Nature Structural & Molecular Biology11, 1060 - 1067 (2004)
Published online: 24 October 2004; | doi:10.1038/nsmb847
Novel roles of TLR3 tyrosine phosphorylation and PI3 kinase in double-stranded RNA signaling
Saumendra N Sarkar1, Kristi L Peters1, Christopher P Elco1, Shuji Sakamoto2, Srabani Pal1
& Ganes C Sen1
1
Department of Molecular Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio
44195, USA.
2
Department of Immunology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, Ohio
44195, USA.
Correspondence should be addressed to Ganes C Sen seng@ccf.org
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.
Viral stress-inducible genes encode proteins that mediate the host innate immune responses against virus infection. Many of these genes, such as ISG56, are induced by dsRNA, a byproduct of replication of some viruses, and interferons (IFNs)1,
2. The promoter of ISG56 contains two ISRE (interferon stimulated response element) but no B sites and its induction by poly(I)poly(C), a widely used synthetic mimic of natural dsRNA, requires the transcription factor IRF3 (refs. 2,
3,
4). We are interested in delineating the relevant signaling pathways, and recent identification of TLR3 as a functional receptor for dsRNA5 has provided an important entry point.
dsRNA signaling through TLR3 leads to the activation of at least three families of transcription factors: IRF-3, NFB and AP-1. This study is focused on the signaling pathway that leads to the activation of IRF-3 and the induction of genes such as ISG56. Important proteins in this pathway are the newly identified Ser/Thr protein kinase TBK1/IKK, which can phosphorylate and activate IRF-3 (refs. 6,7), and TRIF/TICAM-1, a TLR3-specific adaptor protein8,
9. Recently it has been shown that TRIF can directly interact with TBK1 in a ligand-independent manner10, and that the NFB pathway and the IRF-3 pathway bifurcate after TRIF11. However, it is still unclear how these proteins start the signaling cascade upon ligand stimulation, and which other proteins are essential in the complete signaling pathway.
We have recently reported12 that protein tyrosine kinase activity is required for dsRNA signaling by TLR3 because tyrosine residues in the cytoplasmic domain of TLR3 need to be phosphorylated. Here, we report that the activity of phosphatidylinositol-3 kinase (PI3K) is also essential for IRF-3−mediated gene induction by dsRNA. Specific phosphorylated tyrosine residues in the cytoplasmic domain of TLR3 help to recruit PI3K to TLR3 and activate the PI3K-Akt pathway. Our study also reveals that IRF-3 is activated by phosphorylation in two steps, one of which is mediated by the PI3K pathway.
Results Inhibition of PI3K impairs TLR3-mediated signaling The requirement of PI3K activity for the induction of ISG56 transcription by dsRNA was demonstrated by several experiments. In HEK293 cells stably expressing TLR3, dsRNA treatment for 6 h strongly induced P56 mRNA. However, when the same cells were pretreated for 30 min with PI3K inhibitor, LY294002, before treating with dsRNA, P56 mRNA was not induced (Fig. 1a). LY294002 inhibition was specific for the TLR3-mediated gene induction and did not inhibit induction of P56 mRNA by Sendai virus infection (Supplementary Fig. 1 online). The induction of P56, in response to dsRNA, is direct and not mediated by IFNs3. U3A is a cell line missing functional STAT1 and P56 mRNA cannot be induced by IFN in these cells. When TLR3 was expressed in these cells, dsRNA, but not IFN-, could induce P56 mRNA and this induction was also inhibited by LY294002 (Fig. 1b). Expression of a stably integrated luciferase reporter gene driven by the promoter of ISG56 was strongly induced by dsRNA. This induction was also blocked by LY294002 in a dose-dependent fashion, indicating that its effect was at the level of P56 mRNA transcription (Fig. 1c). Another inhibitor of PI3K, wortmannin, had a similar inhibitory effect, at much lower concentrations, on the induction of ISG56-luciferase gene expression by dsRNA treatment (Fig 1d).
Figure 1. Requirement of PI3K activity for ISG56 induction by dsRNA.
(a) Inhibition of P56 mRNA induction by LY294002. Total RNA from 293 cells expressing TLR3 was isolated from untreated cells (lane 1), dsRNA-treated cells (lane 2) and cells treated with dsRNA and 20 M LY294002 (lane 3). RNase protection assays were done to quantify P56 and actin mRNAs. (b) Induction of P56 mRNA by dsRNA in the absence of IFN signaling. P56 mRNA induction was measured as described above in U3A cells expressing TLR3. Cells were left untreated (lane 1), or were treated with IFN- (lane 2), dsRNA (lane 3) or dsRNA and 20 M LY294002 (lane 4). (c)Inhibition of ISG56-luciferase (ISG56-luc) activity by LY294002. Cell line stably transfected with ISG56-luciferase reporter construct was left untreated or treated with dsRNA for 6 h. Cells were pretreated for 30 min with the indicated concentrations of LY294002 before dsRNA treatment. Luciferase activity was measured and expressed as fold induction. (d) Inhibition of ISG56-luciferase activity by wortmannin. The same cell line as above was treated with different doses of wortmannin for 45 min before treating with dsRNA. Luciferase activity was expressed as fold induction.
