NEMO-binding Domains of Both IKKα and IKKβ Regulate IκB Kinase Complex Assembly and Classical NF-κB Activation*

Proinflammatory NF-κB activation requires the IκB (inhibitor of NF-κB) kinase (IKK) complex that contains two catalytic subunits named IKKα and IKKβ and a regulatory subunit named NF-κB essential modulator (NEMO). NEMO and IKKβ are essential for tumor necrosis factor (TNF)-induced NF-κB activation, and we recently demonstrated that NEMO and IKKα are sufficient for interleukin (IL)-1-induced signaling. IKKα and IKKβ both contain a functional NEMO-binding domain (NBD); however, the role of NEMO association with each kinase in NF-κB signaling and IKK complex formation remains unclear. To address this question, we stably reconstituted IKKα−/− and IKKβ−/− murine embryonic fibroblasts (MEFs) with wild-type (WT) or NBD-deficient (ΔNBD) versions of IKKα and IKKβ, respectively. TNF-induced classical NF-κB activation in IKKβ−/− MEFs was rescued by IKKβWT but not IKKβΔNBD, whereas neither IKKβWT nor IKKβΔNBD affected IL-1-induced NF-κB signaling. As previously described, classical NF-κB transcriptional activity was absent in IKKα−/− cells. Reconstitution with either IKKαWT or IKKαΔNBD rescued both IL-1 and TNF-induced transcription, demonstrating that NEMO association is not required for IKKα-dependent regulation of NF-κB-dependent transcription. Stably expressed IKKαWT or IKKβWT associated with endogenous IKKs and NEMO in IKKα−/− or IKKβ−/− MEFs, respectively, resulting in formation of the heterotrimeric IKKα-IKKβ-NEMO complex. In contrast, although the IKKαΔNBD and IKKβΔNBD mutants associated with endogenous IKKs containing an NBD, these dimeric endogenous IKK-IKKΔNBD complexes did not associate with NEMO. These findings therefore demonstrate that formation of the heterotrimeric IKKα-IKKβ-NEMO holocomplex absolutely requires two intact NEMO-binding domains.

NF-B 2 describes a family of transcription factors that regulate the inducible expression of many genes essential for innate and adaptive immunity, inflammation, and cell survival. A wide range of stimuli activates NF-B, including ligation of innate immune receptors (e.g. TLRs), antigen receptor engagement (B cell receptor and T cell receptor), and proinflammatory cytokines (e.g. IL-1 and TNF) (1). NF-B activation by these stimuli is normally rapid and transient; however, constitutive NF-B activity occurs in some chronic inflammatory diseases, solid tumors, leukemias, and lymphomas (1,2). Understanding the molecular mechanisms that regulate NF-B activity will therefore reveal novel targets for blocking pathophysiological NF-B signaling (3)(4)(5).
The five NF-B family members are NF-B1/p105 and NF-B2/p100 that are processed to generate p50 and p52, respectively, p65 (RelA), c-Rel, and RelB (1). These proteins homo-or heterodimerize to form transcriptionally active (e.g. p50-p65) or repressive (e.g. p50-p50) dimers that are retained in the cytosol of resting cells by members of the inhibitory family of IB proteins. The IBs include IB␣, IB␤, IB⑀, and the C termini of p105 and p100. The prototypic NF-B-IB complex expressed in most cell types is a heterodimer of p50 and p65 associated with IB␣ (1). Following cell stimulation, the IBs are rapidly phosphorylated, ubiquitinated, and then degraded by the 26 S proteasome. Free NF-B dimers then migrate to the nucleus, where they bind target gene promoters and regulate transcription (1).
The IB proteins are phosphorylated by the high molecular weight heterotrimeric IB (inhibitor of NF-B) kinase (IKK) complex (1,6,7). The IKK complex contains two kinases named IKK␣ (IKK1) and IKK␤ (IKK2) and a noncatalytic subunit named NEMO (NF-B essential modulator) or IKK␥ (1,6,7). NEMO is critical for proinflammatory IKK activation (8 -12), and we previously identified a domain within the C termini of both IKK␣ and IKK␤ that facilitates their association with NEMO (13,14). A cell-permeable peptide spanning this NEMO-binding domain (NBD) disrupts the IKK complex and blocks proinflammatory NF-B activation, confirming the crucial role of NEMO association for IKK complex activation (13).
Despite their significant structural similarities, genetic analyses of IKK␣ and IKK␤ revealed distinct roles for the kinases during NF-B activation (1,6,7,15). In this regard, TNF-induced IB␣ degradation is dependent upon IKK␤ and also requires NEMO (8,16,17). This mechanism is termed the classical NF-B pathway and is defined as NEMO-and IKK␤-dependent IB phosphorylation and degradation releasing canonical NF-B complexes typified by the ubiquitous p50-p65 heterodimer. IKK␣ plays a separate role in mediating NIK (NF-B-inducing kinase)-dependent processing of NF-B2/p100 to generate p52 (18 -22). This mechanism is activated in the absence of both IKK␤ and NEMO and is named the noncanoni-cal NF-B pathway (15). Ligation of only a subset of receptors, including the lymphotoxin-␤ receptor, CD40, and BAFF-R, activates the IKK␣-dependent noncanonical pathway, and the resulting p52, together with RelB, regulates a panel of chemokine and cytokine genes required for lymphoid organogenesis and B-cell maturation (18 -23). IKK␣ has also been shown to play several roles in regulating the transcriptional activity of classical NF-B that are separate from its upstream signaling function as an IB kinase (24 -29).
Since TNF-induced IB␣ degradation and classical NF-B nuclear translocation occurs in the absence of IKK␣, a model of classical pathway activation has emerged in which IKK␣ is redundant (15). It remains, however, that IKK␣ associates via its NBD with NEMO (14), and we previously questioned whether this association plays a functional role in classical NF-B signaling (30). Surprisingly, we found that although TNF-induced IB␣ degradation requires NEMO and IKK␤, IL-1-induced classical pathway activation is intact in cells lacking IKK␤. Furthermore, IL-1-induced NF-B activation in IKK␤-deficient cells was blocked by the NBD peptide, demonstrating that a complex of only IKK␣ and NEMO is sufficient for IL-1-but not TNF-induced classical NF-B activation (30). Intriguingly, Lam et al. (31) recently demonstrated that IKK␣ plays a crucial compensatory role in regulating constitutive classical NF-B pathway activation in a subset of diffuse large B-cell lymphoma cells in which IKK␤ has been pharmacologically inhibited. These findings therefore identify differences in the absolute requirements for the separate IKK subunits activated in a NEMO-dependent manner by distinct stimuli and suggest that targeting only IKK␤ may not effectively block dysregulated classical NF-B activation. Consequently, determining the role of the interaction of each IKK subunit with NEMO will provide novel insight into the mechanisms that regulate NEMO-dependent classical NF-B activation.
