Nucleic acid sensing TLRs are important for host defense against pathogens but are also potent immunomodulators. For example, the TLR9 ligand CpG DNA is an adjuvant for vaccines in non-human primates (1
). CpG DNAs are also versatile, because depending on the sequence and chemical properties of the CpG DNA, TLR9 signaling can preferentially result in proinflammatory cytokine production and B cell proliferation (CpG DNA-B/D), or in type I IFN immune production (CpG DNA-A/K) (2
The outcome of response to the two different classes of CpG DNAs depends on the endosomal compartment where contact with TLR9 occurs (4
). CpG DNA-A/D and CpG DNA B/K are endocytosed but then are preferentially retained in early endosomes (CpG DNA-A) to elicit IFN production, or lysosomes (CpG DNA-B) to elicit proinflammatory cytokines (4
). TLR9 gains access to these DNAs by trafficking from the endoplasmic reticulum, through the Golgi (5
). Recent data implicate adaptor protein 3 (AP3) in regulation of TLR9 trafficking from the Golgi to lysosome related organelles where IFN production occurs, but whether AP3 is selectively required for IFN production, or is also required for TNF production is controversial (8
). Regardless, distinct regulatory mechanisms selectively governing inflammatory cytokine production have not been identified.
We hypothesized that one of several discrete TLR9 localization motifs was phosphorylated and regulated signaling (YXXΦ, where X= any amino acid, Φ= a bulky hydrophobic amino acid) (10
). We show that TLR9 with a single point mutation at tyrosine 888 (Y→A) selectively impairs TNF production and receptor phosphorylation. Mutation of tyrosine 888 to the structurally conserved phenylalanine (Y→F) retained TNF production, and phosphorylation. We conclude that while not directly phosphorylated, Y888 was structurally required for phosphorylation of TLR9 and thereby regulates TLR9-mediated signal bifurcation.