Analysis of Nuclear Factor-κB (NF-κB) Essential Modulator (NEMO) Binding to Linear and Lysine-linked Ubiquitin Chains and Its Role in the Activation of NF-κB^*

Plasmids, Antibodies, and Reagents

To generate linear ubiquitin-fused NEMO constructs the ORF of Arabidopsis ubiquitin, whose Gly-75 and Gly-76 were replaced by Val (ubiquitin-VV) to create an MluI site, was generated by PCR. Two to seven tandem uncleavable ubiquitin cDNAs were prepared by inserting the mutant ubiquitin encoding MluI∼BssHI into the MluI site of ubiquitin-VV. The whole nucleotide sequence was verified, and the respective tandem ubiquitin cDNA was ligated into FLAG-His₆-human NEMO in pcDNA3.1 vector (Invitrogen). Full-length murine NEMO and human IκBα-WT (amino acids 1–54) and IκBα-AA (amino acid 1–54 S34A/S36A) were cloned into pMAL-C2x (New England Biolabs). Full-length murine NEMO was cloned into pGEX-4T1 (GE Healthcare) using a standard PCR method. Human TAB2-NZF was swapped with NEMO UBAN (amino acids 250–339) or ZnF (amino acids 388–412), in the full-length NEMO cloned into pBABE (described in Ref. 14) or pGEX-4T1, by site-directed mutagenesis method using mega-mutagenesis primers. Mega-mutagenesis primers were produced by PCR using the human Npl4 zinc finger (NZF) domain as template (amino acids 663–693) with oligonucleotides that contain the complementary sequence flanking the site of insertion. F305A and F312A mutations in mouse and human NEMO, respectively, were introduced by site-directed mutagenesis.

Antibodies against following proteins were obtained and used following the manufacturers' protocols: Ser(P)-32/36-IκBα (9246; Cell Signaling), phospho-IKKα/β (2681; Cell Signaling), FLAG (clone M2; Agilent), IKKα/β (sc-7607; Santa Cruz Biotechnology), or IKKα (Imgenex); MBP (sc-13564, Santa Cruz Biotechnology); ubiquitin (sc-8017; Santa Cruz Biotechnology; Fig. 3A only, or 13-1600; Invitrogen), β-actin (C-2; Santa Cruz Biotechnology), GAPDH (14C16; Cell Signaling), PARP (9542; Cell Signaling), NEMO (sc-8330; Santa Cruz Biotechnology). Antibodies against IκBα, phospho-p38, p38, phospho-JNK, JNK, and p65 were as described previously (14). Anti-FLAG M2 was purchased from Sigma. Murine TNFα was purchased from Peprotech. Bovine ubiquitin was purchased from Sigma.

Cell Culture, Immunoprecipitation, Immunoblotting, and Retroviral Transduction

HeLa S3 cells were cultured at 37 °C under 7.5% CO₂ in Spinner flasks using S-MEM (Sigma) supplemented with 10% fetal bovine serum (FBS; Sigma), 100 IU/ml penicillin G, and 100 μg/ml streptomycin with gentle stirring, and HEK293T cells were cultured in DMEM (Sigma) containing 10% FBS, 100 IU/ml penicillin G, and 100 μg/ml streptomycin. Transfections were performed using Lipofectamine2000 (Invitrogen) or ExGen 500 (Thermo) following the manufacturer's protocols. Immunoprecipitation, electrophoresis, and immunoblotting were performed as described previously (20). Wild-type and NEMO-deficient MEF cells were described previously (5). Retroviral transduction of NEMO-deficient MEFs with pBABE NEMO constructs was described elsewhere (14).

Luciferase Assay

The luciferase reporter assay was performed as described (20). Briefly, pSP-6×NF-κB-Luc, pSV40-Renilla-Luc, and various doses of pcDNA3.1-FLAG-His₆-NEMO-[Ub]_0–7 were transfected to HEK293T cells. In NEMO-deficient N-1 cells (21), cells were transfected with luciferase vectors, 1 μg of pcDNA3.1-FLAG-His₆-NEMO or F312A mutant, with or without LUBAC components of pcDNA3.1-myc-HOIP (0.3 μg), pcDNA3.1-HOIL-1L (0.15 μg), and pcDNA3.1-T7-SHARPIN (0.15 μg). After 24 h, cells were lysed, and luciferase activity was measured using Bright-Glo luciferase assay systems (Promega). Some N-1 cells were treated with 10 ng/ml human TNF-α (Promega) for 6 h before harvest.

