IL-17 receptor signaling inhibits C/EBPb by sequential phosphorylation of the regulatory 2 domain

Interleukin-17 (IL-17), the hallmark cytokine of T helper 17 (T _H17) cells, signals through a distinct receptor subclass, yet little is known about the mechanisms involved. IL-17 activates the expression of target genes through the actions of the transcription factors nuclear factor κB (NF-κB), CAAT enhancer binding protein δ (C/EBPδ), and C/EBPβ. The adaptor proteins tumor necrosis factor receptor- associated factor 6 (TRAF6) and Act1 are upstream of NF-κB and C/EBPd, but the regulation of C/EBPβ remains undefined. Here, we show that IL-17 signaling led to phosphorylation of two sites in the regulatory 2 domain of C/EBPβ in a sequential, interdependent fashion. The first was rapid and dependent on extracellular signal-regulated kinase (ERK), whereas the second was dependent on the activity of glycogen synthase kinase 3β (GSK-3β). These pathways were mediated by distinct subdomains within IL-17 receptor A (IL-17RA). Whereas phosphorylation of threonine 188 (Thr¹⁸⁸) was mediated by the previously identified SEF/IL-17R homology domain-Toll-IL-1R-like loop (SEFIR-TILL), phosphorylation of Thr179 occurred through a newly characterized motif located in the distal tail of IL-17RA. Phosphorylated C/EBPβ mediated a negative signal, because blocking ERK and GSK-3β increased expression of IL-17 target genes and a C/EBPβ-Thr¹⁸⁸ mutant enhanced activation of a C/EBP-dependent reporter. Overexpression of GSK-3β inhibited IL-17-induced activation of a C/EBP-dependent reporter, and Thr179 of C/EBPb was not phosphorylated in GSK-3β-deficient cells. Thus, IL-17 triggered the dual phosphorylation of C/EBPβ, which inhibited the expression of proinflammatory genes. This detailed dissection is the first for the IL-17-mediated C/EBP pathway and the first known example of a negative signal mediated by IL-17RA.