Skip Navigation


Human Molecular Genetics Advance Access originally published online on August 29, 2007
Human Molecular Genetics 2007 16(23):2805-2815; doi:10.1093/hmg/ddm237
This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow A corrigendum has been published
Right arrow All Versions of this Article:
16/23/2805    most recent
ddm237v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sebban-Benin, H.
Right arrow Articles by Courtois, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sebban-Benin, H.
Right arrow Articles by Courtois, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?

© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Identification of TRAF6-dependent NEMO polyubiquitination sites through analysis of a new NEMO mutation causing incontinentia pigmenti

Hélène Sebban-Benin1, Alessandra Pescatore2, Francesca Fusco2, Valérie Pascuale1, Jérémie Gautheron1, Shoji Yamaoka3, Anne Moncla4, Matilde Valeria Ursini2,{dagger} and Gilles Courtois1,{dagger},*

1 INSERM U697, Pavillon Bazin, Hôpital Saint-Louis, 1, Avenue Claude Vellefaux, 75010 Paris, France, 2 Institute of Genetics and Biophysics ‘Adriano Buzzati-Traverso’ (CNR), 80131 Naples, Italy, 3 Department of Molecular Virology, Tokyo Medical and Dental University, Tokyo 113-8519, Japan and 4 Département de Génétique Médicale, Hôpital des Enfants de la Timone, 13385 Marseille, France

* To whom correspondence should be addressed. Tel: +33 153722085; Fax: +33 153722051; E-mail: gilles.courtois{at}stlouis.inserm.fr

Received June 21, 2007; Revised August 18, 2007; Accepted August 21, 2007


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
The regulatory subunit NEMO is involved in the mechanism of activation of I{kappa}B kinase (IKK), the kinase complex that controls the NF-{kappa}B signaling pathway. During this process, NEMO is modified post-translationally through K63-linked polyubiquitination. We report the molecular characterization of a new missense mutation of NEMO (A323P) which causes a severe form of incontinentia pigmenti (OMIM#308300), an inherited disease characterized predominantly by skin inflammation. The A323P mutation was found to impair TNF-, IL-1-, LPS- and PMA/ionomycin-induced NF-{kappa}B activation, as well as to disrupt TRAF6-dependent NEMO polyubiquitination, due to a defective NEMO/TRAF6 interaction. Mutagenesis identified the affected ubiquitination sites as three lysine residues located in the vicinity of A323. Unexpectedly, these lysines were ubiquitinated together with two previously identified lysines not connected to TRAF6. Mutation of all these ubiquitination sites severely impaired NF-{kappa}B activation induced by stimulation with IL-1, LPS, Nod2/RICK or serum/LPA. In contrast, mutation at all of these sites had only a limited effect on stimulation by TNF. These findings indicate that post-translational modification of NEMO through K63-linked polyubiquitination is a key event in IKK activation and that perturbation of this step may cause human pathophysiology.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
The family of NF-{kappa}B transcription factors, composed of the evolutionary conserved proteins p50, p52, c-rel, relB and relA, has been shown to play a crucial role in immunity, inflammation, cancerogenesis and apoptosis, by regulating the expression of dozens of genes that control these processes (1). In resting cell, NF-{kappa}B dimers are retained as latent proteins in the cytoplasm through their association with I{kappa}B inhibitory molecules. In response to stimulation, I{kappa}Bs are phosphorylated by the I{kappa}B kinase (IKK) complex at conserved serine residues. This modification induces their K48-linked polyubiquitination and their recognition as substrates for proteolysis by the 26S proteasome. Degradation of I{kappa}Bs allows free NF-{kappa}Bs to translocate into the nucleus, where they interact with a specific DNA motif located in promoters or enhancer elements of target genes (1).

The pivotal kinase complex IKK is composed of two related catalytic subunits IKK-1 (or IKK-{alpha}) and IKK-2 (or IKK-ß), of regulatory subunit NEMO (or IKK-{gamma}) and of accessory proteins such as Hsp90, cdc37 and ELKS, which are less well-characterized (2). Genetic studies have unequivocally demonstrated that the IKK-2 and NEMO subunits of IKK are required for NF-{kappa}B activation by most stimuli (canonical stimulation) (3) whereas a few other may target an IKK-1 containing complex, whose composition is not yet known, through a kinase called NIK (non-canonical stimulation) (4).

Among the inducers of canonical IKK stimulation are pro-inflammatory cytokines such as IL-1ß and TNF-{alpha}; bacterial products such as lipopolysaccharide (LPS) from gram negative bacteria and peptidoglycans from gram positive bacteria; T-cell antigens; viral proteins, such as HTLV-1-derived Tax. In most cases, these molecules act by binding to specific receptors located at the cell surface. Following ligand/receptor interaction, adaptor molecules and kinases are recruited to build up signal transduction modules, which activate various signaling pathways, among them the NF-{kappa}B pathway (5).

Until recently, the precise molecular mechanism for canonical stimulation of IKK was unclear. In particular, the ability of IKK to integrate the various signaling pathways regulating its activity and the exact function of NEMO in this process were not known. Many of these pathways, such as the IL-1R/TLR and TNF-R pathways, were found to use members of the TRAF family of proteins and to require kinase TAK-1 to phosphorylate and activate IKK (6,7). TRAFs have been shown to exhibit ubiquitin ligase (E3) activity which, together with the conjugating enzyme (E2) Ubc13/Uev1, modifies substrates through K63-linked polyubiquitination. In contrast to K48-linked polyubiquitination, which provides a tag for recognition by the 26S proteasome, K63-linked polyubiquitination regulates the activity, but not the half-life, of modified substrates. Among the putative targets of TRAFs are TRAFs themselves (7); components of the NF-{kappa}B signaling pathway, such as RIP, which participates in TNF signaling (8); TAB2 and TAB3, which are regulatory subunits of TAK1 (9); and NEMO itself (10–12). The mechanism by which these modifications trigger IKK activation remains uncertain. In particular, the residues ubiquitinated by TRAFs have not been completely identified, precluding their functional testing. In the case of TCR signaling, ubiquitination of a single lysine residue of NEMO, located in its N-terminal zinc finger, appears to be required for proper NF-{kappa}B activation (13). Although MALT1 may be an E3 ligase for NEMO in this pathway, TRAF6 is also involved in TCR signaling, raising the issue of its specific function (14). A different lysine residue of NEMO has also been shown to be a target for Nod2/RICK (15), but the E3 ligase was not identified.

The exact relationship between NEMO ubiquitination and IKK activation also remains unclear. Ubiquitination may help NEMO to recruit TAK-1 through TAB2/TAB3 recognition of its K63-linked polyubiquitinated chains. Alternatively, since NEMO also exhibits an ubiquitin-binding domain (16,17), ubiquitinated NEMO may help control its oligomeric state. Indeed, forced dimerization of this protein has been shown to induce NF-{kappa}B activation (18,19).