Preparation of Recombinant Proteins and Cellular Extracts

Recombinant E1, UbcH5c, His₆-HOIP-HOIL-1L-Myc-SHARPIN complex, and Myc-IKKα-His₆-IKKβ-FLAG-NEMO complex were prepared as described (13, 20). The MBP-fused IκBα (amino acids 1–54), IκBα-AA (amino acids 1–54, S32A/S36A), and NEMO (expression in Escherichia coli BL21 containing pRARE) were expressed in E. coli and purified using amylose resin (New England Biolabs). Lys-63- and Lys-48-linked ubiquitin chains were prepared as described previously (22). For the preparation of linear ubiquitin chains, GST-di-, tri-, or tetra-ubiquitin was expressed in E. coli BL21, purified, and cleaved with thrombin protease (biotinylated thrombin kit; Novagen) according to the manufacturer's instructions. Thrombin buffer was exchanged with 50 mm Tris-HCl, pH 7.5. Other GST-fused proteins were expressed in E. coli BL21 containing pRARE and induced by isopropyl 1-thio-β-d-galactopyranoside (100 μm) at 16 °C overnight, and purified using glutathione-Sepharose 4B (GE Healthcare).

HeLa S-100 fraction II was prepared according to the method of Hershko and co-workers (23). Briefly, crude HeLa cell extracts in hypotonic buffer (10 mm Tris-HCl, pH 7.5, 1.5 mm MgCl₂, 20 mm NaCl, 0.5 mm DTT, 0.5 mm PMSF) were lysed by a Dounce homogenizer, and a cytosolic fraction was prepared by ultracentrifugation at 100,000 × g for 1 h. The supernatant (S-100) was applied to a DEAE-Sepharose column. The bound fraction (fraction II), which excludes endogenous ubiquitin, was eluted with a buffer (10 mm Tris-HCl, pH 7.5, 1.5 mm MgCl₂, 500 mm NaCl, 0.5 mm DTT, 0.5 mm PMSF), and dialyzed with a re-equilibrated buffer (20 mm Tris-HCl, pH 7.5, 0.1 m NaCl, 0.5 mm DTT, 0.5 mm PMSF, 1 μg/ml leupeptin).

MBP and GST Pulldown Experiments

GST- or MBP-fused proteins immobilized with Sepharose beads were incubated with 1 μg of indicated ubiquitin chains in pulldown buffer (150 mm NaCl, 50 mm Tris-HCl, pH 7.5, 5 mm DTT, 0.1% Nonidet P-40, 0.25 mg/ml BSA) at 4 °C for 16 h on a rotator, or FLAG-tagged proteins were expressed in HEK293-T cells, which were lysed 24 h after transfection and incubated with GST-proteins at 4 °C for 16 h on a rotator. The beads were washed three times with pulldown buffer without BSA. The proteins were then eluted with SDS-sample buffer and boiled at 95 °C for 1 min. The samples were separated on a 12% or 15% SDS-polyacrylamide gels and transferred to PVDF membranes.

In Vitro IKK Kinase Assay

A total of 30 μl of samples containing 10 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 2 mm DTT, 0.5 mm ATP, 10 mm creatine phosphate, 50 μg/ml creatine phosphokinase, 2 mg/ml HeLa-S-100 fraction II, 250 μg/ml ubiquitin, 20 μm ubiquitin aldehyde (Boston Biochem), 100 ng of E1, 200 ng of UbcH5c, 2 μg of LUBAC, and 100 ng of MBP-IκBα or MBP-IκBα-AA were mixed and incubated at 37 °C for 1 h. Effects of di-ubiquitins on IKK activation were examined in total 20 μl of samples containing 10 mm Tris-HCl, pH 7.5, 5 mm MgCl₂, 2 mm DTT, 1 mm ATP, 100 ng of IKK complex, 100 ng of MBP-IκBα or MBP-IκBα-AA, and increasing amounts (0.1, 0.3, and 1 μg) of linear-, Lys-63-, and Lys-48-di-ubiquitins. Samples were incubated at 37 °C for 1 h.