To address this question, we determined the effects of individually deleting the NBD in IKK␣ and IKK␤ on classical NF-B signaling and IKK complex formation. We stably reconstituted IKK␣ Ϫ/Ϫ and IKK␤ Ϫ/Ϫ murine embryonic fibroblasts (MEFs) with wild-type (WT) or NBD-deficient (⌬NBD) versions of IKK␣ and IKK␤, respectively. Reconstitution of IKK␤ Ϫ/Ϫ MEFs with IKK␤ WT but not IKK␤ ⌬NBD rescued TNF-induced classical NF-B activation, confirming the requirement for the NEMO-IKK␤ association for TNF signaling. IL-1 signaling was intact in IKK␤ Ϫ/Ϫ cells, and this was not affected by expression of either IKK␤ WT or IKK␤ ⌬NBD . Classical NF-B transcriptional activity was absent in IKK␣ Ϫ/Ϫ cells, and reconstitution with either IKK␣ WT or IKK␣ ⌬NBD rescued both IL-1-and TNFinduced transcription. This therefore demonstrates that association with NEMO is not necessary for IKK␣-dependent regulation of NF-B-dependent transcriptional activity. Immunoprecipitation analysis and size exclusion chromatography revealed that stably expressed IKK␣ WT or IKK␤ WT associated with endogenous IKKs and NEMO in IKK␣ Ϫ/Ϫ or IKK␤ Ϫ/Ϫ MEFs, respectively. This association resulted in the formation of the heterotrimeric IKK␣-IKK␤-NEMO complex. In contrast, despite the ability of the IKK␣ ⌬NBD and IKK␤ ⌬NBD mutants to associate with endogenous IKKs containing an NBD, these mutants formed dimeric endogenous IKK-IKK ⌬NBD complexes that did not associate with NEMO. These findings therefore demonstrate that formation of the heterotrimeric IKK␣-IKK␤-NEMO holocomplex absolutely requires the presence of two intact NEMO-binding domains.
Generation of Stable Cell Lines-All cloning procedures were performed by PCR using cloned Pfu DNA polymerase (Stratagene, La Jolla, CA). Complementary DNA encoding fulllength IKK␤ or IKK␤ 1-733 (IKK␤ ⌬NBD ) were subcloned into the HindIII and NotI sites of the LZRS-pBMN-lacZ retroviral vector (kindly provided by Dr. Garry Nolan). Resulting LZRS-IKK␤ WT and LZRS-IKK␤ ⌬NBD were transiently transfected using Fugene6 into Phoenix cells and selected for gene expression 24 h after transfection using puromycin (1 g/ml). Puromycin-resistant cells were used to derive conditioned medium to provide a retroviral stock for MEF transduction. For cell transduction, IKK␤ Ϫ/Ϫ MEFs were washed and incubated for 8 h with retrovirus-conditioned medium containing Polybrene (8 g/ml; Sigma). After incubation, retrovirus was removed and replaced with normal growth medium. The transduction process was repeated a further three times until cells became positive for IKK␤ when visualized by immunoblotting.
To generate stably transduced IKK␣ WT and IKK␣ ⌬NBD MEFs, full-length IKK␣ or IKK␣ ⌬NBD cDNA was cloned into the EcoRI and XhoI restriction sites of retroviral GFP-MIGR1 vector (kindly provided by Dr. Warren Pear, University of Pennsylvania, Philadelphia, PA). The resulting MIG-IKK␣ WT and MIG-IKK␣ ⌬NBD were transiently transfected using Fugene6 into Plat-E cells to produce ecotropic virus that was derived from conditioned medium containing Polybrene (8 g/ml). For cell transduction, IKK␣ Ϫ/Ϫ MEFs were washed and incubated for 8 h with retrovirus-conditioned medium. After incubation, retrovirus was removed and replaced with normal growth medium. The transduction process was repeated a further three times until cells became positive for IKK␣, as assessed by FACS analysis using Cell Quest software (FACSort; BD Biosciences).
For cell sorting, transduced cells were trypsinized and washed in FACS buffer (sterile phosphate-buffered saline, 0.5 M EDTA, and 0.5% bovine serum albumin). Evaluation of green fluorescent protein was performed on a three-laser (argon (488 nm), krypton (407 nm), and dye laser (tuned to 600 nm)), 10parameter FACSVantage TM obtained from BD Biosciences. Compensation and data analyses were performed using FlowJo software (Tree Star, Ashland, OR).
Immunoblotting and Immunoprecipitation-Cells were washed once with phosphate-buffered saline and then incubated for 10 min at 4°C in 100 l of TNT lysis buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, and 1% Triton X-100) and a complete miniprotease inhibitor mixture (Roche Applied Science). Samples were then scraped and harvested into 1.5-ml microcentrifuge tubes, vortexed for 30 s, and then centrifuged (425 ϫ g for 10 min). Protein levels in supernatants were determined using a Coomassie protein assay kit (Bio-Rad), and 20 g of protein from each sample was separated by SDS-PAGE (10%) then transferred to a polyvinylidene difluoride membrane (Millipore, Milford, MA) and immunoblotted with primary and horseradish peroxidase-conjugated secondary antibodies. Detection of the bound antibody by enhanced chemiluminescence was performed according to the manufacturer's instructions (Pierce). For immunoprecipitations, cell extracts were incubated with 2 g of primary antibody for 1 h at 4°C, followed by incubation (1 h/4°C) with 50 l of Protein G-Sepharose beads (50% slurry) or 50 l Protein A/G beads (50% slurry). A portion of each sample preimmunoprecipitation (5%) was retained for analysis. The beads were washed three times with lysis buffer, and then samples were analyzed by SDS-PAGE (10%) followed by immunoblotting, as described above.
