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

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ABSTRACT Toll-like receptors (TLRs) are transmembrane proteins that recognize microbial components or cellular danger signals and 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 that 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 that TLR9 was phosphorylated sequentially on Tyr 870 and Tyr 980 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. IMPORTANCE Toll-like receptors (TLRs) are critical components of cellular innate immune responses to microbial infection or tissue damage. TLRs are transmembrane proteins that require activation to mount a successful host response; TLR mutations are associated with human diseases. TLR ligands are also used as vaccine adjuvants to amplify the inflammatory responses of the host, activation of TLRs, and their regulation are essential. Here, we report the molecular mechanisms of TLR9 activation by tyrosine phosphorylation of its cytoplasmic domain. TLR9 interacts with EGFR and Syk, the tyrosine kinases, which phosphorylate two specific tyrosine residues on the TLR9 cytoplasmic domain. Mutation of these tyrosine residues or deficiency of these tyrosine kinases leads to impaired TLR9 signaling. Therefore, our results elucidate the early events of TLR9 signaling with implications in inflammatory diseases.

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