Quantitative PCR

RNA was isolated from MEFs using an RNeasy kit (Qiagen). mRNA (2 μg) was reverse transcribed to cDNA using random primer. Quantitative PCR was performed using SensiMix SYBR and Fluorescein kit (Bioline) and primer pairs specific for mouse A20 (forward primer, ctccagcctcacttccagta; reverse primer, aatacatgcagccagctttc) or mouse actin (forward primer, tgttaccaactgggacgaca; reverse primer, ggggtgttgaaggtctcaaa).

Nuclear Fractionation

Cells were first lysed in hypotonic buffer (20 mm HEPES, pH 7.9, 10 mm KCl, 1 mm EDTA, 0.2% EDTA, 10% glycerol, 1.5 mm MgCl₂, 1 mm DTT, 1 mm PMSF), left on ice for 2 min, and centrifuged for 1 min at 13,000 rpm. Supernatant was collected as cytoplasmic fraction. Pellet was resuspended in 3 × pellet volume of hypertonic buffer (20 mm HEPES, pH 7.9, 10 mm KCl, 0.2 mm EDTA, 20 mm glycerol, 1.5 mm MgCl₂, 400 mm NaCl, 1 mm DTT, 1 mm PMSF), kept on ice for 30 min, and centrifuged for 5 min at 13,000 rpm.

Statistical Analysis

Student's t test was performed using GraphPad Prism software.

Full-length NEMO Preferentially Binds to Linear Ubiquitin Chains

We have shown previously that linear di-ubiquitin binds to NEMO UBAN domain, whereas Lys-63-linked di-ubiquitin could not be detected in GST binding assays (14, 24). However, it was proposed that the C terminus of NEMO, comprising the UBAN and the ZnF, has a higher affinity toward Lys-63- and Lys-48-linked ubiquitin chains consisting of three or more ubiquitin moieties (25). To elucidate the ubiquitin binding preferences of full-length NEMO we performed in vitro binding studies with bacterially purified recombinant full-length NEMO and ubiquitin chains of different linkage types and lengths. Purified NEMO was incubated with equal concentrations of different ubiquitin chain types in the same tube, and binding of NEMO was monitored by washing off unbound chains and Western blot analysis (Fig. 1). Because ubiquitin chains of the same length but with different linkage display different mobility on SDS gels, it is possible to distinguish the different types of chains by using the same anti-ubiquitin antibody (Fig. 1). We first examined the binding preference of full-length MBP-tagged NEMO toward Lys-11-, Lys-63- and linear-tri-ubiquitin (Fig. 1A). Incubation of MBP-NEMO with these different chain types led to a selective binding of linear tri-ubiquitin in competition with the other chain types (Fig. 1A). We have shown previously that only tetra- and longer ubiquitin chains of Lys-63 linkage could be involved in binding to NEMO, and mutational analysis indicated that they bind only to distal patches on the NEMO UBAN domain compared with longitudinal arrangement of linear chains that recognizes both proximal and distal patches (14). Based on this evidence NEMO binding to linear and Lys-63-linked ubiquitin chains is competitive, and it is not possible that both are bound to NEMO at the same time. In accordance, we observed that linear tetra-ubiquitin was able to block binding of Lys-63-linked tetra-ubiquitin with GST-NEMO or MBP-NEMO in vitro (supplemental Fig. S1 and Fig. 1, B and F). In contrast to NEMO, receptor-associated protein 80 (RAP80) selectively bound to Lys-63-linked tetra-ubiquitin (Fig. 1C). This is in accordance with a previous study showing that the two ubiquitin interacting motif domains of RAP80 as a unit selectively bind Lys-63-linked but not Lys-48- or linearly linked di-ubiquitin (26). Because the length of ubiquitin chains might influence the affinity toward NEMO, we set out to analyze the binding of Lys-63-linked hexa- or hepta-ubiquitin to NEMO, in competition with linear tetra-ubiquitin (Fig. 1D). In both cases we found that NEMO selectively bound to linear tetra-ubiquitin compared with the binding to hexa- or to hepta-Lys-63-linked ubiquitin chains. Comparing the ubiquitin binding preference of NEMO with a mixture of long Lys-63-linked ubiquitin chains revealed that NEMO did not pull down Lys-63-linked chains up to at least deca-ubiquitin in the presence of linear tetra-ubiquitin (Fig. 1E). Chains that were longer than deca-ubiquitin showed interaction with NEMO despite the presence of linear tetra-ubiquitin, but because ubiquitin chains longer than deca-ubiquitin were not separated, it is not clear what size of Lys-63-linked chains was necessary for binding and also whether this precipitation of high molecular mass ubiquitin chains is a specific interaction. To get more detailed information about the ubiquitin binding preference of NEMO, we incubated 1 μg of Lys-63-linked tetra-ubiquitin together with different amounts of linearly linked tetra-ubiquitin (Fig. 1F). Interaction of NEMO with linear tetra-ubiquitin alone is rather weak (Fig. 1F). In contrast, already the addition of 20 ng of linear tetra-ubiquitin leads to a strong linear chain binding, emphasizing the high binding preference of NEMO to linear chains. The increase of binding of NEMO to linear chains correlates strongly with an increasing input where already 100 ng of linear ubiquitin chains shows a much stronger binding than Lys-63-linked ubiquitin chains, of which 1 μg was used. Moreover, the linear chains compete out the binding toward Lys-63-linked ubiquitin chains, clearly visible by a decreased Lys-63-linked chain binding, already in the presence of only 200 ng of linear ubiquitin chains. Taken together these results demonstrate the quantitative distinction in ubiquitin chain binding selectivity of full-length NEMO toward linear ubiquitin chains compared with long Lys-63-linked ubiquitin chains.

