Journal of Molecular Biology
TRAF2 Suppresses Basal IKK Activity in Resting Cells and TNFα Can Activate IKK in TRAF2 and TRAF5 Double Knockout Cells
Introduction
Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are characterized by the presence of a TRAF domain at the C-terminus. Except for TRAF1, other TRAFs contain an N-terminal RING finger domain followed by five or seven zinc finger motifs.1, 2 Whereas the RING and zinc finger domains are essential for TRAFs to activate downstream signaling pathways, the TRAF domain is required for their interactions with relevant receptors and downstream effectors.2 TRAF2 is a prototypical member of the TRAF family and transduces signals from the TNFR superfamily members, leading to activation of the c-Jun N-terminal kinase (JNK) and the inhibitor of κB (IκB) kinase (IKK) pathways. JNK activates transcription factors of the AP-1 group (e.g., c-jun/ATF2), and IKK activates those of the nuclear factor κB (NF-κB) group. These transcription factors in turn induce the expression of genes involved in inflammation, the immune response, cell proliferation, cell differentiation, and the suppression of death-receptor-induced apoptosis.3, 4
IKK is a kinase complex that consists of two catalytic subunits (IKKα and IKKβ) and one regulatory subunit (IKKγ). TNFR family members activate NF-κB through a canonical pathway and/or an alternative noncanonical pathway.5 The canonical NF-κB pathway depends on the presence of both IKKβ and IKKγ and is activated by most TNF superfamily members, resulting in the coordinate expression of multiple inflammatory and innate immune genes. The noncanonical pathway depends on NIK and IKKα and is activated by a subset of TNF family members, such as B-cell-activating factor, lymphotoxin α and β heterotrimers, and the CD40 ligand. This pathway triggers the expression of genes involved in adaptive immunity and lymphoid organogenesis.5
Recently, an increasing number of studies have demonstrated that both TRAF2 and TRAF6 possess E3 ligase activity and that they are capable of catalyzing the formation of noncanonical K63-linked polyubiquitin chains on themselves and on their substrates. These studies have also suggested that such K63-linked ubiquitination is essential for activation of the JNK and IKK pathways in response to TNFα and interleukin (IL)-1β stimulation.6, 7, 8 Ubc13/UEV1a is the only E2 ubiquitin-conjugating enzyme complex that is currently known to bind to TRAF2 and TRAF6 and to catalyze K63-linked ubiquitination of these proteins.6 Inhibition of Ubc13 expression by small interfering RNA (siRNA) however results in inhibition of TNFα-induced JNK activation, but not that of IKK activation.9 A recent study has also demonstrated that conditional ablation of Ubc13 results in considerably impaired JNK activation in response to a variety of stimuli in B cells and mouse embryonic fibroblasts (MEFs) without affecting IKK activation under the same conditions. This suggests that either K63-linked polyubiquitination of TRAF2/TRAF6 is not essential for IKK activation or IKK can also be activated by an alternative ubiquitin-independent mechanism in response to cytokine stimulation.10
TRAF2 knockout (T2 KO) MEFs are completely defective in TNFα-induced JNK activation but only partially deficient in NF-κB activation.11 Interestingly, TRAF2-deficient macrophages overproduce TNFα and nitric oxide upon TNFα stimulation. In addition, whereas T2 KO mice die prematurely, mice with double mutants for TRAF2 and either TNFα or TNFR1 survive for several months, suggesting that TRAF2 negatively regulates certain aspects of TNFR1 signaling and thus the canonical NF-κB pathway.12 Also, conditional KO of TRAF2 in B cells results in constitutive activation of the noncanonical NF-κB pathway, suggesting that TRAF2 also negatively regulates this pathway.13 Tada et al. have reported that whereas TRAF5-null MEFs respond normally to TNFα-induced JNK and NF-κB activation, TRAF2 and TRAF5 double knockout (T2/5 DKO) MEFs exhibit an almost complete loss of TNFα-induced NF-κB activation.14 These data suggest that TRAF2 can regulate TNFR1 signaling in either a positive or a negative direction, in a manner that likely varies according to cell type.
We identified a phosphorylation site at the N-terminal region of TRAF2 by a classic phosphopeptide mapping approach.15 While carrying out functional studies investigating the role of TRAF2 phosphorylation in TNFα-induced NF-κB activation in T2/5 DKO cells reconstituted with empty vector, wild type (WT), or phosphomutant TRAF2, we repeatedly observed that control T2/5 DKO MEFs exhibit high basal IKK activity and elevated NF-κB target gene expression in the absence of stimulation with TNFα. Surprisingly, stimulation of these cells with TNFα, which merely activates the canonical NF-κB pathway through the TRAF2/5–RIP1 (receptor-interacting protein 1)–IKK cascade, further increased the expression of NF-κB target genes—to a level higher than that in TNFα-stimulated WT MEFs. To clarify these contradictory observations, we extensively analyzed the activation status of both the canonical and noncanonical NF-κB pathways in T2 KO and T2/5 DKO MEFs. Here, we show that both the canonical and noncanonical NF-κB pathways are constitutively activated in these cells and that the primary function of TRAF2 in TNFR1 signaling is to activate the JNK pathway while inhibiting TNFα-induced cell death by recruiting antiapoptotic proteins to the TNFR1 complex.
Section snippets
TRAF2 negatively regulates basal NF-κB activity
Numerous studies have shown that in HeLa cells, transient TRAF2 expression induces NF-κB and c-Jun activation in the absence of TNFα stimulation.1, 4 As expected, expression of TRAF2 in HeLa cells increased both basal and TNFα-induced NF-κB and c-Jun activation (Fig. S1). Unexpectedly, we found that in T2/5 DKO MEFs, transient expression of TRAF2 significantly reduced basal NF-κB activity, which was otherwise quite high compared with that in WT MEFs (Fig. 1a). In contrast, both basal and
Discussion
Currently, it is widely accepted that TRAF2- and TRAF5-mediated K63-linked RIP1 ubiquitination is essential for TNFα-induced NF-κB activation.22 We report here that (i) TRAF2 suppresses basal IKK complex activity in resting cells by inhibiting NIK activity, (ii) TNFα can also activate the NF-κB pathway in the absence of TRAF2 and TRAF5 expression and RIP1 ubiquitination, and (iii) TRAF2 inhibits TNFα-induced cell death by recruiting cIAP1 to the TNFR1 complex rather than by activating the NF-κB
Cell lines, plasmids, and reagents
HeLa, WT MEF, T2 KO MEF, and T2/5 DKO MEF cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% bovine calf serum and antibiotics. Antibodies and reagents were purchased as follows: anti-TRAF2, anti-JNK, anti-IKKγ, anti-IKKβ, anti-cIAP1, anti-Mn-SOD, and anti-NIK antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-phospho-JNK antibody from Promega; anti-cFLIP Ab from Alexis (San Diego, CA); anti-IκBα, anti-caspase-8, and anti-caspase-2 antibodies from
Acknowledgements
Support by the National Cancer Institute through grant CA78419 (to H.H.) is gratefully acknowledged. We thank Hiroyasu Nakano (Juntendo University School of Medicine, Tokyo, Japan) for providing us with TRAF2/5 DKO cells; Adrian Ting (Mount Sinai Medical Center, New York, NY) for the pMD.G, pMD.OGP, and TRAF5 plasmids; and Gail Bishop and Bruce Hostager (University of Iowa) for the IκBαSR and NIK plasmids and helpful discussions.
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