Several genetic diseases caused by NF-{kappa}B dysfunction have been described (20). The first one, incontinentia pigmenti (IP), is an X-linked dominant and male-lethal disease, which presents in female as a highly heterogeneous, often severe, skin inflammation following birth. Heterozygous IP females survive owing to X-inactivation mosaicism, also presenting abnormalities of the teeth, eyes and central nervous system. The defective gene causing IP has been identified as NEMO (21). In the majority of patients, a similar rearrangement is observed that results in eliminating exons 4–10 of NEMO, which encode critical functional domains of the protein. A second genetic disease associated with NF-{kappa}B dysregulation is anhidrotic ectodermal dysplasia with immunodeficiency (EDA-ID, OMIM#300291), which is caused by hypomorphic mutations of NEMO (22,23) or autosomal dominant mutations of I{kappa}B{alpha} (24,25). Females with this condition are very mildly affected, whereas males suffer from a severe immunodeficiency, characterized by recurrent bacterial or viral infections, associated with impaired development of skin adnexes (sweat glands, hair and teeth).

We have identified a new NEMO mutation responsible for IP. Among the various molecular defects associated with this mutation was impaired TRAF6-dependent NEMO ubiquitination. We therefore searched for the specific lysine residues involved and assessed their participation in the NF-{kappa}B activation process.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
Molecular characterization of a new missense mutation of NEMO causing a severe form of IP
Between 70 and 80% of IP patients exhibit the same genomic rearrangement that deletes the locus-encoding NEMO. Among the remaining patients only very few harbor missense mutations of NEMO, a class of mutations that may provide invaluable information regarding the in vivo function of NEMO in NF-{kappa}B signaling. We sequenced the NEMO gene of a newborn girl exhibiting a severe form of IP associated with neurological abnormalities (see Case Report in Material and Methods). We identified a heterozygous G to C transversion at nucleotide 967 (numbered from the ATG initiation codon), resulting in a missense mutation (A323P) at the beginning of the leucine zipper (Fig. 1A and B).


Figure 1
View larger version (17K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 1. Identification of a missense mutation of NEMO causing a severe form of IP. (A) Sequence analysis. (B) Location of mutation G967C. Coding exons are presented with distinct colors indicating their relationship to the various structural domains of NEMO. N-ter: amino-terminus; CC: coiled coil; Int: intermediate domain; NUB: NEMO ubiquitin-binding domain; LZ: leucine zipper; Pro: proline-rich domain; ZF: zinc finger. (C) Skewed X-inactivation resulting from G967C mutation. The X-inactivation status of the proband (II:1) and her mother (I:2) is indicated in percentage.

 
Similar to most IP patients, X-inactivation skewing in the proband's peripheral blood cells was above 90% (Fig. 1C), precluding any study of cells directly derived from the patient. To evaluate the impact of this mutation on NF-{kappa}B signaling, we transiently complemented several cell lines lacking NEMO with a plasmid expressing A323P NEMO and analyzed their response to several stimuli, including TNF, IL-1, LPS and PMA/ionomycin. Two of these cell lines, 1.3E2 and SVT-2C, are lymphocytes of the murine pre-B and human T-cell lineage, respectively (26,27), whereas NEMO (–) MEFs (Murine embryonic fibroblasts) derived from a mouse model of IP (28).

Transfection of a plasmid expressing A323P NEMO into SVT-2C and NEMO (–) MEFs, resulted in abolished activation of an NF-{kappa}B reporter gene in response to TNF (Fig. 2A). In addition, a strong reduction of activation was detected when NEMO (–) MEFs were transfected with A323P NEMO and stimulated with IL-1. We also found that transfection of A323P mutant in 1.3E2, a cell line responsive to LPS, led to impaired NF-{kappa}B activation in response to this stimulus. Although defective TCR signaling is not observed in IP patients, because of the efficient chromosome X-driven elimination of T-lymphocytes harboring NEMO mutations (20), we assessed whether this pathway was also affected by A323P mutation. Whereas transient complementation of SVT-2C cells with wt NEMO followed by PMA/ionomycin stimulation resulted in potent NF-{kappa}B activation, no such activation was seen upon complementation with A323P mutation (Fig. 2A). These findings indicate that A323P mutation was associated with a strong defect in NF-{kappa}B signaling, affecting several distinct signaling pathways. This was not caused by a major structural alteration of the NEMO protein, since the molecule harboring the A323P mutation did not show defective interaction with previously identified partners such as IKK-2, RIP or TANK, as assessed by co-transfection experiments in 293T cells and immunoprecipitation (Supplementary Material, Fig. S1) (data not shown).


Figure 2
View larger version (30K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 2. Phenotype of A323P mutation. (A) Defective response of A323P NEMO mutant to TNF, IL-1, LPS and PMA/ionomycin. Luciferase assays, using Ig{kappa}-Luc as a reporter plasmid, were carried out after complementing NEMO (–) cells (SVT-2C, 1.3E2 and NEMO (–) MEFs) with an expression vector encoding either wt or A323P NEMO. Twenty-four hours after transfection, the cells were stimulated with either 10 ng/ml of TNF, 10 ng/ml of IL-1, 15 µg/ml of LPS or 50 ng/ml of PMA and 200 ng/ml of ionomycin. The relative luciferase activity of A323P is shown, with 100% corresponding to wt NEMO activity. For each stimulus, the absolute activation index is also indicated. The analysis was performed between two and four times, in duplicate. (B) Stability of A323P mutant. Cytoplasmic extracts derived from Jurkat or SVT-2C cells stably expressing Flag-wt NEMO, Flag-A323P NEMO or mock transfected (pc) were analyzed by western blotting, using an antibody against human NEMO. (C) Integration of A323P mutant into IKK. The same extracts as in (B) were immunoprecipitated with anti-hNEMO and analyzed, after western blotting, with anti-IKK-1.

 
In all the complementation experiments described above, cells were transiently transfected, a method that did not allow us to assay expression of the molecules produced after transfection. To exclude any general impairment in NF-{kappa}B activation resulting from instability of the NEMO protein, we generated stable cell lines expressing wt or A323P NEMO proteins. As shown in Fig. 2B, we were able to produce pools of SVT-2C cells expressing wt or A323P NEMO at similar levels, indicating that the mutated protein was stable (Fig. 2B, compare lane 3 with 2). Moreover, A323P NEMO was normally incorporated into the IKK complex since similar amounts of IKK-1 and IKK-2 were co-immunoprecipitated as with wt NEMO (Fig. 2C) (data not shown). Despite this normal incorporation into IKK, however, NF-{kappa}B activation was not observed in response to TNF (Supplementary Material, Fig. S2). These findings indicate that the A323P mutation was associated with a specific impairment affecting at least two distinct pathways of NF-{kappa}B activation, the TNF and the IL-1/TLR signaling pathways, which are relevant to the phenotype developed by IP patients (20).

