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The Journal of Immunology, 2006, 176: 1072-1080.
Copyright © 2006 by The American Association of Immunologists

The Ret Finger Protein Inhibits Signaling Mediated by the Noncanonical and Canonical I{kappa}B Kinase Family Members1

Jikun Zha*, Ke-Jun Han{dagger}, Liang-Guo Xu{dagger}, Wei He*, Qianhe Zhou*, Danying Chen*, Zhonghe Zhai* and Hong-Bing Shu2,{ddagger}

* Department of Cell Biology and Genetics, College of Life Sciences, Peking University, Beijing, China; {dagger} Department of Immunology, National Jewish Medical and Research Center, Denver, CO 80206; and {ddagger} College of Life Sciences, Wuhan University, Wuhan, China


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
IFN regulatory factor-3 is a transcription factor that is required for the rapid induction of type I IFNs in the innate antiviral response. Two noncanonical I{kappa}B kinase (IKK) family members, IKK{epsilon} and TRAF family-associated NF-{kappa}B activator-binding kinase-1, have been shown to phosphorylate IFN regulatory factor-3 and are critically involved in virus-triggered and TLR3-mediated signaling leading to induction of type I IFNs. In yeast two-hybrid screens for potential IKK{epsilon}-interacting proteins, we identified Ret finger protein (RFP) as an IKK{epsilon}-interacting protein. Coimmunoprecipitation experiments indicated that RFP interacted with IKK{epsilon} and TRAF family-associated NF-{kappa}B activator-binding kinase-1 as well as the two canonical IKK family members, IKK{beta} and IKK{alpha}. RFP inhibited activation of the IFN-stimulated response element and/or NF-{kappa}B mediated by the IKK family members and triggered by TNF, IL-1, polyinosinic-polycytidylic acid (ligand for TLR3), and viral infection. Moreover, knockdown of RFP expression by RNA interference-enhanced activation of IFN-stimulated response element and/or NF-{kappa}B triggered by polyinosinic-polycytidylic acid, TNF, and IL-1. Taken together, our findings suggest that RFP negatively regulates signaling involved in the antiviral response and inflammation by targeting the IKKs.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Induction of type I IFNs after viral infection is critically involved in the innate immune response against viruses (1, 2, 3). Two distinct pathways for activation of innate immune responses by viral infection have been proposed. First, viruses enter cells by membrane fusion at the plasma membrane or through an endocytic process, leading to the release of viral nucleocapsids (or ribonucleoproteins) into the cytoplasm. The viral dsRNA accumulated during viral replication triggers TLR3-independent signaling, leading to the induction of type I IFNs (4, 5). Second, at a late stage of infection, viral-derived dsRNA is released into the extracellular environment due to lysis of infected cells. The released dsRNA binds to TLR3 and triggers signaling, leading to the induction of type I IFNs (6). However, TLR3 is not required for the initial, cell-autonomous recognition of viral infection that induces the first wave of type I IFN production (3, 7, 8).

Transcriptional induction of type I IFN genes requires coordinate and cooperative assembly of multiple transcription factors, including NF-{kappa}B and IFN regulatory factor-3 (IRF-3),3 into transcriptional enhancer complexes in vivo (9, 10). For example, the enhancer of the IFN-{beta} gene contains a {kappa}B site recognized by NF-{kappa}B, a site for ATF-2/c-Jun, and two IFN-stimulated response elements (ISREs) recognized by phosphorylated IRF-3 and/or IRF-7 (9, 10). It has been shown that transcriptional activation of the IFN-{beta} gene requires coordinate and cooperative assembly of an enhanceosome that contains all these transcription factors (9, 10).

Until recently, the molecular events responsible for TLR3- and virus-induced NF-{kappa}B and IRF-3 activation and subsequent transcription of type I IFN genes are largely undefined. However, during the past couple of years major breakthroughs have been achieved with the identification of kinases that phosphorylate IRF-3. It has been shown that two noncanonical I{kappa}B kinase (IKK) family members, TRAF family-associated NF-{kappa}B activator (TANK)-binding kinase-1 (TBK1) and IKK{epsilon}, are critically involved in antiviral responses. Both TBK1 and IKK{epsilon} can phosphorylate IRF-3 and IRF-7 and are required for virus-triggered TLR3-independent production of type I IFNs (11, 12, 13, 14, 15). Both TBK1 and IKK{epsilon} are also associated with TLR3-interacting factor (TRIF), a TIR domain-containing protein that is associated with TLR3 (16, 17, 18, 19, 20). It has been shown that TBK1 and IKK{epsilon} are also involved in TLR3-mediated IFN signaling.

