Role of TIRContaining Adapter Molecules in TLR Signaling

Five TIR-containing intracellular adapter molecules have been identified, and four are known to participate in TLR signaling. MyD88 was the first adapter protein found to mediate IL-1R and TLR signaling (Muzio et al. 1997; Wesche et al. 1997; Burns et al. 1998; Kawai et al. 1999, 2001). MyD88 is comprised of a C-terminal TIR domain involved in interaction with TLRs and an N-terminal death domain that associates with IRAK family members (Muzio et al. 1997; Wesche et al. 1997; Burns et al. 1998). MyD88 is required for responses by most TLRs except TLR3 (Kawai et al. 1999, 2001; Kaisho et al. 2001; Yamamoto et al. 2002a), and is recruited to TLRs via homotypic TIR-TIR domain interactions (Muzio et al. 1997; Wesche et al. 1997; Burns et al. 1998). This triggers the association of MyD88 with IL-1R-associated kinases (IRAK)-4 and IRAK-1, and subsequent phosphorylation reactions by IRAK-4 and IRAK-1 (Muzio et al. 1997; Wesche et al. 1997; Burns et al. 1998; Li et al. 2004; Kollewe et al. 2004), resulting in IRAK-1-mediated phosphorylation of Tollip (Burns et al. 2000), an inhibitory protein that sequesters IRAK-1 in unstimulated cells (Burns et al. 2000). IRAK-1 dissociates from the TLR complex and interacts with TNFR-associated factor 6 (TRAF-6) via a downstream adapter TIFA, leading to TRAF-6 activation (Takatsuna et al. 2003). TRAF-6 exists in a complex with two ubiquitin-conjugating enzymes, UEV1A and UBC13, that activate TGF-P-activated kinase (TAK) 1 through the assembly of a lysine 63-linked polyubiquitin chain and engagement of additional intracellular intermediates Pellino-1 and Pellino-2 (Deng et al. 2000; Jensen and Whitehead 2003; Ea et al. 2004). Activated TAK-1 triggers phosphorylation of IKK-a /p and MAPK kinase (MKK) 3 and 6, leading to activation of MAPKs (e.g., JNK, p38) and transcription factors (e.g., NF-kB, AP-1; Takaesu et al. 2000, 2001; Wang et al. 2001; Jiang et al. 2002, 2003; Jensen and Whitehead 2003; Yoshida et al. 2005). Another downstream intracellular intermediate, evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) was reported to bridge TRAF-6 and MAPK/ERK kinase kinase-1 (MEKK-1) and to regulate MEKK-1 processing and activation of NF-kB and AP-1 (Kopp et al. 1999). IRAK-1 is subsequently ubiquitinated and targeted to the proteosome where it is degraded, which prevents hyperactivation of TLR-stimulated cells (Yamin and Miller 1997; Fig. 2). This MyD88 signaling pathway results in rapid NF-kB and MAP kinase activation, B cell proliferation, and expression of pro-inflammatory cytokines. Studies in MyD88 KO mice revealed the existence of an MyD88-independent pathway initiated by TLR3 and TLR4 that leads to dendritic cell maturation, phosphorylation of interferon (IFN)-regulatory factor (IRF)-3, type I IFN expression, and delayed NF-kB and MAPK activation (Kawai et al. 1999, 2001, Kaisho et al. 2001; Yamamoto et al. 2002b).

The TIR domain-containing adapter protein/MyD88 adapter-like protein (TIRAP/MAL) was initially identified as a second adapter protein involved in TLR4-, but not IL-1R-mediated signaling (Fitzgerald et al. 2001; Horng et al. 2001). Coupled with the observation that TIRAP/MAL-/- mice have an impaired response to TLR2- and TLR4-agonists, while responses to ligands for TLR3, 5, 7, and 9 are preserved (Horng et al. 2002; Yamamoto et al. 2002a, 2004), these findings suggest that TIRAP/MAL may contribute to TLR signaling specificity. Both MyD88-/- and TIRAP/MAL-/- mice exhibit very similar phenotypes in terms of TLR2 and TLR4 signaling, manifested by delayed activation of NF-kB and MAP kinases and the lack of induction of TNF-a, but normal activation of IRF-3 and IFN-P (Horng et al. 2002; Yamamoto et al. 2002a, 2004), indicating that TIRAP/MAL is restricted to the MyD88-dependent pathway. Interestingly, only TLR2 and TLR4 utilize both MyD88 and TIRAP/MAL, whereas TLR3 does not use either adapter (Horng et al. 2002; Yamamoto et al. 2002a, 2004). In contrast, TLR5, 7, 8, and 9 use only MyD88 for triggering both NF-kB activation, production of proinflammatory cytokines, and, surprisingly, induction of type I IFNs (Hemmi et al. 2002; Horng et al. 2002; Yamamoto et al. 2004). However, these responses are triggered by distinct signaling molecules than those involved in TLR3- and TLR4-mediated type I IFN production (Fig. 2, see below). Agonist-induced phosphorylation of two critical tyrosine residues (Y86, Y187) in TIRAP/MAL by Bruton's tyrosine kinase was shown to play a critical role in TLR2 and TLR4 signaling (Gray et al. 2006). In addition, suppressor of cytokine signaling (SOCS)-1 was found to associate with TIRAP/MAL and targets this adapter for polyubiquitination and subsequent