The role of PI3K was tested further by expressing its constitutively active mutant or dominant-negative mutants of PI3K and its downstream target Akt (Fig. 2). When the ISG56-luciferase reporter gene was cotransfected with the expression vector of an inactive catalytic subunit (p110KD) of PI3K, its induction by dsRNA was strongly inhibited (Fig. 2a). Induction of the endogenous P56 protein was inhibited by p110KD as well (Fig. 2b). To examine whether the action of PI3K was mediated by its downstream target enzyme Akt, we measured the effect of expressing a dominant-negative mutant of Akt. Like p110KD, the Akt mutant inhibited ISG56-luciferase induction by dsRNA (Fig. 2c), indicating that both PI3K and Akt are involved in the signaling pathway. However, activation of PI3K, by itself, was not sufficient for ISG56-luciferase induction: a constitutively active form of PI3K (p110*) failed to induce the reporter gene (Fig. 2d, lanes 1 and 2). In contrast, over-expression of transfected TRIF caused robust induction of the reporter gene, which was not inhibited by either p110KD or LY294002 (Fig. 2d, lanes 3−5). The above results indicate that PI3K and Akt activity is necessary, but not sufficient, for P56 induction by dsRNA. Moreover, overexpression of a downstream component of the pathway, TRIF, can bypass the requirements of not only dsRNA but also PI3K.
Figure 2. Impairment of ISG56 induction by dominant-negative inhibitors of PI3K and Akt.
(a) Inhibition of ISG56-luciferase (ISG56-luc) reporter activity by a dominant-negative mutant of PI3K. Cells were transfected with either pcDNA3 (lanes 1 and 2) or p110KD (lanes 3 and 4) along with the ISG56-luciferase reporter construct and a puromycin-resistance plasmid. Puromycin-resistant cells were tested for luciferase activities in dsRNA-treated (lanes 2 and 4) and untreated samples (lanes 1 and 3). (b) Inhibition of endogenous P56 protein induction by a dominant-negative mutant of PI3K. 293 cells were cotransfected with TLR3 and either with pcDNA3 (lanes 1 and 2) or the dominant-negative mutant of PI3K, p110KD (lane 3). Cells were left untreated or treated with dsRNA and induced P56 protein was detected by western blotting with anti-P56. (c) Inhibition of ISG56-luciferase reporter activity by a dominant-negative mutant of Akt. Cells were transfected with either control pcDNA3 (lanes 1 and 2) or Akt dominant negative mutant expression plasmid (lanes 3 and 4); all cells also received the ISG56-luciferase reporter and the normalization control pRL-SV40. Cells were left untreated (lanes 1 and 3) or treated with dsRNA (lanes 2 and 4) for 6 h before measuring luciferase levels. Normalized luciferase levels were expressed as fold inductions. (d) Activation of ISG56-luciferase reporter by ectopic expression of TRIF, but not of constitutively active PI3K. Cells harboring the ISG56-luciferase reporter gene were transfected with lane 1, pcDNA3; lane 2, p110*, a constitutively active form of PI3K; lane 3, TRIF; lane 4, TRIF and the p110KD mutant; lane 5, TRIF along with LY treatment. In this experiment, cells were not treated with dsRNA. Luciferase activities are shown as fold induction over the level in sample 1.
PI3K association with tyrosine-phosphorylated TLR3 Given the observations that TLR3 was tyrosine-phosphorylated in response to dsRNA12 and that PI3K activity was required for gene induction, we investigated whether PI3K could interact with TLR3. Our results demonstrated that PI3K was recruited to TLR3 after ligand addition and that recruitment was dependent on the phosphorylation of a specific tyrosine residue in the cytoplasmic domain of TLR3. TLR3 tyrosine phosphorylation was clearly detected 15 min after dsRNA treatment of cells (Fig. 3a). It peaked around 1 h and started to decline at 2 h. We detected association of TLR3 with the P85 subunit of PI3K in a ligand-dependent fashion. The kinetics of TLR3-PI3K association was very similar to that of TLR3 tyrosine phosphorylation (Fig. 3b). To test the functional contribution of the TLR3-associated PI3K, we assayed for the lipid kinase activity of receptor-associated PI3K. Receptor-associated PI3K enzyme activity increased with dsRNA treatment (Fig. 3c).
Figure 3. Recruitment of PI3K to TLR3 upon ligand-dependent phosphorylation of its tyrosine residues.
(a) Ligand-dependent tyrosine phosphorylation of TLR3. Cells expressing TLR3 were treated with dsRNA for the indicated lengths of time. Lysates were immunoprecipitated with anti-phosphotyrosine (PY20) and western blotted with anti-TLR3. (b) Ligand-dependent interaction of PI3K with TLR3. 293 cells (lane 1) and TLR3-expressing cells (lanes 2−6) were treated with dsRNA for various time periods, cell lysates were immunoprecipitated with anti-FLAG (TLR3 was FLAG-tagged12) and western blotted with anti-PI3K-p85 subunit. Below is the same blot reprobed with TLR3 antibody for immunoprecipitation control. (c) Stimulation of TLR3-associated PI3K enzyme activity after dsRNA treatment of cells. TLR3 was immunoprecipitated from dsRNA-treated (60 min) or untreated cells and PI3K activity was measured. Results are averages from three independent experiments.
