Role of TLR4 tyrosine phosphorylation in signal transduction and endotoxin tolerance.
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 DeltaTLR4 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, IkappaB-alpha degradation, and activation of NF-kappaB 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-kappaB 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.
Medvedev
Andrei E
AE
Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA. amedvedev@som.umaryland.edu
Piao
Wenji
W
Shoenfelt
Joanna
J
Rhee
Sang Hoon
SH
Chen
Haiyan
H
Basu
Subhendu
S
Wahl
Larry M
LM
Fenton
Matthew J
MJ
Vogel
Stefanie N
SN
D019943:Amino Acid Substitution / Q000276:immunology; D000818:Animals; D002460:Cell Line; D006801:Humans; D020672:I-kappa B Proteins / Q000276:immunology / Q000378:metabolism; D007108:Immune Tolerance / Q000187:drug effects / Q000235:genetics; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis / Q000276:immunology; D008070:Lipopolysaccharides / Q000276:immunology / Q000494:pharmacology*; D051218:Lymphocyte Antigen 96 / Q000096:biosynthesis / Q000276:immunology; D048670:MAP Kinase Kinase 4 / Q000276:immunology / Q000378:metabolism; D051379:Mice; D020125:Mutation, Missense / Q000276:immunology; D053594:Myeloid Differentiation Factor 88 / Q000276:immunology / Q000378:metabolism; D000072000:NF-KappaB Inhibitor alpha; D016328:NF-kappa B / Q000276:immunology / Q000378:metabolism; D010766:Phosphorylation; D017386:Sequence Homology, Amino Acid; D015398:Signal Transduction / Q000187:drug effects* / Q000235:genetics / Q000502:physiology; D051195:Toll-Like Receptor 2 / Q000096:biosynthesis / Q000276:immunology; D051197:Toll-Like Receptor 4 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000276:immunology / Q000378:metabolism; D019061:src-Family Kinases / Q000276:immunology / Q000378:metabolism
D020672:I-kappa B Proteins; C495377:LY96 protein, human; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D051218:Lymphocyte Antigen 96; C507779:MYD88 protein, human; D053594:Myeloid Differentiation Factor 88; D016328:NF-kappa B; C000606204:NFKBIA protein, human; C000606205:Nfkbia protein, mouse; C495342:TLR2 protein, human; C495345:TLR4 protein, human; C493487:Tlr4 protein, mouse; D051195:Toll-Like Receptor 2; D051197:Toll-Like Receptor 4; D000072000:NF-KappaB Inhibitor alpha; C060902:lyn protein-tyrosine kinase; D019061:src-Family Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D048670:MAP Kinase Kinase 4