Although the two noncanonical IKK family members phosphorylate IRF-3, the two canonical IKK family members, IKK{alpha} and IKK{beta}, phosphorylate I{kappa}B{alpha}, an inhibitor of NF-{kappa}B (21, 22). Phosphorylation of I{kappa}B{alpha} leads to its degradation by the ubiquitin-proteasome pathways and subsequent activation of NF-{kappa}B. It has been shown that IKK{alpha} and IKK{beta} play important roles in NF-{kappa}B activation triggered by a wide range of stimuli, such as the proinflammatory cytokines, TNF and IL-1, and ligands for the TLRs (21, 22).

Because IKK family members play important roles in inflammatory and innate immune responses, we investigated whether these kinases are regulated by interactions with other proteins. In this report we performed yeast two-hybrid screens for potential IKK{epsilon}-interacting proteins. This effort identified RFP, which is a Ret finger domain-containing protein and a member of the tripartite motif family. In addition to IKK{epsilon}, RFP interacted with TBK1, IKK{alpha}, and IKK{beta}. Our findings suggest that RFP negatively regulates ISRE and NF-{kappa}B activation triggered by several stimuli through its interactions with the noncanonical and canonical IKK family members.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Reagents

Mouse monoclonal and rabbit polyclonal Abs against Flag and hemagglutinin (HA) epitopes (Sigma-Aldrich), rabbit polyclonal Abs against IRF-3 (Santa Cruz Biotechnology), Texas Red-conjugated Affinipure goat anti-mouse IgG and FITC-conjugated Affinipure goat anti-rabbit IgG (Molecular Probes), human rTNF and IL-1 (R&D Systems), polyinosinic-polycytidylic acid (poly(I:C); Amersham Biosciences), and Sendai virus (American Type Culture Collection) were purchased from the indicated manufacturers. The rabbit anti-RFP antiserum was raised against human rRFP peptide 225–519. The mouse anti-IKK{epsilon} antiserum was raised against full-length human rIKK{epsilon} protein.

Yeast two-hybrid screening

To construct an IKK{epsilon} bait vector, a cDNA fragment encoding full-length IKK{epsilon} was inserted in-frame into the Gal4 DNA-binding domain vector pGBT (BD Clontech). The human B cell cDNA library (American Type Culture Collection) was screened as previously described (23).

Constructs

Mammalian expression vector pcDNA3-Flag-IKK{epsilon} was provided by Dr. T. Maniatis (Cambridge, MA). Mammalian expression plasmid for HA-tagged RFP and its mutants was constructed by PCR amplification of the corresponding cDNA fragments from a human B cell cDNA library and subsequently cloned into a CMV promoter-based vector containing an N-terminal HA tag. Mammalian expression plasmids for Flag-IKK{alpha}, Flag-IKK{beta}, Flag-TBK1, Flag-protein kinase A (Flag-PKAc), Flag-TRIF, and Flag-IRF3/5D were previously described (19, 23, 24, 25).

ISRE, c-Jun, and CREB luciferase reporter plasmids were purchased from Stratagene. NF-{kappa}B luciferase reporter plasmid was provided by Dr. G. Johnson (Chapel Hill, NC). The IFN-{beta}-luciferase reporter plasmid was previously described (19).

Cell transfection and reporter gene assays

293 cells (~1 x 105) were seeded on 12-well dishes and transfected the following day by standard calcium phosphate precipitation. Where necessary, empty control plasmid was added to ensure that each transfection received the same amount of total DNA. To normalize for transfection efficiency, 0.1 µg of thymidine kinase (TK)-Renilla luciferase reporter plasmid was added to each transfection. Approximately 16 h after transfection, dual-specific luciferase reporter gene assays were performed using a luciferase assay kit (Promega). Firefly luciferase activities were normalized based on the activities of Renilla luciferase.

Coimmunoprecipitation and Western blot analysis

For transient transfection and coimmunoprecipitation experiments, 293 cells (~1 x 106) were transfected for 24 h. Transfected cells were lysed in 1 ml of lysis buffer (20 mM Tris (pH 7.5), 150 mM NaCl, 1% Triton, 1 mM EDTA, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 1 mM PMSF). For each immunoprecipitation, 0.4-ml aliquots of lysates were incubated with 0.5 µg of the indicated Ab or control IgG and 25 µl of a 1/1 slurry of GammaBind G Plus-Sepharose (Amersham Biosciences) for 2 h. The Sepharose beads were washed three times with 1 ml of lysis buffer containing 500 mM NaCl. The precipitates were fractionated on SDS-PAGE, and Western blot analyses were performed as previously described (19).

