Double-stranded RNA (dsRNA) induces phosphorylation of Toll-like receptor 3 (TLR3) at Tyrosine 759 (Y759) and subsequently triggers signaling pathways to promote interferon-beta (IFN-beta) production. In this study, we found that dsRNA-stimulation induces biphasic TLR3 Y759-phosphorylation in macrophages. In addition to the immediate TLR3 Y759-phosphorylation, we identified a second wave of Y759-phosphorylation accompanied with the increase of both Src and ifn-beta transcription in the later phase of dsRNA-stimulation. Interestingly, Src phosphorylated TLR3 Y759 in vitro and in vivo. However, knockdown of Src abolished the late phase of TLR3 Y759-phosphorlyation and decreased the nuclear accumulation of interferon regulatory factors (IRFs) 3 and 7 and IFN-beta production. Re-introduction of Src restored all these molecular changes. Notably, via down-regulation of Src, dsRNA-elicited TLR3 Y759-phosphorylation, the nuclear accumulation of IRF3/IRF7, and IFN-beta generation were inhibited in inducible nitric oxide synthase (iNOS)-null macrophages. TLR3 knockdown destabilized Src and reduced the nuclear level of IRF3/IRF7 and IFN-beta production in macrophages exposed to LPS (a TLR4 ligand known to induce Src and IFN-beta expression). Ectopic expression of wild type TLR3, but not its 759-phenylalanine mutant, restored Src activity and ifn-beta transcription. Taken together, these results suggested an essential role of iNOS/Src/TLR3 axis in IFN-beta production in macrophages.
Date:
2014-03
Relation:
Journal of Biological Chemistry. 2014 Mar;289(13):9208-9220.
