Tyrosine Phosphorylation of MAL in TLR4 Signaling and Endotoxin Tolerance

TLR4 recognition of LPS triggers signalosome assembly among TLR4, sorting (e.g. MyD88‐adapter‐like, Mal) and signaling (e.g. MyD88) adapters and kinases, resulting in activation of transcription factors and production of inflammatory mediators. Here we examined whether Mal tyrosine phosphorylation regulates Mal‐TLR4 interactions and signaling. In contrast to wild‐type (WT) Mal, overexpression of tyrosine‐deficient Mal variants with mutated Y86, Y106, and Y159 tyrosine residues impaired Mal tyrosine phosphorylation, interactions with Bruton's tyrosine kinase (Btk), phosphorylation of p38, and NF‐κB activation. LPS triggered tyrosine phosphorylation of endogenous Mal and initiated Mal‐Btk interactions in 293/TLR4/MD‐2 cells and human monocytes that were suppressed in endotoxin‐tolerant cells. Compared to WT‐Mal, Y86A‐ and Y159A‐ Mal variants exhibited higher interactions with TLR4 and MyD88, while associations with IRAK‐2 and TRAF‐6 were not affected. Overexpression of Y86A, Y159A and Y106A Mal in 293/TLR4/MD‐2 cells exerted dominant‐negative effects on TLR4‐inducible p38 phosphorylation and NF‐κB reporter activation. In contrast, tyrosine‐deficient Mal species did not affect NF‐κB 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 TLR signaling, regulates Mal interactions with TLR4 and receptor signaling, and is inhibited in endotoxin tolerance. This work was supported by NIH grant AI‐059524 (to A.E.M.)