In vitro kinase assays

Cell transfection and immunoprecipitation were conducted as described above. The immunoprecipitates were washed twice with kinase buffer (25 mM Tris, 5 mM {beta}-glycerophosphate, 2 mM DTT, 0.1 mM Na3VO4, and 10 mM MgCl2 (pH 7.5)) and then incubated in 30 µl of kinase buffer plus 100 µM ATP and 10 µCi of [{gamma}-32P]ATP for 30 min at 30°C. The beads were pelleted by centrifugation and washed once with kinase buffer. The proteins were fractionated by SDS-PAGE. The gel was then dried and subjected to autoradiography.

Immunofluorescent staining

293 cells cultured on glass coverslips were plunged sequentially into methanol and acetone at –20°C, each for 10 min. The cells were rehydrated in PBS, blocked with 1% BSA in PBS for 15 min, and stained with primary Ab (2 µg/ml) in blocking buffer for 1 h at room temperature. The cells were rinsed with PBS and stained with Texas Red-conjugated Affinipure goat anti-mouse IgG (1/200 dilution) or FITC-conjugated Affinipure goat anti-rabbit IgG (1/200 dilution) for 45 min at room temperature. The cells were then rinsed with PBS containing Hercus, mounted in Prolong Antifade (Molecular Probes), and observed with a Leica DMR/XA immunofluorescent microscope using a x100 plan objective.

Sendai virus infection

293 cells (~1 x 105) were seeded in 12-well dishes and transfected the next day with 0.25 µg of ISRE reporter plasmid or 0.25 µg of NF-{kappa}B reporter plasmid together with 0.25 µg of Rous sarcoma virus-{beta}-galactosidase reporter plasmid. Eighteen hours after transfection, cells were washed with medium lacking FCS (washing medium) and then overlaid with washing medium containing Sendai virus. After incubation at 37°C for 60 min, nonadsorbed viruses were removed by repeated washing of the cells. Cells were then cultured in FCS-containing medium for 18 h before reporter gene assays were performed.

RNA interference (RNAi) experiments

Oligonucleotides were cloned into the pSuper.Retro (Oligoengine) plasmid. The RFP RNAi target sequence is GACTCAGTGTGCAGAAAAG.

Detection of IRF-3 dimer by native PAGE

Cells were suspended in lysis buffer (50 mM Tris-HCl (pH 8.0), 150 mM NaCl, 1% Nonidet P-40, 1 mM PMSF, and 1 mM Na3VO4). Cell lysate was cleared by centrifugation. The lysate (10 µg) was fractionated on 7.5% native PAGE, and Western blot analysis was performed with anti-IRF-3 Ab.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
RFP interacts with noncanonical and canonical IKK family proteins

To identify potential IKK{epsilon}-interacting proteins, we used the yeast two-hybrid system to screen a human B cell cDNA library with full-length IKK{epsilon} as bait. We screened a total of ~2 x 106 independent clones and obtained 15{beta}-galactosidase-positive clones. Two of the clones encode the C-terminal fragment (aa 225–513) of RFP.

To determine whether full-length RFP interacts with IKK{epsilon} in mammalian cells, we transfected 293 cells with expression plasmids for HA-tagged RFP and Flag-tagged IKK{epsilon} and performed coimmunoprecipitation experiments. The results indicated that RFP could interact with IKK{epsilon} (Fig. 1A). In the same experiments we found that RFP could interact with TBK1 as well as with IKK{beta} and IKK{alpha} (Fig. 1A). RFP did not interact with PKAc, an unrelated kinase (Fig. 1A). These data suggest that RFP specifically interacts with the noncanonical and canonical IKK proteins.



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  		FIGURE 1. RFP interacts with noncanonical and canonical IKK family members. A, RFP interacts with IKKs, but not PKAc. 293 cells were transfected with the indicated plasmids. Coimmunoprecipitation was performed with anti-HA Ab or control IgG (C). The immunoprecipitates were analyzed by Western blots with anti-Flag Ab (upper panel). The expression levels of transfected proteins in the lysates were analyzed by Western blot analysis with anti-HA and anti-Flag Abs (lower panel). B, RFP colocalizes with IKK{epsilon}. 293 cells were transfected with an expression plasmid for HA-RFP (upper panels) or together with an expression plasmid for Flag-IKK{epsilon} (lower panels). Immunofluorescent stainings were performed with the indicated Abs and 4',6'-diamidino-2-phenylindole. C, Endogenous interaction between RFP and IKK{epsilon}. 293-TLR3 cells (~3 x 107) were treated with poly(I:C) (100 µg/ml) or left untreated for 5 min. The coimmunoprecipitation experiment was performed with rabbit anti-RFP antiserum or preimmune serum. The immunoprecipitates were analyzed by Western blot with mouse anti-IKK{epsilon} antiserum (left panel). The endogenous expression levels of RFP and IKK{epsilon} were detected by Western blot analysis of the lysates with rabbit anti-RFP or mouse anti-IKK{epsilon} antisera, respectively (right panels).