The E3 ligase LUBAC Activates the IKK Complex and NF-κB in a NEMO-dependent Manner

Currently reported evidence indicates that NEMO binding to linear ubiquitin chains is essential for NF-κB activation in cells (14, 17). To assess further whether linear ubiquitin chains can activate the IKK complex we investigated the effect of LUBAC-induced linear ubiquitylation on IKK activation in vitro. To this end we examined the phosphorylation of IκBα using HeLa-S-100 fraction II, lacking endogenous ubiquitin, together with E1, E2 (UbcH5c), and LUBAC, which specifically produces linear ubiquitin chains (Fig. 2A) (27). Both endogenous IκBα and exogenously added MBP-IκBα (amino acids 1–54) were efficiently phosphorylated by addition of E1, E2, and LUBAC to fraction II of HeLa S-100 lysates that contain the IKK complex and IκBα (Fig. 2A). The addition of E1 and E2 alone failed to phosphorylate IκBα (Fig. 2A), indicating a linear ubiquitin chain-dependent activation of the IKK complex.

Next, we wanted to examine whether LUBAC can activate NF-κB and whether this action depends on the ability of NEMO to bind ubiquitin chains. To this end we utilized from Rat-1 cells derived NEMO-deficient N-1 cells and performed a luciferase assay (Fig. 2B) (21). As expected, TNFα treatment of N-1 cells did not activate NF-κB. In contrast, N-1 cells reconstituted with human WT-NEMO showed strong NF-κB activation upon TNFα treatment. However, TNFα treatment did not activate NF-κΒ when N-1 cells were reconstituted with the NEMO-UBAN mutant F312A that cannot bind to ubiquitin. To evaluate the role of LUBAC in the activation of NF-κB in the context of NEMO-ubiquitin binding we overexpressed LUBAC in N-1 cells reconstituted with WT-NEMO or NEMO-F312A (Fig. 2B). LUBAC overexpression in N-1 cells reconstituted with WT-NEMO showed a strong activation of NF-κB, whereas NF-κB did not get activated in NEMO-deficient N-1 cells or in cells that were reconstituted with NEMO-F312A. This indicates that the binding of NEMO to LUBAC-produced linear chains is important for the activation of NF-κB.

Modification of NEMO with Linear Di-ubiquitin Is Sufficient for Full NF-κB Activation in a NEMO-UBAN-dependent Manner

Because LUBAC produces linear chains and its addition to cell lysate activates IKK, we asked whether linear ubiquitin-chains can activate the IKK complex directly and whether other chain types are able to do so. To test this we incubated di-ubiquitin chains with the purified IKK complex and analyzed its activity (Fig. 3A). Addition of purified linearly linked di-ubiquitin to the IKK complex induced phosphorylation of MBP-IκBα (Fig. 3A). This activation was specific for the linear-ubiquitin linkage because when we tested Lys-63- and Lys-48-linked di-ubiquitins, which bind with approximately 100-fold lower affinity to the UBAN domain of NEMO (14, 17), activation of the IKK complex could not be observed (Fig. 3A).

