Research ArticleCell Biology

Cellular Inhibitors of Apoptosis Are Global Regulators of NF-κB and MAPK Activation by Members of the TNF Family of Receptors

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Sci. Signal.  20 Mar 2012:
Vol. 5, Issue 216, pp. ra22
DOI: 10.1126/scisignal.2001878

Abstract

Tumor necrosis factor (TNF) family members are essential for the development and proper functioning of the immune system. TNF receptor (TNFR) signaling is mediated through the assembly of protein signaling complexes that activate the nuclear factor κB (NF-κB) and mitogen-activated protein kinase (MAPK) pathways in a ubiquitin-dependent manner. The cellular inhibitor of apoptosis (c-IAP) proteins c-IAP1 and c-IAP2 are E3 ubiquitin ligases that are recruited to TNFR signaling complexes through their constitutive association with the adaptor protein TNFR-associated factor 2 (TRAF2). We demonstrated that c-IAP1 and c-IAP2 were required for canonical activation of NF-κB and MAPK by members of the TNFR family. c-IAPs were required for the recruitment of inhibitor of κB kinase β (IKKβ), the IKK regulatory subunit NF-κB essential modulator (NEMO), and RBCK1/Hoil1-interacting protein (HOIP) to TNFR signaling complexes and the induction of gene expression by TNF family members. In contrast, TNFRs that stimulated the noncanonical NF-κB pathway triggered translocation of c-IAPs, TRAF2, and TRAF3 from the cytosol to membrane fractions, which led to their proteasomal and lysosomal degradation. Finally, we established that signaling by B cell–activating factor receptor 3 induced the cytosolic depletion of TRAF3, which enabled noncanonical NF-κB activation. These results define c-IAP proteins as critical regulators of the activation of NF-κB and MAPK signaling pathways by members of the TNFR superfamily.

Introduction

Members of the tumor necrosis factor (TNF) superfamily of ligands are homotrimeric type 2 transmembrane proteins that exist either as membrane-embedded or as cleaved, soluble proteins, and they bind to one or more members of the TNF receptor (TNFR) superfamily (1). Most of the TNFRs are type 1 transmembrane proteins that use multiple cysteine-rich domains in their extracellular regions to bind to their respective ligands (2). These ligands and receptors are instrumental in the proper organization and functioning of the immune system, and they are implicated in a number of genetic or acquired human diseases (2). Binding of TNFs to their cognate receptors triggers the assembly of receptor-associated signaling complexes and stimulates activation of multiple signaling pathways, including nuclear factor κB (NF-κB) and the c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinases (MAPKs), and, in some cases, cell death (36). Activation of canonical or noncanonical NF-κB signaling results in the expression of genes that encode proinflammatory and antiapoptotic proteins (7, 8). Most of the members of the superfamily of TNFRs activate both canonical and noncanonical pathways, although TNFR1 and death receptor 3 (DR3) activate only the canonical NF-κB pathway, whereas B cell–activating factor (BAFF) receptor 3 (BR3, also known as BAFFR) activates only the noncanonical NF-κB pathway. In the canonical pathway, inhibitor of κB kinase 2 (IKK2, also known as IKKβ) phosphorylates inhibitor of NF-κB (IκB), which leads to its ubiquitination and subsequent proteasomal degradation (8). This event liberates NF-κB dimers and enables their entry into the nucleus where they activate the expression of target genes. The noncanonical NF-κB pathway is inhibited by continuous proteasomal degradation of NF-κB–inducing kinase (NIK) by a complex containing cellular inhibitor of apoptosis 1 (c-IAP1), c-IAP2, TNFR-associated factor 2 (TRAF2), and TRAF3. Disruption of this protein complex upon stimulation of TNFRs leads to the accumulation of NIK and its subsequent phosphorylation of IKK1 (also known as IKKα). IKK1, in turn, phosphorylates p100 (also known as NF-κB2), triggering its partial proteasomal degradation and the translocation of NF-κB dimers to the nucleus (9, 10).

The assembly of TNFR complexes is a carefully orchestrated process during which ligand binding leads to receptor oligomerization and the subsequent recruitment of signaling adaptor proteins, such as TNFR1-associated death domain (TRADD), TRAF2, or TRAF3 (5, 11). Critical signaling components also include E3 ubiquitin ligases, their substrates, and proteins whose recruitment to the receptor complexes depends on the presence of ubiquitin moieties (12). In the case of TNFR1, TRADD and TRAF2 recruit c-IAP1 and c-IAP2 to the receptor complex where these E3 ligases ubiquitinate the kinase receptor-interacting protein 1 (RIP1), TRAF2, and themselves to enable the recruitment of the IKK complex as well as auxiliary signaling components such as RBCK1/Hoil1-interacting protein (HOIP), a member of the linear ubiquitin chain assembly complex (LUBAC) (1317). The CD40 signaling complex, on the other hand, contains two different classes of ubiquitin ligases, c-IAP1 and/or c-IAP2 and TRAF6, making it potentially less reliant on the exclusive presence of either of these E3 enzymes for efficient signaling (18). However, there are a number of TNFR complexes for which the identity and role of ubiquitin ligases in signaling are not currently known. DR3, the lymphotoxin β receptor (LT-βR), and fibroblast growth factor–inducible-14 (FN-14), for example, engage TRAF2, TRAF3, or both to promote activation of NF-κB and the MAPKs JNK and p38 in a fashion similar to that of TNFR1 (1924). Thus, the ubiquitin ligases c-IAP1 and c-IAP2 could potentially play a prominent role in these receptor-associated signaling complexes.

