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J. Biol. Chem., Vol. 283, Issue 6, 3109-3119, February 8, 2008

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Tyrosine Phosphorylation of MyD88 Adapter-like (Mal) Is Critical for Signal Transduction and Blocked in Endotoxin Tolerance^*

Wenji Piao, Chang Song, Haiyan Chen, Larry M. Wahl, Katherine A. Fitzgerald^¶, Luke A. O'Neill^||, and Andrei E. Medvedev¹

From the Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, NIDCR, National Institutes of Health, Bethesda, Maryland 20892, ^¶Division of Infectious Diseases and Immunology, University of Massachusetts Medical School, Worcester, Massachusetts 01605, and ^||School of Biochemistry and Immunology, Trinity College Dublin, Dublin 2, Ireland

Toll-like receptor 4 (TLR4) recognition of lipopolysaccharide triggers signalosome assembly among TLR4, sorting (e.g. MyD88 adapter-like (Mal)) and signaling (e.g. MyD88) adapters, initiating recruitment and activation of kinases, activation of transcription factors, and production of inflammatory mediators. In this study we examined whether tyrosine phosphorylation of Mal regulates its interactions with TLR4, MyD88, interleukin-1 (IL-1) receptor-associated kinase (IRAK)-2, and tumor necrosis factor receptor-associated factor (TRAF)-6 and is important for signaling. Overexpression of wild-type Mal in human embryonic kidney 293T cells induced its constitutive tyrosine phosphorylation and led to activation of p38, NF-B, and IL-8 gene expression. Mutagenesis of Tyr-86, Tyr-106, and Tyr-159 residues within the Toll-IL-1 receptor domain impaired Mal tyrosine phosphorylation, interactions with Bruton tyrosine kinase, phosphorylation of p38, and NF-B activation. Lipopolysaccharide triggered tyrosine phosphorylation of endogenous Mal and initiated Mal-Bruton-tyrosine kinase interactions in 293/TLR4/MD-2 cells and human monocytes that were suppressed in endotoxin-tolerant cells. Compared with wild-type Mal, Y86A-, Y06A-, and Y159A-Mal variants exhibited higher interactions with TLR4 and MyD88, whereas associations with IRAK-2 and TRAF-6 were not affected. Overexpression of Y86A- and Y106A-Mal in 293/TLR4/MD-2 cells exerted dominant-negative effects on TLR4-inducible p38 phosphorylation and NF-B reporter activation to the extent comparable with P125H-Mal-mediated suppression. In contrast, tyrosine-deficient Mal species did not affect NF-B activation when signaling was initiated at the post-receptor level by overexpression of MyD88, IRAK-2, or TRAF-6. Thus, tyrosine phosphorylation of Mal is required for adapter signaling, regulates Mal interactions with TLR4 and receptor signaling, and is inhibited in endotoxin tolerance.

Received for publication, September 4, 2007 , and in revised form, December 7, 2007.

^* This work was supported by National Institutes of Health Grant RO1 AI-059524 (to A. E. M.). 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.

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

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