Immune Complex Kinase Assay-For immune complex kinase assays, MEF lysates were prepared, and immunoprecipitations were performed as described above. The resulting immunoprecipitates were washed extensively in TNT and then kinase buffer (20 mM HEPES, pH 7.5, 20 mM MgCl 2 , 1 mM EDTA, 2 mM NaF, 2 mM ␤-glycerophosphate, 1 mM dithiothreitol, 10 mM ATP). Precipitates were then incubated for 15 min at 30°C in 20 l of kinase buffer containing glutathione S-transferase-IB␣ (amino acids 1-54; a generous gift from Dr. Serge Fuchs, University of Pennsylvania) and 10 Ci of [␥-32 P]ATP (Amersham Biosciences). The substrate was then precipitated using glutathione-agarose (Amersham Biosciences) and washed extensively with TNT. Beads were then suspended in 20 l of sample buffer, and samples were separated by SDS-PAGE (10%). The resulting gel was stained with Coomassie Blue solution (0.05% Brilliant Blue G250, 40% methanol, 10% acetic acid) and then destained (40% methanol, 10% acetic acid) and dried. Kinase activity was determined by autoradiography.
Generation of Nuclear Lysates for Immunoblotting-MEFs were stimulated with TNF␣ (10 ng/ml) for the indicated times and then scraped into phosphate-buffered saline at 4°C and pelleted (425 ϫ g, 10 min). Pellets were resuspended and swollen for 10 min on ice in 100 l of Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 2 mM NaF, 2 mM ␤-glycerol phosphate, and complete miniprotease inhibitors) plus 0.1% Nonidet P-40 and centrifuged (3800 ϫ g) for 1 min. Supernatants (cytoplasmic fraction) were centrifuged at 20,000 ϫ g for 1 h at 4°C, and the resulting supernatants were snap frozen and retained for analysis. Pelleted nuclei were washed four times with 100 l of Buffer A plus 0.1% Nonidet P-40 buffer before being lysed in 30 l of 1% TNT, 1% SDS buffer plus complete mini protease inhibitors with a 26-gauge (half-inch) needle. Nuclear lysates were then centrifuged for 20 min (20,000 ϫ g) at room temperature and then either used immediately or snap frozen and stored at Ϫ80°C. Lysates were immunoblotted as described above.
Transfections and Luciferase Reporter Assays-WT MEFs and stable cell lines grown in 12-well plates (2.5 ϫ 10 5 /well) were transiently transfected using Fugene6 (Roche Applied Science) following the manufacturer's protocol. Cells were transfected with a total of 0.11 g of DNA/well, consisting of the NF-B-dependent firefly reporter construct pBIIx-luciferase (0.2 g/well) and a Renilla luciferase reporter (0.02 g/ml). Cells were stimulated with TNF␣ or IL-1␣ for 5 h and then lysed in passive lysis buffer (Promega, San Luis Obispo, CA) 24 -36 h after transfection. Samples were assayed using a Luminoscan 96-well automated luminometer (Thermo Labsystems, Franklin, MA), and firefly/Renilla luciferase ratios were calculated using Ascent software (Thermo LabSystems).
Electrophoretic Mobility Shift Assays (EMSAs)-MEFs were stimulated with TNF␣ (10 ng/ml) or IL-1␣ (10 ng/ml) for the indicated times and then scraped into phosphate-buffered saline at 4°C and pelleted (425 ϫ g, 10 min). Pellets were resuspended and swollen for 30 min on ice in 100 l of Buffer A (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 2 mM NaF, 2 mM ␤-glycerol phosphate, and complete mini protease inhibitors), incubated a further 5 min on ice in 0.1% Nonidet P-40, and then vortexed and centrifuged (3800 ϫ g) for 1 min. Supernatants (cytoplasmic fraction) were centrifuged at 20,000 ϫ g for 1 h at 4°C, and the resulting supernatants were snap-frozen and retained for analysis. Pelleted nuclei were washed once with 100 l of Buffer A buffer before being vortexed in 30 l of NarC buffer (20 mM HEPES, pH 7.9, 0.4 M NaCl, pH 8, 1 mM EDTA, pH 8, 2 mM NaF, 2 mM ␤-glycerol phosphate, and complete mini protease inhibitors) for 1 h at 4°C. Nuclear lysates were then centrifuged for 20 min (20,000 ϫ g) at room temperature and then either used immediately or snap-frozen and stored at Ϫ80°C.
Single-stranded complementary oligonucleotides encompassing a consensus NF-B site (upper strand, 5Ј-AGTT-GAGGGGACTTTCCCAGGC-3Ј) or the Oct-1 probe (Santa Cruz Biotechnology) were annealed and then labeled with [␥-32 P]ATP using T4 PNK (New England Biolabs, Beverly, MA). Labeled probe was purified using mini-Quick Spin columns (Roche Applied Science) according to the manufacturer's instructions. For EMSA, 2-5 g of nuclear extracts supplemented with 1 g of poly(dI-dC) (Roche Applied Science) was incubated with an equal volume of 2ϫ binding buffer (40 mM Tris⅐Cl, pH 7.9, 100 mM NaCl, 10 mM MgCl 2 , 2 mM EDTA, 20% glycerol, 0.2% Nonidet P-40, 2 mM dithiothreitol, 100 g/ml bovine serum albumin) on ice for 10 min. After incubation, 1 l of labeled probe was added, and then samples were incubated at room temperature for 20 min. Resulting DNA-NF-B complexes were separated on 5% polyacrylamide nondenaturing gels by electrophoresis, and then gels were dried and visualized by autoradiography.
NBD Peptides-NBD peptides were obtained from the Howard Hughes Medical Institute Biopolymer-Keck Foundation Biotechnology Resource Laboratory at Yale University (New Haven, CT). Immediately prior to use, the peptides were dissolved in dimethyl sulfoxide to a stock of 50 mM. The sequences of the wild-type (NBD WT ) and mutant (NBD MUT ) peptides have been described previously (13,14). The NBD WT peptide contains the region of IKK-␤ from Thr 735 to Glu 745 synthesized in tandem with a membrane permeabilization sequence from the Drosophila antennapedia homeodomain protein. The NBD MUT peptide is identical, except that Trp 739 and Trp 741 are replaced by alanines to render it biologically inactive (13,14).
Fast Protein Liquid Chromatography-Confluent monolayers of MEFs were trypsinized and pelleted after one wash with ice-cold phosphate-buffered saline. To make S100 lysates, cell pellets were swollen on ice for 30 min in 500 l of lysis buffer (10 mM HEPES, pH 7.9, 10 mM KCl, 0.1 mM EDTA). After 30 min, a final volume 0.1% Nonidet P-40 was added to the swollen pellet, and cells were lysed with 15 strikes of a 2-ml Dounce homogenizer on ice. Finally, one-fourth volume of 5ϫ gel filtration buffer (100 mM Tris, pH 7.5, 50% glycerol, 2.5 mM EDTA, 750 mM NaCl) was added to the lysate before being centrifuged for 10 min at 425 ϫ g. The remaining supernatant was then ultracentrifuged 1 h at 100,000 ϫ g.

