We have shown in this study that IRF-5 is targeted for K63-linked polyubiquitination in an IRAK1- and TRAF6-dependent manner and that lysines 410 and 411 in the carboxyl-terminal region of IRF-5 polypeptide are the target residues for TRAF6-mediated ubiquitination. We show that TRAF6 is required for IRF-5 ubiquitination and that this activity depends on the presence of kinase-competent IRAK1. Ubiquitination of IRF-5 facilitates its transport to the nucleus and binding and activation of the promoters of the IFNA and IFNB genes (Fig. ). Mutations of lysines 410 and K411 to arginine localized in a putative TRAF6 consensus binding site, PXEXX(Ar/Ac), prevented ubiquitination and nuclear localization of IRF-5 K410/K411R in response to MyD88 activation while these changes did not affect binding of TRAF6. These findings suggest that the K63-linked ubiquitination of IRF-5 is a critical requirement that regulates the activity of IRF-5 in the MyD88-activated antiviral pathway. However, the ubiquitination of IRF-5 occurs at low levels, and even small degrees of IRF-5 ubiquitination can enhance IRF-5 signaling. We also show that the spliced variant IRF-5-BMv that contains a large internal deletion of 288 nucleotides in the region representing exon 5 and parts of exons 4 and 6 is not ubiquitinated by the MyD88-activated signaling pathway. TRAF6 also failed to bind the IRF-5-BMv variant, thus indicating that the internal deletion in the IRF-5 polypeptide affects its secondary structure and masks the TRAF6 recognition site. We have shown previously that the transcriptional activity of IRF-5-BMv is significantly impaired (23
). In contrast, the MyD88-activated HuIRF-5v4 that contains only 48 nucleotide deletions in exon 6 is still effectively ubiquitinated and is transcriptionally active. Similar to IRF-5, IRF-7 also interacts with TRAF6 and is K63 polyubiquitinated (18
) by the MyD88 signaling pathway. However, the analysis of IRF-7 ubiquitination has shown that the region between amino acids 238 and 285 of IRF-7 is important for both TRAF6 binding and polyubiquitination of IRF-7 (18
). Thus, it is not clear whether the impairment of IRF-7 ubiquitination reflects the inability of TRAF6 to bind IRF-7. Also, the functional consequences of IRF-7 ubiquitination have not yet been established.
Our study provides key new insights into the molecular mechanisms of IRF-5 activation. We along with others have shown previously that IRF-5 is activated by TLR7 and TLR9 signaling, which relies on the adapter molecule MyD88 (28
), but not by the TLR3 or TLR4 MyD88-independent pathway (25
). TLR7 and TLR9 use only MyD88 as an adapter and activate both IRF-7 and IRF-5. MyD88 is also activated by TLR2 and TLR4, but these TLRs induce either very little IFN-β (TLR4) or none at all (TLR2) although they effectively activate NF-κB. Whether the presence of any of the additional adaptors associated with TLR2 or TLR4 (MyD88 and TIRAP with TLR2 and MyD88, TIRAP, TRIF, and Mal with TLR4) interferes with the formation of MyD88, IRAK1, TRAF6, and IRF-5 signaling complex is not known.
TRAFs are major signal transducers of the tumor necrosis factor receptor family. TRAF6 is a ubiquitin ligase-containing RING domain that was shown to be a key factor for the NF-κB activation by interleukin-1receptor, TLR, and nucleotide oligomerization domain 2 signaling pathways (2
). Major biological effects of TRAF signaling are mediated by activation of NF-κB and Ap-1 family members. The unique biological functions of TRAF6 are determined by its C-terminal domain that does not interact with peptides recognized by TRAF1, -2, -3, or -5. Thus, TRAF6 binds to distinct peptides from those binding TRAF2 or TRAF3 (32
). TRAF6 signaling downstream from TLR7 or TLR9 involves association of TRAF6 with IRAK4 and IRAK1. Full-length IRAK1 contains three potential TRAF6 binding sites, suggesting that these TRAF6 binding sites in IRAK1 contribute to TRAF6 activation. Peptides derived from this TRAF6-interacting motif inhibited TRAF6-mediated signal transduction (32
). We have shown that IRAK1, but not an IRAK1 kinase-inactive mutant, regulates TRAF6-mediated ubiquitination of IRF-5, suggesting that the function of IRAK1 is required for the activation of TRAF-6 and consequent TRAF6-mediated ubiquitination of IRF-5.
It was shown that TRAF6 also binds to TRIF (24
), where it recruits TRIF-associated TBK-1 and IκB kinase
). However, the activation of IRF-5 by the TRIF-dependent pathway has not been observed. It was shown that phosphorylation of IRF-7 in the MyD88-mediated pathway does not require TBK-1 and is instead dependent on IRAK1. However, direct phosphorylation of IRF-7 by IRAK1 was not examined (18
). IKKα was also shown to be involved in the TLR7 and TLR9 mediated induction of IFN-α. It was shown that IKKα activates IFNA promoter in synergy with IRF-7, while the induction of IFNB was not greatly modulated. It remains to be clarified whether IKKα cooperates with IRAK-1 (16
) in this pathway or in the activation of IRF-5. Thus, the protein kinase that serves as an IRF-5 kinase in the MyD88-dependent pathway and the role of phosphorylation have yet to be clearly elucidated. Further studies are also required to determine whether the MyD88 and Newcastle disease virus activation of IRF-5 targets the same or distinct serine residues.
Recently another member of the TRAF family, TRAF3, has been implicated as a part of MyD88- and TRIF-dependent signaling complexes (14
). A comparison of the function of TRAF3 and TRAF6 has shown that TRAF6 has a key role in MyD88 signaling but not in TRIF signaling. TLR9-induced activation of inflammatory genes was dependent on TRAF6, while the TLR3 activation was TRAF6 independent. These authors concluded that MyD88 activation recruits both TRAF3 and TRAF6, but the TRAF6-dependent pathway results in the activation of NF-κB and participates in the induction of inflammatory cytokines while the TRAF3 pathway recruits TBK-1 and has an important role in the TRIF-dependent activation of IFN genes. Our data clearly identify IRF-5 as an additional effector of the TRAF6 pathway. Thus, in the TLR7/9 and MyD88 pathways, TRAF6 leads to activation of the IKK, mitogen-activated protein kinase, and IRF-5 pathways. However, one difference between the MyD88-mediated activation of IRF-5 and type I IFN production and NF-κB-mediated activation of inflammatory cytokines is the distinct requirement for IRAK1 in the antiviral pathway. As we have shown in this study, IRAK1 associates with IRF-5 and is required for MyD88-dependent IRF-5 ubiquitination and activation. Similarly, IRAK1 was required for activation of IRF-7 (29
). However, MyD88 activation of mitogen-activated protein kinase was not impaired by IRAK1 deficiency (29
), and the activity of IRAK1 was not required for NF-κB activation (19
). These findings imply that the specificity of TRAF6 recruitment to the antiviral pathway may be determined by the formation of the MyD88, IRAK1, and TRAF6 signaling complex and its association with IRF-5 or IRF-7.
In summary, our study highlights the importance of IRF-5 ubiquitination in the MyD88-mediated antiviral signaling pathway. Further understanding of the molecular mechanisms involved in the IRF-5-induced inflammatory pathway will help to elucidate the role that IRF-5 plays in the pathophysiology of autoimmune diseases such as SLE.