Because linear ubiquitin chains can activate the IKK complex in vitro (Fig. 3A) and di-ubiquitin is sufficient to do so, we asked whether linearly ubiquitylated NEMO can activate NF-κB in vivo and what chain length is required for efficient activation. To this end, we generated FLAG-NEMO fused with one to seven noncleavable linear ubiquitin moieties (FLAG-NEMO-Ub_1–7) at the C terminus of the protein, thus mimicking the linearly ubiquitylated NEMO. Introduction of NEMO together with NF-κB luciferase reporter in HEK293T cells showed no activation of NF-κB, whereas NEMO-Ub₁ partially activated NF-κB (Fig. 3B). Introduction of NEMO having two to seven linearly conjugated ubiquitins potentiated NF-κB strongly and in a similar manner, indicating that the essential unit for full activation of NF-κB is linear di-ubiquitin (Fig. 3B). As a control, GFP fused to seven linearly conjugated ubiquitins at the C terminus could not activate NF-κB (data not shown). To further validate the activation of IKK, we immunoprecipitated endogenous IKKα/β in complex with NEMO or ubiquitin-fused NEMO and checked the phosphorylation levels of IKKs (Fig. 3C). Although similar levels of endogenous IKKα/β were co-immunoprecipitated with NEMO or ubiquitin-fused NEMO, IKKα/β in combination with NEMO-Ub₂ was heavily phosphorylated compared with that with NEMO or NEMO-Ub₁ (Fig. 3C). The proper activation of NF-κB by linear di-ubiquitin attached to NEMO depended on the ability of NEMO to bind to ubiquitin, because when we overexpressed the ubiquitin binding-deficient mutant NEMO-F312A-Ub₂, NF-κB activation was significantly weaker compared with WT-NEMO-Ub₂ (compare Fig. 3, B and D). This residual NF-κB activation in cells expressing NEMO-F312A-Ub_{1, 2–4} was also not specific for the linear linkage as activation with NEMO-F312A-Ub₄ was even weaker than with NEMO-F312A-Ub₁ (Fig. 3D). To examine if the binding of IKKα or IKKβ to linear ubiquitin might be involved in the activation of the IKK-complex we overexpressed FLAG-tagged IKKα, IKKβ, or NEMO in HEK293T cells and performed a pulldown assay with GST-di- or tetra-ubiquitin (supplemental Fig. S2). Although FLAG-NEMO strongly bound to GST-di- and tetra-ubiquitin, there was no interaction of IKKα or IKKβ with linear ubiquitin chains. These results strongly suggest that conjugation of linear di-ubiquitin to NEMO in concert with the interaction of NEMO with linear di-ubiquitin induces efficient canonical IKK activation.

NEMO That Selectively Binds to Lys-63-linked Ubiquitin Chains Weakly Activates NF-κB

Previous studies proposed that complex formation between NEMO and Lys-63-linked ubiquitin chains is important for the TNFα-induced activation of NF-κB signaling (18, 28). To test whether the binding of NEMO to Lys-63-linked ubiquitin chains is sufficient to activate the NF-κB pathway, we engineered NEMO chimeras that contain the NZF domain of TAK1-binding protein (TAB)2, an exclusive binder of Lys-63-linked ubiquitin chains (29) (Fig. 4A). To this end we swapped the NEMO-ZnF with the TAB2-NZF domain in combination with or without UBAN mutation at Phe-305 (F305A-NZF-ΔZnF-K63 or NZF-ΔZnF-K63/M1, Fig. 4A). In addition, we constructed a NEMO chimera, where we replaced the UBAN of NEMO with the NZF (NZF-ΔUBAN-K63; Fig. 4A). With these selective Lys-63 binding NEMO chimeras we set out to analyze the relevance of NEMO binding to Lys-63-linked ubiquitin chains in the activation of NF-κB.

To first confirm the ubiquitin-binding properties of these NEMO variants we purified the respective GST fusion proteins and incubated them with linearly or Lys-63-linked tetra-ubiquitin chains (Fig. 4B). We observed an exclusive binding of the two NZF-containing chimeras F305A-NZF-ΔZnF-K63 and NZF-ΔUBAN-K63 to Lys-63-linked ubiquitin chains. On the other hand, NEMO mutant NZF-ΔZnF-K63/M1 bound to both linearly and Lys-63-linked ubiquitin chains. In contrast, GST-WT-NEMO is a selective linear ubiquitin chain binder, whereas weak binding to Lys-63-linked tetra-ubiquitin was visible only after longer exposures of the film to the Western blot membrane (data not shown). The F305A mutation abolished the binding to ubiquitin, also as shown previously (14).