The IKK␤ NBD Is Required for TNF-but Not IL-1-induced NF-B Activation-
To determine the effects of selectively disrupting the IKK␤-NEMO interaction on classical NF-B signaling and IKK complex formation, we generated retroviral constructs encoding both WT IKK␤ and a truncation mutant (residues 1-733) lacking the NBD (IKK␤ ⌬NBD ) (Fig. 1A). We stably transduced IKK␤-deficient MEFs with these constructs and confirmed by immunoblotting that the cells were reconstituted with IKK␤ WT and IKK␤ ⌬NBD (Fig. 1B). FACS analysis verified that over 95% of the cells were stably transduced with each construct (data not shown). The levels of IKK␤ WT in multiple cell lines that we generated were consistently higher than endogenous IKK␤ in WT MEFs. However, the levels of reconstituted IKK␤ ⌬NBD were comparable with endogenous IKK␤ levels in WT cells (Fig. 1B, compare lanes 1 and 4). To examine the effects of reconstituted IKK␤ WT and IKK␤ ⌬NBD on classical NF-B activation, we incubated WT, IKK␤ Ϫ/Ϫ , IKK␤ WT , and IKK␤ ⌬NBD MEFs for a range of times with TNF or IL-1 and then immunoblotted the resulting lysates using anti-IB␣. Following cytokine treatment, IB␣ degradation and resynthesis was intact in WT MEFs, and consistent with our earlier findings (30), IL-1 but not TNF induced robust IB␣ degradation in IKK␤ Ϫ/Ϫ cells (Fig. 1C). As expected, TNF-stimulated IB␣ degradation was restored in IKK␤ WT -reconstituted MEFs (Fig.  1C). In contrast, TNF did not induce IB␣ degradation in IKK␤ ⌬NBD -reconstituted cells (Fig. 1C), confirming that the IKK␤ NBD is required for TNF-induced classical NF-B pathway activation.
We next examined basal and IL-1-induced NF-B transcriptional activity in the panel of MEFs using an NF-B-specific luciferase reporter assay. Consistent with our previous results (30), IL-1 increased NF-B activity in IKK␤ Ϫ/Ϫ MEFs to a similar level as that observed in WT cells (Fig. 1E). Both basal and IL-1-stimulated levels of NF-B activity were slightly enhanced in IKK␤ WT -reconstituted MEFs, possibly due to the higher levels of IKK␤ expression in these cells compared with WT MEFs (Fig. 1B). However, basal and IL-1-induced NF-B-dependent transcriptional activity was enhanced in IKK␤ ⌬NBD MEFs (Fig.  1E) that contained levels of IKK␤ ⌬NBD comparable with WT MEFs (Fig. 1B). This finding is consistent with our previous observation of enhanced NF-B activation in HeLa cells transiently overexpressing NEMO-binding mutants of IKK␤ (13,14) and supports our earlier conclusion that the IKK␤ NBD plays a role in maintaining the basal activity of the IKK complex.
IKK␤ ⌬NBD Is Not Incorporated into the IKK Holocomplex-Within the tripartite IKK holocomplex, IKK␣ and IKK␤ interact via their respective leucine zipper domains (34). Each IKK subunit in turn associates via its C-terminal NBD with NEMO (13, 14, 35). We therefore questioned whether IKK␤ ⌬NBD was incorporated into a trimeric IKK complex in IKK␤ Ϫ/Ϫ MEFs via interaction with endogenous IKK␣ associated with NEMO. To determine the effects of deleting the IKK␤ NBD on complex formation, we immunoprecipitated IKK complexes from WT, IKK␤ Ϫ/Ϫ , IKK␤ WT , and IKK␤ ⌬NBD MEFs using anti-NEMO, anti-IKK␣, and anti-IKK␤. As shown in Fig. 2A, IKK␣ and IKK␤ both co-immunoprecipitated with NEMO from WT and IKK␤ WT -reconstituted MEFs. In contrast, only IKK␣ associated with NEMO in IKK␤ Ϫ/Ϫ and IKK␤ ⌬NBD -reconstituted cells. When immunoprecipitations were performed using anti-IKK␣, both NEMO and IKK␤ co-precipitated with IKK␣ from WT, IKK␤ WT , and IKK␤ ⌬NBD MEFs, whereas only NEMO associated with IKK␣ in IKK␤ Ϫ/Ϫ cells (Fig. 2B). Immunoprecipitation using anti-IKK␤ pulled down all three IKK complex sub-  (13,14). B, lysates from WT, IKK␤ Ϫ/Ϫ (␤ Ϫ/Ϫ ), IKK␤ WT (␤ WT ), and IKK␤ ⌬NBD (␤ ⌬NBD ) MEFs were immunoblotted using the antibodies indicated (right). C, WT, IKK␤ Ϫ/Ϫ , IKK␤ WT , and IKK␤ ⌬NBD MEFs were incubated with either TNF (10 ng/ml) (left) or IL-1␣ (10 ng/ml) (right) for the times indicated, and then lysates were immunoblotted using anti-IB␣ or anti-tubulin (Tub.) as a loading control. D, the same panel of MEFs was treated with IL-1␣ for the indicated times, and then nuclear extracts were prepared for EMSA. Assays were performed using either a consensus NF-B binding site probe (top) or an Oct-1 probe as a loading control (bottom). E, MEFs were transiently transfected with the NF-B-dependent reporter pBIIx-firefly luciferase together with ␤-actin Renilla luciferase. Twenty-four hours later, cells were either left untreated or treated for a further 5 h with IL-1␣, and then NF-B activity was determined by a dual luciferase assay. units from WT and IKK␤ WT -reconstituted MEFs, but as expected, anti-IKK␤ did not precipitate any of these proteins from IKK␤ Ϫ/Ϫ cells (Fig. 2C, lane 2). Intriguingly, anti-IKK␤ pulled down complexes consisting of IKK␤ ⌬NBD and IKK␣ but not NEMO from IKK␤ ⌬NBD -reconstituted MEFs (Fig. 2C, lane  4). These findings therefore suggest that reconstituted IKK␤ WT is incorporated into the heterotrimeric and IKK complex, whereas IKK␤ ⌬NBD associates only with endogenous IKK␣ in a separate complex that does not contain NEMO.
