TRAF2 must bind to cellular inhibitors of apoptosis for tumor necrosis factor (tnf) to efficiently activate nf-kappab and to prevent tnf-induced apoptosis

: Tumor necrosis factor (TNF) receptor-associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor (TNFR) superfamily, including the TNF-TNFR1 ligand-receptor complex. Here, we define a cIAP1/2-interacting motif (CIM) within the TRAF-N domain of TRAF2, and we use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1-dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-kappaB-inducing kinase stability and suppress constitutive noncanonical NF-kappaB activation. Conversely, following TNFR1 stimulation, cells bearing a CIM-mutated TRAF2 showed reduced canonical NF-kappaB activation and TNF-induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF-induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-kappaB signaling and that efficient activation of NF-kappaB

Running title: Function of the TRAF2-cIAP1/2 interacting motif TNF receptor associated factor-2 (TRAF2) binds to cIAP1 and cIAP2 (cIAP1/2) and recruits them to the cytoplasmic domain of several members of the TNF receptor superfamily (TNFRSF), including the TNF-TNFR1 ligand-receptor complex. Here we define a cIAP1/2 interacting motif (CIM) within the TRAF-N domain of TRAF2, and use TRAF2 CIM mutants to determine the role of TRAF2 and cIAP1/2 individually, and the TRAF2-cIAP1/2 interaction, in TNFR1 dependent signaling. We show that both the TRAF2 RING domain and the TRAF2 CIM are required to regulate NF-B inducing kinase (NIK) stability and suppress constitutive non-canonical NF-B activation. Conversely, following TNFR1 stimulation, cells bearing a CIM mutated TRAF2 showed reduced canonical NF-B activation and TNF induced RIPK1 ubiquitylation. Remarkably, the RING domain of TRAF2 was dispensable for these functions. However, like the TRAF2 CIM, the RING domain of TRAF2 was required for protection against TNF induced apoptosis. These results show that TRAF2 has anti-apoptotic signaling roles in addition to promoting NF-B signaling and that efficient activation of NF-B by TNFR1 requires the recruitment of cIAP1/2 by TRAF2.
Genetic deletion of TRAF2 in the mouse results in early post-natal lethality that is caused by increased NF-B-mediated TNF production and increased cellular sensitivity to TNF killing (12,24,25). Most TRAF family members, including TRAF2, bear RING E3 ubiquitin ligase domains that are believed to catalyze K63-linked ubiquitylation (26,27). One of the proposed targets of TRAF2 RING E3 ligase activity is RIPK1 (28,29), which is modified with K63-linked polyubiquitin chains upon TNFR1 activation. This modified TNFR1 complexed RIPK1 has been proposed to serve as a platform for the recruitment and/or activation of TAB2/TAB3/TAK1 and NEMO/IKK /IKK kinase complexes (30)(31)(32), which target the NF-B inhibitor I B for K48-linked polyubiquitinmediated proteasomal degradation, to initiate canonical p65-dependent NF-B gene transcription.
As in the case of TRAF2, genetic deletion of cIAP1, or IAP antagonist induced loss of cIAP1/2, also sensitizes cells to TNF killing (16,19,(33)(34)(35)(36). Also like TRAF2, both cIAP1 and cIAP2 have been reported to ubiquitylate RIPK1 (10,15,16). Consistent with the idea that cIAP1/2 play an important role in the activation of NF-B by TNF, the removal of cIAP1/2 limits TNF from inducing NF-B by the canonical pathway (10,15,16). While it remains possible that both TRAF2 and cIAP1/2 play similar roles in modulating TNFR1 signaling through RIPK1 ubiquitylation, it is unknown whether cIAP1/2 act indirectly by activating the E3 ligase activity of TRAF2, or if cIAP1/2 act in combination with TRAF2 to directly ubiquitylate RIPK1, or alternatively, whether cIAP1/2 are the major E3 ligases for RIPK1 and TRAF2's role is to recruit them to TNFR1.
