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

Department of Microbiology and Immunology, University of Maryland School of Medicine, Baltimore, Maryland 21201, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 03/2008; 283(6):3109-19. DOI: 10.1074/jbc.M707400200
Source: PubMed


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-kappaB, 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-kappaB 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-kappaB reporter activation to the extent comparable with P125H-Mal-mediated suppression. In contrast, tyrosine-deficient Mal species did not affect NF-kappaB 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.

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Available from: Wenji Piao, May 16, 2014
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    • "Thus, such transient interactions between the receptors and adaptors and the subsequent loss of the interaction may be common to both the TLR and the IL-18 pathways especially because they utilize many of the same intracellular components. For the TLR4 complex, phosphorylation of TLR4 and Mal has been suggested to be involved in the rearrangements [25], [26]. TRAM has been shown to be phosphorylated by Protein kinase C-ε (PKC-ε) upon stimulation by LPS, and the phosphorylation has been implicated in regulating the myristoylation state and thus the membrane targeting [10], [27]. "
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    ABSTRACT: MyD88, a Toll/interleukin-1 receptor homology (TIR) domain-containing adaptor protein, mediates signals from the Toll-like receptors (TLR) or IL-1/IL-18 receptors to downstream kinases. In MyD88-dependent TLR4 signaling, the function of MyD88 is enhanced by another TIR domain-containing adaptor, Mal/TIRAP, which brings MyD88 to the plasma membrane and promotes its interaction with the cytosolic region of TLR4. Hence, Mal is recognized as the "sorting adaptor" for MyD88. In this study, a direct interaction between MyD88-TIR and another membrane-sorting adaptor, TRAM/TICAM-2, was demonstrated in vitro. Cell-based assays including RNA interference experiments and TRAM deficient mice revealed that the interplay between MyD88 and TRAM in cells is important in mediating IL-18 signal transduction. Live cell imaging further demonstrated the co-localized accumulation of MyD88 and TRAM in the membrane regions in HEK293 cells. These findings suggest that TRAM serves as the sorting adaptor for MyD88 in IL-18 signaling, which then facilitates the signal transduction. The binding sites for TRAM are located in the TIR domain of MyD88 and actually overlap with the binding sites for Mal. MyD88, the multifunctional signaling adaptor that works together with most of the TLR members and with the IL-1/IL-18 receptors, can interact with two distinct sorting adaptors, TRAM and Mal, in a conserved manner in a distinct context.
    Full-text · Article · Jun 2012 · PLoS ONE
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    • "The buried surface at the interface of the TLR4-Mal-MyD88 hexameric complex constitutes 649 Å2 from the TLR4 dimer-Mal C complex and 658 Å2 from MyD88. In the docked complex, six residues from TLR4 chain A and 21 residues from Mal chain C make contact with 20 residues from MyD88 chain F. Similarly, six residues from TLR4 chain B and 21 residues from Mal chain D make contact with 20 residues from MyD88 chain E. Previous reports showed that Y86 mutation of Mal significantly alters the affinity of Mal for MyD88 [34], [47]. Furthermore, another recent study showed the importance of the R196 residue via mutation to cysteine in new primary immune-deficiency (MyD88 deficiency) patients. "
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    ABSTRACT: Toll-like receptors (TLRs) activate a potent immunostimulatory response. There is clear evidence that overactivation of TLRs leads to infectious and inflammatory diseases. Recent biochemical studies have shown that the membrane-bound form of ST2 (ST2L), a member of the Toll-like/IL-1 receptor superfamily, negatively regulates MyD88-dependent TLR signaling pathways by sequestrating the adapters MyD88 and Mal (TIRAP). Specifically, ST2L attenuates the recruitment of Mal and MyD88 adapters to their receptors through its intracellular TIR domain. Thus, ST2L is a potent molecule that acts as a key regulator of endotoxin tolerance and modulates innate immunity. So far, the inhibitory mechanism of ST2L at the molecular level remains elusive. To develop a working hypothesis for the interactions between ST2L, TLRs (TLR1, 2, 4, and 6), and adapter molecules (MyD88 and Mal), we constructed three-dimensional models of the TIR domains of TLR4, 6, Mal, and ST2L based on homology modeling. Since the crystal structures of the TIR domains of TLR1, 2 as well as the NMR solution structure of MyD88 are known, we utilized these structures in our analysis. The TIR domains of TLR1, 2, 4, 6, MyD88, Mal and ST2L were subjected to molecular dynamics (MD) simulations in an explicit solvent environment. The refined structures obtained from the MD simulations were subsequently used in molecular docking studies to probe for potential sites of interactions. Through protein-protein docking analysis, models of the essential complexes involved in TLR2 and 4 signaling and ST2L inhibiting processes were developed. Our results suggest that ST2L may exert its inhibitory effect by blocking the molecular interface of Mal and MyD88 adapters mainly through its BB-loop region. Our predicted oligomeric signaling models may provide a basis for the understanding of the assembly process of TIR domain interactions, which has thus far proven to be difficult via in vivo studies.
    Full-text · Article · Aug 2011 · PLoS ONE
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    • "This appears to be an important aspect of SOCS1—mediated inhibition of TLR4 signaling. This phosphorylation of Mal has also been shown to be impaired in cells rendered tolerant to LPS (Piao et al., 2008). An inhibitory role for proteins with an immunoreceptor tyrosine-based activation motif (ITAM) toward TLR signaling has also been suggested (Hamerman et al., 2005). "
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    Preview · Article · Jul 2008 · Immunity
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