Structures and interface mapping of the TIR domain-containing adaptor molecules involved in interferon signaling

TLR4 recruits TRIF-related adaptor molecule (TRAM, also known as TICAM2) as a sorting adaptor to facilitate the interaction between TLR4 and TRIF and then initiate TRIF-dependent IRF3 activation. However, the mechanisms by which TRAM links downstream molecules are not fully elucidated. In this study, we show that TRAM undergoes tyrosine phosphorylation upon TLR4 activation and that is required for TLR4-induced IRF3 activation. Protein tyrosine phosphatase nonreceptor type 4 (PTPN4), a protein tyrosine phosphatase, inhibits tyrosine phosphorylation and subsequent cytoplasm translocation of TRAM, resulting in the disturbance of TRAM–TRIF interaction. Consequently, PTPN4 specifically inhibits TRIF-dependent IRF3 activation and IFN-β production in TLR4 pathway. Therefore, our results provide new insight into the TLR4 pathway and identify PTPN4 as a specific inhibitor of TRIF-dependent TLR4 pathway. Targeting PTPN4 would be beneficial for the development of new strategy to control TLR4-associated diseases without unwanted side effects.

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

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Phosphatase PTPN4 Preferentially Inhibits TRIF-Dependent TLR4 Pathway by Dephosphorylating TRAM

Huai

Song

Wang

et al. 2015

“…TIRAP-dependent binding of MyD88 recruits the signaling molecules IL-1R-associated kinases 1 and 4, which forms so-called myddosomes that activate NF-kB and MAPK signaling cascades, leading to production of proinflammatory cytokines (6,7). While TIRAP-MyD88-dependent signaling occurs at the plasma membrane, Toll-IL-1R domaincontaining adaptor molecule-2 (TICAM-2; also called TRAM)--and TICAM-1 (also called TRIF)-dependent IFN-regulatory factor 3 (IRF3) activation initiates from the endosomes (8)(9)(10)(11)(12)(13)(14). Dissociation from TIRAP-MyD88 and subsequent clathrin-and dynamin-mediated internalization of LPS-TLR4 are prerequisites for binding to the sorting adaptor TICAM-2 that localizes to the endosomal membrane via myristoylation at the N terminus (12,15).…”

mentioning

confidence: 99%

Raftlin Controls Lipopolysaccharide-Induced TLR4 Internalization and TICAM-1 Signaling in a Cell Type–Specific Manner

Tatematsu

Yoshida

Morioka

et al. 2016

Self Cite

The clathrin-dependent endocytic pathway is crucial for endosomal TLR3- and TLR4-mediated Toll–IL-1R domain–containing adaptor molecule-1 (TICAM-1) signaling. TLR4 uses a different signaling platform, plasma membrane and endosomes, for activation of TIRAP-MyD88 and TICAM-2–TICAM-1, respectively. LPS-induced endocytosis of TLR4 is mandatory for TICAM-1–mediated signaling including IFN-β production. Several molecules/mechanisms such as CD14, clathrin, and phosphatidylinositol metabolism have been reported to act as inducers of TLR4 translocation. However, the molecular mechanism of spatiotemporal regulation of TLR4 signaling remains unresolved. We have previously shown that Raftlin is essential for clathrin-dependent endocytosis of TLR3 ligand in human epithelial cells and myeloid dendritic cells (DCs). In this article, we demonstrate that Raftlin also mediated LPS-induced TLR4 internalization and TICAM-1 signaling in human monocyte-derived DCs and macrophages (Mo-Mϕs). When Raftlin was knocked down, LPS-induced TLR4-mediated IFN-β promoter activation, but not NF-κB activation, was decreased in HEK293 cells overexpressing TLR4/MD-2 or TLR4/MD-2/CD14. LPS-induced IFN-β production by monocyte-derived DCs and Mo-Mϕs was significantly decreased by knockdown of Raftlin. Upon LPS stimulation, Raftlin moved from the cytoplasm to the plasma membrane in Mo-Mϕs, where it colocalized with TLR4. Raftlin associated with clathrin-associated adaptor protein–2 in resting cells and transiently bound to TLR4 and clathrin at the cell surface in response to LPS. Thus, Raftlin appears to modulate cargo selection as an accessary protein of clathrin-associated adaptor protein–2 in clathrin-mediated endocytosis of TLR3/4 ligands.

“…According to the structural data, the E87/D88/D89 motif located in the loop region between the bA sheet and the aA helix and S156 in the aC helix region contributes to TICAM-1-TICAM-2 heterodimerization (Supplemental Fig. 1) (33). In multiple alignments of TICAM-2 sequences, these motifs and two other acidic residues, D91 and E92, were highly conserved among various mammalian species (Fig.…”

Section: Resultsmentioning

confidence: 92%

Identification of a Regulatory Acidic Motif as the Determinant of Membrane Localization of TICAM-2

Funami

Matsumoto

Enokizono

et al. 2015

Self Cite

TLR4 triggers LPS signaling through the adaptors Toll/IL-1R domain–containing adaptor molecule (TICAM)-2 (also called TRAM) and TICAM-1 (also called TRIF), together with Toll/IL-1R domain–containing adaptor protein (TIRAP) and MyD88. The MyD88 pathway mediates early phase responses to LPS on the plasma membrane, whereas the TICAM pathway mediates late-phase responses, which induce the production of type I IFN and activation of inflammasomes. TICAM-2 bridges TLR4 and TICAM-1 for LPS signaling in the endosome. Recently, we identified an acidic motif, E87/D88/D89 in TICAM-2, that provides the interaction surfaces between TICAM-2 and TICAM-1. In the present study, we found additional D91/E92 in TICAM-2, conserved across species, that is crucial for TICAM-1 activation. The D91A/E92A mutant protein was distributed largely to the cytosol, despite myristoylation, suggesting its importance for assistance of membrane localization of TICAM-2. An ectopically expressed D91A/E92A mutant per se failed to activate TICAM-1, unlike its wild-type counterpart that forms self-aggregation, but it still retained the ability to pass LPS-mediated IFN regulatory factor (IRF)3 activation. In a TICAM-2 knockout human cell line expressing TLR4/MD-2 with or without CD14, overexpression of the D91A/E92A mutant did not activate IRF3, but upon LPS stimulation, it induced sufficient TLR4-mediated IRF3 activation with high coefficient colocalization. Hence, the D91/E92 motif guides TICAM-2 membrane localization and self-activation for signaling. Our results suggest the presence of two distinct steps underlying endosomal LPS signaling on TICAM-2 for TICAM-1 activation: TICAM-2 assembling in TLR4 and/or TICAM-2 self-activation. D91A/E92A of TICAM-2 selectively associates the TLR4-dependent TICAM-2 assembling, but not cytosolic TICAM-2 self-aggregation, to activate TICAM-1.