Signalosome formation at the receptor complex is thought to involve TLR oligomerization, conformational changes, and post-translational modifications of TLRs and adapters, including phosphorylation events. Agonist engagement of TLR2, TLR3, TLR4 and TLR5 triggers their tyrosine phosphorylation, and mutations of several tyrosines in the TIR domains of these receptors impair TLR signaling (51
). In addition to TLRs, adapter proteins are subject to several posttranslational modifications. For instance, myristoylation and serine phosphorylation of TRAM is important for its membrane anchoring and signaling (56
). LPS triggers tyrosine phosphorylation of MyD88 (60
), and tyrosine phosphorylation of Mal has been reported to be important for TLR2 and TLR4 signaling (55
). Non-classical K63 ubiquitination of TRAF-6, necessary for induction of its functional activities, is another example of post-translational modifications of adapters (61
). However, little is known about mechanisms by which posttranslational modifications of adapters affect signalosome assembly and TLR signaling.
Several lines of evidence shown herein stress an important role for Mal tyrosine phosphorylation in TLR4 signaling. We demonstrate a correlation of signaling incompetence of the P125H Mal mutant with its failure to interact with Btk, a key kinase involved in Mal tyrosine phosphorylation (55
), and to undergo tyrosine phosphorylation. These results are reminiscent of our previous findings of compromised tyrosine phosphorylation of mouse or human TLR4 as a consequence of the P712H or P714H mutations associated with signaling deficiencies of these TLR4 species (51
). This paper also reveals significance of Y86, Y106, and Y159 tyrosine residues within Mal for its interactions with Btk, Mal tyrosine phosphorylation and Mal-mediated activation of p38 phosphorylation, IκB-α degradation, IL-8 mRNA expression, and NF-κB reporter induction. It is plausible that P125, along with Y86, Y106, and Y159 tyrosines, provide docking sites for candidate kinases whose recruitment initiates phosphorylation of Y86, Y106, and Y159 tyrosine residues in a co-operative manner, with each reaction depending on the other. Although previously published suppression of Mal phosphorylation by a Btk inhibitor LFM-A13 (55
) and the interactions studies shown here suggest Btk as the key kinase responsible for tyrosine phosphorylation of Mal, we cannot rule out the involvement of other kinases. Interestingly, protein kinase C (PKC)-δ was also reported to bind Mal within its TIR domain and PKC-δ depletion from RAW cells suppressed TLR2- and TLR4-induced signaling (62
). Thus, the involvement of a multi-kinase complex (e.g.
, Btk-PKC-δ) in Mal phosphorylation is possible, suggesting a cooperative role for P125 and different tyrosine residues to efficiently dock Btk and PKC-δ, allowing tyrosine phosphorylation to take place. Studies are in progress to test this hypothesis and to delineate the role of tyrosine residues within the TIR domain of Mal in Mal associations with PKC-δ.
Induction of endotoxin tolerance re-programs subsequent cellular responses to LPS, suppressing induction of pro-inflammatory cytokines, while not affecting or even enhancing expression of anti-inflammatory mediators and antimicrobial effectors (44
). Several important changes at the level of signalosome assembly have been revealed in endotoxin tolerance, including disrupted recruitment of MyD88 to TLR4 and altered IRAK-1-MyD88 association (49
). In addition, we recently found that LPS-inducible TLR4 tyrosine phosphorylation is necessary for signaling and is drastically compromised in endotoxin-tolerant 293/TLR4/MD-2 cells and human monocytes (51
). Hence, it is possible that dysregulated post-translational modifications of “bridging” and/or “signaling” adapters may play a role in disrupted signalosome complex formation observed in tolerant cells, leading to reprogramming. More specifically, we reasoned that altered tyrosine phosphorylation of Mal may be important for endotoxin tolerization, a possibility that was not addressed previously. To the best of our knowledge, this paper shows for the first time that endotoxin tolerance is associated with compromised associations of Btk with Mal, leading to inhibited Mal tyrosine phosphorylation. Because of the suggested role for Mal as a “bridging” adapter that recruits a “signaling” adapter MyD88 to the TLR4 receptor complex, it is tempting to speculate that impaired recruitment of MyD88 to TLR4 coupled with deficient IRAK-1 activation is associated with compromised tyrosine phosphorylation of both TLR4 (51
) and Mal (shown in this study).
This report demonstrates increased interactions of Y86A, Y106A, and Y159A Mal variants with TLR4 that translate into the ability of these Mal species to act as dominant-negative inhibitors of TLR4-elicited p38 phosphorylation and NF-κB reporter activation. To gain an insight into mechanisms by which Mal tyrosine phosphorylation regulates TLR4 signaling, we examined interactions of WT and signal-incompetent, tyrosine-deficient Mal variants with TLR4, MyD88, IRAK-2, and TRAF-6. The signal-incompetent Y86A, Y106A, and Y159A Mal variants incapable of undergoing tyrosine phosphorylation were found to exhibit significantly higher associations with CD4-TLR4 compared to that observed with WT Mal. These increased associations are functionally significant as the mutagenesis of the Y86, Y106, and Y159 tyrosines rendered Mal into a dominant-negative inhibitor of TLR4 signaling. In contrast, higher interactions of the Y86A and Y106A Mal species with constitutively-active, overexpressed MyD88 are not translated into their ability to inhibit MyD88-induced NF-κB reporter activation. When tested for interactions with IRAK-2 and TRAF-6, both the WT and Y86A Mal variants showed similar associations with these signaling intermediates and moderately enhanced NF-κB reporter activation induced by overexpression of IRAK-2 and TRAF-6. These data suggest that tyrosine phosphorylation of Mal regulates the process of signalosome assembly at the level of TLR4, while failing to affect signaling initiated at the post-receptor levels. Indeed, once overexpressed, activated MyD88, IRAK-2 or TRAF-6 trigger activation of downstream signaling cascades in a TLR- and ligand-independent fashion (most likely, due to their enforced oligomerization) (1
). Under these conditions, tyrosine-deficient Mals (e.g.
