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*

Background: Binding of NEMO to ubiquitin chains is essential for the activation of NF-κB. Results: Full-length NEMO preferentially binds linear ubiquitin chains in competition with lysine-linked ubiquitin chains. NEMO binding linear di-ubiquitin is sufficient for full NF-κB activation. Conclusion: NEMO is a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. Significance: Ubiquitin binding selectivity of NEMO is crucial for understanding the NF-κB activation pathways. Nuclear factor-κB (NF-κB) essential modulator (NEMO), a component of the inhibitor of κB kinase (IKK) complex, controls NF-κB signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells. Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-κB activation in cells. In TNFα-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-κB activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-κB activation patterns in response to numerous cell stimuli.


Nuclear factor-B (NF-B) essential modulator (NEMO), a component of the inhibitor of B kinase (IKK) complex, controls NF-B signaling by binding to ubiquitin chains. Structural studies of NEMO provided a rationale for the specific binding between the UBAN (ubiquitin binding in ABIN and NEMO) domain of NEMO and linear (Met-1-linked) di-ubiquitin chains. Full-length NEMO can also interact with Lys-11-, Lys-48-, and Lys-63-linked ubiquitin chains of varying length in cells.
Here, we show that purified full-length NEMO binds preferentially to linear ubiquitin chains in competition with lysine-linked ubiquitin chains of defined length, including long Lys-63-linked deca-ubiquitins. Linear di-ubiquitins were sufficient to activate both the IKK complex in vitro and to trigger maximal NF-B activation in cells. In TNF␣-stimulated cells, NEMO chimeras engineered to bind exclusively to Lys-63-linked ubiquitin chains mediated partial NF-B activation compared with cells expressing NEMO that binds to linear ubiquitin chains. We propose that NEMO functions as a high affinity receptor for linear ubiquitin chains and a low affinity receptor for long lysine-linked ubiquitin chains. This phenomenon could explain quantitatively distinct NF-B activation patterns in response to numerous cell stimuli.
NF-B transcription factors play a crucial role during development as well as in various biological functions including innate and adaptive immunity, inflammation, and cell survival (1,2). The NF-B signaling pathway is activated by various stimuli including LPS, TNF␣, IL-1, and UV irradiation (1). It is well established that the kinase complex IKK 5 is critical for the mediation of its downstream signal by phosphorylating inhibitor of B (IB), which leads to its subsequent degradation by the ubiquitin-proteasome system (2). The IKK complex consists of two kinases, IKK␣ and IKK␤, and a regulatory subunit called NEMO (3). It has been shown that NEMO is a critical component for cell survival and activation of the canonical NF-B pathway (4,5).
In such signaling cascades, the binding of the adaptor protein NEMO to ubiquitin chains appears to be a critical node in linking upstream ubiquitin signals with the activation of the IKK complex and subsequently the NF-B pathway (10, 14 -17).
NEMO is readily precipitated in protein complexes containing highly Lys-63-polyubiquitylated substrates (18); however, these complexes involved multiple interaction surfaces, and it was not clear whether interactions between NEMO and long Lys-63-linked ubiquitin chains are direct. More recent structural and functional studies indicate that the isolated NEMO UBAN domain preferentially binds to linear ubiquitin chains, but it can also bind with lower affinity to other types of ubiquitin chains including Lys-11, Lys-48, and Lys-63, presumably when they form longer chains (10,14). This led to the hypothesis that in addition to the linkage type also the length of the chains might be a critical determinant in NF-B activation (19). To address these issues we have undertaken a systematic and thorough biochemical approach by purifying full-length NEMO and analyzing its binding to ubiquitin chains with different linkages and length. In addition, by utilizing engineered NEMO chimeras that recognize exclusively Lys-63-linked ubiquitin chains as well as NEMO that is permanently linearly ubiquitylated we have tested the contribution of the linkage and length of specific ubiquitin chains in the activation of the NF-B pathway upon TNF␣ stimulation in vivo.