Role of specific tyrosines in the TIR domain The mechanism by which tyrosine-phosphorylated TLR3 associates with PI3K was investigated using cell lines expressing various mutant forms of TLR3 (Fig. 4a). In our previous study, we had screened different cytoplasmic TyrPhe mutants of TLR3 for their gene activation potentials12. The results (Fig. 4b) indicated that Tyr759 and Tyr858 are the most important tyrosines in TLR3 because a mutant (YW) that retained only two (residues 759 and 858) out of five tyrosines behaved like the wild-type protein, whereas another mutant (YM) containing Tyr733 and Tyr858 behaved like the 5F mutant, in which all five tyrosines were mutated to phenylalanine. Tyr759 was necessary, but not sufficient, for P56 mRNA induction because neither the Y759F mutant, carrying a single TyrPhe mutation at 759, nor the 4F759Y mutant, in which all the other four tyrosine residues had been mutated to phenylalanine, could support induction of ISG56 (Fig. 4b) or the gene encoding IFN- (Supplementary Fig. 2 online).
Figure 4. Specific tyrosine residues of TLR3 are required for mediation of dsRNA signaling.
(a)Expression levels of wild-type and mutant TLR3 proteins. Comparable levels of wild-type and mutant TLR3 proteins were expressed in different cell lines. Cell lysates containing 40 g of total protein for each cell line were analyzed by SDS-PAGE and western blotted with anti-TLR3. (b) Induction of P56 mRNA in cells expressing wild-type or mutant TLR3. RNase protection assays were done with RNA extracted from cell lines expressing wild-type and mutant TLR3 left untreated or treated with dsRNA for 6 h. (c) Tyrosine phosphorylation of TLR3 mutants. Cell lines expressing wild-type TLR3 (WT), all five TyrPhe mutated TLR3 (5F), three (733, 756 and 764) TyrPhe mutated TLR3 (YW), a different set of three (756, 764 and 759) TyrPhe mutated TLR3 (YM), only one (759) TyrPhe mutated TLR3 (Y759F) and only one Tyr (759) restored with four other TyrPhe mutated TLR3 (4F759Y) were treated with dsRNA (+) or left untreated (-). Cell lysates were immunoprecipitated with anti-phosphotyrosine followed by western blotting with TLR3 antibody. (d) TLR3-PI3K interaction in cells expressing wild-type or mutant TLR3. Cells were treated with dsRNA (+) or left untreated (-). Cell lysates were immunoprecipitated with anti-FLAG (TLR3) antibody and western blotted with anti-PI3K p85 subunit. Lanes are in the same order as in c. Below are the same blots reprobed with TLR3 antibody for control.
In the next series of experiments we attempted to determine the role of specific tyrosine residues in PI3K binding. Wild-type TLR3 was tyrosine-phosphorylated in response to dsRNA treatment, whereas the mutant (5F) was not (Fig. 4c). As expected, the YW mutant behaved like the wild-type protein, whereas the YM mutant behave like the 5F mutant. The Y759F and 4F759Y mutants were both tyrosine-phosphorylated in dsRNA-treated cells (Fig. 4c). As anticipated, the p85 subunit of PI3K was coimmunoprecipitated with wild-type and YW proteins, but not 5F and YM mutant proteins, indicating that the interaction is dependent on receptor phosphorylation (Fig. 4d). Notably, the Y759F mutant, although tyrosine-phosphorylated in response to dsRNA, did not bind the p85 subunit of PI3K, whereas the 4F759Y mutant did. These results indicate that the recognition of phosphorylated Tyr759 in the cytoplasmic domain of TLR3 is required for PI3K binding to TLR3.
Role of TLR3-PI3K interaction in IRF-3 activation IRF-3 is the sole transcription factor necessary and sufficient for ISG56 induction by dsRNA4. IRF-3 is activated by phosphorylation, which leads to its dimerization and nuclear translocation13,
14. In the next series of experiments, we examined these steps of IRF-3 activation in cell lines expressing TLR3 tyrosine mutants. When IRF-3 activation was monitored by examining its dimerization, as expected, IRF-3 dimers were formed in dsRNA-treated cells expressing wild-type or YW proteins, but not in those expressing 5F or YM proteins (Fig 5a). Notably, IRF-3 dimers were also present in dsRNA-treated cells expressing the Y759F mutant protein and in the LY294002-treated cells expressing wild-type protein. We next investigated whether dimeric IRF-3 translocated to the nucleus by western blotting the nuclear fractions for IRF-3. In cells expressing wild-type TLR3, the nuclear abundance of IRF-3 was strongly increased after dsRNA treatment, irrespective of LY294002 treatment of the cells (Fig. 5b, lanes 1−3). Similarly, dsRNA treatment caused nuclear translocation of IRF-3 in cells expressing theY759F or YW mutant proteins (Fig. 5b, lanes 6−7 and 10−11). There was no translocation of IRF-3 in cells expressing the 5F or YM mutant TLR3 proteins (Fig. 5b, lanes 4−5 and 8−9). The above conclusions were confirmed by visualizing the subcellular location of IRF-3 by immunofluorescence (Fig. 5c). IRF-3 was mostly cytoplasmic in untreated cells expressing the wild-type protein (Fig. 5c, panel 1), but it translocated to the nucleus after dsRNA treatment (panel 2). Similar translocation of IRF-3 after dsRNA treatment was observed in LY294002-treated cells (panel 3) or in cells expressing the Y759F mutant protein (panel 4). These results demonstrated that IRF-3 was dimerized and translocated to the nucleus, without any resultant gene induction, under conditions that blocked the activity of PI3K (LY294002) or eliminated its association with TLR3.