Defective NEMO polyubiquitination caused by A323P mutation
We and others have previously shown that co-transfecting plasmids expressing NEMO and ubiquitin in 293T cells results in NEMO K63-linked polyubiquitination and that this kind of modification does not regulate the stability of this protein but may regulate its function (10,11). Interestingly, using this assay, a dramatic reduction in A323P NEMO ubiquitination was observed (Fig. 3A, compare lanes 7,8 with lanes 3,4). We therefore attempted to identify the pathway responsible for this basal ubiquitination process. We found that co-transfection of a dominant negative version of TRAF6, but not TRAF2, diminished NEMO ubiquitination (Fig. 3B) (data not shown), suggesting that a TRAF6-dependent pathway was responsible for NEMO ubiquitination. When TRAF6 was co-expressed with both wt NEMO and ubiquitin, there was a strong increase in NEMO ubiquitination; in contrast, the A323P mutation exhibited defective ubiquitination (Fig. 3C, compare lane 6 with lane 3).


Figure 3
View larger version (63K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 3. Defective ubiquitination of A323P mutant. (A) Impaired basal ubiquitination of A323P NEMO. HEK 293T cells were transfected with wt NEMO or A323P NEMO alone (lanes 1,2 and 5,6) or with HA-Ub (lanes 3,4 and 7,8). Cell extracts were prepared 24 h later and either analyzed, after western blotting, with anti-hNEMO (left panel) or immunoprecipitated with anti-hNEMO and then analyzed, after western blotting, with anti-HA. Retarded ubiquitinated forms of NEMO are indicated by Ub-NEMO. (B) Basal ubiquitination of NEMO is TRAF6-dependent. HEK 293T cells were co-transfected with wt NEMO and HA-Ub, without (lane 1) or with (lane 2) a dominant negative version of TRAF6 ({Delta}N-TRAF6). After 24 h, cell extracts were prepared and analyzed, after western blotting, with anti-hNEMO, anti-HA or anti-TRAF6. (C) Impaired TRAF6-dependent NEMO ubiquitination caused by A323P mutation. HEK 293T cells were transfected with wt NEMO (lanes 1–3) or A323P NEMO (lanes 4–6). In lanes 2 and 5, NEMO proteins were co-transfected with HA-Ub alone, whereas in lanes 3 and 6 TRAF6 was also included.

 
Since TRAF6 has been proposed to act as an E3 ligase for NEMO (14), we examined the step at which the A323P mutation may perturb the ubiquitination process. Following co-transfection of TRAF6 and wt NEMO into 293T cells, we observed a specific interaction between these two molecules (Fig. 4). Worth noting, we could also see in the same immunoprecipitate the presence of ubiquitinated NEMO, since TRAF6 and NEMO were tagged with the same Flag epitope. With the A323P mutant, however, immunoprecipitation with an anti-NEMO antibody pulled down a decreased amount of TRAF6, and there was little ubiquitinated NEMO, indicating that the primary recognition of NEMO by TRAF6 was defective, most likely causing the subsequent defect in ubiquitination.


Figure 4
View larger version (31K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 4. Defective interaction between TRAF6 and A323P NEMO. (A) Analysis of TRAF6/NEMO interaction. HEK 293T cells were transfected with Flag-TRAF6 alone (lanes 1,2), with Flag-TRAF6 and wt Flag-NEMO (lanes 3,4) or with Flag-TRAF6 and A323P Flag-NEMO (lanes 5,6). Whole-cell extracts were prepared 24 h later, immunoprecipitated with anti-hNEMO and analyzed, after western blotting, with anti-Flag. Ub-F-NEMO: ubiquitinated Flag-NEMO; IgG: immunoglobulin. (B) TRAF6-dependent NEMO ubiquitination. The experiment was carried out as described in (A), using an anti-ubiquitin antibody to detect ubiquitinated NEMO.

 
Identification of TRAF6-dependent NEMO ubiquitination sites
The observation that the A323P mutation caused an impairment in the TRAF6-dependent ubiquitination of NEMO suggested that this may provide a clue to identifying the specific lysine residues targeted by TRAF6. As shown in Fig. 5A, two conserved lysines (human coordinates hK321 and hK326; mouse coordinates mK314 and mK319) are located in close proximity to hAla 323 (corresponding to mVal 316) and may be affected by the appearance of a proline nearby. To check for this possibility, we performed experiments similar to those above, boosting NEMO ubiquitination with addition of exogenous ubiquitin, or only co-transfected NEMO and TRAF6. In this latter case, the only visible modification was the appearance of the first ubiquitin adduct as a single major retarded band, allowing a more quantitative evaluation of the ubiquitination process. Moreover, for practical reasons, all the following experiments were performed using murine NEMO, after verifying that V316P mutation behaved equivalently to human A323P mutation (Supplementary Material, Fig. S3). Individual mutations of conserved lysines mK314 and mK319, or even mK318, did not substantially reduce TRAF6-induced NEMO (Fig. 5B) (data not shown). In contrast, a double mutation targeting both mK318 and mK319 (mutant 2R) resulted in decreased ubiquitination, which was further decreased but not abolished by mutating mK314 (mutant 3R) (Fig. 5B and C). These findings confirmed that the lysines modified by TRAF6 were located close to hA323/mV316. Importantly, our observation that the triple-mutation mK314/318/319R in mutant 3R did not completely abolish TRAF6-induced NEMO ubiquitination suggested the existence of additional acceptor sites.


Figure 5
View larger version (32K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 5. Identification of TRAF6-dependent NEMO ubiquitination sites. (A) Conserved lysine residues located in the vicinity of A323. NEMO sequences from various species are aligned to indicate the existence of two conserved lysines (asterisks). N- and C-terminal truncations of mNEMO used in this study are also indicated. (B, C). Mutagenesis analysis of NEMO ubiquitination sites. Various wt and mutated versions of HA-NEMO were co-transfected with HA-Ub alone or with HA-Ub and Flag-TRAF6 (B) or were transfected without or with TRAF6 (C). After 24 h, whole-cell extracts were prepared and analyzed, after western blotting, with anti-NEMO. (D) Identification of NEMO ubiquitination sites using C-terminal truncations of NEMO. The analysis was carried out as in (B). (E) Identification of NEMO ubiquitination sites using an N-terminal truncation of NEMO. The analysis was carried out as in (B) but using, in this specific case, an antibody directed against the C-terminus of NEMO.