The c-IAP proteins are members of the IAP family of cell death regulators that were originally identified through their interaction with TRAF2 (25). c-IAP1 and c-IAP2 are really interesting new gene (RING) domain–containing ubiquitin ligases that promote the assembly of polyubiquitin chains on several signaling molecules, including NIK, RIP1, TRAF2, and the c-IAP proteins themselves (13, 16, 2628). c-IAP1 and c-IAP2 are recruited to TNFR1 signaling complexes through their constitutive association with TRAF2. They regulate TNFR1-mediated activation of the canonical NF-κB pathway, typically by ubiquitinating RIP1 (13, 16, 29). c-IAP1 and c-IAP2 are also implicated in FN-14 and CD40 signaling, although the precise role of cellular IAPs in TNF-like weak inducer of apoptosis (TWEAK)– or CD40 ligand (CD40L)–stimulated canonical NF-κB or JNK signaling is not clear (3033). Binding of TWEAK to its receptor triggers the degradation of the c-IAP proteins, with direct consequences on the stability of NIK and the activation of the noncanonical NF-κB pathway (26, 32), but it is not clear whether a similar mechanism operates downstream of CD40 because putative roles for the E3 ubiquitin ligase activity of TRAF2 and the degradation of TRAF3 have been reported (30, 31).

Here, we defined the roles of the c-IAPs in signaling by members of the TNF superfamily and demonstrated their importance for the activation of NF-κB and JNK signaling pathways. Our investigation of TNFR-associated complexes revealed the critical importance of c-IAP1 and c-IAP2 for the recruitment of the distal signaling components NF-κB essential modulator (NEMO, also known as IKKγ), IKK2, and HOIP to these complexes. In the absence of c-IAP proteins, signaling by TNF family members was blunted, with diminished kinase activation and gene expression. Activation of the noncanonical NF-κB pathway by the TNFRs that we investigated also stimulated the intracellular translocation and subsequent proteasomal and lysosomal degradation of the c-IAP proteins, TRAF2, and TRAF3. Lastly, we established that BR3-mediated signaling promoted the sequestration of TRAF3, which led to activation of the noncanonical NF-κB pathway. Collectively, our data demonstrate the crucial role of c-IAP proteins in TNFR family–mediated activation of NF-κB and MAPK signaling.

Results

c-IAP proteins are required for activation of canonical NF-κB and JNK signaling by TNFR family members

The ubiquitin ligase activities of the c-IAP proteins play a critical role in the stimulation of canonical NF-κB signaling by TNF-α and in the activation of JNK by CD40L (13, 16, 29, 30, 34). These ligands induce the formation of protein signaling complexes that engage c-IAP proteins through their constitutive association with the adaptor protein TRAF2. Given that most TNFR family members engage TRAF2, we examined the role of c-IAP proteins in the activation of NF-κB and MAPK by several members of the TNF family of ligands (11). To optimize protein detection, we tested the sensitivities and specificities of a number of commercially available antibodies against c-IAPs, TRAFs, IKK2, NEMO, and HOIP. Specificity was validated by analysis of cells treated with short interfering RNAs (siRNAs) or with gene-targeted knockout cells (fig. S1 and table S1). We also examined the cell surface abundance of various TNFRs to identify suitable cell lines for our study (fig. S2 and table S2).

TNF-α, TNF ligand–related 1 (TL1A), TWEAK, and LIGHT (LT-βR ligand) each induced activation of the canonical NF-κB and MAPK pathways, as demonstrated by degradation of the NF-κB inhibitory protein IκB and the phosphorylation of JNK and p38 (Fig. 1 and fig. S3). Preincubation of cells with the IAP antagonist BV6 (26), which induces degradation of the c-IAP proteins, reduced the extent of IκB phosphorylation and degradation as well as of the phosphorylation of JNK and p38 in response to the aforementioned ligands (Fig. 1 and fig. S3). Although the absence of c-IAP proteins profoundly diminished the degradation of IκB induced by most of the receptors, CD40-induced IκB degradation was only mildly affected by pretreatment with BV6 (Fig. 1E), suggesting functional redundancy of the E3 ubiquitin ligase activities of TRAF6 and the c-IAP proteins. Indeed, knockdown of TRAF6 or the c-IAPs individually only partially reduced IκB degradation in response to the stimulation of CD40 (fig. S4); however, the combined knockdown of TRAF6 and that of c-IAP1 and c-IAP2 or treatment with BV6 almost completely prevented IκB degradation (fig. S4). Of note, the abundance of c-IAP proteins was substantially reduced after treatment with antibodies against TWEAK, LIGHT, CD27L, or CD40 (Fig. 1 and fig. S3).

Fig. 1

c-IAP proteins are required for canonical NF-κB, JNK, and p38 signaling by members of the TNFR family. (A to E) The indicated cell lines were pretreated with dimethyl sulfoxide (DMSO) as a control or with BV6 (2.5 μM) for 15 hours, after which they were treated with (A) TNF-α (20 ng/ml), (B) TL1A (100 ng/ml), (C) TWEAK (100 ng/ml), (D) LIGHT (100 ng/ml), or (E) CD40L (400 ng/ml) for the indicated times. The abundances of c-IAP1, c-IAP2, IκB, pJNK, and pp38 proteins were analyzed by Western blotting with the indicated antibodies. Actin was used as a loading control. Data are representative of at least five experiments.

To further explore the necessity for c-IAP proteins in signaling by TNF family members, we examined TNF-α–, TWEAK-, and LIGHT-dependent NF-κB and JNK activation in HT1080 cells after siRNA-mediated knockdown of c-IAP1 and c-IAP2. As was seen in the experiments with BV6 pretreatment, simultaneous knockdown of c-IAP1 and c-IAP2 resulted in blunted NF-κB and JNK signaling, whereas control siRNAs did not alter these signaling events (fig. S5). Because the activation of NF-κB and MAPK signaling induces the expression of multiple genes, we assessed the role of the c-IAP proteins in the stimulation of gene expression by TNFR family members by real-time polymerase chain reaction (PCR) analysis. TNF-α, TL1A, TWEAK, and LIGHT induced a robust increase in the abundances of TNFα, MCP1, IL-8, and RelB mRNAs compared to those in untreated cells (Fig. 2 and fig. S6). Although less markedly, cells treated with antibodies against CD40 or CD27L also exhibited increases in the amounts of TNFα and RelB mRNAs (Fig. 2 and fig. S6). Preincubation of cells with BV6 attenuated TNF-α–, TWEAK-, LIGHT-, and CD27L-stimulated gene expression. Consistent with the moderate effects of BV6 on CD40-dependent activation of NF-κB (Fig. 1E), loss of c-IAP proteins only modestly affected the abundance of TNFα mRNA in cells treated with antibody against CD40, and the expression of RelB was not affected at all (Fig. 2E and fig. S6D). Together, these data suggest that the c-IAP proteins are critical mediators of the activation of NF-κB and MAPK signaling by TNF family members.