Figure 5. PI3K activity is not required for dimerization and nuclear translocation of IRF-3.
(a) IRF-3 dimerization in cells expressing wild-type (WT) or mutant TLR3 after dsRNA treatment. Where indicated, cells were treated with dsRNA and LY294002 for 2 h. Cell extracts were electrophoresed on nondenaturing native gels, followed by western blotting with anti-IRF-3. (IRF-3)2 denotes the position of dimeric IRF-3. (b) Nuclear translocation of IRF-3 after dsRNA treatment of cells. Nuclear fractions from treated or untreated cells were western blotted with IRF-3 antibody: untreated wild-type TLR3 cells (lane 1), wild-type TLR3 cells treated with dsRNA (lane 2), wild-type TLR3 cells pretreated with 20 M LY294002 before treating with dsRNA (lane 3), untreated 5F cells (lane 4), 5F cells treated with dsRNA (lane 5), untreated YW cells (lane 6) and YW cells treated with dsRNA (lane 7), untreated YM cells (lane 8), YM cells treated with dsRNA (lane 9), untreated Y759F cells (lane 10) and Y759F cells treated with dsRNA (lane 11). Below are the same blots reprobed with DRBP76 (a nuclear protein) antibody for loading control. (c) Visualization of IRF-3 nuclear translocation by immunofluorescence with IRF-3 antibody. Wild-type cells were untreated (panel 1), treated with dsRNA for 2 h (panel 2) or pretreated for 30 min with LY294002 before treating with dsRNA for 2 h (panel 3). Additionally, Y759F cells were treated with dsRNA for 2 h (panel 4). As indicated, phase contrast, DAPI fluorescence and IRF-3 immunofluorescence for each field are shown. Magnification bar, 40 m.
The reason nuclear IRF-3 failed to activate gene transcription was investigated further by examining the extent of its association with the coactivator CBP and the ISG56 promoter. Coimmuno- precipitation experiments with nuclear fractions of wild-type and Y759F cells revealed that dsRNA treatment of the cells induced association of IRF-3 with CBP in wild-type cells, but not in Y759F cells (Fig. 6a). Occupancy of the ISRE elements in the ISG56 promoter by IRF-3 was monitored by chromatin immunoprecipitation experiments. Proteins occupying specific DNA sites in the chromatin were crosslinked to DNA in situ, IRF-3 was immunoprecipitated and its associated DNA was used to detect the presence of the P56 promoter by PCR amplification (Fig. 6b). Quantification and normalization of the data revealed that IRF-3 in the nucleus of dsRNA-treated wild-type cells was strongly associated with the P56 promoter (Fig. 6c, lane 2), whereas little such association was observed in Y759F cells (lane 5) or in wild-type cells treated with LY (lane 3). These data demonstrate that the lack of P56 gene induction in cells with defective PI3K activity was due to the failure of nuclear IRF-3 to bind the promoter in the chromatin and the coactivator CBP.
Figure 6. Requirement of PI3K action for IRF-3 association with CBP and ISG56 promoter.
(a)IRF-3−CBP interaction. CBP was immunoprecipitated from nuclear extracts and associated IRF-3 was detected by western blotting in wild-type cells (lanes 1 and 2) and Y759F cells (lanes 3 and 4). Cells in lanes 2 and 4 were treated with dsRNA. (b) Occupancy of the ISG56 promoter by IRF-3 was assayed by chromatin immunoprecipitation assay. Top, amounts of ISG56 promoter DNA associated with IRF-3; bottom, amounts of total ISG56 promoter DNA before IRF-3 immunoprecipitation in untreated wild-type cells (lane 1), wild-type cells treated with dsRNA (lane 2), wild-type cells treated with dsRNA and LY294002 (lane 3), untreated Y759F cells (lane 4) and Y759F cells treated with dsRNA (lane 5). (c) Occupancy of the ISG56 promoter by IRF-3. The data in b were quantified and normalized with respect to the total DNA inputs. The designations of the lanes are as in b.