To further explore the nature of the IKK complexes in the reconstituted cell lines, we performed size exclusion chroma-tography of S100 lysates. As shown in Fig. 3A (top), and consistent with previous reports (10,34,36,37), IKK␣, IKK␤, and NEMO in WT MEFs co-eluted in fractions corresponding to a predicted molecular mass of 600 -900 kDa. Immunoprecipitation of complexes from these fractions using anti-NEMO or anti-IKK␣ confirmed that all three subunits co-precipitated, with the majority of the IKK holocomplex appearing in fractions 18 and 19 (Fig. 3A, bottom). Fractionation of lysates from IKK␤ Ϫ/Ϫ cells showed that IKK␣ and NEMO elute together in fractions 18 -21, suggesting a complex of ϳ500 -900 kDa ( Fig. 3B; top). In addition, IKK␣ eluted in fractions 22-24, which did not contain any NEMO. Immunoprecipitation of complexes from fractions 17-23 using anti-NEMO demonstrated association of IKK␣ with NEMO in fractions 18 -21 (Fig.  3B, bottom). These data therefore demonstrate that in IKK␤ Ϫ/Ϫ MEFs, an IKK complex consisting of only NEMO and IKK␣ exists that is similar in size to the tripartite complex in WT cells. In addition, a portion of IKK␣ does not associate with NEMO in the absence of IKK␤ but instead elutes in fractions corresponding to a lower molecular weight NEMO-independent complex.
All three IKK complex subunits eluted in high molecular weight fractions (fractions [17][18][19][20] in IKK␤ WTreconstituted IKK␤ Ϫ/Ϫ MEFs (Fig.  3C, top), and immunoprecipitation demonstrated that IKK␣, IKK␤, and NEMO associate in these fractions (Fig. 3C, bottom). Similar to IKK␤ Ϫ/Ϫ cells, IKK␣ also appeared in fractions 21-24, which did not contain NEMO, and NEMO-IKK␣ complexes could not be immunoprecipitated in these fractions. Hence, reconstituted IKK␤ WT incorporates into the high molecular weight tripartite IKK holocomplex in IKK␤ Ϫ/Ϫ MEFs, and lower molecular weight endogenous IKK␣-containing complexes are also detected in these cells. We also consistently observed IKK␤ in later eluting fractions up to sample number 27. IKK␤ in these fractions was not associated with either IKK␣ or NEMO (Fig. 3C, bottom) (data not shown), suggesting that it is an IKK␤ homodimeric complex possibly resulting from the high level of overexpression in these cells (Fig. 1B). Similar to IKK␤ Ϫ/Ϫ MEFs, IKK␣ and NEMO eluted together in fractions 17-21 in IKK␤ ⌬NBD cell lysates, suggesting that they exist as a complex comparable in size with native IKK in WT MEFs (Fig. 3D, top). IKK␤ ⌬NBD did not co-elute with the majority of IKK␣ and NEMO but instead eluted in two pools with peaks in fractions 20 -22 and 26 -27, respectively. The first pool of IKK␤ ⌬NBD co-eluted with IKK␣ in fractions 19 -23, whereas the second pool did not co-elute with either IKK␣ or NEMO. When immunoprecipitations were performed using anti-NEMO (Fig. 3D, bottom), IKK␣ but not IKK␤ ⌬NBD associated with NEMO in the high molecular weight fractions. Similarly, NEMO co-precipitated with anti-IKK␣ in fractions 17-21, but IKK␤ ⌬NBD only associated with IKK␣ in fractions 20 -23.
Collectively, these data support our findings in Fig. 2 and demonstrate that reconstituted IKK␤ ⌬NBD does not incorporate into the tripartite IKK complex in IKK␤ Ϫ/Ϫ MEFs. In these cells, IKK␣ and NEMO form the high molecular weight IKK complex, whereas IKK␤ ⌬NBD associates with IKK␣ in a separate NEMO-independent complex. In addition, IKK␤ ⌬NBD appears in a third, lower molecular weight complex that does not contain either IKK␣ or NEMO.
IKK␣ ⌬NBD Restores Noncanonical NF-B Signaling in IKK␣ Ϫ/Ϫ MEFs-To establish the effects of selectively disrupting the IKK␣-NEMO interaction on NF-B signaling and IKK complex formation, we generated retroviral constructs encoding both WT IKK␣ and a truncation mutant (residues 1-745) lacking the NBD (IKK␣ ⌬NBD ) (Fig. 4A) and used these to stably transduce IKK␣-deficient MEFs. Immunoblotting demonstrated that the cells were reconstituted with IKK␣ WT and IKK␣ ⌬NBD (Fig. 4B), and FACS analysis confirmed that over 95% of the cells were stably transduced with each construct (not shown). The levels of IKK␣ WT and IKK␣ ⌬NBD in multiple stable cells lines generated were consistently similar to those of endogenous IKK␣ in WT MEFs.
Since the ability of IKK␣ to regulate NF-B-dependent transcription has been shown to involve phosphorylation of p65 at Ser 536 (28,38,39), we immunoblotted cell lysates using an antibody that recognizes phosphorylated Ser 536 on p65. As shown in Fig. 5E, Ser 536 phosphorylation in response to TNF was severely diminished in IKK␣ Ϫ/Ϫ cells, whereas re-expression of either IKK␣ WT or IKK␣ ⌬NBD restored TNF-induced p65 phosphorylation to levels similar to that in WT MEFS (Fig. 5E).
In addition to IKK␣, NEMO has also been shown to shuttle between the cytoplasm and nucleus (40 -43). We therefore questioned whether the ability of IKK␣ to enter the nucleus, where it regulates NF-B transcriptional activity, requires its association with NEMO. As shown in Fig. 5F (top), IKK␣ was present in the nucleus of untreated WT, IKK␣ WT , and IKK␣ ⌬NBD MEFs, and neither TNF nor IL-1 (not shown) stimulation further enhanced its nuclear localization. Furthermore, NEMO was present in the nucleus in WT, IKK␣ Ϫ/Ϫ , and IKK␣ WT MEFs but was severely depleted in the nucleus of IKK␣ ⌬NBD cells. As previously shown (24,29), IKK␤ was absent in the nucleus of WT cells. Surprisingly, however, we detected elevated nuclear IKK␤ in IKK␣ Ϫ/Ϫ MEFs that was significantly diminished following reconstitution with either IKK␣ WT or IKK␣ ⌬NBD . Notably, NEMO was also present in the nucleus of IKK␣ Ϫ/Ϫ MEFs.
To determine whether nuclear IKK␣ in the reconstituted cells is activated in response to proinflammatory stimuli, we immunoblotted nuclear lysates using a phosphospecific antibody that recognizes both IKK␣ and IKK␤ only when they are phosphorylated on crucial serines within their catalytic activation loops. As shown in Fig. 5G, this antibody detected phosphorylated IKK␣ in WT, IKK␣ WT , and IKK␣ ⌬NBD cells, demonstrating that IKK␣ in the nuclei of these cells is activated in response to TNF. However, although IKK␤ was present in the nucleus of IKK␣ Ϫ/Ϫ cells (Fig.  5G, lanes 3 and 4), we did not detect its phosphorylation, suggesting that nuclear IKK␤ is not activated in response to TNF.
