FIGURE 3.

Pro⁴³⁴ in the TIR domain and the C-terminal RHIM domain are required for full activation of TICAM-1. A, constructs of various TICAM-1 mutants were used. The mutant P434H construct contains a mutated TIR domain in which Pro⁴³⁴ is substituted with His. The mutant RHIM construct contains a mutated RHIM domain (⁵⁸⁷VQLG⁶⁹⁰ are replaced with four Ala). The mutant P434H-RHIM construct has two mutated sites, Pro⁴³⁴ and RHIM. The mutant N+TIR (1-566 aa) contains the N-terminal region and the TIR domain and the mutant N+(TIR-P434H) contains the N-terminal region with the mutated TIR-P434H domain. B, Pro⁴³⁴ in the TIR domain and the RHIM domain play an important role in TICAM-1-mediated NF-κB and IFN-β promoter activation. HEK293 cells in 96-well plates were transfected with the indicated expression plasmids (0.2, 2.0, and 20.0 ng) together with the IFN-β promoter reporter (upper panel, 100 ng) or NF-κB reporter plasmids (lower panel, 100 ng), and phRL-TK (1.0 ng). Twenty-four hours after transfection, the luciferase reporter activities were measured. The average activities from three independent assays are shown as relative stimulation. C, HEK293 cells in 24-well plates were transfected with expression plasmids for HA-tagged wild-type TICAM-1 (20 and 200 ng) or each TICAM-1 mutant (20 ng) in the presence of an apoptosis inhibitor. Protein expression levels were determined by immunoblotting using anti-HA pAb. The protein levels of the TICAM-1 mutants are significantly higher than that of wild-type TICAM-1 using the same concentration of DNA plasmid. Lower panel, long exposed membrane. D, the homodimerizing ability of the TICAM-1 mutants was assessed using a co-immunoprecipitation assay. HEK293 cells were transfected with the indicated plasmids for HA- or FLAG-tagged TICAM-1 mutants, and cell lysates were immunoprecipitated with anti-FLAG mAb. Immunoprecipitants were resolved on SDS-PAGE under reducing conditions followed by immunoblotting with anti-HA pAb or anti-FLAG mAb. The protein expression in the total cell lysates was analyzed by immunoblotting with anti-HA pAb or anti-FLAG mAb (IB). Only the N+(TIR-P434H) mutant did not homodimerized (upper panel). Closed arrowheads indicate full-length TICAM-1, and open arrowheads indicate the truncated form of TICAM-1 (N+TIR).