 
To independently confirm that the region of NEMO containing A323P mutation indeed provided ubiquitination sites for TRAF6, we performed deletion analyses, using N- or C-terminal truncations of the murine protein. Removing the zinc finger of NEMO (construct dC385) and upstream sequences, as far as amino acid 343 (construct dC343), did not substantially reduce NEMO ubiquitination (Fig. 5D). In contrast, further deletions, up to amino acid 299 (construct dC299), strongly reduced but did not totally abolish NEMO ubiquitination. This observation confirmed the existence of ubiquitination sites between amino acid 343 and amino acid 299, in which mK314, mK318 and mK319 are located, as well as suggesting the presence of (an) additional ubiquitination site(s) upstream of amino acid 299.

Removing the first 241 N-terminal amino acid of NEMO (construct dN241), we were still able to detect TRAF6-dependent ubiquitination. In contrast, introduction of the mK318/319R mutations into dN241 slightly decreased ubiquitination whereas introduction of mK314/318/319R mutations strongly decreased ubiquitination, confirming that residues 314 and 318/319 are ubiquitination sites in this part of the molecule (Fig. 5E) (data not shown).

Interestingly, hK285 is a lysine residue of NEMO that is modified by the Nod2/RICK signaling pathway, which is perturbed in Crohn's disease, but the E3 ligase used by Nod2/RICK to ubiquitinate this site has not been identified (15). Since we had observed residual NEMO ubiquitination upstream of amino acid 299, we tested whether hK285 is also a target of TRAF6. We found that mutation of mK278 (the murine equivalent of hK285) slightly diminished TRAF6-induced NEMO ubiquitination when the process was boosted by adding exogenous Ub (data not shown) but severely reduced NEMO ubiquitination when only TRAF6 and NEMO were co-transfected (Fig. 5C). Moreover, the residual ubiquitination of 3R was almost totally abolished upon mutating mK278 (construct 4R) (Fig. 5B and C).

Essential role of NEMO ubiquitination in signaling pathways activating NF-{kappa}B through TRAF6
Having identified several lysines of NEMO that may be sites of TRAF6-dependent polyubiquitination, we investigated the impact of their mutation on NF-{kappa}B signaling. We first analyzed the effect of individual mutations at mK278, mK314, mK318 and mK319 on the activation of NF-{kappa}B by IL-1, which is a prototypical cytokine using TRAF6 E3 ligase (5). All the mutants behaved similarly to wt NEMO upon transient complementation of NEMO (–) MEFs (data not shown). Since defective TRAF6 ubiquitination of NEMO requires the mutation of several of these residues, we assessed whether the 2R, 3R or 4R mutants would affect NF-{kappa}B signaling. Whereas 2R was as active as wt NEMO (data not shown), 3R showed a slight (~15%) reduction in NF-{kappa}B activation, and 4R showed a greater reduction (~40%) (Fig. 6A). Activation of NF-{kappa}B by TNF was not significantly affected by mutation 4R, indicating that the newly identified sites of ubiquitination were specifically required by a stimulus using TRAF6.


Figure 6
View larger version (21K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 6. Functional analysis of NEMO ubiquitination. (A) Participation of NEMO ubiquitination in TNF and IL-1 signaling. The analysis was carried out as in Fig. 2A, using complementation of NEMO (–) MEFs with the indicated NEMO proteins. Cells were serum-starved for 16 h, then stimulated for 4 h with either 10 ng/ml of TNF or IL-1 and luciferase activity determined. The experiments were carried out in duplicate, and each mutant was analyzed between two and five times. (B) Participation of NEMO ubiquitination in LPS signaling. The analysis was carried out as in (A), using 15 µg/ml of LPS. (C) Participation of NEMO ubiquitination in Nod2/RICK signaling. NEMO (–) MEFs were co-transfected with NEMO-expressing plasmids and either empty plasmid or RICK and Nod2-expressing plasmids. Luciferase activity was measured 24 h later. The experiment was repeated twice, in duplicate. (D) Role of TRAF6 in NF-{kappa}B activation by RICK. HEK 293T cells were transfected with reporter plasmid Ig{kappa}-Luc and RICK expression vector alone or together with an expression vector encoding a dominant negative version of TRAF6 ({Delta}N-TRAF6). Expression of RICK was analyzed after western blotting (lower panel). (E) Participation of NEMO ubiquitination in NF-{kappa}B activation by serum and LPA. The analysis was carried out as in (A), using 20% serum or 10 µM LPA.

 
Since complementation of NEMO (–) cells followed by stimulation with IL-1 confirmed the requirement for the zinc finger domain of NEMO (29) (data not shown), we assessed whether the lysine residue contained in the zinc finger (mK392R), which has been reported to be involved in TCR signaling (13), represents a target for TRAF6 and control activation of NF-{kappa}B by IL-1. We found that the mK392R NEMO mutant was 40% less active than wt NEMO and that mutating residue mK392 in construct 4R (mutant 5R) resulted in strongly defective NF-{kappa}B activation (Fig. 6A). These findings indicated that this specific lysine participates in IL-1 signaling, in cooperation with upstream lysines located at mK278, mK314 and mK318/K319. Importantly, although a slight (~25%) reduction in NF-{kappa}B activation was observed when cells transfected with mK392R NEMO were treated with TNF, this defect was not further amplified in the 5R mutant.

We next assessed whether other signaling pathways known to activate NF-{kappa}B through TRAF6 were also dependent upon NEMO ubiquitination. Activation of NF-{kappa}B by LPS, which acts through a TLR-dependent signaling pathway was reduced more than 60% when NEMO (–) MEFs were complemented with the 5R mutant (Fig. 6B). We also analyzed the contribution of NEMO ubiquitination to pathways that remain less well-characterized but might also require TRAF6. The first one, the Nod2/RICK pathway, was checked because it has been associated with a lysine residue (hK285/mK278) that we have identified as a target for TRAF6 (see above). Importantly, in the publication reporting ubiquitination of lysine 285 by Nod2/RICK, no functional data were provided supporting its specific involvement in NF-{kappa}B activation (15). We found that NF-{kappa}B activation by Nod2/RICK overexpression was not inhibited by complementing NEMO (–) MEFs with the mK278R mutant or 4R but was reduced 20% by the mK278R mutant and almost abolished when complementation was carried out with mutant 5R (Fig. 6C). In addition, NF-{kappa}B activation by overexpressed RICK in 293T cells was strongly reduced by co-expressing a dominant negative version of TRAF6 (Fig. 6D). These results indicate that NF-{kappa}B activation by Nod2/RICK requires the ubiquitination of several lysine residues of NEMO, not only of hK285/mK278, and that the E3 ligase involved is indeed TRAF6.