In this study we have identified TRAF2's cIAP1/2 interaction motif (CIM), and used TRAF2 CIM mutants defective in cIAP1/2 binding to determine how cIAP1/2 and TRAF2 coordinately suppress non-canonical NF-B activation and regulate signaling from TNFR1.

EXPERIMENTAL PROCEDURES
Cell Culture, Transfections, Constructs and Lentiviral Infections. All cell lines were maintained at 37˚C, 10% CO 2 in DMEM supplemented with 8% FBS and passaged twice weekly. Transient transfections using 1μg DNA per 10cm tissue culture plate were performed with Effectene™ (Qiagen) using manufacturer's protocols. The NF-B lentiviral reporter vector pTRH1 mCMV NF-B dscGFP was purchased from System Biosciences. Cre-recombinase and SV40 Large T antigen were cloned into the lentiviral vector pFU. Mouse TRAF2 and the CIM and RING domain TRAF2 mutants were cloned into pEF-NFLAG and the 4-hydroxy tamoxifen inducible lentiviral vector pF 5x UAS, which we have described in detail elsewhere (19,45). Full length cIAP1 and the BIR1 constructs were cloned into pEF-NHA. Complete sequence of all constructs can be obtained upon request. Lentiviral particles were generated as previously described (17,19).
Generation of MEFs. Knock-out MEFs were generated from E15 embryos using standard procedures and infected with SV40 Large T antigen expressing lentivirus. TRAF2 conditional knock-out MEFs were similarly generated from TRAF2 LoxP/LoxP E15 embryos. To delete TRAF2 the transformed MEFs were infected with a cre expressing lentivirus (pFU cre SV40 Hygro) and deletion was confirmed by PCR and Western blotting.
Death Assays. Cells were seeded on 12 well tissue culture plates at approximately 70 percent confluency and allowed to adhere. Compound A (500 nM), described in (19), or human Fc-TNF (60 ng/ml) were added to cells for 24 hrs and cell death measured by Propidium Iodide (PI) staining and flow cytometry. In each sample 10,000 events were measured, and the cell death (% PI positive cells) quantified.
Western Blotting and Immunoprecipitations.
Immunoprecipitations were performed as previously described (17,19,46) with some minor alterations. Briefly, 4x10 7 MEFs were treated in the presence or absence of human FLAG-TNF at 1 g/ml for 5 minutes. Cells were lysed in IP-lysis buffer (30 mM Tris-HCl, pH 7.4, 120 mM NaCl, 2 mM EDTA, 2 mM KCl, 1 % TX100, Roche complete protease-inhibitor cocktail) at 4 °C for 30 minutes. The lysates were centrifuged at 15,000g for 30 minutes. 0.5 g of FLAG-TNF was added to the non-stimulated (time=0) control and the TNF-R1 complex was precipitated using M2 beads (Sigma) for 16 hours. The beads were washed five times with IP-lysis buffer and eluted with 2x LDS buffer (NuPAGE, Invitrogen). Proteins were separated by SDS-PAGE (NuPAGE, Invitrogen) and analyzed by Western blotting. Membranes were stripped with 50 mM Glycine (pH 2.3) and reprobed with other antibodies.
Immunofluorescence. MEFs grown overnight on glass coverslips were treated with Fc-TNF (60ng/ml) for the indicated times, immediately fixed with 3.2% paraformaldehyde for 20 minutes, washed in PBS, and permeabilized with 0.5% TritonX-100 for 5 minutes. Cells were blocked, incubated with p65 antibody, washed four times with PBS, and then incubated with anti-rabbit Alexafluor488 conjugated secondary antibody (Invitrogen) and washed four times again. All blocking steps and antibody incubations were performed with PBS containing 1% BSA for 30 minutes. Cells were viewed on a Olympus BX50 fluorescence microscope and SPOT RT imaging software using a x40 objective.

