IL-17 Receptor Signaling Inhibits C/EBPβ by Sequential Phosphorylation of the Regulatory 2 Domain

Interleukin-17 (IL-17), the hallmark cytokine of T helper 17 (T(H)17) 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 kappa B (NF-kappa B), CAAT enhancer binding protein delta (C/EBPd), and C/EBP beta. The adaptor proteins tumor necrosis factor receptor-associated factor 6 (TRAF6) and Act1 are upstream of NF-kappa B and C/EBP delta, but the regulation of C/EBP beta remains undefined. Here, we show that IL- 17 signaling led to phosphorylation of two sites in the regulatory 2 domain of C/EBP beta 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 3 beta (GSK-3 beta). These pathways were mediated by distinct subdomains within IL-17 receptor A (IL-17RA). Whereas phosphorylation of threonine 188 (Thr(188)) was mediated by the previously identified SEF/IL-17R homology domain-Toll-IL-1R-like loop (SEFIR-TILL), phosphorylation of Thr(179) occurred through a newly characterized motif located in the distal tail of IL-17RA. Phosphorylated C/EBP beta mediated a negative signal, because blocking ERK and GSK-3 beta increased expression of IL-17 target genes and a C/EBP beta-Thr(188) mutant enhanced activation of a C/EBP-dependent reporter. Overexpression of GSK-3 beta inhibited IL-17-induced activation of a C/EBP-dependent reporter, and Thr(179) of C/EBPb was not phosphorylated in GSK-3 beta-deficient cells. Thus, IL- 17 triggered the dual phosphorylation of C/EBP beta, 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.