IKKi: A novel regulator of Act1, IL-17 signaling and pulmonary inflammation

TH17 inflammatory helper cells constitute an important subset of T lymphocytes that secrete the cytokine interleukin 17 (IL-17) and thereby drive inflammation in response to microbial antigens. Dysregulation of TH17 cells, characterized by increased IL-17 production, is a hallmark of many inflammatory and autoimmune diseases such as asthma, multiple sclerosis and rheumatoid arthritis.1 The adaptor molecule and E3 ubiquitin ligase Act1 is a key signaling molecule downstream of the IL-17 receptor (IL-17R) that triggers activation of the NF-κB and mitogen-activated protein kinase (MAPK) signaling pathways, which in turn coordinate the induction of cytokine and chemokine genes that are central to the inflammatory response.2 However, the details of IL-17 signaling and precisely how Act1 is regulated have been poorly understood. In a recent issue of Nature Immunology, Bulek et al. have identified the kinase I kappa B kinase i (IKKi, also known as IKKe) as a critical regulator of Act1 and IL-17 signaling.3 The binding of IL-17 to the heteromeric IL-17 receptor complex (composed of IL-17RA and IL-17RC) leads to the recruitment of Act1 via conserved SEFIR domains present within the receptor complex and Act1. Following the recruitment of Act1, the kinase TAK1 and the E3 ubiquitin ligase TRAF6 are recruited to the receptor and facilitate activation of the IKK complex and the transcription factor NF-κB.2 IL-17 and Act1 also activate the MAPK pathway and promote the stabilization of certain chemokine mRNAs such as CXCL1 mRNA.4 In addition to its function as an adaptor molecule, Act1 functions as a U-box E3 ubiquitin ligase whose activity is essential for IL-17 signaling.5 However, the mechanisms of Act1 activation and downstream signal transmission have been unclear. The recent findings by Bulek et al. describe a novel mechanism of IL-17-induced Act1 activation for MAPK activation and chemokine mRNA stabilization.3 The authors found an IL-17-dependent interaction between Act1 and IKKi concomitant with the phosphorylation of Act1.3 IKKi and the related kinase TBK1 are best known as key signal transducers in innate immune pathways triggered by viral infection that lead to the activation of interferon regulatory factors and Ifn gene induction.6 Using airway epithelial cells from IKKi-deficient mice, the authors demonstrated that IKKi is required for the induction of proinflammatory cytokines and MAPK activation but not NF-κB activation upon IL-17 stimulation.3 The effects of IKKi on inflammatory pathways appear to be specific to IL-17 signaling because no difference in MAPK and NF-κB activation was observed upon tumor-necrosis factor stimulation of airway epithelial cells. Because Act1 is important for IL-17-dependent neutrophilia and pulmonary inflammation, the authors next examined the role of IKKi in IL-17-induced pulmonary inflammation. Indeed, after IL-17 intranasal injection, there were significantly fewer infiltrating neutrophils, diminished lung inflammation, and a lower abundance of the chemokine CXCL1 in the bronchoalveolar lavage fluid and lungs of IKKi-deficient mice.3 Because IKKi deficiency did not affect IL-17-dependent NF-κB activation, the authors explored other facets of IL-17 signaling and found that IKKi was essential for IL-17 to promote the stabilization of chemokine mRNAs.3 Thus, IKKi bifurcates IL-17 signaling to activate Act1-dependent MAPK signaling and chemokine mRNA stabilization. How does Act1 phosphorylation by IKKi regulate IL-17 signaling? The authors confirmed that IKKi was indeed a kinase for Act1 in airway epithelial cells stimulated with IL-17, and recombinant IKKi directly phosphorylated Act1.3 IL-17 stimulation enhanced IKKi autophosphorylation, which, surprisingly, was diminished in Act1-deficient cells, suggesting that Act1 may be involved in the activation of IKKi. IKKi mRNA was previously shown to be upregulated by stimulation with lipopolysaccharide.7 Interestingly, IL-17 also upregulated the expression of IKKi, suggesting a possible feed-forward mechanism of IL-17 signaling. The authors then identified Ser311 as the IL-17-inducible Act1 phosphorylation site, using a mass spectrometry approach.3 This phosphorylation site is functionally important because reconstitution of Act1-deficient cells with an Act1 phosphorylation defective mutant (Ser311→Ala) impaired IL-17-induced MAPK signaling and chemokine stabilization but had no effect on NF-κB activation.3 Although the authors did not generate a phospho-mimetic Act1 mutant (for example, Ser→Glu substitution), it will be interesting to determine whether such a mutant could bypass the requirement of IKKi for triggering IL-17-mediated MAPK activation. The authors proceeded to examine the mechanism of IL-17-mediated chemokine stabilization by IKKi and Act1 in a series of elegant experiments. A previous study by this group demonstrated that IL-17-mediated NF-κB activation depends on Act1 and TRAF6, whereas chemokine stabilization occurs independently of TRAF6.2, 8 Indeed, Act1 phosphorylated by IKKi preferentially interacted with TRAF2 and TRAF5 compared to TRAF6.3 Additionally, there was no effect on the interaction between Act1 and TRAF6 in IKKi–/– fibroblasts stimulated with IL-17. These experimental findings were also supported by molecular modeling experiments using known crystal structures of TRAF2 and TRAF6.3 Thus, the phosphorylation of Act1 by IKKi triggers a conformational change in Act1 that enhances its affinity for TRAF2/5 and diminishes its binding to TRAF6, ultimately leading to MAPK activation and chemokine stabilization (Figure 1). Figure 1 Schematic of IL-17 signaling pathways. The binding of IL-17 to its receptor leads to the recruitment of the adaptor molecule and E3 ligase Act1. TRAF6 and TAK1 cooperate with Act1 to activate NF-κB, which is important for the induction of proinflammatory ... In conclusion, the study by Bulek et al. has identified IKKi as a novel regulator of Act1 and the MAPK pathway in IL-17 signaling. However, several questions remain unanswered and should be addressed in future studies. How is IKKi activated in response to stimulation with IL-17, and how does Act1 facilitate this process? What determines the specificity of IKKi for its substrates, such as Act1, IκBα, interferon regulatory factor-3, STAT1 and c-Jun?9 The activating stimulus likely dictates the specificity of IKKi for its substrates, but what are the underlying mechanisms? The E3 ubiquitin ligase TRAF3 is a key molecule in innate immune-mediated IKKi/TBK1 activation, probably through its catalysis of lysine 63 (K63)-linked polyubiquitin chain formation on the kinases, which then serve as signaling scaffolds.10 Perhaps TRAF3 or another TRAF (TRAF2 or TRAF5) ubiquitinates IKKi downstream of IL-17R. Alternatively, an upstream kinase such as AKT may trigger the activation of IKKi, similar to what occurs in oncogenic settings.11 Furthermore, because IKKi and TBK1 play overlapping roles in antiviral signaling pathways, it will be interesting to determine whether TBK1 also functions downstream of IL-17. Remarkably, IL-17 has been shown to have no effect on interferon regulatory factor activation despite the well-known role of IKKi in antiviral signaling.3 The study by Bulek et al. suggests that inhibitors of IKKi may be useful for the treatment of certain inflammatory diseases. In addition, a better mechanistic understanding of IL-17 signaling may lead to the development of new therapies to inhibit the growth of tumors that are fueled by IL-17.