Identification of a cIAP1 and cIAP2
interacting motif (CIM) in TRAF2. The cIAP1 BIR1 domain and several key residues within this region have been shown to mediate cIAP1 binding to TRAF2 (17,22,23). However, the region of TRAF2 required for cIAP1 and cIAP2 binding has not been clearly defined since yeast two hybrid experiments suggested the TRAF-N domain was important (9). Our own yeast two hybrid analysis suggested that a TRAF2-N domain containing fragment, residues 262-349, is sufficient for cIAP1/2 binding (Fig. 1A, 1C and not shown). To verify the ability of this region to bind cIAP1 in vivo, we performed coimmunoprecipitation experiments in 293T cells. Immunoprecipitation of full length FLAG-TRAF2 confirmed that it interacts with the cIAP1 BIR1 domain and can also bind two BIR1 domain truncation mutants lacking the first 22 and 39 residues (Fig. 1B) (17,22,23). Furthermore, a FLAG-TRAF2-N (aa262-349) construct was sufficient by itself to immunoprecipitate HA tagged full length cIAP1 or the cIAP1 BIR1 domain alone (Fig. 1C). Experiments using purified cIAP1 BIR1 and TRAF2-N domains demonstrated that the binding between these two regions was direct (Fig. 2D).
While the crystal structure of truncated TRAF2 is available, only the C-terminal part of the TRAF-N domain is present (47,48) making it difficult to identify the potential interaction interface between TRAF2-N and cIAP1 BIR1. We therefore examined the conservation of the TRAF2-N domain from a number of organisms to predict which residues might be important for the TRAF2-cIAP1 interaction (Fig. 1D). TRAF1 has also been shown to interact with cIAP1/2 (9,49) and was also therefore included in the alignment. This analysis revealed a cluster of residues spanning amino acids 283 to 294 that were highly conserved across all TRAF2 species examined and also TRAF1 (Fig. 1D). Importantly, this region showed little conservation with TRAF3 or Drosophila TRAF2 ( Fig. S1A and S1B), none of which bind cIAP proteins. We therefore created several alanine point substitutions between or near mouse TRAF2 residues 283-294, a TRAF2 deletion spanning the residues 283-293, and a TRAF2 truncation by introducing a stop codon at residue D310 (Fig. 1D).
TRAF2 residues 283-293 are required for binding to cIAP1 and cIAP2. To determine which residues of TRAF2 are important for binding cIAP1 and cIAP2 we transiently transfected the indicated FLAG-TRAF2 constructs ( Fig. 1D and 2A) into 293T cells and examined their binding to endogenous cIAP1 and over expressed cIAP2 by FLAG immunoprecipitation and Western blot. As before TRAF2-N (262-349) and wildtype (WT) TRAF2 bound cIAP1 and, as expected, TRAF1 also immunoprecipitated cIAP1. The TRAF2 deletion, 283-293 did not bind detectable amounts of cIAP1 or cIAP2, and the double alanine point substitution EVE292AVA mutant showed drastically reduced binding to both cIAP1 and cIAP2 ( Fig. 2A-2D). Single alanine point substitution of either E292 or E294 was less disruptive of cIAP1 and cIAP2 binding than mutation of both residues in the EVE292AVA mutant (Fig. 2B). TRAF2 E283A (and EN283AA) showed slightly reduced cIAP1 binding, but the other TRAF2 mutants, including the TRAF2 D310 truncation mutant and a RING mutant lacking the first 87 amino acids, all interacted with cIAP1 like wild type TRAF2 ( Fig. 2A, 2B). Similar decreases in TRAF2 mutant binding to cIAP1 were also observed in other cell types, such as D645 glioma cells (Fig.  S1C). These mutations are unlikely to affect TRAF2 trimerization because the cIAP1/2 interacting motif was not present in the trimeric TRAF2 crystal structure (47,48). Consistent with the idea that the TRAF2 mutations do not affect TRAF2 structure dramatically, the TRAF2 mutants with reduced binding to cIAP1/2 interacted with endogenous TRADD and RIPK1 to the same extent as the wild type protein ( Fig.  2C) (50). Experiments using purified cIAP1 or cIAP2 BIR1 domain proteins with purified wild type, EVE292AVA or 283-293 TRAF2-N domain proteins confirmed that BIR1 binding to TRAF2-N was direct and that mutant TRAF2-N binding was reduced (EVE292AVA) or abolished ( 283-293) (Fig. 2D). The cIAP1/2 interacting motif (CIM) of TRAF2 therefore spans residues 283-294, and mediates the direct interaction between the TRAF-N domain of TRAF2 and the BIR1 domain of cIAP1.