Taken together, the findings in Fig. 5 strongly support a model in which the transcriptional regulatory activity of IKK␣ is independent of its function as a NEMO-associated IB kinase. Our data also suggest that the ability of IKK␣ to enter the nucleus and become activated in response to proinflammatory cytokines is NEMOindependent, whereas normal nuclear localization of NEMO requires an intact IKK␣ NBD. Finally, these results indicate that IKK␤ can enter the nucleus but only in the absence of IKK␣.
To further investigate the complexes formed in the reconstituted cell lines, we performed size exclusion chromatography. Following separation of lysates from IKK␣ Ϫ/Ϫ cells, IKK␤ and NEMO eluted together in fractions 18 -21 (Fig. 6C,  top), and immunoprecipitations using anti-NEMO demonstrated that IKK␤ associated with NEMO in these fractions (Fig. 6C, bottom). This profile suggests a NEMO-IKK␤ complex of similar size (500 -900 kDa) to the tripartite IKK complex in wild-type MEFs that predominantly eluted in fractions 18 and 19 (Fig. 3A). When we reconstituted IKK␣ Ϫ/Ϫ MEFs with IKK␣ WT , the bulk of IKK complexes eluted in fractions 17-20 (Fig. 6D, top), and immunoprecipitation using either anti-NEMO or anti-IKK␣ confirmed that these complexes contain IKK␣, IKK␤, and NEMO (Fig. 6D, bottom). Thus, IKK␣ WT is incorporated into the high molecular weight tripartite IKK complex in IKK␣ Ϫ/Ϫ cells.
Fractionation of lysates from IKK␣ ⌬NBD -reconstituted MEFs showed that IKK␣ ⌬NBD , IKK␤, and NEMO elute in a broad range of fractions spanning predicted molecular masses from ϳ250 to 900 kDa (Fig. 6E, top). When immunoprecipitations were performed using anti-NEMO, IKK␤ but not IKK␣ ⌬NBD associated with NEMO in fractions 19 -24 (Fig. 6E, bottom). In contrast, when anti-IKK␣ was used to immunoprecipitate complexes, only IKK␤ associated with IKK␣ ⌬NBD in fractions 18 -25, and NEMO was not co-precipitated in these samples. Hence, these data support our findings in Fig. 6, A and B, and demonstrate that reconstituted IKK␣ ⌬NBD is not incorporated into the tripartite complex. Instead, similar to IKK␤ ⌬NBD in IKK␤ Ϫ/Ϫ MEFs (Figs. 2 and 3), endogenous IKK␤ and NEMO form a high molecular weight IKK complex, whereas IKK␣ ⌬NBD associates with IKK␤ in a distinct NEMO-independent complex.
In addition to these complexes, our analysis of nuclear extracts demonstrated that IKK␣ ⌬NBD exists alone in the nucleus separate from either IKK␤ or NEMO (Fig. 5F). Furthermore, nuclear IKK␣ ⌬NBD was active as measured using an anti-phospho-IKK␣/␤ antibody (Fig. 5G). Since NEMO is absolutely required for activation of the cytoplasmic IKK complex leading to IB␣ phosphorylation (8 -10, 12), we questioned whether the NEMO-independent IKK␤-IKK␣ ⌬NBD complex could be activated in response to IL-1␣ or TNF. To test this, we immunoprecipitated IKK complexes from cytoplasmic extracts of IKK␣ ⌬NBD and IKK␣ WT MEFs using either anti-IKK␣ or anti- . Immunoprecipitated material was immunoblotted using anti-IKK␣, anti-IKK␤, and anti-NEMO as indicated (right). Samples of lysates saved prior to immunoprecipitation (Pre-IP) were immunoblotted using anti-IKK␣, anti-IKK␤, and anti-NEMO as shown. C-E, S100 extracts from IKK␣ Ϫ/Ϫ (C), IKK␣ WT (D), and IKK␣ ⌬NBD (E) MEFs were fractionated by size exclusion chromatography. Fractions were immunoblotted using the antibodies indicated (left). The column was precalibrated, and the molecular weights of standard proteins are indicated above the appropriate fractions in C. Fractions containing the high molecular weight IKK complex were immunoprecipitated using either anti-NEMO or anti-IKK␣. The resulting immunoblots from these immunoprecipitations are displayed below the fractionation profile for each cell type in C-E.
NEMO and then performed immune complex kinase assays using glutathione S-transferase-IB␣ 1-54 as a substrate. As shown in Fig. 7A, immunoprecipitation using anti-NEMO pulled down active IKK complexes from both cell types treated with either TNF or IL-1␣ (Fig. 7A, lanes 2, 3, 8, and 9). In contrast, anti-IKK␣ only precipitated active IKK from IKK␣ WT MEFs despite the fact that NF-B signaling was intact in IKK␣ ⌬NBD cells, as shown by degradation of IB␣ (lanes 4 -6). These findings therefore suggest that the cytoplasmic IKK␤-IKK␣ ⌬NBD complex in IKK␣ ⌬NBD cells is not activated in response to cytokines, and it is instead the NEMO-IKK␤ alone complex (Fig. 6) that is activated.
To further test this concept, we treated IKK␣ ⌬NBD cells with the NBD peptide that disrupts the association of NEMO with the IKKs (13,14). As shown in Fig. 7B, the mutant NBD peptide did not affect TNF-or IL-1␣-induced IB␣ degradation, whereas the wild-type peptide completely blocked IB␣ degradation in response to both cytokines. The data in Fig. 7 therefore confirm that NEMO is absolutely required for IB␣ degradation in IKK␣ ⌬NBD cells and demonstrate that the cytoplasmic IKK␤-IKK␣ ⌬NBD complexes in these cells are not activated by proinflammatory cytokines.

DISCUSSION
The prevailing model of NF-B signaling suggests that the association of IKK␣ with NEMO plays no role in classical NF-B pathway activation (15). However, we demonstrated recently that IKK complexes consisting of only IKK␣ and NEMO activate classical NF-B in response to IL-1 (30). In contrast, TNF signaling absolutely requires IKK␤, and the classical NF-B pathway cannot be activated by TNF in IKK␤-deficient cells (16,17). Since both IKK␣ and IKK␤ associate with NEMO via their C-terminal NBDs, these findings suggest that the ability of both kinases to bind NEMO regulates their function within the classical signaling pathway. We therefore undertook this study to determine the effects of selectively deleting the NBD in IKK␣ and IKK␤ on TNF-and IL-1-induced NF-B activation and IKK complex formation. Our findings are summarized in the model depicted in Fig. 8.