 
Previous reports suggest that RFP localizes in either the cytoplasm or the nucleus in different cell types (26, 27, 28, 29). We transfected 293 cells with HA-tagged RFP and performed immunofluorescent staining with anti-HA mAb. The results indicated that overexpressed RFP was mostly located in the cytoplasm, and only a small fraction was localized in the nucleus in 293 cells (Fig. 1B). To determine whether RFP colocalizes with IKK{epsilon}, we transfected 293 cells with HA-tagged RFP and Flag-tagged IKK{epsilon} and performed double-immunofluorescent staining with anti-HA and anti-Flag Abs. The results indicated that RFP was colocalized with IKK{epsilon} in the cytoplasm (Fig. 1B).

To determine whether endogenous RFP associates with endogenous IKK{epsilon}, we raised rabbit anti-RFP and mouse anti-IKK{epsilon} Abs and performed coimmunoprecipitation experiments. 293-TLR3 cells treated with poly(I:C) or left untreated for 5 min were lysed, and the lysates were immunoprecipitated with rabbit anti-RFP antiserum. The immunoprecipitates were analyzed by Western blot with mouse anti-IKK{epsilon} antiserum. The results indicated that the association of endogenous RFP with IKK{epsilon} was poly(I:C) dependent (Fig. 1C).

RFP is a substrate of IKKs

Because RFP interacts with IKKs, we determined whether RFP is a substrate for IKKs. We transfected expression vectors for HA-tagged RFP together with empty vector, Flag-IKK{alpha}, Flag-IKK{beta}, Flag-IKK{epsilon}, or Flag-TBK1 into 293 cells and performed in vitro kinase assays. The results indicated that RFP was strongly phosphorylated by IKK{epsilon} and TBK1, but only weakly phosphorylated by IKK{alpha} and IKK{beta} (Fig. 2).



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  		FIGURE 2. RFP is a substrate of the IKKs. 293 cells (~1 x 106) were cotransfected with expression plasmids for HA-tagged RFP and Flag-tagged IKK{alpha}, IKK{beta}, IKK{epsilon}, and TBK1 as indicated. The cell lysates were immunoprecipitated with anti-HA Ab, and the immunoprecipitates were subjected to in vitro kinase assays and autoradiography. The expression levels of the transfected proteins were analyzed by Western blot with anti-HA and anti-Flag Abs (lower panel).

 
RFP inhibits NF-{kappa}B and ISRE activation mediated by overexpression of IKKs

Because RFP interacts with IKKs, we determined the effects of RFP on signaling mediated by IKKs. Previously, it has been shown that IKK{epsilon}, TBK1, IKK{alpha}, and IKK{beta} can activate NF-{kappa}B, whereas IKK{epsilon} and TBK1 can activate ISRE (11, 12, 13, 14, 15, 16, 17, 18, 19, 20). As shown in Fig. 3, RFP potently inhibited NF-{kappa}B activation mediated by IKK{epsilon}, TBK1, IKK{beta}, and IKK{alpha} as well as ISRE activation mediated by IKK{epsilon} and TBK1. In these experiments, RFP did not affect the expression levels of the IKKs (Fig. 3H), suggesting that the inhibitory effects of RFP on IKK signaling were not at the protein expression level. Consistent with the observation that RFP did not interact with PKAc, RFP did not affect PKAc-mediated activation of CREB in reporter gene assays (Fig. 3G). These data suggest that RFP specifically inhibits signaling mediated by IKKs.