A functional TRAF2 CIM and RING domain are required to inhibit non-canonical NF-B. In unstimulated WT cells TRAF3 binding to NIK recruits a TRAF2-cIAP1/2 complex to allow cIAP1/2 mediated NIK degradation and inhibition of spontaneous noncanonical NF-B (11,12,33,51). Deletion of TRAF2 or cIAP1 therefore results in constitutive non-canonical NF-B signaling (Fig. 3A, 3C) (17,19,52). Complementation of TRAF2 knockout MEFs with the EVE292AVA TRAF2 mutant, that is still able to bind low amounts of cIAP1/2 (Fig. 2B, 2D), was able to partially restore NF-B activity in TRAF2 knockout cells to the basal levels observed in WT cells (Fig.  3B, 3C), while the TRAF2 283-293 CIM mutant completely failed to do so, as measured by increased NIK levels, p100 processing to p52 and NF-B GFP reporter activity (Fig. 3B, 3C). The TRAF2 RING domain mutant also failed to reduce TRAF2 knockout NIK levels and p100 processing (Fig. 3C) despite the fact that it binds cIAP1 and cIAP2 (Fig. 2B). This shows that cIAP1/2 mediated K48-linked ubiquitylation and degradation of NIK relies on TRAF2 as an adaptor protein, and that the TRAF2 RING domain is also essential for the ability of cIAP1/2 to degrade NIK.
The TRAF2 CIM is required for resistance to TNF induced cell death. TRAF2 knockout MEFs immortalized with the SV40 large T antigen are highly susceptible to cell death induced by TNF treatment alone (17) (Fig.  4A). These TRAF2 knockout cells showed no significant difference in their ability to be killed by TRAIL and only a slight, potentially insignificant, difference in their ability to be killed by FasL (Fig. 4A), suggesting TRAF2 primarily provides protection against TNF induced apoptosis. Similarly, SV40 large T immortalized wild type (WT) MEFs that have been treated with the IAP antagonist Compound A to remove cIAP1, cIAP2 are killed by TNF treatment within 24 hrs (19) (Fig. 4A). Because cIAP1/2 are recruited by TRAF2, these data indicate that either cIAP1/2 or TRAF2, or both, confer protection against TNF killing but do not distinguish between these possibilities.
Reintroduction of inducible WT TRAF2, or the E283A TRAF2 mutant that is able to bind cIAP1/2, into TRAF2 knockout MEFs almost fully restored protection against TNF killing (Fig. 4B, 4C). However, induction of the TRAF2 CIM 283-293, EVE292AVA or RING domain mutants in TRAF2 knockout MEFs failed to prevent TNF induced cell death (Fig. 4C). These data demonstrate that cIAP1/2 binding to TRAF2 is crucial to prevent apoptosis induced by TNF and suggests that any interaction cIAP1/2 may have with RIPK1 (14) is not sufficient for TNF resistance in the absence of their binding TRAF2. Significantly, these results indicate that the RING domain of TRAF2 is also required for TNF resistance (Fig.  4C), even though this construct retains the ability to bind both cIAP1 and cIAP2 (Fig. 1B). Therefore both TRAF2 and cIAP1/2 each play important roles in protecting cells from TNF induced death, and they must do so bound to each other.
