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J. Biol. Chem., Vol. 282, Issue 22, 16042-16053, June 1, 2007

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Role of TLR4 Tyrosine Phosphorylation in Signal Transduction and Endotoxin Tolerance^*

Andrei E. Medvedev¹², Wenji Piao, Joanna Shoenfelt, Sang Hoon Rhee, Haiyan Chen, Subhendu Basu, Larry M. Wahl^¶, Matthew J. Fenton^||3, and Stefanie N. Vogel²

From the Departments of Microbiology and Immunology and ^||Medicine, University of Maryland School of Medicine, Baltimore, Maryland 21201, the Division of Gastroenterology, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts 02215, and the ^¶NIDCR, National Institutes of Health, Bethesda, Maryland 20892

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 TLR4 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, IB- degradation, and activation of NF-B 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-B 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.

Received for publication, July 17, 2006 , and in revised form, February 27, 2007.

^* This work was supported by National Institutes of Health Grants AI-059524 (to A. E. M.), AI-057490, and AI-44936 (to S. N. V.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

² Both senior authors contributed equally to this work.

³ Current address: National Institute of Allergy and Infectious Diseases, 6610 Rockledge Dr., Bethesda, MD 20892.

¹ To whom correspondence should be addressed: Dept. of Microbiology and Immunology, University of Maryland School of Medicine, 660 West Redwood St., Rm. 324, Baltimore, MD 21201. Tel.: 410-706-5854; Fax: 410-706-2129; E-mail: amedvedev{at}som.umaryland.edu.

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