T cells were transfected with myc-RIP1, HA-tagged ubiquitin constructs (WT, K11-, K48-, or K63-only), with or without Flag-c-IAP1 for 24 h. Cells were pretreated with MG132 (20 μM) for 1 h, lysates were boiled in NP40 lysis buffer containing 1% SDS for 10 min, diluted 10-fold and immunoprecipitated using anti-myc or anti-Flag beads. Flag c-IAP1 and myc-RIP1 were detected in immunoprecipitated material and in input lysates using anti-HA, anti-myc, and anti-Flag antibodies. (B) Recombinant RIP1 was incubated for 45 min in a ubiquitination reaction in the absence or the presence of recombinant c-IAP1, UbcH5a, and wild-type or K11-only ubiquitin proteins. RIP1 and c-IAP1 modifications were determined with anti-RIP1 and anti-c-IAP1 antibodies. (C) Determination of c-IAP1-mediated polyubiquitin linkages on RIP1. Recombinant RIP1 (5 μg) was incubated for 45 min in ubiquitination reactions in the presence of recombinant c-IAP1 (1.25 μg), UbcH5a (1.25 μg), and wild-type ubiquitin. Following ubiquitination reactions, samples were incubated in 6 M Urea at room temperature for 30 min with rocking, diluted 15 times and immunoprecipitated using anti-RIP1 antibodies. Coomassie-stained gel containing equivalent amounts of immunoprecipitated RIP1 (left side): the red lines mark the boundaries of gel regions that were excised and subjected to in-gel trypsin digestion, followed by Ubiquitin-AQUA analysis using OrbiTrap (bottom and right). Extracted ion chromatograms of indicated peptides from gel region A are shown where heavy peaks (red), corresponding to isotope labelled internal standard peptides, and light peaks (blue), corresponding to digested analyte peptides, are labelled with retention time and m/z ratio. The relative abundance for light peptide chromatograms (y axis) has been zoomed 2X relative to corresponding heavy peptides. Quantification of the amount of c-IAP1, RIP1 protein, total ubiquitin, and K11-linked ubiquitin for each corresponding gel region for each sample is shown on the bottom.

NEMO binds K11-linked ubiquitin chains. Ub binding measured by biolayer interferometry (A–D). (A) Direct binding of biotinylated NEMO_UBD and c-IAP1_BIR3-RING to K11- and K63-linked ubiquitin dimers are shown as raw sensograms (response in nm plotted as a function of time) for a range of ubiquitin dimer concentrations. (B) Saturation response values from the sensograms in (A) are plotted as a function of ubiquitin dimer concentration. The resulting binding curves were fit to a simple 1:1 binding model to determine equilibrium-binding constants (K_d). (C) A summary of the determined K_d values is shown with s.d. (n=3). (D) NEMO binds K11-linked ubiquitin chains in vivo. 293T cells were transfected with Myc-RIP1, FLAG-c-IAP1, HA-tagged ubiquitin constructs (K11- or K63-only), and with or without FLAG-NEMO for 40 h. Protein samples were immunoprecipitated using anti-NEMO antibodies and western blotting of cellular lysates and immunoprecipitates was performed with the indicated antibodies.