Fig. 2

Loss of c-IAP proteins results in reduced expression of genes regulated by members of the TNF family. (A to F) Quantitative real-time PCR analysis of the abundances of the indicated mRNAs was performed with RNA samples derived from the indicated cell lines that had been left untreated or were treated overnight with BV6 (4 μM) before being incubated with (A) TNF-α (20 ng/ml) for 4 hours, (B) TL1A (100 ng/ml) for 4 hours, (C) TWEAK (100 ng/ml) for 7 hours, (D) LIGHT (100 ng/ml) for 4 hours, (E) antibody against CD40 (500 ng/ml) for 4 and 7 hours, or (F) CD27L (100 ng/ml) for 7 hours. The abundances of the indicated mRNAs were normalized to that of RPL19, which was an internal control. Error bars represent the SD. *P < 0.05; **P < 0.01; ns, not significant. Data are representative of at least two experiments.

c-IAP proteins enable the assembly of TNFR family complexes

Next, we investigated the importance of c-IAP proteins for the assembly of complexes associated with TNFR family members. We treated cells with TNF family members or with agonistic antibodies against TNFR family members after preincubation with BV6 to eliminate the c-IAP proteins, and then we immunoprecipitated complexes containing ligated TNFR family members (Fig. 3). We confirmed the BV6-induced degradation of c-IAP1 and c-IAP2 and the stimulation of signaling for all tested cell lines (fig. S7). We found that TRADD, TRAF2, and RIP1 were recruited to the TNFR1 complex irrespective of the presence of c-IAP1 (Fig. 3A); however, RIP1 was polyubiquitinated only in the presence of c-IAP1 (Fig. 3A). Notably, NEMO, IKK2, and TAK1, which are components of the canonical NF-κB signaling complex, were present in the TNFR1 complex only if c-IAP1 was also present. In addition, the E3 ubiquitin ligase HOIP, a key component of LUBAC, was also recruited to the receptor complex in a c-IAP1–dependent fashion (Fig. 3A). These data confirm those from another study on the importance of c-IAP1 for TNFR1-mediated signaling and complex assembly and validate our experimental approach (13, 14, 16). The DR3-associated complex had a composition similar to that of the TNFR1 complex (Fig. 3A). TRADD and TRAF2 were recruited to DR3 independently of c-IAP1, whereas RIP1 ubiquitination and the recruitment of NEMO, IKK2, TAK1, and HOIP to the receptor complex were dependent on the presence of c-IAP1 (Fig. 3A).

Fig. 3

c-IAP proteins regulate the assembly of signaling complexes of TNFR family members. (A to C) HT1080, Ku812F, HT29, and Daudi cells, as indicated, were pretreated with DMSO or BV6 (3 μM) for 15 hours before being treated with FLAG–TNF-α (1 μg/ml), TL1A (1 μg/ml), His-LIGHT (1 μg/ml), FLAG-TWEAK (1 μg/ml), or antibody against CD40 for the indicated times. Cell lysates were subjected to immunoprecipitation (IP) with the indicated antibodies, and the amounts of protein in the cellular lysates and in the ligand-associated complexes were determined by Western blotting with the indicated antibodies. Data are representative of four experiments.

The binding of LIGHT to LT-βR or of TWEAK to FN-14 resulted in the recruitment of TRAF2 and TRAF3 independently of the presence of c-IAP1 (Fig. 3B). In contrast, NEMO, IKK2, and HOIP were recruited to LT-βR or FN-14 only when c-IAP1 was present (Fig. 3B). Therefore, although LT-βR and FN-14 recruited proteins that were different from those recruited to the TNFR1 and DR3 complexes, they still required c-IAP proteins for the recruitment of distal signaling molecules, such as NEMO, IKK2, and HOIP. Experiments involving knockdown of NEMO or HOIP confirmed their importance for the activation of canonical NF-κB signaling by TNF-α, TWEAK, and LIGHT (fig. S8).

Next, we investigated CD40 as an example of a receptor that engages two different classes of ubiquitin ligases, the c-IAPs and TRAF6 (Fig. 3C). Treatment of cells with agonistic antibody against CD40 triggered the recruitment of three TRAF proteins to the receptor complex, namely, TRAF2, TRAF3, and TRAF6. Through their association with TRAF2, c-IAP1 and c-IAP2 were also recruited to the receptor complex. Depletion of c-IAP1 and c-IAP2 with BV6 prevented the recruitment of HOIP to the CD40 complex, but it did not interfere with the recruitment of NEMO or IKK2 (Fig. 3C). These data suggest that in cases where the c-IAP proteins are the major E3 ubiquitin ligases, they are critical for TNFR family signaling and complex assembly. If, on the other hand, the receptor complex contains another functional E3 ubiquitin ligase, such as TRAF6 in the case of the CD40 complex, then the c-IAP proteins do not seem to be essential for downstream signaling.

TNFR family–mediated signaling has different effects on the stabilities of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins