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  		FIGURE 3. RFP inhibits NF-{kappa}B and/or ISRE activation mediated by the IKKs. A–F, RFP inhibits activation of NK-{kappa}B (A, C, E, and F) or ISRE (B and D) mediated by IKK{epsilon} (A and B), TBK1 (C and D), IKK{beta} (E), and IKK{alpha} (F) in a dose-dependent manner. 293 cells (~1 x 105) were transfected with 0.1 µg of NF-{kappa}B (A, C, E, and F) or ISRE (B and D) luciferase reporter plasmid; 0.1 µg of TK-Renilla luciferase reporter plasmid; 0.2 µg of an expression plasmid for IKK{epsilon} (A and B), TBK1 (C and D), IKK{beta} (E), or IKK{alpha} (F); and increased amounts of an expression plasmid for RFP as indicated. Sixteen hours after transfection, firefly luciferase activities were measured and normalized based on the activities of Renilla luciferase. G, RFP does not inhibit PKAc-mediated CREB activation. 293 cells (~1 x 105) were transfected with 0.5 µg of 5xGAL4-Luc reporter plasmid; 0.1 µg of TK-Renilla luciferase reporter plasmid; expression vectors for GAL4-c-CREB1–280 (0.1 µg), Flag-tagged PKAc (1 µg), HA-tagged RFP, or empty control plasmid (1 µg); and increased amounts of HA-tagged RFP as indicated. Reporter assays were performed 16 h after transfection. H, RFP does not affect the expression levels of the IKKs. 293 cells (~3 x 105) were transfected with HA-RFP and Flag-tagged IKKs (1 µg each) as indicated. Cell lysates were analyzed by Western blot with anti-HA and anti-Flag Abs. I, A schematic presentation of the structure and mutants of RFP. J, Effects of RFP mutants on IKK{epsilon}-mediated ISRE activation. 293 cells (~1 x 105) were transfected with 0.1 µg of ISRE-luciferase reporter plasmid, 0.1 µg of TK-Renilla luciferase reporter plasmid, 0.2 µg of an expression plasmid for IKK{epsilon}, and 0.4 µg of the indicated RFP mutant plasmids. Reporter assays were performed 16 h after transfection. For luciferase assays, data shown are the average ± SE.

 
RFP is a member of the tripartite motif family, which is characterized by a conserved Ring finer, a B box, and a coiled-coil domain (together called RBCC) (30, 31). In addition to these characteristic features, RFP contains a unique C-terminal region known as the RFP domain. We made a series of RFP deletion mutants and examined their abilities to inhibit IKK{epsilon}-mediated ISRE activation. As shown in Fig. 3, I and J, we found that the C-terminal RFP domain is required for RFP-mediated inhibition of ISRE activation triggered by IKK{epsilon}.

RFP Inhibits NF-{kappa}B and ISRE activation triggered by multiple pathways

Because RFP can inhibit IKK{epsilon}- and TBK1-mediated NF-{kappa}B and ISRE activation, we determined whether RFP inhibits signaling induced by viral infection and poly(I:C) treatment. As shown in Fig. 4, A and B, RFP inhibited activation of ISRE and the IFN-{beta} promoter induced by Sendai virus infection and poly(I:C) stimulation in 293-TLR3 cells in a dose-dependent manner.



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  		FIGURE 4. RFP inhibits signaling triggered by viral infection, poly(I:C), IL-1, and TNF. A, RFP inhibits poly(I:C)- and Sendai virus-induced ISRE activation. 293-TLR3 cells (~1 x 105) were transfected with 0.1 µg of ISRE-luciferase reporter plasmid and increased amounts of an expression plasmid for RFP as indicated. Eighteen hours after transfection, cells were infected with Sendai virus, treated with poly(I:C), or left untreated for 8 h before luciferase assays were performed. B, RFP inhibits poly(I:C)- and Sendai virus-induced activation of the IFN-{beta} promoter. The experiments were performed as described in A, except that IFN-{beta} promoter reporter plasmid was used. C, RFP inhibits NF-{kappa}B activation triggered by IL-1 and TNF. 293 cells (~1 x 105) were transfected with 0.1 µg of NF-{kappa}B-luciferase reporter plasmid and increased amounts of an expression plasmid for RFP as indicated. Eighteen hours after transfection, cells were treated with IL-1 (10 ng/ml) or TNF (10 ng/ml) or were left untreated for 8 h before luciferase assays were performed. For luciferase assays, data shown are the average ± SE.

 
Because RFP interacts with IKK{beta} and inhibits IKK{beta}-mediated NF-{kappa}B activation, we determined whether RFP inhibits TNF- and IL-1-induced NF-{kappa}B activation. As shown in Fig. 4C, RFP inhibited TNF- and IL-1-induced NF-{kappa}B activation in a dose-dependent manner.

