TRAF6 and IRAK-1 are proximal and essential signaling components in IL-1R/TLR-mediated NF-κB and MAP kinase activation (13
). TRAF6 contains a RING domain that confers E3 activity, and its autoubiquitination with K63-linked polyubiquitin chains is necessary for TAK1 activation and ligand-induced NF-κB activation (19
). IRAK-1, on the other hand, is a serine/threonine kinase and is thought to be an adaptor important for recruitment of TRAF6 to liganded IL-1R. Because IRAK-1 in many ways resembles RIP (both contain death domains, intermediate domains, and kinase activities that are dispensable for NF-κB activation and are recruited to their respective receptors and ubiquitinated), we considered the possibility that it, too, has the capacity to bind NEMO and activate IKK. Our findings show that although IRAK-1 and TRAF6 are ubiquitinated with K63-linked chains following IL-1 treatment, surprisingly, NEMO binds only to ubiquitinated IRAK-1. The reason for this specificity is not clear, but it may reflect a low but meaningful affinity of NEMO for unubiquitinated IRAK-1. Because the avidity of NEMO for polyubiquitin increases as a function of chain length (52
), it is also possible that in stimulated cells the much longer polyubiquitin chain length observed with IRAK-1 compared to TRAF6 makes it a better binding partner for NEMO. The binding of NEMO to ubiquitinated IRAK-1 was confirmed by the observation that an IRAK-1 mutant, which cannot be ubiquitinated, bound poorly to NEMO and failed to promote receptor-mediated NF-κB activation.
The apparent reduction in IRAK-1 levels following IL-1 treatment is due to a shift to higher molecular weights resulting from its recruitment to the cytoplasmic tail of IL-1R and phosphorylation, possibly by IRAK-4 and/or autophosphorylation, and unmodified IRAK-1 can be recovered by phosphatase treatment of IRAK-1 immunoprecipitates from IL-1-stimulated cells (4
). Whether there is subsequent degradation of IRAK-1 is unclear, as we were unable to prevent the decrease in the unmodified band with proteasome inhibitors (unpublished observation). Our finding that IRAK-1 and all its lysine mutants were equally reduced after stimulation indicates that the lysine mutants were, in fact, recruited to the IL-1R signaling complex. This is also supported by the finding that IRAK-1K134/180R
fully restored IL-1-induced p38 activation.
Substitution of arginine for lysine 134 has been reported to impair IRAK-1 ubiquitination but not its ability to restore IL-1-induced NF-κB activation (54
). In contrast, we observed only a small and variable reduction in IRAK-1K134R
ubiquitination and binding to NEMO. It is possible that this discrepancy may be due to the differences in cells used. We instead found that mutation of both lysines, 134 and 180, markedly impaired IRAK-1 ubiquitination and function. Our finding that mutation of either lysine alone had a modest (K134) or very little (K180) effect on ubiquitination may reflect the semiquantitative and perhaps nonlinear nature of the assay, or possibly that loss of ubiquitination at one site enhances ubiquitination at the other. The small amount of residual ubiquitination of the K134/180R mutant indicates that there must be other, minor, sites of ubiquitination, but the ubiquitin linkage is unknown. Regardless, this ubiquitination does not appear to be physiologically relevant, because the K134/180R mutant was unable to restore NF-κB activation in IRAK-1-deficient cells.
Pellino is a family of E3s that bind IRAK-1 (39
). It was recently proposed that Pellino catalyzes the K63-linked polyubiquitination of IRAK-1, based on in vitro and overexpression studies (30
). Our results using TRAF6-deficient cells, however, indicate that TRAF6 is both required for IRAK-1 ubiquitination and that its E3 activity is necessary for generating the high-molecular-weight ubiquitinated IRAK-1, NEMO binding, and activation of NF-κB. Therefore, although it may be possible to enforce ubiquitination of IRAK-1 by Pellino, in cells it appears that TRAF6 is the major functionally relevant E3. It is an interesting speculation that Pellino family members may be responsible for the residual low-molecular-weight ubiquitination of IRAK-1 found in TRAF6-deficient cells.
Our data show that the E3 activity of TRAF6 is not only required for IL-1-induced IRAK-1 ubiquitination and NF-κB activation, but also p38 activation (Fig. ). In addition, results obtained with the IRAK-1 double lysine mutant argue that IRAK-1 ubiquitination is not required for the latter (Fig. ). Therefore, there must be another target for TRAF6's E3 activity that couples to MAP kinase activation. The kinase TAK1 is required for activation of both IKK and p38 in response to IL-1 and TLR ligands (38
). The ability of TAK1 to activate IKK and MKK6, an upstream activator of p38, is thought to result from K63-linked polyubiquitination of TRAF6 and recruitment of TAB2/TAB3/TAK1 complexes (48
). It is therefore possible that ubiquitinated TRAF6, or perhaps another substrate, recruits TAK1 through interactions with TAB2 and TAB3, and ubiquitinated IRAK-1 recruits IKK through an interaction with NEMO. Once in proximity, TAK1 could phosphorylate IKKβ and MKK6, leading to the activation of NF-κB and p38, respectively.
The kinase activity of IRAK-4 is required for the recruitment of IRAK-1 to the cytoplasmic tail of IL-1R (18
). Patients harboring mutations that disrupt either IRAK-4 expression or the CC2-LZ region of NEMO are susceptible to recurrent bacterial infections (8
). Furthermore, knock-in mice containing mutations that disrupt the kinase activity of IRAK-4 are resistant to TLR-induced septic shock (15
). Our finding that IRAK-1 ubiquitination is required for normal IL-1/TLR ligand-induced NF-κB activation suggests that patients with IRAK-4 and NEMO CC2-LZ mutations may fail to respond to pathogenic organisms because of an inability to recruit IRAK-1 or NEMO, respectively.
The K63-linked polyubiquitin-binding activity of NEMO has emerged as a mechanism for recruiting IKK to receptor signaling complexes and activating NF-κB (9
). Our findings show that this activity is also required for IL-1R/TLR-induced NF-κB activation, demonstrating that NEMO-polyubiquitin binding is a broadly used and highly conserved pathway for the activation of NF-κB by a wide variety of physiologic and pathological extracellular stimuli.