The TNF family member TWEAK reduces the amounts of c-IAP proteins in treated cells (26, 32). We also observed lower amounts of c-IAP1 and c-IAP2 proteins in cells treated with TWEAK, LIGHT, or antibody against CD40, but not in cells treated with TNF-α or TL1A (Figs. 1 and 4 and figs. S3, S9, and S10). When cells were preincubated with the proteasome inhibitor MG132 before receptor stimulation, the extent of degradation of IκB was blunted, but the amounts of c-IAP proteins were not substantially stabilized in response to LIGHT, TWEAK, or antibody against CD40 (Fig. 4, C to E, and fig. S9, B and C). CD40L, TWEAK, and antibody against CD40 stimulate the proteasomal or lysosomal degradation of TRAF2 and TRAF3 proteins in a c-IAP–dependent fashion, with the proteasomal degradation of TRAF3 potentially playing a central role in the activation of JNK by CD40 (31, 32). Whereas we found that the abundance of TRAF3 protein was substantially reduced in mouse B cells after treatment with CD40, we observed a slight reduction in the amount of TRAF3 in Daudi and Ku812F cells, but no loss of TRAF3 protein in Ramos lymphoma cells, despite observing robust JNK activation in all of these cell lines (Fig. 4D and fig. S9C). In contrast, all of the cell lines treated with the agonistic antibody against CD40 contained less TRAF2 than did untreated cells, and the magnitude and kinetics of the loss of TRAF2 protein correlated with the loss of c-IAP proteins. In agreement with previous studies, we found that preincubation of cells with the IAP antagonist BV6 stabilized TRAF2 and TRAF3, further implicating c-IAP proteins as critical regulators of TRAF2 and TRAF3 protein stability in the context of CD40 and TWEAK signaling (Fig. 4 and fig. S9). However, MG132 did not effectively block the loss of TRAF2 or TRAF3 proteins in cells stimulated with antibody against CD40 or with CD40L (Fig. 4). In contrast to the effects of MG132 on IκB degradation, we found that inhibition of the proteasome prolonged the phosphorylation of JNK after stimulation with TNF-α, TL1A, LIGHT, agonistic antibody against CD40, CD40L, or TWEAK, whereas pretreatment with BV6 abolished JNK phosphorylation (Fig. 4 and fig. S9, A to C). These results suggest that the proteasomal machinery regulates c-IAP–dependent activation of JNK by agonists of TNFR family members.

Fig. 4

Differential effects of the signaling of TNFR family members on the stabilities of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins. (A to E) The indicated cell lines were pretreated with BV6 (3 μM) for 12 hours or MG132 (20 μM) for 30 min, after which they were incubated with (A) TNF-α (20 ng/ml), (B) TL1A (100 ng/ml), (C) LIGHT (100 ng/ml), (D) antibody against CD40 (500 ng/ml), or (E) TWEAK (100 ng/ml). The abundances of c-IAP1, c-IAP2, TRAF2, TRAF3, IκB, and pJNK proteins were determined by Western blotting analysis with the indicated antibodies. Data are representative of three experiments.

Because MG132 was not effective in blocking the loss of c-IAP, TRAF2, or TRAF3 proteins after ligation of TNFR family members (Fig. 4 and fig. S9), we investigated whether these proteins were relocalized within cells so that they were revealed in their entirety only after solubilization and were not released after relatively mild lysis in 1% Triton X-100 detergent. We found that TWEAK, LIGHT, and CD40L led to the quick and marked reduction in the abundance of c-IAP1 (and of c-IAP2 in the case of CD40 signaling) whether cells were lysed in Triton or SDS buffers (Fig. 5 and fig. S10). In addition, these ligands stimulated a substantial reduction in the amount of TRAF3 protein, with slower kinetics. The amount of TRAF2 protein was reduced in response to TWEAK and CD40L, as well as in some LIGHT-treated cell lines (Fig. 5 and fig. S10). We next examined whether the combination of proteasome and lysosome inhibitors could stabilize c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins in SDS-lysed cells treated with agonists for members of the TNFR family. Whereas both proteasome and lysosome inhibitors provided some stabilization, the combination of both inhibitors completely restored the amounts of c-IAP, TRAF2, and TRAF3 proteins (Fig. 5 and fig. S10).

Fig. 5

Proteasome and lysosome inhibitors stabilize c-IAP and TRAF proteins in cells treated with TNFR agonists. (A to C) The indicated cell lines were pretreated with MG132 (20 μM), CA-074Me (20 μM), or a combination of both inhibitors for 30 min, after which they were incubated with (A) TWEAK (100 ng/ml), (B) LIGHT (100 ng/ml), or (C) CD40L (400 ng/ml) for the indicated times. Cells were collected and lysed in 1% Triton X-100 buffer (Tr), followed by extractions of the remaining cellular pellets in 1% SDS buffer (SDS). The abundances of c-IAP1, c-IAP2, TRAF2, TRAF3, IκB, and p100 proteins were analyzed by Western blotting. Loading controls included cadherin for (A) and (B) and HSP90 for (C). Data are representative of at least three experiments.

With the exceptions of TNFR1 and DR3, stimulation of TNFR family members activates the noncanonical NF-κB pathway. This signaling pathway is regulated by the E3 ubiquitin ligase activities of c-IAP1 and c-IAP2 and leads to the processing of the NF-κB2 precursor protein p100 to its active p52 form. We found that accumulation of p52 in all of the cell lines that we examined occurred concomitantly with a decline in the amounts of c-IAP, TRAF2, and TRAF3 proteins (Fig. 5 and fig. S10). Collectively, these data suggest that agonists of TNFR family members that stimulate noncanonical NF-κB signaling cause the translocation of c-IAP, TRAF2, and TRAF3 proteins from Triton buffer–soluble cellular compartments to membrane-containing, SDS-soluble fractions, culminating in their degradation by the proteasome or lysosome.

Membrane targeting and dimerization reduce the amounts of soluble c-IAP1 and TRAF2 proteins

TNF family members and agonistic antibodies trigger the oligomerization of TNFR family members, which provides a platform for the recruitment of downstream adaptor proteins. To investigate whether membrane targeting or dimerization were sufficient to induce loss of soluble c-IAP1 protein, we generated c-IAP1 variants with an Fpk dimerization domain and a membrane-directed myristoylation signal (Fig. 6 and fig. S11) (35, 36). The addition of the dimerization agent FK1012H2 to cells expressing Fpk–c-IAP1 caused a loss of c-IAP1 protein that was inhibited by MG132 (Fig. 6A). A ubiquitin ligase–inactivating mutation in the RING domain of c-IAP1 (H588A) prevented FK1012H2-induced loss of c-IAP1 protein, suggesting that the E3 ligase activity of c-IAP1 was important for its dimerization-dependent degradation (Fig. 6A). Similarly, a membrane-targeting myristoylation signal fused to c-IAP1 also caused a substantial reduction in the amount of c-IAP1 protein but not when the RING domain of c-IAP1 was mutated (Fig. 6B). In addition, expression of a myristoylated variant c-IAP1 in multiple myeloma cells with genetic ablation of c-IAP proteins, KMS-28PE, did not affect the abundance of nuclear p52, whereas a c-IAP1 variant that was not targeted to the membrane fraction remained cytoplasmic, which resulted in diminished noncanonical NF-κB signaling compared to that in cells expressing a non–membrane-targeted version of c-IAP1 (fig. S11A).