TNF induced RIPK1 modification is dependent on a functional TRAF2-cIAP1/2 interaction but the TRAF2 RING domain is dispensable. Recent work has suggested that cIAP1/2 act as the E3 ubiquitin ligases to ubiquitylate RIPK1 following TNFR1 activation (14)(15)(16). However, it has also been proposed that the E3 ubiquitin ligase activity of TRAF2 mediates ubiquitylation of RIPK1 following TNFR1 signaling (28,29). Combined deletion of TRAF2 and TRAF5 (TRAF2/TRAF5 DKO) significantly prevented RIPK1 ubiquitylation in response to TNF stimulation and dramatically reduced cIAP1/2 binding to TNFR1 (Fig. 5). Thus the phenotype of the TRAF2/TRAF5 knock-out cells is consistent with both models and cannot distinguish between either. Restoration of WT TRAF2 expression in TRAF2/TRAF5 DKO MEFs restored RIPK1 ubiquitylation and cIAP1/2 recruitment to TNFR1, as expected (Fig. 5). However, TRAF2/5 DKO cells reconstituted with the TRAF2 CIM 283-293 mutant did not recruit cIAP1 and did not restore RIPK1 ubiquitylation in response to TNF (Fig. 5). Reintroduction of TRAF2 RING, which is still able to bind cIAPs ( Fig.2) but lacks E3 ligase acivity, also restored RIPK1 ubiquitylation and cIAP1/2 recruitment (Fig. 5). These data are consistent with the idea that cIAP1/2 are the most important E3 ligases for RIPK1, and fit the model whereby if TRAF2 does not recruit cIAP1/2 then RIPK1 fails to undergo TNF induced ubiquitylation and associates with FADD and caspase-8 to induce cell death. Intriguingly, although TNF induced RIPK1 ubiquitylation (Fig. 5), and NF-B function (see below) are retained upon loss of the TRAF2 RING domain, these cells are still susceptible to TNF killing (Fig. 4C).
The TRAF2 CIM, but not the TRAF2 RING domain, is required for efficient TNF induced NF-B activation. As previously described (24,53), TNF induced NF-B activation in TRAF2 knockout cells was similar to wildtype cells, as determined by NF-B reporter assays and Western blotting for p65 phosphorylation and I B (Fig. 3A, 6A). Interestingly, we consistently observed that cIAP1-/-, TRAF2-/-, and TRAF2/TRAF5 DKO MEFs that have been immortalised with the large SV40T antigen have increased levels of basal phosphorylated p65 when compared to WT cells (Fig. 6A, 6B) (17,19). However, this does not correlate with increased basal nuclear p65 levels in any of these cell lines (Fig. 6C, 6D, 7D) (17).
TRAF2 and TRAF5 are redundant with respect to TNF induced activation of NF-B, because deletion of both genes is required to prevent it (54). To examine the role of the TRAF2-cIAP1/2 interaction in TNF induction of NF-B we therefore complemented TRAF2/TRAF5 double knockout (DKO) MEFs with TRAF2 WT, CIM mutant, and RING domain deletion constructs. TRAF2/TRAF5 DKO cells were significantly impaired in TNF induced p65 phosphorylation, I B degradation and translocation of p65 to the nucleus (Fig. 6B,  6C), and complementation with WT TRAF2 restored all these NF-B associated functions (Fig. 6B, 6C). While the TRAF2 CIM mutant EVE292AVA, that retains weak cIAP1/2 binding (Fig. 2B, 2D), partially restored TNF induced NF-B function (Fig. 6B, 6C) the TRAF2 283-293 CIM mutant was unable to restore either I B degradation or nuclear translocation of p65 in response to TNF (Fig.  6B, 6C).