Consistent with the stringent requirement for NEMO and IKK␤ in TNF signaling (8 -12), IKK␤ ⌬NBD did not rescue defective TNF-induced IB␣ degradation in IKK␤ Ϫ/Ϫ cells. This conclusively demonstrates that direct association of NEMO with IKK␤ is absolutely necessary for TNF-induced classical NF-B activation. Confirming our previous study (30), IL-1induced IB␣ degradation and NF-B activation was intact in IKK␤ Ϫ/Ϫ MEFs. In light of the failure of IKK␤ ⌬NBD to rescue TNF signaling and previous studies demonstrating that IKK␤ is a more efficient kinase for IB␣ than IKK␣ (32,33), we speculated that IKK␤ ⌬NBD might function as a dominant negative and block IL-1 signaling. However, reconstitution of IKK␤ Ϫ/Ϫ MEFS with IKK␤ ⌬NBD did not affect IL-1-induced IB␣ degradation or NF-B activation. This is in line with our earlier finding that a catalytically inactive IKK␤ mutant that blocks TNF signaling did not affect IL-1-induced classical NF-B activation (30). We therefore conclude that IL-1-induced NEMOdependent IKK␣ activation overcomes the potential dominant negative effects of catalytically inactive or NBD-deficient IKK␤. These data underscore the ability of IKK␣ to either compensate in the absence of IKK␤ or function in the presence of defective IKK␤ in IL-1-but not TNF-induced classical pathway activation.
Although IKK␤ ⌬NBD did not affect IL-1-induced IB␣ degradation and NF-B activation in IKK␤ Ϫ/Ϫ MEFs, NF-B transcriptional activity was markedly enhanced in IKK␤ ⌬NBD cells. IKK␤ NEMO binding mutants were shown previously to be constitutively active when overexpressed in HeLa cells (13,14), and certain deubiquitinases and protein phosphatases that inhibit IKK activity have been reported to associate with NEMO following IKK activation (44 -49). Hence, the inability of IKK␤ ⌬NBD to recruit these enzymes via NEMO might lead to its increased basal activity. We previously proposed that phosphorylation within the IKK␤ NBD is a negative regulatory signal that affects the function of NEMO (13,14). Several lines of biochemical evidence support this hypothesis (13, 14, 50 -53), and Higashimoto et al. (51) demonstrated recently that phosphorylation within the NBD by Polo-like kinase down-regulates IKK activity. Notably, however, basal NF-B activation measured by EMSA was not enhanced in IKK␤ ⌬NBD cells, suggesting that additional regulatory signals control IKK and NF-B transcriptional activity. In this regard, CUEDC2 was shown recently to directly associate with IKK␣ and IKK␤ to inhibit their activity, and this probably occurs in the absence of NEMO (54). Clearly, further work is required to understand fully the mechanisms that deactivate the IKK complex; however, these accumulated findings suggest that both NEMO-dependent and -independent mechanisms exist to accomplish this. The noncanonical NF-B pathway requires IKK␣ and can be activated in the absence of either IKK␤ or NEMO (18 -20, 22). We confirmed this by rescuing defective lymphotoxin-␤ receptor-induced p100 processing in IKK␣ Ϫ/Ϫ MEFs with IKK␣. Moreover, IKK␣ ⌬NBD also rescued p100 processing, providing further evidence that the association of IKK␣ with NEMO plays no role in noncanonical pathway activation. We therefore conclude that disrupting the interaction of NEMO with the IKKs selectively blocks the classical pathway and does not affect noncanonical NF-B signaling. This strongly supports the potential use of the NBD peptide or other pharmacological strategies targeting this interaction as highly specific inhibitors of the classical NF-B pathway (5,13,14,35).
Consistent with our earlier findings (30) and those of others (17,55), IL-1 and TNF-induced IB␣ degradation was intact in IKK␣-deficient cells. Furthermore, IKK␣ ⌬NBD did not block either TNF-or IL-1-induced IB␣ degradation, demonstrating that IKK␣ ⌬NBD does not function as a dominant negative inhibitor of IKK␤. Together with the effects of re-expressing IKK␤ ⌬NBD in IKK␤ Ϫ/Ϫ MEFs described above, this provides further support for our previously proposed model of classical pathway activation (30). In this model, TNF signaling absolutely requires IKK␤ associated with NEMO, whereas IL-1 is less stringent and utilizes NEMO with either IKK␣ or IKK␤ to phosphorylate IB␣ (30).
Although IL-1 and TNF both induced IB␣ degradation in IKK␣ Ϫ/Ϫ cells, neither cytokine activated NF-B transcrip-tional activity in the absence of IKK␣. In addition, DNA binding of nuclear NF-B was significantly reduced in IKK␣-deficient cells. These observations confirm previous reports describing critical roles for IKK␣ in directly phosphorylating and regulating the transcriptional activity of NF-B proteins as well as transcriptional co-activators, co-repressors, and histones (24 -28, 56). Consistent with these functions, IKK␣ WT rescued full DNA binding and transcriptional activity in IKK␣ Ϫ/Ϫ cells, and intriguingly, activity was also rescued by IKK␣ ⌬NBD . Furthermore, both IKK␣ WT and IKK␣ ⌬NBD rescued TNF-and IL-1␣-induced phosphorylation of p65, which is severely diminished in IKK␣ Ϫ/Ϫ cells. This therefore demonstrates that association with NEMO is not required for IKK␣ to regulate the transcriptional activity of NF-B that occurs, at least in part, via phosphorylation of p65.
The ability of IKK␣ to regulate NF-B transcriptional activity depends on its capacity to enter the nucleus (24 -28, 56). It has also been shown that NEMO shuttles in and out of the nucleus and plays a key role in DNA damage-induced NF-B activation (40 -42, 57). In addition, NEMO regulates transcription by binding to CBP at NF-B-dependent promoters (43). We therefore questioned whether nuclear translocation of NEMO requires its association with IKK␣ and whether nuclear IKK␣ activity requires NEMO. As previously described, IKK␣ and NEMO, but not IKK␤, were present in the nuclei of WT MEFs (24,29); however, cytokine-stimulation did not affect the nuclear levels of either of these proteins. TNF stimulation activated nuclear IKK␣ in WT MEFs and also activated IKK␣ that was present in the nuclei of both IKK␣ WT and IKK␣ ⌬NBD cells. Since our immune complex kinase analysis demonstrated that cytoplasmic IKK␣ ⌬NBD complexes cannot be activated, these findings strongly suggest that IKK␣ activation occurs in the nucleus. Further work is clearly required to determine the precise mechanisms of activation of nuclear IKK␣; however, our data demonstrate that this occurs independently of its association with NEMO.
