The human toll signaling pathway: divergence of nuclear factor kappaB and JNK/SAPK activation upstream of tumor necrosis factor receptor-associated factor 6 (TRAF6)

Abstract

The human homologue of Drosophila Toll (hToll) is a recently cloned receptor of the interleukin 1 receptor (IL-1R) superfamily, and has been implicated in the activation of adaptive immunity. Signaling by hToll is shown to occur through sequential recruitment of the adapter molecule MyD88 and the IL-1R-associated kinase. Tumor necrosis factor receptor-activated factor 6 (TRAF6) and the nuclear factor kappaB (NF-kappaB)-inducing kinase (NIK) are both involved in subsequent steps of NF-kappaB activation. Conversely, a dominant negative version of TRAF6 failed to block hToll-induced activation of stress-activated protein kinase/c-Jun NH2-terminal kinases, thus suggesting an early divergence of the two pathways.

Figure 1

hToll expression and induction of NF-κB. (A) Human monocytes were treated with LPS (100 ng/ml) for different periods of time and analyzed for their hToll mRNA content by Northern blotting. Two distinct transcripts specific for hToll are detected and induced by LPS stimulation. (B) Ectopic expression of hToll-Flag and CD4/Toll but not the mutant version ΔhToll-Flag activate NF-κB in 293T cells in a dose-dependent manner, as measured by NF-κB reporter gene activity. (C) Equal amounts of hToll-Flag and ΔhToll-Flag are produced upon ectopic expression in 293T cells (3.2 μg of each expression construct were used for this experiment). IP, Immunoprecipitation. BLOT, Immunoblotting analysis.

Figure 1

hToll expression and induction of NF-κB. (A) Human monocytes were treated with LPS (100 ng/ml) for different periods of time and analyzed for their hToll mRNA content by Northern blotting. Two distinct transcripts specific for hToll are detected and induced by LPS stimulation. (B) Ectopic expression of hToll-Flag and CD4/Toll but not the mutant version ΔhToll-Flag activate NF-κB in 293T cells in a dose-dependent manner, as measured by NF-κB reporter gene activity. (C) Equal amounts of hToll-Flag and ΔhToll-Flag are produced upon ectopic expression in 293T cells (3.2 μg of each expression construct were used for this experiment). IP, Immunoprecipitation. BLOT, Immunoblotting analysis.

Figure 1

hToll expression and induction of NF-κB. (A) Human monocytes were treated with LPS (100 ng/ml) for different periods of time and analyzed for their hToll mRNA content by Northern blotting. Two distinct transcripts specific for hToll are detected and induced by LPS stimulation. (B) Ectopic expression of hToll-Flag and CD4/Toll but not the mutant version ΔhToll-Flag activate NF-κB in 293T cells in a dose-dependent manner, as measured by NF-κB reporter gene activity. (C) Equal amounts of hToll-Flag and ΔhToll-Flag are produced upon ectopic expression in 293T cells (3.2 μg of each expression construct were used for this experiment). IP, Immunoprecipitation. BLOT, Immunoblotting analysis.

Figure 2

Functional and structural evidence of MyD88 recruitment to the hToll signaling complex. (A) Sequence alignment of human MyD88 (amino acids 152–296), hToll (667– 840), hIL-1RAcP (391–570), and hIL-1RI (381–569). Alignment was performed with Clustall software. Shading, Identical amino acids with a score <3. Boxes, Identical amino acids with a score = 0. Dots, Conserved amino acids that are essential for IL-1RI to signal (reference 20). (B) MyD88 associates with hToll but not with a truncated version of hToll (ΔhToll) lacking the cytoplasmic region sharing sequence similarity with MyD88. 293T cells were transfected with hToll-Flag, ΔhToll-Flag, or IL-1RAcP–Flag as a positive control together with AU1-tagged MyD88. The presence of MyD88 that coprecipitated with the receptors was detected by immunoblotting with a rabbit polyclonal antiserum to MyD88. (C) ΔMyD88 inhibits hToll-induced but not the unrelated TNFR-2–induced NF-κB activity. 1 μg of receptors and 1.5 μg of ΔMyD88 were transfected. Data are expressed as the percentage of relative receptor-induced NF-κB activity.

Figure 2

Functional and structural evidence of MyD88 recruitment to the hToll signaling complex. (A) Sequence alignment of human MyD88 (amino acids 152–296), hToll (667– 840), hIL-1RAcP (391–570), and hIL-1RI (381–569). Alignment was performed with Clustall software. Shading, Identical amino acids with a score <3. Boxes, Identical amino acids with a score = 0. Dots, Conserved amino acids that are essential for IL-1RI to signal (reference 20). (B) MyD88 associates with hToll but not with a truncated version of hToll (ΔhToll) lacking the cytoplasmic region sharing sequence similarity with MyD88. 293T cells were transfected with hToll-Flag, ΔhToll-Flag, or IL-1RAcP–Flag as a positive control together with AU1-tagged MyD88. The presence of MyD88 that coprecipitated with the receptors was detected by immunoblotting with a rabbit polyclonal antiserum to MyD88. (C) ΔMyD88 inhibits hToll-induced but not the unrelated TNFR-2–induced NF-κB activity. 1 μg of receptors and 1.5 μg of ΔMyD88 were transfected. Data are expressed as the percentage of relative receptor-induced NF-κB activity.

Figure 3

NF-κB activation by hToll occurs through IRAK, TRAF6, and NIK. (A) IRAK is recruited to hToll but not to the inactive truncated version of hToll (ΔhToll). IL-1RAcP served as a positive control. Cells were transfected and analyzed as in Fig. 2 B. (B) IRAK-2 binds very weakly to hToll. IL-1RI served as a positive control. Cells were transfected and analyzed as in A. (C) ΔTRAF6 but not the unrelated ΔTRAF2 attenuates CD4/Toll-induced NF-κB activity. A dominant negative mutant version of the downstream molecule NIK [NIK(KK-AA)] also inhibits CD4/hToll-induced NF-κB activity.

Figure 4

hToll-induced SAPK activation requires MyD88 but not TRAF6. (A) hToll activates SAPK/JNK in a dose-dependent manner, as determined by the presence of the active phosphorylated form of JNK (pJNK). F.I., Fold induction calculated by normalizing pJNK with JNK. HC, IgG heavy chain. (B) MyD88 (152–296) (ΔMyD88) but not TRAF6 (289–522) (ΔTRAF6) abolishes hToll-induced SAPK/JNK phosphorylation. F.I., Fold induction calculated by normalizing pJNK with JNK. HC, IgG heavy chain.

Figure 4

hToll-induced SAPK activation requires MyD88 but not TRAF6. (A) hToll activates SAPK/JNK in a dose-dependent manner, as determined by the presence of the active phosphorylated form of JNK (pJNK). F.I., Fold induction calculated by normalizing pJNK with JNK. HC, IgG heavy chain. (B) MyD88 (152–296) (ΔMyD88) but not TRAF6 (289–522) (ΔTRAF6) abolishes hToll-induced SAPK/JNK phosphorylation. F.I., Fold induction calculated by normalizing pJNK with JNK. HC, IgG heavy chain.

Figure 5

Overview of hToll and TNFR-1 signaling pathways. The diagram shows that the bifurcation of NF-κB and JNK/SAPK activation by hToll or TNFR-1 occurs at different levels with respect to the specific TRAFs. TRADD, TNFR-1–associated death domain. RIP, Receptor-interacting protein.