TLR9 signaling requires ligand-induced phosphorylation of two specific tyrosine residues by EGFR and Syk

Toll-like receptors (TLRs) are transmembrane proteins required for recognizing microbial components or cellular danger signals to activate intracellular signaling pathways leading to induction of anti-microbial and inflammatory genes. Inactive TLRs require ligand-induced activation to recruit adaptor proteins, e.g., MyD88, to trigger the synthesis of cytokines and interferons. TLR9 is an endosomal membrane-bound protein, which binds to CpG-containing microbial DNA or endogenous signals from dead cells or tissue damage. We showed that TLR9 activation requires EGFR, a tyrosine (Tyr) kinase, which interacts with and phosphorylates the cytoplasmic domain of TLR9. Blocking EGFR activity pharmacologically, or knocking out EGFR gene in myeloid cells, suppressed lethal TLR9-induced hepatotoxicity. Here, we reveal that TLR9 required two Src family of kinases, Syk and Lyn, which, together with EGFR, led to phosphorylation and activation of TLR9. Lack of either of these kinases inhibited TLR9-MyD88 interaction, thereby inhibiting TLR9-mediated gene induction. Unlike EGFR, which constitutively binds TLR9, activated Syk interacted with TLR9 in a CpG-dependent manner. Activated Syk interacted with TLR9 and was critical for activating TLR9-bound EGFR. Quantitative mass spectrometric analyses revealed TLR9 was phosphorylated, sequentially, on Tyr870 and Tyr980 by Syk and EGFR, respectively. Mutation of either of these tyrosines led to complete loss of TLR9-induced cytokine production. For activation, Syk was phosphorylated by Lyn, which was activated by CpG-mediated scavenger-receptor A, and surprisingly, independent of TLR9. In summary, our results uncovered the molecular details of TLR9 activation by its Tyr-phosphorylation, which is critical for TLR9-mediated intracellular signaling.