Phosphorylation status of IRF-3 To investigate the biochemical basis for the observed functional defect in transcriptional activity of nuclear dimeric IRF-3, we examined its phosphorylation status. Because activation of IRF-3 has been correlated with phosphorylation of Ser396 (ref. 15), we investigated whether this residue was phosphorylated in the inactive IRF-3 species. Using an antibody specific for phosphoserine 396 (pSer396) of IRF-3, we demonstrated that even incompletely activated IRF-3 contained pSer396 (Fig. 7a, top panel). Overall phosphorylation of IRF-3 was also indicated by the upward shift of the position of IRF-3 in these samples (Fig. 7a, bottom panel). This result demonstrated that Ser396 was phosphorylated by the TLR3 signaling pathway activated by dsRNA even in the absence of PI3K activation. Because IRF-3 is thought to be phosphorylated at multiple sites, we examined the complete phosphorylation status of IRF-3 by two-dimensional gel analysis, which revealed that in untreated cells IRF-3 exists as a heterogeneous population suggesting multiple and incomplete phosphorylation states (Fig. 7b, panels 1 and 4).The multiple spots collapsed into one major spot after phosphatase treatment (Fig. 7b, panels 2 and 3) confirming that they represented different phosphorylated species. The nuclear IRF-3 from dsRNA-treated cells expressing wild-type TLR3 was heterogeneous as well (Fig 7b, panel 5) but all species had lower isoelectric points (moved to the left) compared with the IRF-3 from untreated cells (panel 4). Nuclear IRF-3 isolated from dsRNA- and LY294002-treated cells expressing wild-type TLR3 had intermediate isoelectric points (panel 6), as did IRF-3 from dsRNA-treated cells expressing the Y759F mutant (panel 7). These results demonstrated that the observed incomplete activation of IRF-3 was due to its incomplete phosphorylation, and the activity of TLR3-associated PI3K was necessary for full phosphorylation of additional residue(s) of IRF-3, a step that was essential for complete functional activation of this transcription factor.
Figure 7. Incomplete phosphorylation of IRF-3 in the absence of PI3K action.
(a) Phosphorylation of Ser396 of IRF-3. Top, western blotting with IRF-3−pSer396-specific antibody of whole-cell extract of untreated wild-type cells (lane 1), and nuclear fractions of dsRNA-treated wild-type cells (lane 2), dsRNA-treated Y759F cells (lane 3) and LY294002 and dsRNA-treated wild-type cells (lane 4). Bottom, same samples probed with IRF-3 antibody. (b) Two-dimensional gel analysis of IRF-3. IRF-3 phosphorylation status was monitored by two-dimensional gel analysis of cell extracts followed by western blotting with anti-IRF-3. Samples were electrofocused under a pH gradient of 4 (left) to 7 (right). Panels 1−3 are from one experiment and panels 4−7 are from another. In the first experiment, whole-cell extracts of untreated wild-type cells (panel 1) or the whole-cell extracts of untreated wild-type cells after treatment with 100 U of calf intestine alkaline phosphatase for 4 h at 37 °C (panel 2) were compared with nuclear extracts of dsRNA-treated (2 h) wild-type cells after treatment with phosphatase (panel 3). The second experiment compared whole-cell extract of untreated wild-type cells (panel 4) with nuclear extracts of wild-type cells treated with dsRNA for 2 h (panel 5), or treated with LY294002 (30 min) followed by dsRNA (2 h) (panel 6) and nuclear extract of dsRNA-treated (2 h) Y759F cells (panel 7).
Discussion Tyrosine phosphorylation in TLR3 signaling The results presented here have revealed several new features of the signaling pathway that is triggered by the dsRNA-TLR3 interaction and leads to the induction of IRF-3−driven genes (Fig. 8). Although inhibitors of tyrosine kinases had been shown to inhibit signaling by dsRNA12,
16, the underlying mechanism was not known. Here, we have connected that observation to the requirement of phosphorylation of two specific tyrosine residues in the cytoplasmic domain of TLR3 for productive signaling. dsRNA rapidly triggers their phosphorylation, presumably by activating a latent protein tyrosine kinase that may be constitutively bound to the cytoplasmic domain of TLR3. Alternatively, it is possible that the kinase is recruited to the receptor after a dsRNA-elicited conformational change of its cytoplasmic domain. Phosphorylation of Tyr759 leads to the recruitment of PI3K to TLR3, as demonstrated by the behavior of the 4F759Y mutant (Fig. 4d), whereas phosphorylation of Tyr858 is presumably involved in TBK1 activation. It is conceivable that these interactions are indirect and mediated by unidentified adaptor proteins. It is also likely, given the information in the literature, that dynamic assembly and disassembly of a multiprotein complex containing TLR3, TBK1, IRF-3 and PI3K are essential steps in the signaling pathway17. Although the association of PI3K with TLR3 required ligand-induced tyrosine phosphorylation of the receptor, the same may not be true for all components of the signaling complex; some proteins, such as TRIF, may be constitutively bound to TLR3 (refs. 8,9). The involvement of PI3K in other TLR signaling pathways has been reported18,
19,
20. However, the mechanism of PI3K activation and its functional importance in activ-ating specific transcription factors have remained unclear.
Figure 8. Model for IRF-3 activation in two steps.
PTK is the protein tyrosine kinase responsible for TLR3 tyrosine phosphorylation. TRIF, an essential adaptor protein, is recruited to phosphorylated TLR3, but it may be bound to unactivated TLR3 as well. TBK1 recruitment through TRIF leads to phosphorylation of (IRF-3)M, the IRF-3 monomer, at Ser396, causing the formation of (IRF-3)ID, the IRF-3 inactive dimer, which can translocate to the nucleus but cannot activate gene transcription because of inefficient promoter binding and CBP interaction. (IRF-3)AD is the IRF-3 active dimer, which is formed by additional phosphorylation by the PI3K pathway. The dotted line denotes multiple steps between PI3K, Akt and (IRF-3)ID phosphorylation. ISRE is the IFN-stimulated response element in the promoter of ISG56. The discontinuous curved double line denotes the nuclear membrane.