Surprisingly, we found IKK␤ and NEMO in the nucleus of IKK␣ Ϫ/Ϫ cells. Unlike IKK␣, IKK␤ does not contain a nuclear localization sequence, suggesting that in the absence of IKK␣, NEMO chaperones IKK␤ into the nucleus (28). Notably, and in contrast to nuclear IKK␣, we did not detect any TNF-induced activation of nuclear IKK␤ in IKK␣ Ϫ/Ϫ cells. When we re-expressed IKK␣ WT in IKK␣ Ϫ/Ϫ cells, the cytoplasmic and nuclear distribution of IKK␣, IKK␤, and NEMO was identical to that in Reconstituted IKK␤ WT forms a heterotrimeric IKK complex with endogenous NEMO and IKK␣ and rescues TNF signaling (middle). In contrast, IKK␤ ⌬NBD neither affects IL-1 signaling nor rescues TNF signaling (right). IKK␤ ⌬NBD is not incorporated into the heterotrimeric IKK complex but forms complexes with endogenous IKK␣ that do not contain NEMO. B, IKK␣ Ϫ/Ϫ MEFs contain NEMO-IKK␤ complexes that activate classical signaling (i.e. IB␣ degradation and NF-B nuclear localization) in response to TNF and IL-1; however, NF-B transcriptional activity is defective in these cells (left). IKK␣ WT (middle) and IKK␣ ⌬NBD (right) both rescue transcriptional activity, demonstrating that association with NEMO is not required for the ability of IKK␣ to regulate transcription. IKK␣ WT forms a heterotrimeric IKK complex with endogenous NEMO and IKK␤, and a portion of IKK␣ WT enters the nucleus (middle). IKK␣ ⌬NBD is not incorporated into the heterotrimeric IKK complex but instead forms NEMOindependent complexes with endogenous IKK␤ (right). Nuclear localization of IKK␣ does not require NEMO association as IKK␣ ⌬NBD enters the nucleus.
WT MEFs, demonstrating that IKK␣ maintains these homeostatic levels. Furthermore, in IKK␣ ⌬NBD -reconstituted MEFs we found IKK␣ in the nucleus in the absence of significant levels of NEMO. Taken together, these findings demonstrate that although NEMO binding is not required for IKK␣ to enter the nucleus and regulate NF-B transcription, the IKK␣ NBD is critical for NEMO nuclear localization in cells containing IKK␣. Future efforts to fully dissect the mechanisms regulating nuclear localization of the separate IKK complex subunits are necessary, but our findings suggest that these mechanisms are not mutually exclusive.
IKK␣ and IKK␤ associate via interactions between their respective leucine zipper domains (34), and the most abundant form of the IKK complex is a heterodimer of IKK␣ and IKK␤ associated with NEMO (1,6,7). This suggests that IKK heterodimerization is the preferential conformation for the IKKs, although IKK homodimeric complexes have been reported (9,58,59). We predicted that ⌬NBD versions of IKK␣ and IKK␤ re-expressed in knock-out MEFs would heterodimerize with the endogenous partner kinase containing an intact NBD. We further reasoned that NEMO would associate via this endogenous NBD and that the resulting complex would consist of the re-expressed IKK ⌬NBD , the endogenous IKK, and endogenous NEMO. Consistent with this notion, immunoprecipitation and gel filtration analysis confirmed that IKK␣ WT and IKK␤ WT reexpressed in IKK␣ Ϫ/Ϫ and IKK␤ Ϫ/Ϫ MEFs, respectively, formed heterotrimeric complexes with endogenous IKKs and NEMO (see model in Fig. 8). In contrast, although IKK␣ ⌬NBD and IKK␤ ⌬NBD associated with endogenous IKK␤ and IKK␣, respectively, these heterodimers did not bind to NEMO despite the presence of an intact NBD on the endogenous kinase. Furthermore, our immune complex kinase assay and NBD peptide experiments clearly demonstrate that in IKK␣ ⌬NBD cells, the IKK␤-IKK␣ ⌬NBD complex is not activated by IL-1␣ or TNF, and it is the NEMO-IKK␤ complex that responds to stimulation. Consequently, these findings demonstrate that in the absence of one NBD, the heterotrimeric IKK holocomplex cannot assemble, and the resulting NEMO-independent complexes do not respond to proinflammatory cytokine signaling.
Recent reports have revealed that NEMO forms dimers through interactions between specific domains within the N-terminal region of the protein (35,60). Furthermore, this portion of NEMO is necessary and sufficient for association with the NBDs of IKK␣ and IKK␤ (13,35,52,60). Recent elegant crystallographic studies revealed that this dimeric conformation of the NEMO N terminus forms two parallel 800-Å ␣-helical IKK-binding pockets into which the NBDs insert (35). Since the heterodimeric complexes formed between endogenous IKK and re-expressed IKK ⌬NBD did not associate with NEMO, we conclude that the presence of two NBDs is essential for stable formation of this interaction with NEMO. It is intriguing to speculate that the single NBD IKK heterodimer cannot "clamp" into the IKK binding pocket formed by the NEMO dimers; however, extensive structural analysis of these ⌬NBD complexes is required to draw any definitive conclusions. Nevertheless, our data suggest that separately targeting the interaction of NEMO with either IKK will disrupt the entire IKK complex.
In conclusion, we have demonstrated that the interaction of NEMO with both IKKs is necessary for classical NF-B pathway activation and IKK complex assembly. We have further established that IL-1-induced classical NF-B activation remains intact in cells lacking functional IKK␤. Tremendous effort has been directed toward developing specific inhibitors of IKK␤ (3,4); however, our findings suggest that this strategy may not effectively block all classical NF-B activation. Importantly, Lam et al. (31) demonstrated recently that selective inhibition of IKK␤ blocks classical pathway-dependent cell survival in a subtype of diffuse large B-cell lymphoma cells only when IKK␣ is concomitantly ablated. These accumulated findings therefore support efforts to develop NBD-targeting small molecule inhibitors. Such drugs are predicted to inhibit all classical NF-B activation while retaining both the noncanonical pathway and the NEMO-independent transcriptional regulatory roles of IKK␣ intact.