It has recently been suggested that TNF induced K63-linked ubiquitylation of TRAF2 on lysine 31 regulates binding of TAB2/TAB3 to TRAF2 and is required for proper TNF induced IKK and NF-B activity (55). If this model is correct then complementation of TRAF2/TRAF5 DKO cells with a RING TRAF2 mutant, which lacks residues 1-87, should fail to restore TNF induced NF-B function. However, we observed that TRAF2/TRAF5 DKO MEFs complemented with RING TRAF2 restored TNF induced p65 phosphorylation, I B degradation and translocation of p65 to the nucleus to a similar level as observed when TRAF2/TRAF5 DKO MEFs were complemented with WT TRAF2 (Fig. 6B, 6C).
Although rapid TNF induced nuclear translocation of p65 is mostly lost in TRAF2/TRAF5 DKO MEFs when compared to WT cells, some p65 nuclear localization could be detected after 60 minutes of TNF stimulation (Fig. 6D). Because immunoprecipitated TNFR1 from TRAF2/ TRAF5 DKO cells still bound low levels of cIAP1/2 (Fig. 5), we speculated that the late nuclear translocation of p65 in TRAF2/TRAF5 DKO cells observed by immunofluorescence could be caused cIAP1/2 function. Consistent with this idea, when we depleted cIAP1/2 from TRAF2/TRAF5 DKO cells using the IAP antagonist Compound A (19), p65 translocation to the nucleus failed to occur, even 60 minutes after TNF stimulation (Fig. 6D).
Deletion of TRAF2 results in enhanced TNF induced c-FLIP degradation. Previous work has demonstrated that in the absence of NF-B signaling in TRAF2/TRAF5 DKO or p65 knockout cells the lack of TNF induced c-FLIP L expression sensitises to TNFR1 killing (56). While it has previously been proposed that TRAF2 knockout cells are also sensitive to TNF due to reduced basal levels of c-FLIP L (57) our Western blots suggested that both unstimulated TRAF2-/-and TRAF2/TRAF5 DKO MEFs contain almost equivalent levels of c-FLIP L when compared to WT cells (Fig. 7). However, when stimulated with TNF, both TRAF2-/-and TRAF2/TRAF5 DKO cells displayed a dramatic loss of c-FLIP L when compared to WT cells (Fig. 7). Hence despite the normal NF-B activation in TRAF2-/-MEFs, c-FLIP L is still efficiently depleted upon TNF stimulation and this probably accounts, at least partially, for their increased sensitivity to TNFR1 induced death.

DISCUSSION
Our study identifying and characterizing the role of the cIAP1/2 interacting motif of TRAF2 reveals that a key role of TRAF2 is in the recruitment of cIAP1/2 into signaling platforms. One of these is the NIK kinase complex that is essential for non-canonical NF-B activation. TRAF2 is a key molecule in the regulation of NIK stability as loss of TRAF2 results in NIK stabilisation, activation, and constitutive non-canonical NF-B signaling (17,52,58). Using TRAF2 CIM and RING mutants we complemented TRAF2 knockout cells and found that restoration of the normal low level of NIK and reduction of spontaneous NF-B activity required both the TRAF2 CIM and RING domain. These findings are consistent with recent reports showing that repression of non-canonical NF-B activity requires a complex of TRAF3, TRAF2 and cIAP1/2, with each component performing a distinct function (11,12,19,33). TRAF3 interacts with NIK and also binds the TRAF2-cIAP1/2 module, thereby bringing cIAP1/2 into the proximity of NIK, resulting in cIAP1/2 mediated K48-linked ubiquitylation of NIK and its proteasomal degradation (11,12,33). These studies and our work here demonstrate that in addition to cIAP1/2, both the TRAF2 and TRAF3 RING domains are required for NIK degradation.
