TIRAP Functions as an Adaptor Protein for RAGE Figure 2. TIRAP and MyD88 function as adaptor proteins for RAGE. (A) Co-precipitation of endogenous TIRAP and MyD88 but not TRAM with recombinant RAGE(wt). HEK293 cells were transfected with RAGE(wt) and RAGE(DCyt) tagged with -Myc-HA-Flag-6His, [32P]phosphate-labeled, and treated with AGE at 100 mg/ml or with other ligands at 10 ng/ml for 1 h. TLR2/4 blocking mixture (Mixture, see Materials and Methods) was preincubated with the ligands for 30 min at room temperature. Cell extracts were analyzed by Western blotting with or without prior immunoprecipitation and by autoradiography. The results were confirmed by 7 related experiments. (B and C) AGE (100 mg/ml)-induced phosphorylation of RAGE at Ser391 is essential for recruitment of endogenous TIRAP and MyD88 (B: The results were confirmed by an independent and two related experiments.) and downstream signal transduction (C: The results were confirmed by an independent and two related experiments.) in HEK293 cells. Full-length 6His-2HA-RAGE constructs were used. SSST, wild-type; ASST, Ser391 to Ala. (D and E) Abortive signal transduction from activated endogenous RAGE in MyD88-/- or TIRAP-down regulated mouse fibroblasts was rescued by forced expression of the corresponding human genes. Mouse TIRAP siRNA and expression vectors were transfected 48 h before treatment with AGE (100 mg/ml, 1 h). Experiments were performed under the conditions similar to those described in (A) except for immunoprecipitation using a biotinylated antibody against endogenous RAGE. 32P shows the results of autoradiography of the immunoprecipitated RAGE. The results were confirmed by an independent and two related experiments. doi:10.1371/journal.pone.0023132.g002 Discussion The findings described above clearly demonstrate that TIRAP and MyD88 function as adaptor proteins for RAGE as well as for TLR2/4 (Figure 5). The recruitment of the adaptor proteins induced further recruitment of IRAK4, which in turn activated the downstream effecter kinases such as Akt, p38, JNK and IKKs and ultimately led to the induction or inflammatory cytokines, IL-6, IL-8, and TNFa through activation of NFkB. On the other hand, IFNb was not induced by RAGE ligands. This result may be due to the lack of interaction between the cytoplasmic domain of RAGE and TRAM (Figure 2A). We previously showed that S100A11 could activate Akt via RAGE in keratinocytes [15]. This is probably mediated by MyD88, since MyD88 is known to directly interact with and activate the downstream effector kinase Akt [28]. Ligand-induced phosphorylation of RAGE at Ser391 is a prerequisite for efficient recruitment of the adaptor proteins. The phosphorylation was mediated by the PKCf (Figure 1) through binding with the cytoplasmic domain of RAGE (Figure S1G). Kinase activity of PKCf was also remarkably upregulated by stimulation of HEK293 cells with diverse RAGE ligands and of primary normal human endothelial cells (HUVECs) with AGE. AGEs are non-enzymatical adducts of proteins formed in pro- oxidant and hyperglycemic environments [2,3,23]. Treatment of the endothelial cells with AGE induced apoptosis (Figure S3I) PLoS ONE | www.plosone.org 4 August 2011 | Volume 6 | Issue 8 | e23132