EXPERIMENTAL PROCEDURES
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 MluIϳBssHI into the MluI site of ubiquitin-VV. The whole nucleotide sequence was verified, and the respective tandem ubiquitin cDNA was ligated into FLAG-His 6 -human NEMO in pcDNA3.1 vector (Invitrogen). Full-length murine NEMO and human IB␣-WT (amino acids 1-54) and IB␣-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. Megamutagenesis 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.
MBP and GST Pulldown Experiments-GST-or MBP-fused proteins immobilized with Sepharose beads were incubated with 1 g 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 FLAGtagged 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.
Quantitative PCR-RNA was isolated from MEFs using an RNeasy kit (Qiagen). mRNA (2 g) 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).
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 fulllength 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 fulllength MBP-tagged NEMO toward Lys-11-, Lys-63-and lineartri-ubiquitin (Fig. 1A). Incubation of MBP-NEMO with these different chain types led to a selective binding of linear triubiquitin 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 heptaubiquitin 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 g 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 g 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-B in a NEMO-dependent Manner-Currently reported evidence indicates that NEMO binding to linear ubiquitin chains is essential for NF-B 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 IB␣ 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 IB␣ and exogenously added MBP-IB␣ (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 IB␣ ( Fig. 2A). The addition of E1 and E2 alone failed to phosphorylate IB␣ ( Fig.  2A), indicating a linear ubiquitin chain-dependent activation of the IKK complex.
Next, we wanted to examine whether LUBAC can activate NF-B 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, TNF␣ treatment of N-1 cells did not activate NF-B. In contrast, N-1 cells reconstituted with human WT-NEMO showed strong NF-B activation upon TNF␣ treatment. However, TNF␣ treatment did not activate NF-〉 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-B 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-B, whereas NF-B 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-B.

Modification of NEMO with Linear Di-ubiquitin Is Sufficient for Full NF-B 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 diubiquitin to the IKK complex induced phosphorylation of MBP-IB␣ (Fig. 3A). This activation was specific for the linearubiquitin linkage because when we tested Lys-63-and Lys-48linked 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-B 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-B luciferase reporter in HEK293T cells showed no activation of NF-B, whereas NEMO-Ub 1 partially activated NF-B (Fig. 3B). Introduction of NEMO having two to seven linearly conjugated ubiquitins potentiated NF-B strongly and in a similar manner, indicating that the essential unit for full activation of NF-B is linear di-ubiquitin (Fig. 3B). As a control, GFP fused to seven linearly conjugated ubiquitins at the C terminus could not activate NF-B (data not shown). To further validate the activation of IKK, we immunoprecipitated endogenous IKK␣/␤ in complex with NEMO or ubiquitin-fused NEMO and checked the phosphorylation levels of IKKs (Fig.  3C). Although similar levels of endogenous IKK␣/␤ were co- immunoprecipitated with NEMO or ubiquitin-fused NEMO, IKK␣/␤ in combination with NEMO-Ub 2 was heavily phosphorylated compared with that with NEMO or NEMO-Ub 1 (Fig. 3C). The proper activation of NF-B 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-Ub 2 , NF-B activation was significantly weaker compared with WT-NEMO-Ub 2 (compare Fig. 3, B and D). This residual NF-B activation in cells expressing NEMO-F312A-Ub 1, 2-4 was also not specific for the linear linkage as activation with NEMO-F312A-Ub 4 was even weaker than with NEMO-F312A-Ub 1 (Fig. 3D). To examine if the binding of IKK␣ or IKK␤ to linear ubiquitin might be involved in the activation of the IKK-complex we overexpressed FLAG-tagged IKK␣, IKK␤, or NEMO in HEK293T cells and performed a pulldown assay with GST-di-or tetraubiquitin (supplemental Fig. S2). Although FLAG-NEMO strongly bound to GST-di-and tetra-ubiquitin, there was no interaction of IKK␣ or IKK␤ 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-B-Previous studies proposed that complex formation between NEMO and Lys-63-linked ubiquitin chains is important for the TNF␣-induced activation of NF-B signaling (18,28). To test whether the binding of NEMO to Lys-63-linked ubiquitin chains is sufficient to activate the NF-B 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-⌬ZnF-K63 or NZF-⌬ZnF-K63/M1, Fig. 4A). In addition, we constructed a NEMO chimera, where we replaced the UBAN of NEMO with the NZF (NZF-⌬UBAN-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-B.
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-⌬ZnF-K63 and NZF-⌬UBAN-K63 to Lys-63-linked ubiquitin chains. On the other hand, NEMO mutant NZF-⌬ZnF-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-63linked 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
Next, we examined whether these Lys-63-selective NEMO chimeras are able to mediate TNF␣-induced NF-B 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). TNF␣-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-⌬ZnF-K63/M1, we observed degradation of IB␣ after treatment with TNF␣ for 15 min (Fig. 5A). Interestingly, MEFs, which express either NEMO-NZF-⌬UBAN-K63 or NEMO-F305A-NZF-⌬ZnF-K63, showed only partial degradation of IB␣ 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 TNF␣-induced activation of NF-B signaling. To examine whether the signaling defects in NEMO mutant-expressing cells were specific for NF-B 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 TNF␣-stimulated cells to analyze NF-B activation in these MEFs downstream of IB␣ degradation (Fig. 5B). MEFs reconstituted with WT-NEMO or NEMO-NZF-⌬ZnF-K63/M1 showed strong activation of NF-B as determined by the amount of nuclear p65 after 15 and 30 min of TNF␣ 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 chainbinding NEMO, nuclear translocation of p65 was markedly impaired in MEFs expressing NEMO-NZF-⌬UBAN-K63 or NEMO-F305A-NZF-⌬ZnF-K63 (Fig. 5B).
To further assess the NF-B activation potential of Lys-63selective NEMO chimeras, we analyzed the induction of the NF-B target gene A20 (Fig. 5C). After 30 min of TNF␣ treatment, A20 gene expression was induced at 24.8 Ϯ 2.33-fold in WT-NEMO-expressing cells and 19.1 Ϯ 4.6-fold in NEMO-NZF-⌬ZnF-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-B properly, the Lys-63 binding-selective NEMO chimera NEMO-F305A-NZF-⌬ZnF-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-B when they have a lower affinity toward Lys-63linked ubiquitin chains (15).