The Bcl10/MALT1 complex, which is involved in the NF-{kappa}B activation by lysophosphatidic acid (LPA), a major component of serum exhibiting mitogenic properties, appears to act through TRAF6 (30,31). We therefore analyzed whether TRAF6-dependent NEMO ubiquitination was involved in serum- and LPA-induced NF-{kappa}B activation. With both stimuli, we observed a reduction in NF-{kappa}B activation when NEMO (–) MEFs were complemented with 5R mutant (Fig. 6E), indicating the essential role of NEMO ubiquitination in serum-induced NF-{kappa}B activation.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
Although recent publications have shown that NEMO can be ubiquitinated upon cell stimulation, the exact function of this modification remains elusive. Several lines of evidence suggest that this specific type of post-translational modification may be important in NF-{kappa}B activation. First, polyubiquitinated chains appended to NEMO are K63-linked and IKK activation has been shown to require K63 polyubiquitination, a process carried out by TRAFs, in concert with Ubc13/Uev1 (6,7). Second, CYLD, an enzyme exhibiting deubiquitinase activity and an affinity restricted to K63-linked ubiquitin chains, has been shown to directly bind to NEMO and to deubiquitinate this protein, down-regulating NF-{kappa}B activation (10,32). Third, two regulatory subunits of the TAK1 complex, TAB2 and TAB3, possess ubiquitin-binding domains that appear to specifically recognize K63-linked chains (33). Although these proteins may interact with ubiquitinated RIP or TRAF molecules during the NF-{kappa}B activation process, they may also bind ubiquitinated NEMO (18,19). Fourth, oligomerization of NEMO may participate in IKK activation, and NEMO itself contains a domain, the NUB domain, with affinity for K63-linked polyubiquitinated chains (16,17). NEMO ubiquitination may therefore trigger this oligomeric change. To sort out the precise sequence of post-translational modification that takes place upon cell stimulation, triggering NF-{kappa}B activation, and the exact role of ubiquitination, it is necessary to identify the modified lysines in their respective proteins. We have identified several of the lysines involved in TRAF6-dependent NEMO ubiquitination, and have also analyzed their participation in several NF-{kappa}B activation pathways.

IP is the disorder that provides the best insight into the physiological role of NEMO. Characterization of IP-related NEMO mutants may therefore represent a unique opportunity to elucidate the molecular mechanism of IKK regulation. In this study, we identified and characterized one of the very few missense mutations of NEMO, A323P, which causes a severe form of IP. Moreover, this mutation was shown to severely affect NF-{kappa}B activation by several distinct signaling pathways. Although this general impairment may have been due to a destabilization of NEMO, we have shown that this was not the case. In addition, mutated NEMO normally integrates into the IKK complex. Thus, the question arises of the molecular explanation for the defective activation of NF-{kappa}B caused by the A323P mutation upon stimulation by TNF, IL-1, LPS or PMA/ionomycin.

In the TNF-dependent signaling pathway, IKK activation has been shown to require an interaction between NEMO, through its NUB domain, and TNF-R1-associated ubiquitinated RIP. Since A323P is located in the vicinity of the NUB domain and introduces a destabilizing Pro residue, it is likely that the abolished NF-{kappa}B activation in response to TNF is caused by the impaired recruitment of IKK to TNF-R1. This is supported by findings showing that mutations of residues Y308, D311, F312 and L329, which are located in the same region as A323, affect the recognition of ubiquitinated RIP by NEMO (16,17).

Both IL-1 and LPS pathways require TRAF6. We have shown that an impaired interaction between NEMO and TRAF6 results in defective NEMO ubiquitination and affects the NF-{kappa}B activation process. No interaction between NEMO and TRAF6 has as yet been reported, although TRAF6 is considered to be a NEMO E3 ligase (14). The amino acid sequences participating in this interaction and whether binding is direct or requires (an) additional partner(s) remain to be determined and may help design new inhibitors of NF-{kappa}B activation. The defect in the IL-1 pathway caused by the A323P mutation may also apply to the TCR signaling, since TRAF6 has been shown to participate in both (14).

The impaired TRAF6-induced NEMO ubiquitination observed in the A323P mutant prompted us to define which lysine residues were involved. Using mutagenesis and deletion analyses, we have identified two (or three) new lysines, located around A323, which are targeted by TRAF6. Although we demonstrated the ubiquitination of mK314, it is much more difficult to establish whether only mK318, only mK319 or both are modified in vivo, given their close proximity and their compensation for each other upon mutagenesis (see Fig. 5B).

Residues mK314 and mK318/319 are clearly not the only lysines modified by TRAF6. We demonstrate in this study that two previously identified ubiquitinated residues, mK278/hK285 and mK392/hK399, are also targets of TRAF6 and participate in NF-{kappa}B activation by stimuli using this E3 ligase. Lysine 285 (K278 in mouse) in NEMO was previously reported to be modified upon co-transfection with RICK, a component of the Nod2 signaling pathway. Nod2, a member of the NOD-LRR family, has been shown to be mutated in Crohn's disease, a non-specific chronic inflammatory condition that can affect the entire gastrointestinal tract. The report describing hK285 as a new NEMO ubiquitination site did not identify the E3 enzyme carrying out the reaction, and hK285 was not directly shown to be important for NF-{kappa}B activation. We demonstrate here that TRAF6 is the specific E3 used by RICK to ubiquitinate NEMO, a not unexpected finding given the known interaction between these two proteins (34). Moreover, since TRAF6 targets several distinct lysines, the single hK285R substitution would be unlikely to have any impact on NF-{kappa}B activation by Nod2/RICK.

A new pathway that shows striking similarities to the TCR pathway has been recently identified. It is used by the family of G protein-coupled receptors (GPCRs) and requires CARMA-3, Bcl10, MALT-1 and TRAF6 to activate IKK through NEMO ubiquitination (30,31). Among the stimuli acting through GPCRs is LPA, a major mitogenic component of serum. We have demonstrated in this study that mutating the TRAF6-dependent NEMO ubiquitination sites affects NF-{kappa}B stimulation by serum and LPA, expanding the participation of these sites to NF-{kappa}B activation by stimuli acting through GPCRs.

In conclusion, we have identified critical TRAF6-dependent ubiquitinated sites on NEMO through the molecular characterization of a new NEMO mutation causing IP. These sites are required for NF-{kappa}B activation and appear to integrate signaling pathways used by a variety of stimuli, including IL-1, LPS, Nod2/RICK and LPA (Fig. 7). It remains essential to fully understand how their modification by ubiquitination influences the IKK activation process, in particular, if they provide a tag for enhancing interactions among components regulating IKK activity or represent the final step, triggering its catalytic activation. Answering these important questions will undoubtedly provide new clues for designing more specific and efficient agents to treat various pathologies caused by NF-{kappa}B dysfunction.


Figure 7
View larger version (12K):
[in this window]
[in a new window]
[Download PowerPoint slide]
  		Figure 7. Signaling pathways connecting to TRAF6-dependent NEMO ubiquitination. The various TRAF6-dependent signaling pathways which require the lysine residues defined in this study to activate NF-{kappa}B are indicated. Only the C-terminal-half of mouse NEMO protein is presented. The equivalent lysine residues in human NEMO are K285, K321, K325, K326 and K399, respectively.