To further test how these chimeras bind to different ubiquitin chain types, we performed binding competition assays with the different GST-NEMO chimeras and linearly and Lys-63-linked tetra-ubiquitin chains (Fig. 4C). Although WT-NEMO and the NZF-containing but UBAN-deficient chimeras selectively bound linearly and Lys-63-linked tetra-ubiquitin chains respectively, as expected, the NEMO mutant NZF-ΔZnF-K63/M1 bound to linearly and Lys-63-linked tetra-ubiquitin chains with similar strength.

Next, we examined whether these Lys-63-selective NEMO chimeras are able to mediate TNFα-induced NF-κB signaling by generating reconstituted NEMO-deficient MEF lines with the respective NEMO mutants or WT-NEMO (Fig. 5). In these cell lines WT-NEMO and mutants were expressed at similar levels and could form an IKK complex (supplemental Fig. S3). TNFα-induced IKK activation was then examined using the reconstituted MEFs (Fig. 5A). In NEMO-deficient MEFs reconstituted with WT-NEMO or the mutant NEMO-NZF-ΔZnF-K63/M1, we observed degradation of IκBα after treatment with TNFα for 15 min (Fig. 5A). Interestingly, MEFs, which express either NEMO-NZF-ΔUBAN-K63 or NEMO-F305A-NZF-ΔZnF-K63, showed only partial degradation of IκBα with both mutants. The impaired activation of IKK in NEMO knock-out MEFs reconstituted with NEMO-F305A mutant has been shown previously (14). These observations suggest that binding of NEMO to Lys-63-linked ubiquitin chains is not sufficient to efficiently regulate TNFα-induced activation of NF-κB signaling. To examine whether the signaling defects in NEMO mutant-expressing cells were specific for NF-κB activation, we tested the TNFR-dependent activation of MAP kinase signaling pathways (supplemental Fig. S4). As indicated by their phosphorylation, both MAP kinases p38 and JNK are activated in cells expressing WT-NEMO and NEMO chimeras, suggesting that NEMO chimeras do not nonspecifically affect other signaling functions of the TNFR.

Next, we examined the nuclear translocation of p65 in TNFα-stimulated cells to analyze NF-κB activation in these MEFs downstream of IκBα degradation (Fig. 5B). MEFs reconstituted with WT-NEMO or NEMO-NZF-ΔZnF-K63/M1 showed strong activation of NF-κB as determined by the amount of nuclear p65 after 15 and 30 min of TNFα treatment. On the other hand, MEFs reconstituted with the ubiquitin binding-deficient F305A mutant did not mediate efficient p65 translocation. In contrast to MEFs with linear ubiquitin chain-binding NEMO, nuclear translocation of p65 was markedly impaired in MEFs expressing NEMO-NZF-ΔUBAN-K63 or NEMO-F305A-NZF-ΔZnF-K63 (Fig. 5B).

To further assess the NF-κB activation potential of Lys-63-selective NEMO chimeras, we analyzed the induction of the NF-κB target gene A20 (Fig. 5C). After 30 min of TNFα treatment, A20 gene expression was induced at 24.8 ± 2.33-fold in WT-NEMO-expressing cells and 19.1 ± 4.6-fold in NEMO-NZF-ΔZnF-K63/M1 cells compared with NEMO-deficient cells (Fig. 5C). In contrast, induction of A20 gene expression was significantly impaired in cells with Lys-63-binding NEMO or NEMO-F305 mutant (Fig. 5C). Although only WT-NEMO or the chimera that binds linear ubiquitin chains activated NF-κB properly, the Lys-63 binding-selective NEMO chimera NEMO-F305A-NZF-ΔZnF-K63 was also able to induce A20 expression at a certain level, which is significantly higher then with the non-ubiquitin-binding NEMO-F305A mutant (Fig. 5C). This is in agreement with a recent study showing that linear chain binding-deficient NEMO mutants could still partially activate NF-κB when they have a lower affinity toward Lys-63-linked ubiquitin chains (15).