To determine the role of endogenous RFP in NF-{kappa}B and ISRE signaling, we determined the effects of knockdown of RFP on the respective signaling pathways. We constructed an RFP RNAi vector that could dramatically knockdown the expression levels of transfected RFP (Fig. 5A) and endogenous RFP (Fig. 5B). In reporter gene assays, the RFP RNAi vector could enhance NF-{kappa}B activation triggered by TLR3 engagement, TNF and IL-1 (Fig. 5, C, E, and F), as well as ISRE-dependent transcription triggered by TLR3 engagement in 293-TLR3 cells (Fig. 5D). In contrast, RFP RNAi did not affect MAPK kinase kinase 3-induced c-Jun activation (Fig. 5G). These results suggest that RFP is a physiological inhibitor of signaling pathways triggered by TLR3, TNF, and IL-1.



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  		FIGURE 5. Knockdown of RFP expression enhances signaling triggered by poly(I:C), IL-1, and TNF. A, RFP RNAi inhibits the expression of transfected RFP, but not a control protein. 293 cells (~3 x 105) were cotransfected with 1 µg of HA-tagged RFP plasmid, 1 µg of Flag-tagged PKAc plasmid, and 4 µg of RFP or control RNAi plasmid as indicated. Twenty-four hours after transfection, cell lysates were analyzed by Western blot with anti-HA and anti-Flag Abs. B, RFP RNAi inhibits the expression of endogenous RFP. 293-TLR3 cells were transfected with control or RFP RNAi plasmid. Transfected cells were selected with puromycin (10 µg/ml) for 4 wk. Cell lysates were analyzed by Western blots with rabbit anti-RFP and mouse anti-IKK{epsilon} antisera. C and D, RFP RNAi enhances activation of NF-{kappa}B (C) and ISRE (D) triggered by poly(I:C) in a dose-dependent manner. 293-TLR3 cells (~1 x 105) were transfected with 0.1 µg of NF-{kappa}B (C) or ISRE (D) luciferase reporter plasmid and increased amounts of RFP RNAi plasmid as indicated. Sixteen hours after transfection, cells were treated with poly(I:C) (100 µg/ml) or were left untreated for an additional 6 h before luciferase assays were performed. E and F, RFP RNAi enhances activation of NF-{kappa}B triggered by IL-1 (E) and TNF (F) in a dose-dependent manner. 293 cells (~1 x 105) were transfected with 0.1 µg of NF-{kappa}B luciferase reporter plasmid and increased amounts of RFP RNAi plasmid as indicated. Sixteen hours after transfection, cells were treated with IL-1 (10 ng/ml; D) or TNF (10 ng/ml; E) or were left untreated for an additional 6 h before luciferase assays were performed. G, RFP RNAi does not inhibit activation of c-Jun mediated by MAPK kinase kinase 3 (MEKK3). 293 cells (~1 x 105) were transected with 0.5 µg of 5xGAL4-Luc reporter plasmid, 0.1 µg of TK-Renilla luciferase reporter plasmid, and expression vectors for GAL4-c-Jun1–223 (0.1 µg), Flag-tagged MEKK3 (1 µg), and increased amounts of HA-tagged RFP as indicated. Reporter assays were performed 16 h after transfection. For luciferase assays, data shown are the average ± SE.

 
RFP inhibits IKK{epsilon}-induced translocation of IRF-3 into the nucleus

The results from the studies described above indicate that RFP interacts with and is a substrate of IKK{epsilon} and inhibits ISRE-dependent transcription triggered by TLR3 engagement and virus infection. One of the possible explanations for these observations is that RFP competes with IRF-3 for interaction with and phosphorylation by IKK{epsilon}. To test this possibility, we transfected 293 cells with constant amounts of expression vectors for IKK{epsilon} and IRF-3 and increased amounts of an expression plasmid for RFP. Coimmunoprecipitation and in vitro kinase experiments indicated that RFP could not compete with IRF-3 for interaction with and phosphorylation by IKK{epsilon} (data not shown). Consistent with these observations, RFP did not inhibit IKK{epsilon}-induced dimerization of IRF-3 (Fig. 6A), a process that requires IRF-3 phosphorylation (32).