Fig. 6

Engineered membrane targeting and dimerization reduce the amounts of soluble c-IAP1 and TRAF2 proteins. (A and B) HT1080 (A) and HEK 293T (B) cells were transiently transfected with (A) control plasmid or plasmids encoding Fpk–c-IAP1 or Fpk–c-IAP1m (RING domain mutant H588A) or (B) control plasmid or plasmids encoding Myr-Fpk–c-IAP1 or Myr-Fpk–c-IAP1m. Forty-eight hours after transfection, cells were left untreated or were treated with MG132 for 30 min, after which they were treated with (A) FK1012H2 (250 nM, FK) for 1 or 3 hours or (B) were left untreated. Cellular lysates were analyzed by Western blotting with an antibody against the FLAG tag (to detect c-IAP1 constructs) and with an antibody against actin. The asterisk denotes a nonspecific band. (C and D) HEK 293T cells were transiently transfected with (C) plasmid encoding c-IAP1 together with empty plasmid (vec + c1), plasmid encoding Fpk-TRAF2 (Fpk-T2 + c1), or plasmid encoding Fpk-TRAF2ΔR (RING domain deletion) (Fpk-T2ΔR + c1) or (D) with plasmid encoding c-IAP1 together with empty plasmid (vec + c1), plasmid encoding Myr-Fpk–TRAF2 (Myr-3xFpk-T2 + c1), or plasmid encoding Myr-Fpk–TRAF2ΔR (Myr-3xFpk-T2ΔR + c1). Forty-eight hours later, cells were treated for 1 or 3 hours with FK1012H2 (250 nM, FK). Cellular lysates were analyzed with antibody against the FLAG tag (to detect c-IAP1 constructs), antibody against the Myc tag (to detect TRAF2 constructs), and antibody against actin as a loading control. Data are representative of at least three experiments.

Previous studies suggested the possible involvement of the RING domain of TRAF2 in activation of the ubiquitin ligase activity of c-IAP1 (31). To examine the role of the TRAF2 RING domain in modulating c-IAP1 protein stability, we expressed constructs of wild-type TRAF2 or a mutant TRAF2 from which the RING domain was deleted that contained the Fpk dimerization domain, the membrane-directed myristoylation signal, or both. The coexpression of c-IAP1 with Fpk-TRAF2 resulted in a loss of c-IAP1 after addition of FK1012H2, even when the RING domain of TRAF2 was deleted (Fig. 6C). Likewise, membrane targeting of TRAF2 constructs led to a reduction in the amounts of c-IAP1 and TRAF2 proteins independently of the RING domain in TRAF2 (Fig. 6D and fig. S11B). Further emphasizing the lack of an active role for the TRAF2 RING domain, expression of TRAF2 or RING-deleted TRAF2, but not of myristoylated TRAF2 variants, caused a reduction in noncanonical NF-κB signaling and consequent MCP1 expression in TRAF2-knockout mouse embryo fibroblasts (MEFs) (fig. S11, C and D). The addition of the dimerization agent in the presence of the myristoylation signal caused a substantial loss in the amount of wild-type c-IAP1 protein, but not in the amount of the c-IAP1 RING mutant (Fig. 6D). These results emphasize that the ubiquitin ligase activity of c-IAP1, but not that of TRAF2, was required for the efficient degradation of c-IAP1 and TRAF2 proteins. Instead, TRAF2 seemed to serve as an adaptor to facilitate the recruitment of c-IAP proteins to members of the TNFR family.

BR3 signaling activates the noncanonical NF-κB pathway by triggering the translocation of TRAF3 to membranes

BR3 is unique among TNFRs because it activates exclusively noncanonical NF-κB signaling (37, 38). BR3 also binds only to TRAF3 and not to any of the other TRAFs (39, 40). We studied BR3 signaling in NALM-6 and WSU-FSCCL B cell lines, which express BR3 and exhibit noncanonical NF-κB signaling in response to BAFF or agonistic antibody against BR3 (αBR3) (fig. S12A and table S2). Treatment of NALM-6 and WSU-FSCCL cells with BAFF or αBR3 depleted soluble cytoplasmic TRAF3 protein concomitant with the accumulation of insoluble, membrane-associated TRAF3 protein (Fig. 7A and fig. S12). At the same time, the amounts of cytoplasmic or insoluble c-IAP1 and TRAF2 proteins were unaffected by the stimulation of BR3 (Fig. 7A and fig. S12). Preincubation of cells with proteasome or lysosome inhibitors did not prevent this reduction in the amount of soluble TRAF3 protein, suggesting that its disappearance was not a result of proteasomal or lysosomal degradation (Fig. 7 and fig. S12C). Treatment of freshly isolated B cells from human tonsils also caused a redistribution of TRAF3 to insoluble fractions without affecting the amounts of c-IAP1 or TRAF2 proteins (Fig. 7B). As before, proteasomal or lysosomal inhibitors did not affect the relocalization of TRAF3 in these primary B cells (Fig. 7B).