Role of PI3K in TLR3 signaling The results presented above provide important clues to the nature of the involvement of PI3K in IRF-3 activation. On one end of the signaling pathway, recruitment of PI3K to TLR3 was shown to be essential for its action in this context. On the other end, phosphorylation of specific residues in IRF-3 seems to be the mechanism for its full activation. It is quite likely that PI3K itself does not phosphorylate IRF-3, but initiates the activation of a kinase cascade, the last member of which phosphorylates IRF-3. We can place the kinase Akt in this pathway downstream of PI3K because dsRNA activated Akt in wild-type cells, but not in 5F or Y759F cells (Supplementary Fig. 3 online), and a dominant-negative mutant of Akt blocked the induction of ISG56 transcription (Fig. 2c). However, none of the known phosphorylation sites of IRF-3 matches the putative consensus sequence of Akt targets, indicating the involvement of other unidentified kinases downstream of Akt. TBK1 activation does not require this pathway, as evidenced by its activation and the resultant partial phosphorylation, dimerization and nuclear translocation of IRF-3 after dsRNA treatment of Y759F or LY-treated wild-type cells. However, it remains possible that TBK1 itself has two activation states. In this scenario, the first state can activate IRF-3 only partially. Upon further activation through additional phosphorylation by the PI3K/Akt pathway, the more active form of TBK1 can phosphorylate additional residues of IRF-3. Assessment of TBK1 phosphorylation states in the presence and absence of the PI3K pathway would resolve this issue. In the future, PI3K ablation experiments using short interfering RNA or genetically altered mouse cells should further support our conclusion.
As constitutively active PI3K (p110*) failed to activate ISG56 gene expression (Fig. 2d), probably because the other arm of the signaling pathway, mediated by TBK1, was not activated without dsRNA treatment, we proposed in the model that activation of the PI3K pathway is necessary, but not sufficient, to mediate TLR3 signaling. TRIF overexpression bypassed the need for dsRNA and TLR3, because TRIF can activate IRF-3 even in cells not expressing TLR3 (ref. 9). We observed that when TRIF was overexpressed, the need for PI3K was obviated as well (Fig. 2d), probably because it activated a downstream kinase in the PI3K pathway directly. TRIF can also function as an adaptor molecule for TLR4, and it is required for IRF-3 activation by LPS-mediated TLR4 signaling9,
21. In that context, it has been reported that PI3K can downregulate TLR4 signaling in the NFB pathway22. This raises the possibility that activation of IRF-3 by TLR3 and TLR4 may be differentially regulated by PI3K.
Two-step phosphorylation of IRF-3 Our studies not only reveal essential roles of PI3K and TLR3 tyrosine phosphorylation in dsRNA-mediated signaling, but also reveal multiple stages of IRF-3 activation. IRF-3 phosphorylation, dimerization and nuclear translocation were previously equated with its functional activation13,
15,
23,
24. Our results clearly show that the above paradigm needs to be examined more closely. When PI3K was taken out of the signaling cascade by the TLR3 Y759F mutation or by LY294002 treatment, dsRNA could activate IRF-3 only partially through the action of the TBK1 pathway. Consistent with published results6,
15, IRF-3 activation by the TBK1 pathway caused phosphorylation of Ser396 (Fig. 7a). However, that phosphorylation was clearly not sufficient to impart transcriptional activity to IRF-3, although it was sufficient for IRF-3 dimerization and nuclear translocation. Full activation of IRF-3 required further phosphorylation by the PI3K pathway, presumably of other specific Ser/Thr residue(s). A similar two-step phosphorylation and activation model for IRF-3 has been proposed from its recently determined crystal structure25. In this study, two other potential clusters of Ser/Thr residues have been proposed to be involved in its activation: one is the Ser402-Thr404-Ser405 cluster, and the other is the Ser385-Ser386 cluster, whose mutations to alanine are known to block gene induction by IRF-3 (ref. 13). Our preliminary results with IRF-3−overexpressing cells suggest that the Ser385-Ser386 cluster could be the target of phosphorylation by the PI3K pathway (Supplementary Fig. 4 online).
Similar two-step activation of transcription factors involving PI3K has been reported for other signaling pathways. One example is STAT1, whose full activation requires phosphorylation of both Tyr701 and Ser727. Phosphorylation of the latter residue in response to IFN-, but not IL-1 or TNF-, requires the action of the PI3K/Akt pathway26. Similarly, activation of NFB by IL-1 requires the action of PI3K27. Although in the absence of PI3K activation the p65 subunit of NFB is released from IB in IL-1 treated cells, it remains transcriptionally inactive without its own phosphorylation, which requires PI3K, Akt and IKK28.