DISCUSSION
Because direct NEMO-ubiquitin interactions have only been determined by using isolated NEMO fragments, we examined how full-length NEMO interacts with different types of ubiquitin chain linkages. By applying competition assays using fulllength NEMO, we found that NEMO preferably binds linear ubiquitin chains compared with Lys-63-or Lys-11-linked chains, even when lysine-linked ubiquitin chains were longer than the linear chains (Fig. 1). The preference for linear ubiquitin chains is in agreement with previous studies, which showed that the NEMO-UBAN has an up to 100ϫ higher affinity to linear di-ubiquitin over Lys-63-, or Lys-48-linked di-ubiquitin (14,16,17). This might suggest that in a cellular environment where different ubiquitin chain types exist, e.g. at the activated TNFR complex (10,11,30), NEMO will preferentially bind to linear ubiquitin chains, whereas in the absence of linear ubiquitin chains other lysine-linked chains can engage in binding to NEMO and the activation of NF-B. In line with this, recent studies provided several lines of evidence that linear ubiquitylation by LUBAC E3 ligases is essential for the control of NF-B signaling (11)(12)(13)20). In the case of endogenous TNF␣-receptor complexes, linear ubiquitylation was detected on RIP1 and NEMO molecules (11). The absolute quantification of ubiquitin amounts using the absolute quantification of proteins method revealed a very low abundance of linear ubiquitin chains upon overexpression of LUBAC ligase, comprising Ͻ3% of the total ubiquitin pool in untreated cells (12). This suggests that linear ubiquitin chains are tightly regulated in their production, e.g. in a compartment-specific manner and/or are forming preferentially shorter chains due to their rapid cleavage in the cellular cytosol. In this context it is interesting that linear ubiquitin chains, as short as dimer, fused to NEMO can activate NF-B to a full extent compared with longer chains (Fig. 3). Thus, linearly linked di-ubiquitin conjugated to NEMO, due to its rather high affinity to the UBAN, constitutes an efficient and robust signal for IKK activation. On the other hand, MEFs reconstituted with NEMO chimeras harboring the highly Lys-63-selective NZF and unable to bind to linear chains led to only partial activation of NF-B signaling (Fig. 5). This partial activation confirms that binding of NEMO to Lys-63linked ubiquitin chains can have an effect on NF-B activation that is quantitatively different from linear chain-dependent activation.
These results collectively suggest that NEMO can act as bifunctional ubiquitin receptor: on one side a high affinity linear ubiquitin decoder leading to an efficient activation of NF-B and a lower affinity receptor for lysine-linked chains leading to quantitatively different patterns of NF-B activation. Moreover, the length of the ubiquitin chains appears to be important in such pathways: linear di-ubiquitin is a sufficient signal for the full activation of NF-B, whereas tetra-or longer lysine-linked ubiquitin chains are required for TNF␣-induced activation of the NF-B pathway. The future challenge in the field is to monitor endogenous ubiquitylation events at the activated TNFRs by using ubiquitin sensors that recognize specific chains in cells, e.g. fluorophore-tagged ubiquitin chain type-selective ubiquitin binding domains. Current data suggest that Lys-63and Lys-11-linked ubiquitin chains are the most proximal signals conjugated to the receptor-associated complexes, whereas linear ubiquitin chains are formed following LUBAC recruitment to the TNFR complexes leading to modification of NEMO and subsequent activation on NF-B. Dissection of dynamic changes in ubiquitin networks that control the NF-B pathway are important for better understanding of numerous cellular processes as well as pathogenesis of human diseases including autoimmunity, cancer, and inflammation.