Fig. 7

BR3 signaling triggers the translocation of TRAF3 to the SDS-soluble fraction. (A) NALM-6 cells were treated with recombinant BAFF (2 μg/ml) or agonistic antibody against BR3 (αBR3, 2 μg/ml) for the indicated times. Cells were collected and lysed in CEB (Cyt.) followed by extractions of the remaining cellular pellets in 1% SDS buffer (1% SDS). The abundances of c-IAP1, TRAF2, TRAF3, HSP90, SP1, and calreticulin (Calret.) were determined by Western blotting analysis with the appropriate antibodies. (B) Purified human tonsillar B cells were pretreated with MG132 (20 μM) and CA-074Me (20 μM) for 30 min, after which they were incubated with recombinant BAFF (2 μg/ml) or agonistic antibody against BR3 (2 μg/ml) for the indicated times. Cells were collected and lysed as described for (A), and the abundances of c-IAP1, TRAF2, TRAF3, p100/p52, HSP90, SP1, and calreticulin were determined by Western blotting analysis with the appropriate antibodies. (C) WSU-FSCCL cells were treated for the indicated times with recombinant FLAG-tagged BAFF (2 μg/ml). Cell lysates were subjected to immunoprecipitation with antibody against BAFF, and protein abundances in cellular lysates and in the BAFF- and BR3-associated complexes were determined by Western blotting with the indicated antibodies. The asterisk indicates a nonspecific cross-reacting band. (D) RPMI-8226 cells were transiently transfected with empty plasmid (vector) or with plasmid encoding the indicated TRAF3 constructs. Forty-eight hours after transfection, nuclear, cytoplasmic, and membrane cellular fractions were analyzed by Western blotting with the indicated antibodies. Data are representative of three experiments.

Having established the redistribution of TRAF3 as a critical event in BR3 signaling, we next explored the assembly of the BR3-associated receptor complex. Binding of BAFF to BR3 resulted in the recruitment of TRAF3, whereas TRAF2, TRAF6, and c-IAP1 were not recruited to BR3 (Fig. 7C). In a similar fashion, our immunofluorescence studies showed the aggregation of TRAF3 in cells in a BAFF-dependent fashion (fig. S13). At the same time, and in agreement with experiments analyzing endogenous receptor complexes, we found that the subcellular localization of TRAF2 was affected by CD40 signaling but not by BR3 signaling (fig. S13). We also explored how membrane targeting and dimerization affected the abundance of TRAF3 protein. Myristoylation of TRAF3 excluded it from the soluble cellular compartment and resulted in the enrichment of TRAF3 in the membrane fraction (fig. S14A). Introduction of the Fpk dimerization domain and treatment with FK1012H2 did not destabilize TRAF3 protein, implying that auto-ubiquitination of TRAF3 was likely not instrumental in the regulation of its stability or in BR3 signaling (fig. S14A). The multiple myeloma cell line RPMI-8226 harbors a mutation that causes expression of a truncated TRAF3 protein and the constitutive activation of the noncanonical NF-κB pathway (41). Ectopic expression of TRAF3 in RPMI-8226 cells prevented p100 processing and NF-κB–regulated expression of the gene encoding Bcl-2 (42), whereas the expression of membrane-targeted myristoylated TRAF3 did not block noncanonical NF-κB signaling (Fig. 7D). In a similar fashion, expression of the TRAF3 RING mutant blocked the appearance of nuclear p52, as well further demonstrating the lack of TRAF3-mediated ubiquitination in these signaling pathways (fig. S14B). In conclusion, our data suggest that BR3 signaling triggers the translocation of TRAF3 to the insoluble membrane compartment and that the sequestration of TRAF3 from soluble cellular signaling complexes promotes activation of the noncanonical NF-κB pathway.

Discussion

Members of the TNF superfamily of ligands and their receptors are responsible for development, immunity, and homeostasis in metazoan organisms (2); thus, signals emanating from these receptors are tightly regulated. TRAF proteins have crucial roles in the assembly of TNFR-associated protein complexes (11). TRAF2, TRAF3, and TRAF6 are recruited to TNFR family members in different combinations. BR3 binds only to TRAF3 (39), whereas FN-14, LT-βR, and CD40 recruit multiple TRAF proteins (43). Although TRAF2, TRAF3, and TRAF6 were proposed to be the E3 ubiquitin ligases responsible for the ubiquitination of RIP1, NIK, and themselves, studies suggest that only TRAF6 is a functional ubiquitin ligase (44, 45). TRAF2 and TRAF3 appear to be adaptor proteins that facilitate ubiquitination by recruiting the substrates RIP1 or NIK within the proximity of the E3 ligases c-IAP1 and c-IAP2 (16, 26, 29, 33); however, to date, the roles of the c-IAP proteins in the signaling of TNFR superfamily members have not been well defined.

Here, we provide evidence that c-IAP proteins are important regulators of canonical and noncanonical NF-κB signaling, as well as of MAPK signaling, that emanates from members of the TNFR superfamily. In experiments involving c-IAP knockdown or a small-molecule IAP antagonist that activates the autoubiquitination and subsequent proteasomal degradation of c-IAPs (26, 46), we demonstrated that c-IAP proteins were necessary for TNF family members or agonistic antibodies against TNFR family members to efficiently activate canonical NF-κB or MAPK signaling. Accordingly, induction of gene expression by TNFR agonists was also severely reduced in the absence of c-IAP proteins. In addition, without c-IAP proteins, distal signaling components such as NEMO, IKK2, and HOIP were not recruited to TNFR-associated protein complexes. The role of these proteins in TNFR family signaling is well demonstrated (4, 14, 47, 48), so it is not surprising that exclusion of these critical signaling molecules from TNFR-associated complexes had a profound effect on downstream signaling. The recruitment of TRAF2, TRAF3, TRAF6, or other proximal signaling adaptors to receptor-associated complexes was not affected by the absence of c-IAP proteins. Therefore, the E3 ubiquitin ligases c-IAP1 and c-IAP2 are key regulators of the activation of NF-κB and MAPK signaling by TNFR family members.

Our findings led us to propose a new classification of the TNFR superfamily based on the proteins that they recruit to their complexes and on the E3 ubiquitin ligase(s) that propagates the signaling initiated by binding of their cognate ligands (fig. S15). The first group represents the death domain–containing receptors TNFR1 and DR3, which recruit TRAF2 and, through TRAF2, the c-IAP proteins as essential E3 ubiquitin ligases to their signaling complexes. The second proposed group of receptors includes FN-14, LT-βR, CD27, and many others that engage TRAF2 and TRAF3 in their receptor-associated complexes and that still depend on c-IAP proteins as critical E3 ubiquitin ligases. The third group is embodied by CD40, which associates with TRAF2, TRAF3, and TRAF6, and relies on c-IAP proteins, TRAF6, or both to promote ubiquitination. Finally, BR3 is a clear outlier that does not depend on ubiquitin ligases, but promotes the removal of TRAF3 from its cytoplasmic inhibitory complex, which then enables activation of noncanonical NF-κB signaling (fig. S15).