Results obtained from biochemical analysis of IRF-3 (Fig. 6) clearly show the nature of the functional defects of partially phosphorylated IRF-3. Although present in the nucleus, it failed to stably bind to the ISRE element of the P56 promoter. This failure was most probably due to the lack of formation of a stable transcription- initiation complex through the interaction of IRF-3 with other components of this complex. Indeed, the partially phosphorylated form of IRF-3 did not coimmunoprecipitate with CBP, a transcriptional coactivator, whereas the fully phosphorylated form did. It has been shown that even when bound to a promoter, IRF-3 cannot serve as a transcription factor unless it is activated by phosphorylation16. Thus it is apparent that PI3K pathway−mediated phosphorylation of IRF-3 is essential for its interaction with other components of the transcriptional initiation machinery and the resultant induction of transcription of target genes.
Methods Cells, reagents and plasmids. HEK293 and its derivative cell lines were maintained in high-glucose DMEM supplemented with 10% (v/v) heat- inactivated FCS (v/v), 2 mM l-glutamine, 50 units ml-1 penicillin, 50 g ml-1 streptomycin and 400 g ml-1 G418 where appropriate. Development of the 293 wild-type TLR3 and 293 mutant TLR3 cell lines has been described12. The 293 TLR3 cell line stably expressing ISG56-luciferase was isolated by cotransfecting the parental line with the pGL3Basic ISG56-luciferase reporter and pBabePuro selection plasmids and selecting for puromycin (1 g ml-1) resistant clones. Clones were screened for optimal luciferase inducibility by dsRNA. U3A-TLR3 cells were generated by transfecting cells with the TLR-3 expression plasmid12 and selecting for neomycin-resistant clones. Individual clones were expanded and screened for TLR3 expression by western analysis. The PI3K inhibitor LY294002 was purchased from Alexis Biochemicals and Wortmannin was obtained from Calbiochem. Human anti-IRF-3 and IRF-3 pSer396-specific polyclonal antibodies were gifts from M. David29 (University of California, San Diego) and J. Hiscott15 (McGill University). The expression plasmids for constitutively active (p110*) and kinase dead (p110KD) PI3K catalytic subunit were provided by D. Cantrell30 (University of Dundee). The expression plasmids for AktKD and TRIF were obtained from J. Downward31 (London Research Institute) and X. Li11 (Cleveland Clinic Foundation), respectively.
Expression assays. Preparation and treatment of cells with dsRNA (100 g ml-1 poly(I)poly(C)) has been described4. Luciferase assays of transiently transfected cells were done as described12. Constitutively active TRIF and p110 were transfected 6 h before the cells were harvested for the luciferase assays. Ribonuclease protection assays (RPA) were done using the Ambion RPA III kit. RPA probes have been described4.
TLR3 coimmunoprecipitation assays. For TLR3 coimmunoprecipitations, 2 107 cells were lysed in buffer containing 150 mM NaCl, 20 mM HEPES, pH 7.4, 1.5 mM MgCl2, 2 mM DTT, 0.5% (v/v) Triton X100, 2 mM EGTA, 10 mM NaF, 12.5 mM -glycerophosphate, 1 mM Na3VO4 and 1 protease inhibitor (complete EDTA-free protease inhibitor tablets, Roche Molecular Biochemicals). Extracts containing 5 mg of protein were immunoprecipitated overnight with 13 l of anti-FLAG M2 agarose beads in 1 ml volume. Beads were washed five times with the lysis buffer, boiled in 1 SDS-PAGE loading buffer, separated by SDS-PAGE and a western blot was done with an antibody against the p85 subunit of PI3K (Upstate Biotechnology). For immunoprecipitation with the PY20 antibody, lysates were precleared for 2 h with 7 l protein G Sepharose (prewashed and resuspended in the lysis buffer at 1:1). Precleared lysates were incubated for 1 h with 1 g PY20 antibody (Pharmingen), followed by incubation with 7 l protein G Sepharose overnight. After incubation, beads were washed and the tyrosine phosphorylation status of TLR3 was analyzed by immunoblotting with a TLR3 antibody (Biocarta).
PI3 kinase assay. After treatment with dsRNA for 1 h, cells were lysed in RIPA buffer supplemented with 25 mM NaF, 0.4 mM PMSF, 0.5 mM Na3VO4, 10 g ml-1 pepstatin A, 10 g ml-1 aprotinin and 10 g ml-1 leupeptin. Following the above procedure for TLR3 immunoprecipitation, beads were washed three times with RIPA lysis buffer followed by three washes with kinase buffer (20 mM MOPS, pH 7.6, 200 mM sucrose, 5 mM MgCl2, 1 mM EDTA, with the phosphatase and protease inhibitors listed above). Immune complex kinase reactions were carried out by incubating the beads with 50 Ci [-32P] ATP, 10 M cold ATP and 20 g phosphoinositide (Sigma) at 30 °C for 20 min. The reaction was stopped by adding chloroform/methanol/HCl (50:100:1), and phosphoinositides were isolated by extraction with chloroform. Products were analyzed by TLC (Silica gel H with 1% (w/v) potassium oxalate) using a chloroform/methanol/3.3 N ammonium hydroxide (43:38:12) buffer. Phosphorylated, radiolabeled phosphoinositides were visualized and quantified with a Molecular Dynamics phosphorimager.
