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Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling

Abstract

Double-stranded RNA (dsRNA), a frequent byproduct of virus infection, is recognized by Toll-like receptor 3 (TLR3) to mediate innate immune response to virus infection. TLR3 signaling activates the transcription factor IRF-3 by its Ser/Thr phosphorylation, accompanied by its dimerization and nuclear translocation. It has been reported that the Ser/Thr kinase TBK-1 is essential for TLR3-mediated activation and phosphorylation of IRF-3. Here we report that dsRNA-activated phosphorylation of two specific tyrosine residues of TLR3 is essential for initiating two distinct signaling pathways. One involves activation of TBK-1 and the other recruits and activates PI3 kinase and the downstream kinase, Akt, leading to full phosphorylation and activation of IRF-3. When PI3 kinase is not recruited to TLR3 or its activity is blocked, IRF-3 is only partially phosphorylated and fails to bind the promoter of the target gene in dsRNA-treated cells. Thus, the PI3K-Akt pathway plays an essential role in TLR3-mediated gene induction.

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Figure 1: Requirement of PI3K activity for ISG56 induction by dsRNA.
Figure 2: Impairment of ISG56 induction by dominant-negative inhibitors of PI3K and Akt.
Figure 3: Recruitment of PI3K to TLR3 upon ligand-dependent phosphorylation of its tyrosine residues.
Figure 4: Specific tyrosine residues of TLR3 are required for mediation of dsRNA signaling.
Figure 5: PI3K activity is not required for dimerization and nuclear translocation of IRF-3.
Figure 6: Requirement of PI3K action for IRF-3 association with CBP and ISG56 promoter.
Figure 7: Incomplete phosphorylation of IRF-3 in the absence of PI3K action.
Figure 8: Model for IRF-3 activation in two steps.

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Acknowledgements

We are grateful to J. Hiscott for providing the pSer396-specific antibody. We thank A. Larner, G. Stark, X. Li, B. Williams, D. Cantrell, T. Fujita and M. David for important reagents and helpful discussions and H. Smith and T. Rowe for technical assistance. This study was supported in part by US National Institutes of Health grants CA62220 and CA68782.

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Correspondence to Ganes C Sen.

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Supplementary information

Supplementary Fig. 1

PI3K-dependence of P56 mRNA induction by Sendai virus or double-stranded RNA. (PDF 632 kb)

Supplementary Fig. 2

Induction of IFNβ mRNA in wild-type and Y759F cell lines. (PDF 574 kb)

Supplementary Fig. 3

Phosphorylation status of Akt in Y759F cells. (PDF 594 kb)

Supplementary Fig. 4

Phosphorylation of Ser386 of IRF-3. (PDF 701 kb)

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Sarkar, S., Peters, K., Elco, C. et al. Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling. Nat Struct Mol Biol 11, 1060–1067 (2004). https://doi.org/10.1038/nsmb847

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