 

   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
Case report
Patient KB is a 12-year-old female who developed cutaneous rash with vescicles on the trunk and the limbs (first stage of IP) at 1 month of age. Those vescicles then became hyperkeratotic warty lesions (second stage) at 3 months of age. In the third stage, pigmented spots appeared in linear and whorled pattern on the trunk and the limbs (at 1 year), while the fourth stage had not been reached at the time of the NEMO molecular analysis. Skin biopsies performed at the first stage confirmed marked dyskeratosis and eosinophilic infiltration consistent with IP.

No alterations were observed in the hair, fingernails and eyes. In contrast, neurological alterations were present: microcephaly and brain atrophy were diagnosed at birth, and at the same time CT-scan analysis showed severe cortico-subcortical atrophy, mostly in the left hemisphere. Neonatal seizures were observed at the time of IP diagnosis (1 month of age). Mental retardation and learning difficulties were diagnosed at 3 years.

The patient is the second child in the family with four siblings. However, the other members of the family, parents and three siblings, were examined and nobody was IP affected.

Reagents
The following antibodies were used: anti-human NEMO (#3328, a kind gift of Dr N. Rice), sc-8330 (Santa-Cruz, Santa Cruz, CA, USA) and Ref. 559675 (BD Pharmingen, San Diego, CA, USA), anti-mouse NEMO (#3329, a kind gift of N. Rice), anti-IKK-1 (Imgenex, San Diego, CA, USA), anti-IKK-2 (Imgenex), anti-RIP (Imgenex), anti-TRAF6 (sc-7221, Santa-Cruz), anti-ubiquitin (sc-9133, Santa-Cruz), anti-RICK (sc-22763, Santa-Cruz) and anti-phospho-JNK (Cell Signaling, Danvers, MA, USA). TNF-{alpha} and IL-1ß were from Roche (Basel, Switzerland) and Peprotech (Rocky Hill, NJ, USA), LPS and LPA were from Sigma-Aldrich (St. Louis, MO, USA).

Mutation detection and X-inactivation analysis
Mutation G967C of NEMO was identified and X-inactivation skewing was measured using protocols described previously for other NEMO mutations (35).

Plasmid construction
Mutation A323P was introduced into the hNEMO cDNA (35), previously inserted into a pc-DNA3-Flag expression vector, by using the Quikchange mutagenesis Kit (Invitrogen, Carlsbad, CA, USA) and checked by sequencing. The same procedure was used to prepare all the point mutations in the full-length version of mNEMO, previously inserted into a pcDNA3-HA expression vector (10). The truncated versions of mNEMO (dN241, dC385, dC343 and dC299) were generated by PCR and inserted into pcDNA3-HA.

Transfections
1.3E2 cells were transfected using DEAE-Dextran, as previously described (26). Transfection of SVT-2C cells was performed similarly. Briefly, 5 x 106 cells were washed in TBS (25 mM Tris–HCl (pH 7.4), 137 mM NaCl, 5 mM KCl, 0.6 mM Na2HPO4, 0.7 mM CaCl2, 0.5 mM MgCl2), resuspended in 250 µl of TBS, and NEMO expression plasmid (200 ng), Ig{kappa}-Luc reporter plasmid (1 µg) and internal control eF1-LacZ (200 ng) were added. Cells were finally mixed with 250 µl of DEAE-Dextran (1 mg/ml, diluted in TBS). After 45 min of incubation at room temperature, cells were diluted with 5 ml of TBS, centrifuged and resuspended in 5 ml of RPMI/10% FCS. Twenty-four hours later, cells were recovered in 100 µl of lysis buffer (LB) (25 mM Tris-phosphate (pH 7.8), 8 mM MgCl2, 1 mM DTT, Triton X-100, 25% glycerol) and luciferase activity was assessed using the Luciferase Assay system (Promega, Madison, WI, USA). Transfection of NEMO (–) MEFs was carried out by electroporation. Briefly, the cell equivalent of a confluent 10 cm2 dish was resuspended in 300 µl of PBS and 100 µl was electroporated (500 µF, 160V, Gene Pulser X-cell Electroporation System, BioRad, Hercules, CA, USA) after adding 1 µg of reporter plasmid, 200 ng of eF1-LacZ and 500 ng of NEMO expression vector. After electroporation, cells were resuspended in 9 ml of DMEM/10% FCS and used to fill 3 wells of a 6-well dish. The morning after, the medium was changed. In the evening, cells were serum-starved by adding DMEM without FCS. After 16 h of starvation, cells were mock-, TNF- (10 ng/ml), IL-1- (10 ng/ml) or LPS- (15 µg/ml) stimulated. Four hours later, cells were lysed with LB and luciferase was measured as described above. Transfection of HEK 293T cells in 6-well dishes was carried out using a standard calcium phosphate procedure (10). In each transfection protocol, an internal control (eF1-lacZ) was included to correct for variations between the samples. ß-Galactosidase activity was measured using Luminescent ß-galactosidase Reporter System 3 (Clontech, Mountain View, CA, USA).

Immunoprecipitation and western blotting
HEK 293T cells were recovered by gentle pipetting in 1 ml of PBS. They were centrifuged at 6000 rpm for 1 min in a microfuge and resuspended in 50–100 µl of TNT buffer [200 mM NaCl, 1% Triton X-100, 20 mM Tris–HCl (pH 7.5)]. After incubation on ice for 10 min and centrifugation at 14 000 rpm for 10 min, the supernatant was recovered and the protein concentration determined. Proteins (100–200 µg in 200 µl of TNT) were either directly analyzed, after electrophoresis, by western blotting (see below) or immunoprecipitated. In this latter case, extracts were incubated under rotation for 2 h at 4°C with the relevant antibody. Protein A- or protein G-Sepharose was then added and the mixture incubated for a further 1 h at 4°C. Sepharose beads were quickly centrifuged in a microfuge (30s at 14 000 rpm) and washed four times with TNF. After the final wash, the beads were resuspended in 30 µl of buffer A (10 mM KCl, 2 mM MgCl2, 0.1 mM EDTA, 1 mM DTT, 10 mM Hepes (pH 7.8))/loading dye and electrophoresed through a 10% acrylamide gel. Gels were transferred at 100 V for 1 h onto Immun-Blot PVDF membranes (BioRad), which were blocked in 4% dry milk/PBS for 1 h. Incubation with the relevant Ab was carried out in 0.4% dry milk/PBS for 1 h. The membrane was washed three times in 0.05% Tween/PBS for 10 min and incubated with the secondary HPR-linked antibody for 45 min in 0.4% dry milk/PBS. After the final washes, membranes were analyzed using either ECL + (Amersham, Little Chalfont, UK) or SuperSignal West Pico Chemiluminescent Kit (Pierce, Rockford, IL, USA).


   SUPPLEMENTARY MATERIAL
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 
Supplementary Material is available at HMG Online.