IRF-3 activation assays. IRF-3 dimerization was analyzed by native PAGE and immunoblotting with IRF-3 antibody32. Cells were lysed in a buffer containing 75 mM NaCl, 50 mM Tris-Cl, pH 7.5, 1 mM EDTA, 1% (v/v) NP40, protease and phosphatase inhibitors as described above. Protein (15 mg) for each sample was mixed with 2¥ loading buffer (0.125 mM Tris-Cl, pH 6.8, 20% (v/v) glycerol and 0.1 mg ml-1 bromophenol blue) and electrophoresed by native PAGE.
Detection of nuclear translocation of IRF-3 by immunofluorescence has been described4. IRF-3 translocation was also monitored by measuring IRF-3 levels in the nuclear fractions of cells. For this, 2 ¥ 106 cells were washed and collected in PBS. Nuclei were isolated as described33 with the exception that nuclei were pelleted by centrifugation at 1,000g for 5 min at 4 °C. After the wash in glycerol storage buffer (50 mM Tris-Cl, pH 8.3, 5 mM MgCl2, 0.1 mM EDTA and 40% (v/v) glycerol), nuclei were lysed in the appropriate buffer for further analysis.
CBP−IRF-3 interactions were examined by coimmunoprecipitation. After treatment, nuclei were isolated as described above and lysed in NP40 lysis buffer (20 mM Tris-Cl, pH 8.0, 10% (v/v) glycerol, 100 mM KCl, 1 mM EDTA, 5 mM MgCl2, 1 mM PMSF, 1 mM Na3VO4, 1 mM b-glycerophosphate, 1 mM DTT and 5 mg ml-1 each of aprotinin, leupeptin and pepstatin). Lysates were sonicated and clarified by centrifugation. Nuclear extract (250 mg) was immunoprecipitated with 2 mg CBP antibody and 15 ml Protein A/G Plus (both from Santa Cruz Biotechnology) overnight at 4 °C. Lysates were washed with NP40 lysis buffer, separated by SDS-PAGE and transferred to PVDF. A western blot was done to detect coimmunoprecipitated IRF-3.
Chromatin immunoprecipitation assays were based on the protocol accompanying the Upstate ChIP assay kit. Briefly, cells were left untreated or treated with dsRNA for 4 h. Where indicated, LY pretreatment was for 30 min. Chromatin and protein were crosslinked with formaldehyde, and cells were washed and subsequently lysed in SDS lysis buffer. Samples were sonicated, clarified by centrifugation and precleared with salmon sperm DNA and protein A agarose. IRF-3 was immunoprecipitated from 200 mg of lysate. Immunoprecipitates were washed consecutively with low-salt buffer, high-salt buffer, LiCl wash buffer and 1¥ TE. The protein−DNA complexes were eluted from the beads, and the crosslinks were reversed. The protein was digested with proteinase K, and the DNA was isolated by phenol-chloroform extraction. PCR reactions were carried out with primers specific for the gene encoding the P56 promoter where the reactions were spiked with [a-32P]dCTP. The samples were resolved on a gel, and radioactive PCR products were visualized and quantified using a Molecular Dynamics phosphorimager.
Two-dimensional gel analysis of IRF-3 was done on either the whole-cell lysate (untreated) or nuclear fractions (dsRNA treated). Nuclei isolated from treated cells were lysed in 750 l rehydration buffer (8 M urea, 2% (w/v) CHAPS, trace bromophenol blue). Alternatively, untreated cells were washed twice with PBS and directly lysed in 1−2 ml rehydration buffer. Cell lysates were sonicated for 5 s, and debris was removed by centrifugation at 13,000 r.p.m. (16,000 g) for 10 min. A western blot was done to determine the relative amount of IRF-3 in each sample. Lysates containing equivalent amounts of IRF-3 were separated in the first dimension by isoelectric focusing on Immobiline dry strips, pH 4−7 (Amersham). Focused proteins were separated in the second dimension by SDS-PAGE. The gels were transferred to PVDF and western blotted with an IRF-3 antibody.
Received 22 March 2004; Accepted 8 September 2004; Published online: 24 October 2004.
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Acknowledgments 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.
Competing interests statement:
The authors declare that they have no competing financial interests.
B sites and its induction by poly(I)
poly(C), a widely used synthetic mimic of natural dsRNA, requires the transcription factor IRF3 (refs. 
, which can phosphorylate and activate IRF-3 (refs. 
, could induce P56 mRNA and this induction was also inhibited by LY294002 (
M LY294002 (lane 3). RNase protection assays were done to quantify P56 and actin mRNAs. (b) Induction of P56 mRNA by dsRNA in the absence of IFN signaling. P56 mRNA induction was measured as described above in U3A cells expressing TLR3. Cells were left untreated (lane 1), or were treated with IFN-
Phe mutants of TLR3 for their gene activation potentials
, but not IL-1 or TNF-
, requires the action of the PI3K/Akt pathway
107 cells were lysed in buffer containing 150 mM NaCl, 20 mM HEPES, pH 7.4, 1.5 mM MgCl2, 2 mM DTT, 0.5% (v/v) Triton X100, 2 mM EGTA, 10 mM NaF, 12.5 mM 