Fig. 2 MyD88-dependent and MyD88-independent TLR signaling pathways. TLRs respond to PAMPs or endogenous TLR ligands either directly or in co-operation with depicted co-receptors, leading to TLR oligomerization, conformational changes within the TIR domain, and recruitment of adapter proteins and kinases. TLR2 utilizes exclusively TIRAP/Mal and MyD88 adapter proteins and kinases IRAK-4 and IRAK-1 for triggering proinflammatory cytokine production via engagement of downstream adapter TRAF-6 and kinases TAK-1, IKKa/p, MEKK-1, and MKK3/6. The TLR4-mediated MyD88 signaling pathway is initiated by engagement of TIRAP/ Mal and association of MyD88 with the cytoplasmic region of TLR4 via their TIR domain interactions, followed by recruitment of IRAK-4 and IRAK-1-Tollip complex. This triggers phosphor-ylation of IRAK-1, dissociation of Tollip, interaction of IRAK-1 with TRAF-6, and stimulation of MAP kinases and transcription factors via the engagement of the downstream adapters (e.g., ECSIT) and activation of IKK, TAK-1, MEKK-1, and MKK-3,4,6 kinases. TLR4 stimulates MyD88-independent pathway via adapter proteins TRAM and TRIF that signal activation of IRF-3 via stimulation of two non-typical IKKs, IKKe and TBK-1 and the induction of IFN-a and IFN-P-dependent genes. Engagement of the MyD88-independent pathway also mediates dendritic cell maturation and delayed activation of MAPK and NF-kB. TLR3 signaling uses the adapter protein TRIF that activates NF-kB and production of proinflammatory cytokines via TRAF6-TAK-1 and RIP-1 and induction of type I IFN expression via IKKe/TBK-1 and IRF-3. TLR7/9 signaling occurs by utilization of the MyD88/IRAK-4/IRAK-1/TRAF-6 module of adapter proteins and kinases whereby TRAF-6 (by still poorly understood mechanisms) activates proinflammatory cytokine production via NF-kB and IRF-5 and type I IFN expression via IRF-7

degradation (Mansell et al. 2006), representing a rapid and selective means of limiting the innate immune response. Whereas TIRAP/MAL has been postulated to serve as a bridging adapter between TLR2, TLR4 and MyD88, it cannot be ruled out that it also mediates specific signaling functions. In this respect, Horng et al. (2002) showed that PKR is a downstream target of TIRAP/Mal and Kagan and Medzhitov (2006) recently reported that TIRAP/Mal is recruited to the plasma membrane lipid rafts through a phosphoinositol-4,5-bisphosphate binding site where it facilitates MyD88 recruitment to TLR4.