Our data also shows that TNF induced nuclear translocation of the canonical NF-B subunit p65 is significantly delayed in TRAF2/TRAF5 DKO cells, which agrees with previously reported NF-B electrophoretic mobility gel shift assays and in vitro IKK activity assays performed on TRAF2/TRAF5 DKO cells (54,55). cIAP1/2 have also been implicated in canonical NF-B signaling following TNFR1 activation, and both cIAP1/2 and TRAF2 have been suggested to be the E3 ligases required for ubiquitylation of RIPK1 (10,(14)(15)(16)28,29). Ubiquitylated RIPK1 is believed to be the key signaling platform required for correct TNFR1 induced NF-B and a TRAF2 CIM mutant was unable to restore either RIPK1 ubiquitylation or normal NF-B signaling in response to TNF. Recently however we have shown that RIPK1 knockout MEFs are able to induce NF-B in response to TNF (L.W. and J.S, submitted). We therefore believe that RIPK1 ubiquitylation serves as a marker for TNF induced activation of NF-B but is not a requirement. While our work here and recent studies therefore suggest a role for cIAP1/2 in normal canonical NF-B responses initiated by TNF stimulation (10,15,16), it remains unclear what their key targets might be given that RIPK1 itself is dispensable for TNF induced NF-B.
The ubiquitylation of RIPK1 by cIAP1/2 has been shown to prevent it associating with FADD and caspase-8, thereby limiting TNFR1 induced cell death (10,15,16). Consistent with this model, cIAP1 knockout MEFs have increased RIPK1 recruitment to TNFR1 and increased sensitivity to TNF killing compared to wild type MEFs (10,19). Similarly, IAP antagonist treatment that depletes cIAP1/2 causes a loss of RIPK1 modification following TNF stimulation (10,16 While we show that TRAF2 must recruit cIAP1/2 in order to properly activate NF-B, TRAF2 is not just a passive adaptor, because TRAF2's RING domain is required for imparting resistance to induction of apoptosis by TNF. Our results with TRAF2 -/cells and those reconstituted with a TRAF2 RING mutant, demonstrates an apparently normal NF-B induction combined with sensitivity to TNF killing. While decreased cIAP1/2 recruitment into the TNFR1 signalling complex in TRAF2-/-MEFs may lead to less RIPK1 ubiquitylation and theoretically promote TNF induced cell death, our data shows that TRAF2 RING restores cIAP1/2 binding to TNFR1 and RIPK1 ubiquitylation, yet fails to restore TNF resistance. The almost normal canonical NF-B response on the background of increased noncanonical NF-B signalling in TRAF2 -/-MEFs ((17) and this work) may render them sensitive to TNF but this remains to be tested. Alternatively, an enhancement in c-FLIP L degradation may render TRAF2-/-cells more susceptible to TNFR1 induced death. To this end, we observed a dramatic decrease in c-FLIP L levels in TRAF2-/-cells upon TNF stimulation which is likely to contribute to their enhanced TNF sensitivity. While this data fits current models where active caspase-8 has been shown efficiently cleave c-FLIP L (59), it remains to be determined how c-FLIP L levels are regulated by the presence of TRAF2.
Our results here define a TRAF2-c1AP1/2 interacting motif and provide independent support for recent studies with IAP antagonist compounds that demonstrate a fundamental role for cIAP1/2 in regulating TNF signaling. Reconstitution of TRAF2 and TRAF2/TRAF5 DKO cells with a TRAF2 RING mutant has however raised new questions. This mutant was able to recapitulate wild type TRAF2's ability to activate canonical NF-B in response to TNF in TRAF2/TRAF5 DKO cells but was unable to protect TRAF2 knock-out cells from the cytotoxic activity of TNF. It will therefore be interesting to determine what the additional cytoprotective function of TRAF2 is.

FOOTNOTES
We would like to thank Hiroyasu Nakano for TRAF2/TRAF5 double knock-out MEFs, David Baltimore for pFU and lentiviral packaging constructs, Jurg Tschopp for generous gifts of materials and Robert Brink (Garvan Institute) for critically reading the manuscript.
JS is supported by NHMRC grants #433013, 541901 and 541902. DV is an Australian Fellow, funded by the Leukemia and Lymphoma Society and NHMRC grant #461221. PDM is a recipient of a Health Sciences Career Development Award (University of Otago). EFL was funded by The Cancer Council Victoria. JEV is supported by a Human Frontiers Science Program Fellowship.