Role of TLR4 Tyrosine Phosphorylation in Signal Transduction and Endotoxin Tolerance

• Published in: The Journal of biological chemistry 2007-06, Vol.282 (22), p.16042-16053
• Main Authors: Medvedev, Andrei E., Piao, Wenji, Shoenfelt, Joanna, Rhee, Sang Hoon, Chen, Haiyan, Basu, Subhendu, Wahl, Larry M., Fenton, Matthew J., Vogel, Stefanie N.
• Format: Article
• Language: English
• Subjects: Amino Acid Substitution - immunology; Animals; Cell Line; Humans; I-kappa B Proteins - immunology; I-kappa B Proteins - metabolism; Immune Tolerance - drug effects; Immune Tolerance - genetics; Lipopolysaccharide Receptors - biosynthesis; Lipopolysaccharide Receptors - immunology; Lipopolysaccharides - immunology; Lipopolysaccharides - pharmacology; Lymphocyte Antigen 96 - biosynthesis; Lymphocyte Antigen 96 - immunology; MAP Kinase Kinase 4 - immunology; MAP Kinase Kinase 4 - metabolism; Mice; Mutation, Missense - immunology; Myeloid Differentiation Factor 88 - immunology; Myeloid Differentiation Factor 88 - metabolism; NF-kappa B - immunology; NF-kappa B - metabolism; NF-KappaB Inhibitor alpha; p38 Mitogen-Activated Protein Kinases - immunology; p38 Mitogen-Activated Protein Kinases - metabolism; Phosphorylation; Sequence Homology, Amino Acid; Signal Transduction - drug effects; Signal Transduction - genetics; Signal Transduction - physiology; src-Family Kinases - immunology; src-Family Kinases - metabolism; Toll-Like Receptor 2 - biosynthesis; Toll-Like Receptor 2 - immunology; Toll-Like Receptor 4 - genetics; Toll-Like Receptor 4 - immunology; Toll-Like Receptor 4 - metabolism
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M606781200

In this study, we examined whether tyrosine phosphorylation of the Toll-IL-1 resistance (TIR) domain of Toll-like receptor (TLR) 4 is required for signaling and blocked in endotoxin tolerance. Introduction of the P712H mutation, responsible for lipopolysaccharide (LPS) unresponsiveness of C3H/HeJ mice, into the TIR domain of constitutively active mouse ΔTLR4 and mutation of the homologous P714 in human CD4-TLR4 rendered them signaling-incompetent and blocked TLR4 tyrosine phosphorylation. Mutations of tyrosine residues Y674A and Y680A within the TIR domains of CD4-TLR4 impaired its ability to elicit phosphorylation of p38 and JNK mitogen-activated protein kinases, IκB-α degradation, and activation of NF-κB and RANTES reporters. Likewise, full-length human TLR4 expressing Y674A or Y680A mutations showed suppressed capacities to mediate LPS-inducible cell activation. Signaling deficiencies of the Y674A and Y680A TLR4s correlated with altered MyD88-TLR4 interactions, increased associations with a short IRAK-1 isoform, and decreased amounts of activated IRAK-1 in complex with TLR4. Pretreatment of human embryonic kidney (HEK) 293/TLR4/MD-2 cells with protein tyrosine kinase or Src kinase inhibitors suppressed LPS-driven TLR4 tyrosine phosphorylation, p38 and NF-κB activation. TLR2 and TLR4 agonists induced TLR tyrosine phosphorylation in HEK293 cells overexpressing CD14, MD-2, and TLR4 or TLR2. Induction of endotoxin tolerance in HEK293/TLR4/MD-2 transfectants and in human monocytes markedly suppressed LPS-mediated TLR4 tyrosine phosphorylation and recruitment of Lyn kinase to TLR4, but did not affect TLR4-MD-2 interactions. Thus, our data demonstrate that TLR4 tyrosine phosphorylation is important for signaling and is impaired in endotoxin-tolerant cells, and suggest involvement of Lyn kinase in these processes.