   FUNDING
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
NOTE ADDED IN PROOF
 REFERENCES
 
FIRB grant from Ministry of Education and Research, Italy; EU project GENE SKIN (contract no 512117 to M.V.U.); Fondation pour la Recherche Médicale and Ligue Nationale contre le Cancer (to G.C.).


   NOTE ADDED IN PROOF
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
REFERENCES
 
While this work was under review, Abbott et al. (2007) (Coordinate regulation of Toll-like receptor and NOD2 signaling by K63-linked polyubiquitin chains. Mol. Cell. Biol, 27, 6012-6025) reached essentially similar conclusions regarding the participation of TRAF6 in NEMO ubiquitination by Nod2/RICK and the role played by residues hK285 and hK399 of NEMO in NF-kB activation by LPS and MDP, an activator of Nod2.


   ACKNOWLEDGEMENTS
 
We thank Dr S.C. Sun for the kind gift of SVT-2C cells, Dr X. Xu for preparation of mutant K278R and Dr M.G. Miano for helpful discussion.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the last two authors should be regarded as joint First Authors. Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 SUPPLEMENTARY MATERIAL
 FUNDING
 NOTE ADDED IN PROOF
 REFERENCES
 

  1. Karin M., Ben-Neriah Y. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. (2000) 18:621–663.[CrossRef][Web of Science][Medline]

  2. Scheidereit C. I{kappa}B kinase complexes: gateways to NF-{kappa}B activation and transcription. Oncogene (2006) 25:6685–6705.[CrossRef][Web of Science][Medline]

  3. Pasparakis M., Luedde T., Schmidt-Supprian M. Dissection of the NF-{kappa}B signalling cascade in transgenic and knockout mice. Cell Death Differ. (2006) 13:861–872.[CrossRef][Web of Science][Medline]

  4. Pomerantz J.L., Baltimore D. Two pathways to NF-{kappa}B. Mol. Cell (2002) 10:693–695.[CrossRef][Web of Science][Medline]

  5. Hayden M.S., Ghosh S. Signaling to NF-{kappa}B. Genes Dev. (2004) 18:2195–2224.[Abstract/Free Full Text]

  6. Deng L., Wang C., Spencer E., Yang L., Braun, You J., Slaughter C., Pickart C., Chen Z.J. Activation of the I{kappa}B kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. Cell (2000) 103:351–361.[CrossRef][Web of Science][Medline]

  7. Wang C., Deng L., Hong M., Akkaraju G.R., Inoue J., Chen Z.J. TAK1 is a ubiquitin-dependent kinase of MKK and IKK. Nature (2001) 412:346–351.[CrossRef][Medline]

  8. Li H., Kobayashi M., Blonska M., You Y., Lin X. Ubiquitination of RIP is required for tumor necrosis factor {alpha}-induced NF-{kappa}B activation. J. Biol. Chem. (2006) 281:13636–13643.[Abstract/Free Full Text]

  9. Ishitani T., Takaesu G., Ninomiya-Tsuji J., Shibuya H., Gaynor R.B., Matsumoto K. Role of the TAB2-related protein TAB3 in IL-1 and TNF signaling. EMBO J. (2003) 22:6277–6288.[CrossRef][Web of Science][Medline]

  10. Kovalenko A., Chable-Bessia C., Cantarella G., Israël A., Wallach D., Courtois G. The tumour suppressor CYLD negatively regulates NF-{kappa}B signalling by deubiquitination. Nature (2003) 424:801–805.[CrossRef][Medline]

  11. Tang E.D., Wang C.Y., Xiong Y., Guan K.L. A role for NF-{kappa}B essential modifier/I{kappa}B kinase-{gamma} (NEMO/IKK{gamma}) ubiquitination in the activation of the I{kappa}B kinase complex by tumor necrosis factor-{alpha}. J. Biol. Chem. (2003) 278:37297–37305.[Abstract/Free Full Text]

  12. Qin J., Yao J., Cui G., Xiao H., Kim T.W., Fraczek J., Wightman P., Sato S., Akira S., Puel A., et al. TLR8-mediated NF-{kappa}B and JNK activation are TAK1-independent and MEKK3-dependent. J. Biol. Chem. 281:21013–21021.

  13. Zhou H., Wertz I., O'Rourke K., Ultsch M., Seshagiri S., Eby M., Xiao W., Dixit V.M. Bcl10 activates the NF-{kappa}B pathway through ubiquitination of NEMO. Nature (2004) 427:167–171.[CrossRef][Medline]

  14. Sun L., Deng L., Ea C.K., Xia Z.P., Chen Z.J. The TRAF6 ubiquitin ligase and TAK1 kinase mediate IKK activation by BCL10 and MALT1 in T lymphocytes. Mol. Cell (2004) 14:289–301.[CrossRef][Web of Science][Medline]

  15. Abbott D.W., Wilkins A., Asara J.M., Cantley L.C. The Crohn disease's protein, Nod2, requires RIP2 in order to induce ubiquitinylation of a novel site on NEMO. Curr. Biol. (2004) 14:2217–2227.[CrossRef][Web of Science][Medline]

  16. Wu C.J., Conze D.B., Li T., Srinivasula S.M., Ashwell J.D. NEMO is a sensor of Lys 63-linked polyubiquitination and functions in NF-{kappa}B activation. Nat. Cell Biol. (2006) 8:398–406.[CrossRef][Web of Science][Medline]

  17. Ea C., Deng L., Xia Z.P., Pineda G., Chen Z.J. Activation of IKK by TNF{alpha} requires site-specific ubiquitination of RIP1 and polyubiquitin binding by NEMO. Mol. Cell (2006) 22:245–257.[CrossRef][Web of Science][Medline]

  18. Inohara N., Koseki T., Lin J., del Peso L., Lucas P.C., Chen F.F., Ogura Y., Nunez G. An induced proximity model for NF-{kappa}B activation in the Nod1/RICK and RIP signaling pathways. J. Biol. Chem. (2000) 275:27823–27831.[Abstract/Free Full Text]

  19. Poyet J.L., Srinivasula S.M., Lin J.H., Fernandes-Alnemri T., Yamaoka S., Tsichlis P.N., Alnemri E.S. Activation of the I{kappa}B kinases by RIP via IKK{gamma}/NEMO-mediated oligomerization. J. Biol. Chem. (2000) 275:37966–37977.[Abstract/Free Full Text]

  20. Courtois G., Smahi A. NF-{kappa}B-related genetic diseases. Cell Death Differ. (2006) 13:843–851.[CrossRef][Web of Science][Medline]

  21. Smahi A., Courtois G., Vabres P., Yamaoka S., Heuertz S., Munnich A., Israël A., Heiss N.S., Klauck S.M., Kioschis P., et al. Genomic rearrangement in NEMO impairs NF-{kappa}B activation and is a cause of incontinentia pigmenti. Nature (2000) 405:466–472.[CrossRef][Medline]