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  		FIGURE 6. RFP inhibits IKK{epsilon}-induced translocation of IRF-3 into the nucleus. A, RFP does not inhibit IKK{epsilon}-induced dimerization of IRF-3. 293 cells (~3 x 105) were transfected with the indicated expression plasmids (2 µg each). Twenty-four hours after transfection, cell lysates were prepared and subjected to native PAGE or SDS-PAGE. Western blots were performed with the indicated Abs. B, RFP inhibits IKK{epsilon}-induced translocation of IRF-3 into the nucleus. 293 cells were transfected with the indicated expression plasmids. Immunofluorescent stainings were performed with anti-Flag Ab and 4',6'-diamidino-2-phenylindole. C, RFP interacts with IRF-3. 293 cells (~1 x 106) were transfected with the expression plasmids for HA-tagged RFP and Flag-tagged IRF-3. The cell lysates were immunoprecipitated with anti-HA ({alpha}HA) Ab or control IgG (C). The transfected proteins in lysates (L) and immunoprecipitates were analyzed by Western blot with anti-Flag Ab.

 
Because RFP does not inhibit IKK{epsilon}-mediated phosphorylation and dimerization of IRF-3, we determined whether RFP inhibits IKK{epsilon}-induced translocation of IRF-3 into the nucleus. As shown in Fig. 6B, transiently transfected IRF-3 was localized mostly in the cytoplasm in 293 cells. When cotransfected with IKK{epsilon}, the majority of IRF-3 was translocated into the nucleus. However, with the addition of RFP, this translocation was blocked (Fig. 6B). These data suggest that RFP inhibits IRF-3-mediated transcription by sequestering active IRF-3 in the cytoplasm. Consistent with this conclusion, we found that RFP could interact with IRF-3 in 293 cells in coimmunoprecipitation experiments (Fig. 6C).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
In this study we identified RFP as an IKK{epsilon}-interacting protein by the yeast two-hybrid system. RFP is localized predominantly in the cytoplasm in NIH-3T3 and mouse A9 cells, whereas in other cell lines, such as HepG2 and HeLa, RFP is a normal component of PML nuclear bodies and interacts directly with another family member called PML (26, 27, 28, 29). The biological function of RFP is largely unknown. It has been shown that RFP is involved in the regulation of gene expression and cell proliferation. RFP becomes oncogenic when its RBCC moiety is fused to the Ret tyrosine kinase (33). A recent report suggests that overexpression of RFP causes apoptosis (34).

Two RBCC family members, MID1 and pyrin, have been reported to participate in the regulation of development, inflammation, and immune responses. MID1 is an E3 ubiquitin ligase that associates with the catalytic subunit of protein phosphatase 2A and targets it for ubiquitin-dependent degradation. Mutation of MID1 causes X-lined Opitz/BBB syndrome (35). Mutation of pyrin causes familial Mediterranean fever, a recessively inherited disorder characterized by dramatic episodes of fever and serosal inflammation (36). In this report we provide evidences that RFP, another member of the RBCC family, participates in the negative regulation of innate immunity and inflammation through targeting the noncanonical and canonical IKKs.

In coimmunoprecipitation experiments, RFP was not only associated with IKK{epsilon}, but was also associated with TBK1, IKK{alpha}, and IKK{beta}. Reporter gene assays indicated that RFP could inhibit signaling mediated by these kinases as well as that triggered by viral infection, TLR3 engagement, TNF, and IL-1 (Figs. 3 and 4). In contrast, knockdown of RFP expression by RNAi enhanced signaling triggered by these stimuli (Fig. 5). These results demonstrate that RFP negatively regulates signaling triggered by these stimuli through targeting the IKK family members.

Using IKK{epsilon} as a model, we explored the mechanisms of how the IKK-mediated signaling is inhibited by RFP. RFP did not affect the expression levels of the IKK family members (Fig. 3). Therefore, inhibition of signaling by RFP is not caused by down-regulation of expression of IKKs. Although RFP interacted with and was a substrate of IKK{epsilon}, RFP did not compete with IRF-3 for interaction with and phosphorylation by IKK{epsilon} (data not shown). Consistently, RFP did not affect dimerization of IRF-3 induced by IKK{epsilon}, a process required for its phosphorylation. However, coimmunoprecipitation experiments indicated that RFP could interact with IRF-3 (Fig. 6C), and overexpression of RFP blocked IKK{epsilon}-induced translocation of IRF-3 into nucleus (Fig. 6B). Taken together, these data suggest that RFP does not affect IRF-3 phosphorylation and dimerization. Instead, RFP inhibits IRF-3-mediated transcription by interaction with both IKK{epsilon} and IRF-3 and sequestering active IRF-3 in the cytoplasm. In our domain-mapping experiments, we found that the C-terminal RFP domain is required for the inhibitory effect of RFP on IKK{epsilon}-mediated ISRE activation. The simplest explanation for this observation is that the C-terminal RFP domain is responsible for sequestering IRF-3 in the cytoplasm.