The effects of signaling of TNFR family members on the stability of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins were also noted. Apart from TNFR1 and DR3, all of the other TNFR superfamily members that stimulate activation of the noncanonical NF-κB pathway decreased the stability of the c-IAP proteins, and, to a lesser extent, the stabilities of TRAF2 and TRAF3 proteins as well. As before, BR3 is different because it does not bind to TRAF2; thus, c-IAP1 and c-IAP2 are not recruited to this receptor. A previous study suggested that stimulation of CD40 does not affect the abundances of c-IAP1 or c-IAP2 and that c-IAP proteins are ubiquitinated by TRAF2 in an activating fashion that would enable c-IAP1 or c-IAP2 to promote the degradative ubiquitination of TRAF3 (31). In contrast, we found that CD40-, TWEAK-, and LIGHT-stimulated activation of noncanonical NF-κB signaling correlated with a pronounced decline in c-IAP protein abundance. In addition, our data indicate that the stimulus-dependent membrane association of c-IAP proteins depletes them from cytoplasmic complexes in which they serve as E3 ubiquitin ligases responsible for the degradative ubiquitination of NIK. Once relocated to membrane fractions, c-IAP proteins are subject to proteasomal and lysosomal degradation. Similar observations have been made for TWEAK-stimulated signaling (32) and for the CD40-stimulated membrane translocation and degradation of TRAF2 (49, 50). Collectively, these findings underscore the mechanistic aspects of noncanonical NF-κB activation by TNF family members where receptor-mediated signaling depletes cytoplasmic pools of c-IAPs, TRAF2, and TRAF3 by promoting the membrane association and consequent degradation of these proteins (51).

Whereas the intact RING domain was required for the degradation of c-IAP1, the RING domain of TRAF2 was completely dispensable for the decrease in c-IAP1 protein abundance after the dimerization or membrane targeting of TRAF2. TRAF2 was presumed to be the critical E3 ubiquitin ligase in TNFR superfamily signaling for a long time (52), but recent data indicate that, unlike TRAF6, TRAF2 and TRAF3 do not interact with ubiquitin-conjugating enzymes or promote the assembly of polyubiquitin chains (29, 33, 44, 45). In addition, structural studies of the interaction between TRAF2 and c-IAP1 and c-IAP2 revealed that a TRAF2 trimer interacts with a single molecule of c-IAP1 or c-IAP2 (53, 54). Therefore, further receptor-mediated aggregation of TRAF2 could result in the dimerization of otherwise monomeric c-IAP proteins, thereby inducing their E3 ubiquitin ligase activity (55). Here, we found that TRAF2 promoted the dimerization and membrane targeting of c-IAP1 in a manner independent of the TRAF2 RING domain. Similarly, the recruitment of TRAF3 to the BR3 complex and the consequent membrane targeting of TRAF3 were critical for BR3-mediated activation of noncanonical NF-κB signaling independently of the function of the TRAF3 RING domain. Thus, our results further establish TRAF2 and TRAF3 as adaptor proteins that juxtapose c-IAP proteins and their substrates to enable efficient signaling (16, 26, 29).

In summary, our study provides evidence for the involvement of c-IAP proteins in the signaling of TNFR superfamily members. c-IAP1 and c-IAP2 are attractive targets for the development of anticancer therapeutics, and clinical trials are currently under way to explore the safety and pharmacokinetics of IAP antagonists (56). Increased understanding of the functions of IAP proteins within signal transduction pathways and of their turnover will aid these efforts to provide new therapeutics for cancer patients.

Materials and Methods

Cell lines and reagents

A2058 human melanoma cells, HT1080 human fibrosarcoma cells, HT29 and Ku812F human chronic myelogenous leukemia cells, Daudi and Ramos human Burkitt’s lymphoma cells, the Jurkat human T cell leukemia cell line, human embryonic kidney (HEK) 293T cells, multiple myeloma RPMI-8226 cells, and the HKB-11 cell line (an epithelial somatic cell hybrid of kidney and B cells) were obtained from the American Type Culture Collection. The EFM-192A human breast carcinoma, NALM-6 human B cell precursor leukemia, WSU-FSCCL B cell lymphoma, and SU-DHL-1 anaplastic large cell lymphoma cell lines were obtained from DSMZ (German Collection of Microorganisms and Cell Cultures). KMS-28PE cells were obtained from the Japanese Collection of Research Bioresources. TRAF2-deficient MEFs were provided by D. Goeddel. Normal human dermal fibroblasts (HNDFs) and prostate epithelial cells (PrECs) were obtained from Lonza Walkersville Inc. and maintained according to the manufacturer’s instructions. Primary human mononuclear cells (MNCs) were isolated from human tonsils (BIO Options) as previously described (57). Primary human and mouse B cells were purified from tonsil MNCs or mouse spleens with the human- or mouse-specific B Cell Isolation Kit II (Miltenyi Biotec) according to the manufacturer’s instructions. To generate HKB-11–BR3 stable clones, we transfected HKB-11 cells with the pIRES-hyg2 (Clontech) plasmid, which contains the coding region for human BR3, and we selected and maintained clones with hygromycin. HKB-11–CD40 cells were described previously (58). Adherent cancer cell lines were grown in 50:50 Dulbecco’s modified Eagle’s medium and FK12 medium, whereas suspension cell lines were grown in RPMI medium supplemented with 10% fetal bovine serum, penicillin, and streptomycin. Human recombinant soluble TNF-α, TWEAK, and BAFF were from Genentech Inc. Recombinant mouse CD27 ligand, human LIGHT, human TL1A, human CD40L, mouse CD40L, agonistic antibody against human CD40, biotinylated antibody against human CD40, biotinylated antibody against human LT-βR, antibody against human DR3, biotinylated antibody against human DR3, antibodies against human TNFR1, and biotinylated antibody against human TNFR1 were obtained from R&D Systems. FLAG-tagged recombinant TNF-α was purchased from Alexis. Antibodies against phosphorylated and total forms of IκB, antibodies against phosphorylated and total p38, antibodies against p100/p52, antibody against Bcl-2, antibody against heat shock protein of 90 kD (HSP90), antibody against all cadherins, and antibody against SP1 were from Cell Signaling. Antibody against pJNK was obtained from BD Biosciences. Antibody against tubulin was from MP Biomedicals, and antibody against actin was from Sigma. Antibody against human BR3 and agonist antibody against TACI were from Genentech Inc. (59, 60). Information about the antibodies against c-IAP1, c-IAP2, TRAF2, TRAF3, TRAF3, TRAF6, IKK2, NEMO, TAK1, and HOIP can be found in fig. S1 and table S1. MG132, CA-074Me, and z-VAD-Fmk were purchased from Calbiochem, and lactacystin and epoxomicin were obtained from Sigma. The synthetic cell-permeable dimerizer FK1012H2 (35) and BV6 were synthesized at Genentech Inc.

