MyD88 Adapter-like (Mal)/TIRAP Interaction with TRAF6 Is Critical for TLR2- and TLR4-mediated NF-κB Proinflammatory Responses

Abstract

Toll/interleukin-1 (TIR)receptor-containing adapters are critical in orchestrating the different signal transduction pathways following Toll-like receptor (TLR) activation. MyD88 adapter-like (Mal), also termed TIRAP, is involved in bridging MyD88 to the receptor complex for TLR-2 and TLR4 signaling in response to bacterial infection. We have previously reported an interaction between Mal and tumor necrosis factor receptor-associated factor 6 (TRAF6) via a TRAF6-binding motif, the disruption of which inhibited TLR-mediated NF-B-luciferase reporter activity. Given the recent report of intracellular TRAM localization promoting sequential signaling in TLR4 responses, we further characterized Mal interaction with TRAF6, the cellular localization, and the outcomes of disrupting this association on TLR inflammatory responses. We found that Mal and TRAF6 directly interact in response to TLR2 and TLR4 stimulation, although membrane localization is not necessary to facilitate interaction. Critically, reconstitution of murine Mal-deficient macrophages with MalE190A, containing a mutation within the TRAF6-binding motif, fails to reconstitute the proinflammatory response to TLR2 and TLR4 ligands compared with wild type Mal. Furthermore, Mal interaction with TRAF6 mediates Ser phosphorylation of the p65 subunit of NF-B and thus controls transcriptional activation but not nuclear translocation of NF-B. This study characterizes the novel role for Mal in facilitating the direct recruitment of TRAF6 to the plasma membrane, which is necessary for TLR2-and TLR4-induced transactivation of NF-B and regulation of the subsequent pro-inflammatory response.

ERK5 Protein Promotes, whereas MEK1 Protein Differentially Regulates, the Toll-like Receptor 2 Protein-dependent Activation of Human Endothelial Cells and Monocytes

“…In addition to acting through AP-1 transcription factors, p38-MAPK promotes mRNA stabilization, influences NF-B activity, modulates gene expression by regulating chromatin modifiers, and directly influences protein translation downstream of inflammatory stimuli (25,31,(65)(66)(67)(68)(69). Further studies will be required to define which of these many different p38-MAPK-dependent mechanisms are responsible for TLR2 signaling in ECs.…”

“…Activation of the canonical NF-B pathway results in the degradation of bound IB␣, IB␤, or IB⑀ in the cytoplasm, which leads to the translocation of NF-B to the nucleus to mediate transcriptional events (22,23). Proinflammatory stimuli can also induce the transactivation of p65 through phosphorylation, such as occurs after the TLR2-dependent activation of macrophages (24,25). The MAPK family members p38-MAPK, JNK, and ERK1/2 participate in the inflammatory response and are induced by TLR2 activation in macrophages (26 -28).…”

The Small GTPase Arf6 Is Essential for the Tram/Trif Pathway in TLR4 Signaling

“…Cell (33) and display decreased TNF␣ production upon LPS or Pam2 stimulus (32). We previously transduced these immortalized macrophages with wild-type Mal, which increases their responsiveness toward TLR4 and TLR2 ligands (31,34).…”

Poxviral Protein A46 Antagonizes Toll-like Receptor 4 Signaling by Targeting BB Loop Motifs in Toll-IL-1 Receptor Adaptor Proteins to Disrupt Receptor:Adaptor Interactions

“…4D, first and second panels). Studies have shown that Mal and TRAF6 directly interact in response to TLR4 stimulation (48,49), and hence the effect of A46 on the Mal-TRAF6 interaction was also assessed. Similar to the case for MyD88:IRAK2 and Mal:IRAK2, the Mal: TRAF6 interaction was unimpaired by A46 (Fig.…”