FIGURE 1.

TAK1 Ser-412 phosphorylation is essential for TLR signaling and induction of proinflammatory cytokines. A, IL-1R-stably expressing HEK293T cells (C6), RAW264.7 cells, and HEK293T cells were treated by IL-1β (10 ng/ml), LPS (100 ng/ml), or TNF-α (10 ng/ml), respectively, for the indicated time. Cell lysates were then used for immunoblotting with the indicated antibodies. B, RAW264.7 cells were transiently transfected with 4 μg of empty vector, FLAG-tagged TAK1 wild type (TAK1-WT), TAK1 Thr-187 to alanine mutant (T187A), or TAK1 Ser-412 to alanine mutant (S412A) vector. After 48 h, cells were treated with LPS (100 ng/ml) for the indicated time. Proteins in the NF-κB and MAPK pathways were detected by immunoblotting. C, Cre-mediated TAK1 knock-out MEF cells were transfected with empty vector or TAK1-WT, T187A, or S412A vector by using the MEF Nucleofector Kit. After 24 h, cells were treated with LPS (1 μg/ml) for the indicated time. Cell lysates were then used for immunoblotting with the indicated antibodies. D, Cells were transfected as in B but treated with LPS (100 ng/ml), poly(I:C) (20 μg/ml), or CpG ODN (0.3 μm) for longer duration. Secretion of IL-6 and TNF-α in the supernatant was analyzed by ELISA. Data are plotted as means ± S.E. (error bars); *, p < 0.05; **, p < 0.01; ***, p < 0.001 versus controls. ns, no significance. All experiments were performed at least three times. E, C6 cells were transiently transfected with 1 μg of empty vector or vectors expressing FLAG-tagged TAK1, K63W, or S412A. After 48 h, cells were treated with IL-1β (10 ng/ml) for the indicated time. Total RNA were then extracted and used to determine induction of IL-8, TNF-α, MCP-1, and RANTES by using quantitative real-time PCR (Q-RT-PCR). Data are plotted as means ± S.E.; *, p < 0.05; **, p < 0.01 versus controls. All experiments were performed at least three times. p-, phosphorylated.