Two other adapter proteins are involved in MyD88-independent signaling triggered by TLR3 and TLR4. TIR domain-containing adapter inducing IFN-P (TRIF), also called TICAM-1, was first identified as an adapter protein that mediates the MyD88-independent signaling pathway elicited by TLR3 (Yamamoto et al. 2002a, 2003a, b; Oshiumi et al. 2003a, b). The functional importance of TRIF was first demonstrated in TRIF KO mice that exhibited impaired IFN-P induction and IRF-3 activation in response to TLR3 and TLR4 agonists (Hoebe et al. 2003; Oshiumi et al. 2003a, b; Yamamoto et al. 2003a, b). TRIF mediates downstream signaling by interacting with IKKe and TANK-binding kinase 1 (TBK1), resulting in phosphorylation and nuclear translocation of IRF-3 (Sato et al. 2003; Oganesyan et al. 2006). In addition to IRF-3 activation, TRIF was also shown to cause NF-kB activation via its interactions with downstream adapters, TRAF6 and receptor-interacting protein (RIP) 1 (Yamamoto et al. 2002a, b; Meylan et al. 2004). Ultimately, a co-ordinated activation of NF-kB and IRF-3 leads to expression of type I IFNs (Nusinzon and Horvath 2006). TLR3 was shown to associate directly with TRIF (Oshiumi et al. 2003a, b), whereas another adapter, TRIF-related adapter molecule (TRAM), also known as TICAM-2, is required for TLR4 engagement of TRIF (Fitzgerald et al. 2003; Oshiumi et al. 2003a, b; Yamamoto et al. 2003a, b). TRAM interacts with TLR4 and TRIF, but not with TLR3, and is involved in TLR4-medi-ated MyD88-independent signaling (Fitzgerald et al. 2003; Oshiumi et al. 2003a, b; Yamamoto et al. 2003a, b). TRAM is myristoylated and localized to the plasma membrane in unstimulated cells (Rowe et al. 2006). LPS triggers transient phosphorylation of TRAM by PKCe on serine-16, which results in its translocation from the membrane in a PKCe-dependent manner (McGettrick et al. 2006). Overexpression of a TRAMS16A mutant failed to activate NF-kB- and ISRE-dependent reporters and showed an impaired ability to reconstitute signaling in TRAM-deficient cells (McGettrick et al. 2006), indicating a critical role for serine-16 phosphorylation in signaling. Moreover, TRAM-dependent activation of IRF-3 and induction of RANTES were attenuated in PKCe-deficient cells (McGettrick et al. 2006), suggesting that TRAM is the target for PKCe. It is tempting to speculate that PKCe-dependent TRAM phosphorylation and translocation from the membrane into the cytoplasm enables TRAM interactions with downstream adapters (e.g., TRIF) or kinases (e.g., TBK1/IKKe). Further studies will be needed to address this hypothesis.

Interestingly, TRIF, IKKe, and TBK1 are not required for TLR7/9-mediated induction of type I IFNs, although they are necessary for TLR4-induced IFN-P expression (for a review, see Kaishi and Akira 2006). While MyD88 and TIRAP/ MAL are not involved in type I IFN induction by TLR3 or TLR4, TLR7- and TLR9-mediated activation of IFN-a requires MyD88 and involves IRAK-4/IRAK-1, TRAF-6, and IRF-7 (Heil et al. 2003; Honda et al. 2004; Uematsu et al. 2005). Recently, complex molecular pathways involved in MyD88-dependent production of inflammatory cytokines and expression of type I IFNs via TLR7/9 were revealed. MyD88 was shown to co-localize in endosomes with IRF-7, but not IRF-3, and IRF-7 can directly associate with IRAK-1 and TRAF6, resulting in type I IFN production in plasmacytoid DC (Honda et al. 2004). Interestingly, plasmacytoid DC obtained from IRF-7-KO mice exhibit a severely impaired ability to produce type I IFNs, but not other cytokines (Honda et al. 2005), indicating selective involvement of IRF-7 in type I IFN expression. Conversely, IRF-5 also associates with MyD88 and TRAF6 and is required for induction of inflammatory cytokines, but not type I IFN, in response to TLR7/9 engagement (Takaoka et al. 2005). Another IRF family member, IRF-4, also interacts with MyD88 and negatively regulates TLR7/TLR9-mediated, MyD88-dependent NF-kB and MAPK activation and production of inflammatory cytokine, while not affecting the ability of TLR7/9-stimulated pDCs to secrete IFN-a (Negishi et al. 2005). Thus, distinct members of the IRF families have important, yet distinct functions in regulation of TLR7/9-induced, MyD88-dependent signaling cascades leading to pro-inflammatory cytokine production and secretion of type I IFNs (Fig. 2). Further studies will determine how the same signaling components (e.g., MyD88-IRAK-4/IRAK-1-TRAF-6) can impart activation of both proinflammatory cytokine release and type I IFNs expression (e.g., TLR7/ TLR9), or only proinflammatory cytokine production (e.g., TLR4-mediated MyD88-dependent signaling).

Sterile a and Armadillo motifs (SARM) is the fifth putative adapter that contains a TIR domain. SARM is a 690-amino-acid protein that expresses two sterile a motif (SAM) domains and an Armadillo repeat motif (ARM) and exhibits a high degree of sequence similarity to proteins in Drosophila melanogaster and Caenorhabditis ele-gans. SAM domains can homo- and hetero-oligomerize and mediate protein-protein interactions. The Armadillo repeat mediates the interaction of P-catenin with its lig-ands and is involved in protein-protein interactions with the small GTPase Ras (for a review, see O'Neill et al. 2003). Thus, the structural organization and domain functions of SARM are consistent with its regulatory role in signaling. Of note, a nema-tode ortholog of SARM, TIR-1, was found to be critical for TLR-independent innate immunity (Couillault et al. 2004). Recently, SARM was shown to negatively regulate TRIF-mediated, MyD88-independent signaling (Carty et al. 2006).

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