Induction of ISGs by TLR3 Signaling

TLR3 is present mostly on the endosomal membrane, although in some cell types its presence on the plasma membrane has been noted (Matsumoto et al. 2003). Its ectodomain specifically recognizes endosomal dsRNA through ionic interactions between the negatively charged ligand and positively charged amino acid residues present on both sides of a canyon in which the dsRNA perfectly fits (Bell et al. 2005; Choe et al. 2005; Bell et al. 2006). Extracellular dsRNA has to be endocytosed to reach TLR3, as revealed by the chloroquine sensitivity of the process (de Bouteiller et al. 2005). Because many viruses enter the cell through endocytosis, their genomic RNAs may encounter TLR3 in the endosome. The first step in TLR3 signaling is its dimerization, which presumably leads to a conformational change of its cytoplasmic domain to initiate the signaling process (Fig. 2).

The most novel feature of TLR3 signaling is the need for receptor Tyr-phos-phorylation (Sarkar et al. 2003, 2004). Although phosphorylation of specific Tyr residues located in the cytoplasmic domains of receptors for growth factors and cytokines is quite common, this feature is unique for TLR3 among the Toll-like receptors. There are five Tyr residues in the cytoplasmic domain of human TLR3 and several of these residues, if not all, are phosphorylated at the beginning of the signaling process. The functional roles of these residues have been assessed by mutating them, individually or in combinations. At least two of the five are essential for signaling; one of them has to be Tyr759, the other one can be Tyr858 or Tyr733. Tyr-phosphorylation of TLR3 is a ligand-depen-dent process, but the responsible protein kinase has not yet been identified. Tyr759 or 858, after phosphorylation, can recruit the signaling complex. The main adaptor protein is TRIF, but TRAF3 is needed as well (Hacker et al. 2006; Oganesyan et al. 2006). The different branches of signaling bifurcate from TRIF (Jiang et al. 2004). A complex containing TRAF6, TAB1, TAB2, and TAK1 activates the protein kinases JNK, P38, and IKK (Jiang et al. 2003). Another adaptor, RIP-1, is also recruited by TRIF and it is required for NFkB activation (Meylan et al. 2004). JNK, P38, and IKK activate the transcription factors c-Jun, ATF2, and NFkB, respectively. A separate branch of signaling originating from TRIF is triggered by the recruitment of the protein kinases TBK1 or IKKe,

Pathway Neutrophils

ISRE

Fig. 2 Signaling pathways activated by viruses and dsRNA. Depending on cell type, viral dsRNA can signal either through Toll-like receptor 3 or RNA helicases: RIG-I/mda-5. Through different sets of adaptors, the signal causes activation of two major transcription factors, IRF-3 and NF-kB, followed by induction of specific sets of genes

ISRE

Fig. 2 Signaling pathways activated by viruses and dsRNA. Depending on cell type, viral dsRNA can signal either through Toll-like receptor 3 or RNA helicases: RIG-I/mda-5. Through different sets of adaptors, the signal causes activation of two major transcription factors, IRF-3 and NF-kB, followed by induction of specific sets of genes which directly phosphorylate IRF-3 (Fitzgerald et al. 2003; Sharma et al. 2003). IRF-3 phosphorylation leads to its dimerization and translocation to the nucleus where it binds to the ISRE sites in the promoters of the target genes and induces their transcription. The histone deacetylase, HDAC6, is required for IRF-3 to function as a transcription factor (Nusinzon and Horvath 2006).

As mentioned above, Tyr759 of TLR3 is absolutely needed for complete signaling by this receptor. When this residue is mutated to Phe, NFkB- and IRF-3-driven genes are not induced by dsRNA. Surprisingly, in dsRNA-treated cells expressing the mutant receptor, NFkB is released from IkB and translocated to the nucleus but it does not drive gene transcription (our unpublished observation); similarly, IRF-3 is dimerized and translocated to the nucleus but it is transcriptionally inactive (Sarkar et al. 2004). Investigation of the underlying molecular mechanisms has revealed that the activation of both transcription factors, IRF-3 and NFkB, is a two-step process and the second step is defective in cells expressing the Y759F mutant of TLR3. Phosphorylated Tyr759 recruits PI3 kinase, probably indirectly, to the TLR3 complex, PI3 kinase is activated, it phosphorylates Akt, which leads to additional phosphorylation of the TBK1-activated IRF-3. The tyrosine kinase, Src, which is known to be activated by Akt, may be a participant in this pathway, because Src is activated by TLR3 signaling and its presence is needed for gene induction by TLR3 (our unpublished observation). As expected from the above description, inhibitors of PI3 kinase, Akt and Src, both block IRF-3-mediated gene induction by TLR3. They have the same effect as the Tyr759 mutation of TLR3, namely incomplete phosphorylation of IRF-3, as revealed by two-dimensional gel analysis of nuclear IRF-3 isolated from dsRNA-treated cells expressing Y759F TLR3. Chromatin immu-noprecipitation assays demonstrate that unlike fully phosphorylated IRF-3, underphosphorylated IRF-3 cannot bind tightly to the promoter and interact with co-activators, such as CBP (Sarkar et al. 2004).

In the NFkB pathways, the first step of activation is mediated by the phosphorylation of IkB by the IKK complex and the consequent release of NFkB and its translocation to the nucleus. TLR3 Tyr759 is not required for the above process or for the phosphorylation of NFkB P65 protein in Ser276 and Ser536 resides. However, it is required for additional phosphorylation of P65 as revealed by two-dimensional gel analysis of nuclear P65. Underphosphory-lated P65 cannot bind to the promoters of the target genes tightly and drive their transcription. Surprisingly, the PI3 kinase pathway is not required for the second step of P65 phosphorylation (our unpublished observation). The above studies highlight the two-step nature of the activation of both IRF-3 and NFkB, although the details are different. The first step is initiated by the phosphory-lation of Tyr858 of TLR3, leading to the release of NFkB from IkB and the dimerization of IRF-3 as a result of its partial phosphorylation. The second step is initiated by the phosphotyrosine 759 of TLR3. It leads to further phosphorylation of IRF3 and its full activation and complete phosphorylation of NFkB P65 and its full activation.

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