Western blotting analysis

Western blotting analysis was performed as described previously with the following lysis buffer: 20 mM tris-HCl (pH 7.5), 150 mM NaCl, 1 mM EDTA, and 1% Triton X-100 (26). In some experiments (as indicated in the figure legends), the remaining cellular pellets were solubilized in the same buffer containing 1% SDS. Where indicated, cancer cell lines and primary human B cells stimulated with BAFF or agonistic antibody against BR3 were lysed initially with Cytoplasmic Extraction Buffer (CEB, Thermo Scientific) according to the manufacturer’s instructions, after which the remaining cellular pellets were solubilized with buffer containing 1% SDS. To analyze NF-κB and MAPK signaling pathways, we added 1× Phosphatase Inhibitor Cocktail 2 (Sigma) to the lysis buffer.

Gene silencing and siRNAs

The sequences of the siRNA oligonucleotides were designed with the Dharmacon siDESIGN Center software (Dharmacon Research Inc.) and synthesized at Genentech Inc. or were obtained from Dharmacon. The following siRNA pairs were used for gene knockdown experiments: c-IAP1: 5′-TCGCAATGATGATGTCAAATT-3′ and 5′-TTTGACATCATCATTGCGATT-3′ (pair 12) and 5′-GAATGAAAGGCCAAGAGTTTT-3′ and 5′-AACTCTTGGCCTTTCATTCTT-3′ (pair 13); c-IAP2: 5′-TCTAACACAAGATCATTGATT-3′ and 5′-TCAATGATCTTGTGTTAGATT-3′ (pair 5) and 5′-ATTCGGTACAGTTCACATGTT-3′ and 5′-CATGTGAACTGTACCGAATTT-3′ (pair 10). Dharmacon Smart siRNA duplexes #9 and #11 against NEMO, #7 against TRAF2, #9 and #10 against TRAF3, #9 to #12 against TRAF6, #11 to #14 against IKK2, #5 to #8 against HOIP, and the scrambled control were as described previously (26).

Supplementary Materials

www.sciencesignaling.org/cgi/content/full/5/216/ra22/DC1

Materials and Methods

Fig. S1. Characterization of commercial antibodies against c-IAP1, c-IAP2, TRAF2, TRAF3, TRAF6, IKK2, NEMO, TAK1, and HOIP.

Fig. S2. Characterization of the cell surface expression of members of the TNFR family.

Fig. S3. c-IAPs are required for canonical NF-κB, JNK, and p38 activation by TNF-α, TWEAK, LIGHT, CD27L, and CD30L.

Fig. S4. TRAF6 and c-IAP proteins mediate CD40-dependent activation of the canonical NF-κB pathway.

Fig. S5. TNF-α–, TWEAK-, and LIGHT-stimulated NF-κB and JNK activation depends on c-IAP1 and c-IAP2.

Fig. S6. Loss of c-IAP proteins results in reduced expression of genes regulated by members of the TNF family.

Fig. S7. c-IAP proteins regulate the function of complexes involving TNFR family members.

Fig. S8. TNF-α–, TWEAK-, and LIGHT-stimulated NF-κB and JNK activation depends on NEMO and HOIP.

Fig. S9. Differential effects of the signaling of TNFR family members on the stability of c-IAP1, c-IAP2, TRAF2, and TRAF3 proteins.

Fig. S10. Proteasome and lysosome inhibitors stabilize c-IAP and TRAF proteins during signaling by TNF family members.

Fig. S11. Membrane targeting of TRAF2 or c-IAP1 reduces their cytoplasmic abundance and enables noncanonical NF-κB signaling.

Fig. S12. BR3 signaling triggers the translocation of TRAF3 to the insoluble fraction.

Fig. S13. BAFF- and BR3-dependent signaling regulates the cellular localization of TRAF3.

Fig. S14. The RING domain of TRAF3 is not critical for TRAF3-mediated regulation of noncanonical NF-κB signaling.

Fig. S15. Signaling pathways of members of the TNFR superfamily.

Table S1. Summary of the antibodies against c-IAP1, c-IAP2, TRAF2, TRAF6, TRAF3, IKK2, NEMO, TAK1, and HOIP.

Table S2. Summary of the cell surface expression of TNFR family members.

References

References and Notes

Acknowledgments: We thank W. Fairbrother, D. Seshasayee, K. Newton, N. Kayagaki, I. Wertz, V. Dixit, K. O’Rourke, S. Marsters, M. Starovasnik, C. de Almagro, members of the departments of Early Discovery Biochemistry and Physiological Chemistry, and the Oligo Synthesis, FACS, and Sequencing facilities at Genentech that provided help with insightful discussions, suggestions, and reagents. Author contributions: E.V. and D.V. conceived and planned the study; E.V., T.G., and D.V. performed signaling studies including Western blotting, immunoprecipitations, and cloning; H.M. performed flow cytometry assays and provided primary mouse tissues; K.Z. and K.D. produced and purified BV6 and FK1012H2; L.G.K. and T.G. performed immunofluorescence staining; and D.V. supervised the study and wrote most of the manuscript with input from E.V. and the other co-authors. Competing interests: All of the authors are employees of Genentech Inc., which funded this research. E.V., K.D., and T.G. hold stock in Roche.
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