, Y86A) do not suppress cell stimulation, suggesting their failure to sequester signaling-competent downstream intermediates.
It remains unknown how tyrosine phosphorylation of Mal regulates signalosome formation and signaling at the level of TLR4. Studies with molecularly-engineered MyD88 species demonstrated the importance of the delivery of MyD88 to the plasma membrane, showing a possibility to bypass the requirement for Mal if MyD88 could be plasma-membrane redirected due to the presence of grafted membrane-anchoring domains (33
). However, a key question that remains unknown is how Mal carries out MyD88 recruitment. One possible mechanism is that Mal acts exclusively at the plasma membrane, by associating with TLR4 and creating docking platforms to recruit cytoplasmic MyD88. This possibility is supported by the membrane localization of Mal and generation of phosphatidylinositol-4,5-biphosphate (PIP2) by agonist-stimulated CD11b/CD18 that could anchor Mal at the plasma membrane through the PIP2-binding domain of Mal (33
). This process is thought to facilitate Mal re-localization to membrane microdomains enriched with TLR4, leading to Mal-TLR4 interactions and co-localization (33
). Despite a relative distance of the conserved Y86 from the PIP2 domain (aa 15
), Y86 tyrosine phosphorylation may initiate conformational changes affecting the PIP2 domain and, hence, influencing Mal microdomain localization/TLR4 association within the plasma membrane. This could eventually affect the assembly of docking platforms formed by TLR4/Mal that are necessary for recruitment of downstream signaling adapters and kinases.
Alternatively, Mal could recruit MyD88 to TLR4 by acting as a shuttle molecule between the plasma membrane and the cytoplasm. Interestingly, serine phosphorylation of another “bridging” adapter, TRAM initiates its translocation from the plasma membrane, supposedly leading to dissociation of TRAM-TRIF complex to the cytoplasm where it triggers signaling (57
). Likewise, a similar regulation of Mal translocation from the cell membrane by its tyrosine phosphorylation could be envisioned, due to a lower interaction of tyrosine-phosphorylated, constitutively-active WT Mal with TLR4. In contrast to WT Mal, stronger associations of tyrosine-deficient Y86A, Y106A, or Y159A Mal variants with TLR4 may suggest retention of tyrosine-deficient Mal at the plasma membrane. This increased association of tyrosine-deficient, signaling-incompetent Mal species with TLR4 is different than the failure of the serine-deficient, signal-compromised TRAM variant to interact with TLR4 in either constitutive or LPS-inducible manners (57
). Thus, despite Mal and TRAM are both considered as “bridging” adapters, phosphorylation of these adapters seems to differently regulate their localization and signalosome formation.
Tyrosine phosphorylation of Mal targets it for ubiquitination by suppressor of cytokine signaling (SOCS)-1 and subsequent proteosomal degradation (63
), leading to down-regulation of TLR4 signaling due to unavailability of Mal. However, it is also plausible that Mal degradation at early stages of signal transduction promotes a release of activated components of the MyD88/IRAK-4/IRAK-1/TRAF-6 signalsome from the receptor. Hence, impaired tyrosine phosphorylation of Mal could suppress its degradation, preventing dissociation of signaling components MyD88, IRAK-2, and TRAF-6 from the receptor complex, and, as a result, inhibiting signaling. Mal was also found to be cleaved by caspase-1 that generates signaling-competent Mal species necessary for signal transduction (60
). Although Y86 and Y106 are situated distantly from the D198 residue that is critical for caspase-1-mediated cleavage of Mal (60
), tyrosine phosphorylation of Y159 could potentially be involved in changes of Mal conformation and its molecular electrostatic charge that may promote Mal-caspase-1 interactions. Thus, phosphorylation of different tyrosine residues within Mal could regulate its functions at several levels by non-redundant mechanisms.
In spite of uncompromised abilities of tyrosine-deficient Mal species to interact with constitutively-active MyD88, IRAK-2 and TRAF-6, Mal tyrosine phosphorylation could confer upon Mal the ability to shuttle from the membrane and to bind non-activated downstream signaling intermediates, delivering them to TLR4. This would initiate orchestrated signalosome assembly, leading to MyD88/IRAK-2/TRAF-6 complex formation, posttranslational modifications, and activation of downstream signaling. In line with this hypothesis, tyrosine de-phosphorylated Mal would be incapable of translocating from the membrane to the cytoplasm and/or would not be able to bind inactive, non-oligomerized cytoplasmic intermediates and recruit them to TLR4, failing to initiate competent signalosome formation. Studies employing cell fractionation, biotinylation and confocal microscopy are in progress to further elucidate molecular mechanisms by which Mal posttranslational modifications affect TLR4 signalosome formation and activation of signaling intermediates at the receptor complex.