Two tyrosine residues of Toll-like receptor 3 trigger different steps of NF-κB activation

Innate immune response to viral infection is often triggered by Toll-like receptor 3 (TLR3)-mediated signaling by double-stranded (ds) RNA, which culminates in the activation of the transcription factor NF-kappa B and induction of NF-kappa B-driven genes. We demonstrated that dsRNA-induced phosphorylation of two specific tyrosine residues, 759 and 858, of TLR3 was necessary and sufficient for complete activation of the NF-kappa B pathway. When Tyr-759 of TLR3 was mutated, gene induction was inhibited, although NF-kappa B was partially activated. It was released from I kappa B and translocated to the nucleus but failed to bind to the kappa B site of the target A20 gene promoter. This defect could be attributed to incomplete phosphorylation of the RelA (p65) subunit of NF-kappa B, as revealed by two-dimensional gel analyses of p65, isolated from dsRNA-treated cells expressing either wild type TLR3 or the Tyr-759 -> Phe mutant TLR3. Thus, two phosphotyrosine residues of TLR3 activate two distinct pathways, one leading to NF-kappa B release and the other leading to its phosphorylation.