are known to be adaptor proteins for TLR2/4, bound to the phosphorylated RAGE and transduced a signal to downstream molecules. The findings indicate that functional interaction between RAGE and TLRs coordinately regulates inflammation, immune response and other cellular functions. Results Cytoplasmic Domain of RAGE Is Phosphorylated by PKCf upon Ligand Binding We first examined whether the cytoplasmic domain of RAGE is phosphorylated. Overexpression of full-length RAGE but not that of a cytoplasmic domain-deleted construct resulted in phosphor- ylation in HEK293 cells, to which known RAGE ligands including S100A11, S100A12 and HMGB1 were applied (Figure 1A). Extent of the phosphorylation was S100A11 dose- and time- dependent (Figures S1A and S1B). Phosphorylation of RAGE was observed also upon stimulation of another RAGE ligands, S100A8/A9 (Figure S1C). Since the cytoplasmic domain of RAGE lacks any potential kinase domain, we screened for a protein kinase(s) that can phosphorylate recombinant RAGE in vitro and identified PKCa among representative kinases examined (Figures 1B and S1D). Screening of PKC isotypes resulted in identification of PKCf to be the most probable candidate for phosphorylation of RAGE (Figure 1C). Down regulation of endogenous PKCf but not that of PKCa, PKCe and PKCm abrogated S100A11-induced phosphorylation of RAGE in 32P-orthophosphate-labeled HEK 293 cells (Figure 1D). The specific siRNA against PKCf also effectively inhibited S100A8/A9-induced phosphorylation of RAGE (Figure S1H). Endogenous PKCf was activated by RAGE ligands such as S100A11, S100A12, HMGB1 and AGE in RAGE- transfected HEK293 cells as demonstrated by an in vitro assay showing phosphorylation of a PKCf-specific substrate (Figure S1E) by the cell extracts (Figure 1E). Activity of PKCf was dependent on dose of the ligands (Figure S1F). Extracts prepared from HEK293 cells transfected with cytoplasmic domain-deleted, hence signal transduction-deficient, RAGE did not show such an activity (Figure 1E). The cytoplasmic domain of human RAGE has 4 potential phosphorylation sites, i.e., Ser391, Ser399, Ser400, and Thr401. Among the 4 potential phosphorylation sites of the cytoplasmic domain of RAGE, only Ser391 is conserved among humans, mice, Guinea pigs, rats, rabbits, dogs, and cats. Replacement of Ser391 with Ala, but not any of the other 3 residues, resulted in abrogation of phosphorylation by PKCf in vitro (Figure 1F). Overexpressed wild-type RAGE but not the Ser391-replaced variant was phosphorylated in HEK293 cells when S100A11 was applied, while binding of PKCf to RAGE was independent of the phosphorylation status (Figure 1G and S1G). These results indicated that RAGE is phosphorylated at Ser391 by PKCf upon ligand binding. TIRAP binds to cytoplasmic domain of RAGE and transduces a signal from ligand-activated RAGE The nature and functional mode of possible adaptor proteins for RAGE are not well understood. Our trial screening for proteins that bind to the cytoplasmic domain of RAGE by immunopre- cipitation followed by mass spectrometry failed in identification of any promising candidates. We changed the strategy to a candidate-based screening partly because of implicated functional similarity between RAGE and TLRs [20]. Use of a potent expression vector and optimization of tags to express the short TIRAP Functions as an Adaptor Protein for RAGE Figure 1. RAGE is phosphorylated at Ser391 by PKCf upon ligand binding. (A) Phosphorylation of full-length RAGE (wt, closed arrowhead) but not cytoplasmic domain-deleted RAGE (DCyt: open arrowhead). HEK293 cells were transfected with epitope-tagged RAGE constructs (RAGE-Myc-HA-Flag-6His), [32P]phosphate-labeled, and treat- ed with the ligands (10 ng/ml, 1 h), and then RAGE proteins were pulled down with anti-HA antibody (clone HA-7)-agarose before Western blotting and autoradiography. The results were confirmed by five similar experiments. (B and C) Phosphorylation of RAGE(Cyt) in vitro. 6His-2HA- RAGE(Cyt) purified from transfected HEK293 cells by anti-HA antibody (clone HA-7)-agarose was incubated with constitutively active recombi- nant kinases of commercial source. Western blotting with anti-HA and anti-6His antibodies was performed for determination of amounts of RAGE(Cyt) and kinase proteins, respectively. The results were confirmed by five similar experiments (B) or by an independent and four related experiments (C). (D) Down-regulation of endogenous PKCf but not of other isoforms by siRNA abrogated S100A11 (10 ng/ml, 6 h)-induced phosphorylation of co-transfected RAGE-Myc-HA-Flag-6His in HEK293 cells. The results are representative of three independent experiments. (E) Activation of endogenous PKCf by RAGE ligands. HEK293 cells transfected with RAGE-Myc-HA-Flag-6His (wt or DCyt) were treated with 10 ng/ml of GST, S100A11, S100A12, and HMGB1 or 100 mg/ml of AGE for 1 h. Cell extracts were incubated with a specific PKCf-substrate in the presence of [c-32P]-ATP. The experiment was performed in quadruplicate, and the results were confirmed by three related experiments. (F) Determination of phosphorylation site of RAGE(Cyt). Recombinant PKCf- 6His (top panel) was incubated with isolated variants of 6His-2HA- RAGE(Cyt) [SSST (wild-type), ASST (Ser391 to Ala), SAST (Ser399 to Ala), SSAT (Ser400 to Ala), SSSA (Thr401 to Ala)] and assayed as for (B) and (C). The results were confirmed by an independent and two related experiments. (G) S100A11 (10 ng/ml)-dependent phosphorylation of transfected 6His-2HA-RAGE(wt) at Ser391 in HEK293 cells. [32P]Phos- phate-labeled cell extracts were pulled down with anti-HA agarose and immunoblotted with anti-6His antibody (top) or analyzed by autoradi- ography (middle). Co-precipitated endogenous PKCf was detected with anti-PKCf antibody (bottom). The results were confirmed by an independent and five related experiments. doi:10.1371/journal.pone.0023132.g001 PLoS ONE | www.plosone.org 2 August 2011 | Volume 6 | Issue 8 | e23132