  22. Zonana J., Elder M.E., Schneider L.C., Orlow S.J., Moss C., Golabi M. A novel X-linked disorder of immune deficiency and hypohidrotic ectodermal dysplasia is allelic to incontinentia pigmenti and due to mutations in IKK{gamma}(NEMO). Am. J. Hum. Genet. (2000) 67:1555–1562.[CrossRef][Web of Science][Medline]

  23. Döffinger R., Smahi A., Bessia C., Geissmann F., Feinberg J., Durandy A., Bodemer C., Kenwrick S., Dupuis-Girod S., Blanche S., et al. X-linked anhidrotic ectodermal dysplasia with immunodeficiency is caused by impaired NF-{kappa}B signaling. Nature Genet. (2001) 27:277–285.[CrossRef][Web of Science][Medline]

  24. Courtois G., Smahi A., Reichenbach J., Döffinger R., Cancrini C., Bonnet M., Puel A., Chable-Bessia C., Yamaoka S., Feinberg J., et al. A hypermorphic I{kappa}B{alpha} mutation is associated with autosomal dominant anhidrotic ectodermal dysplasia and T cell immunodeficiency. J. Clin. Invest. (2003) 112:1108–1115.[CrossRef][Web of Science][Medline]

  25. Janssen R., van Wengen A., Hoeve M.A., ten Dam M., van der Burg M., van Dongen J., van de Vosse E., van Tol M., Bredius R., Ottenhoff T.H., et al. The same I{kappa}B{alpha} mutation in two related individuals leads to completely different clinical syndromes. J. Exp. Med. (2004) 200:559–568.[Abstract/Free Full Text]

  26. Courtois G., Whiteside S.T., Sibley C.H., Israël A. Characterization of a mutant cell line that does not activate NF-{kappa}B in response to multiple stimuli. Mol. Cell Biol. (1997) 17:1441–1449.[Abstract]

  27. Harhaj E.W., Good L., Xiao G., Uhlik M., Cvijic M.E., Rivera-Walsh I., Sun S.C. Somatic mutagenesis studies of NF-{kappa}B signaling in human T cells: evidence for an essential role of IKK{gamma} in NF-{kappa} B activation by T-cell costimulatory signals and HTLV-I Tax protein. Oncogene (2000) 19:1448–1456.[CrossRef][Web of Science][Medline]

  28. Schmidt-Supprian M., Bloch W., Courtois G., Addicks K., Israël A., Rajewsky K., Pasparakis M. NEMO/IKK {gamma}-deficient mice model incontinentia pigmenti. Mol. Cell (2000) 5:981–992.[CrossRef][Web of Science][Medline]

  29. Makris C., Roberts J.L., Karin M. The carboxyl-terminal region of I{kappa}B kinase {gamma} (IKK{gamma}) is required for full IKK activation. Mol. Cell Biol. (2002) 22:6573–6581.[Abstract/Free Full Text]

  30. Klemm S., Zimmermann S., Peschel C., Mak T.W., Ruland J. Bcl10 and Malt1 control lysophosphatidic acid-induced NF-{kappa}B activation and cytokine production. Proc. Natl. Acad. Sci. USA (2007) 104:134–138.[Abstract/Free Full Text]

  31. Grabiner B.C., Blonska M., Lin P.C., You Y., Wang D., Sun J., Darnay B.G., Dong C., Lin X. CARMA3 deficiency abrogates G protein-coupled receptor-induced NF-{kappa}B activation. Genes Dev. (2007) 21:984–996.[Abstract/Free Full Text]

  32. Trompouki E., Hatzivassiliou E., Tsichritzis T., Farmer H., Ashworth A., Mosialos G. CYLD is a deubiquitinating enzyme that negatively regulates NF-{kappa}B activation by TNFR family members. Nature (2003) 424:793–796.[CrossRef][Medline]

  33. Kanayama A., Seth R.B., Sun L., Ea C.K., Hong M., Shaito A., Chiu Y.H., Deng L., Chen Z.J. TAB2 and TAB3 activate the NF-{kappa}B pathway through binding to polyubiquitin chains. Mol. Cell (2004) 15:535–548.[CrossRef][Web of Science][Medline]

  34. McCarthy J.V., Ni J., Dixit V.M. RIP2 is a novel NF-{kappa}B-activating and cell death-inducing kinase. J. Biol. Chem. (1998) 273:16968–16975.[Abstract/Free Full Text]

  35. Fusco F., Bardaro T., Fimiani G., Mercadante V., Miano M.G., Falco G., Israël A., Courtois G., D'Urso M., Ursini M.V. Molecular analysis of the genetic defect in a large cohort of IP patients and identification of novel NEMO mutations interfering with NF-{kappa}B activation. Hum. Mol. Genet. (2004) 13:1763–1773.[Abstract/Free Full Text]


Add to CiteULike CiteULike   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us    What's this?


This article has been cited by other articles:


Home page
 Mol. Cell. Biol.Home page
S. Paz, M. Vilasco, M. Arguello, Q. Sun, J. Lacoste, T. L.-A. Nguyen, T. Zhao, E. A. Shestakova, S. Zaari, A. Bibeau-Poirier, et al.
Ubiquitin-Regulated Recruitment of I{kappa}B Kinase {varepsilon} to the MAVS Interferon Signaling Adapter
Mol. Cell. Biol., June 15, 2009; 29(12): 3401 - 3412.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. E. Keating and A. G. Bowie
Role of Non-degradative Ubiquitination in Interleukin-1 and Toll-like Receptor Signaling
J. Biol. Chem., March 27, 2009; 284(13): 8211 - 8215.
[Full Text] [PDF]

Home page
 Cancer Res.Home page
H.-S. Jin, D.-H. Lee, D.-H. Kim, J.-H. Chung, S.-J. Lee, and T. H. Lee
cIAP1, cIAP2, and XIAP Act Cooperatively via Nonredundant Pathways to Regulate Genotoxic Stress-Induced Nuclear Factor-{kappa}B Activation
Cancer Res., March 1, 2009; 69(5): 1782 - 1791.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow FREE Full Text (PDF) Freely available
Right arrow Supplementary Data
Right arrow A corrigendum has been published
Right arrow All Versions of this Article:
16/23/2805    most recent
ddm237v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in ISI Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Add to My Personal Archive
Right arrow Download to citation manager
Right arrow Search for citing articles in:
ISI Web of Science (8)
Right arrowRequest Permissions
Google Scholar
Right arrow Articles by Sebban-Benin, H.
Right arrow Articles by Courtois, G.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Sebban-Benin, H.
Right arrow Articles by Courtois, G.
Social Bookmarking
Add to CiteULike   Add to Connotea   Add to Del.icio.us  
What's this?