Currently, we do not know how RFP inhibits the IRF-3 activation pathway under physiological conditions. One possibility is that RFP is associated with IRF-3 under physiological conditions. Upon viral infection, TBK1 or IKK{epsilon} is activated, which then phosphorylates RFP and IRF-3 and causes dissociation of RFP and IRF-3 and translocation of the phosphorylated IRF-3 to the nucleus.

In our preliminary studies we found that RFP could also interact with p65 and inhibit p65-mediated NF-{kappa}B activation. Whether RFP inhibits NF-{kappa}B activation through a mechanism similar to its inhibition of the IRF-3 activation pathway requires additional experiments.

Excessive immune responses or unwanted high levels of basal activity cause diseases such as allergy, inflammation, and autoimmune diseases. Therefore, negative regulation of the active signaling pathways is important for protecting the host from damage induced by excessive immune responses. The identification of RFP as an inhibitor of IKK-mediated signaling provides a novel mechanism of regulation of the signaling involved in inflammation and innate immune responses against viruses.


   Acknowledgments
 
We thank Lixia Li, Jiancheng Hu, Zhiqing Li, Lianyun Li, Xiaoqin Su, and Lin Luo for their help and discussion.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This work was supported by a grant from the Chinese High-Technology Program (2003AA221030). Back

2 Address correspondence and reprint requests to Dr. Hong-Bing Shu, College of Life Sciences, Wuhan University, Wuhan 430072, China. E-mail address: shuh{at}whu.edu.cn Back

3 Abbreviations used in this paper: IRF-3, IFN regulatory factor-3; HA, hemagglutinin; IKK, I{kappa}B kinase; ISRE, IFN-stimulated response element; PKAc, protein kinase A; poly(I:C), polyinosinic-polycytidylic acid; RBCC, conserved Ring finer, B box, and coiled-coil domain; RFP, Ret finger protein; TBK1, TANK-binding kinase-1; TIR, Toll/IL-1 receptor; TK, thymidine kinase; TANK, TRAF family-associated NF-{kappa}B activator; RNAi, RNA interference. Back

Received for publication July 15, 2005. Accepted for publication October 26, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 

  1. Akira, S., H. Hemmi. 2003. Recognition of pathogen-associated molecular patterns by TLR family. Immunol. Lett. 85: 85-95. [Medline]
  2. Durbin, J. E., A. Fernandez-Sesma, C. K. Lee, T. D. Rao, A. B. Frey, T. M. Moran, S. Vukmanovic, A. Garcia-Sastre, D. E. Levy. 2000. Type I IFN modulates innate and specific antiviral immunity. J. Immunol. 164: 4220-4228. [Abstract/Free Full Text]
  3. Levy, D. E., I. J. Marie. 2004. RIGging an antiviral defense—it’s in the CARDs. Nat. Immunol. 5: 699-701. [Medline]
  4. Levy, D. E., A. Garcia-Sastre. 2001. The virus battles: IFN induction of the antiviral state and mechanisms of viral evasion. Cytokine Growth Factor Rev. 12: 143-156. [Medline]
  5. tenOever, B. R., M. J. Servant, N. Grandvaux, R. Lin, J. Hiscott. 2002. Recognition of the measles virus nucleocapsid as a mechanism of IRF-3 activation. J. Virol. 76: 3659-3669. [Abstract/Free Full Text]
  6. Alexopoulou, L., A. C. Holt, R. Medzhitov, R. A. Flavell. 2001. Recognition of double-stranded RNA and activation of NF-{kappa}B by Toll-like receptor 3. Nature 413: 732-738. [Medline]
  7. Yoneyama, M., M. Kikuchi, T. Natsukawa, N. Shinobu, T. Imaizumi, M. Miyagishi, K. Taira, S. Akira, T. Fujita. 2004. The RNA helicase RIG-I has an essential function in double-stranded RNA-induced innate antiviral responses. Nat. Immunol. 5: 730-737. [Medline]
  8. Honda, K., S. Sakaguchi, C. Nakajima, A. Watanabe, H. Yanai, M. Matsumoto, T. Ohteki, T. Kaisho, A. Takaoka, S. Akira, et al 2003. Selective contribution of IFN-{alpha}/{beta} signaling to the maturation of dendritic cells induced by double-stranded RNA or viral infection. Proc. Natl. Acad. Sci. USA 100: 10872-10877. [Abstract/Free Full Text]
  9. Maniatis, T., J. V. Falvo, T. H. Kim, T. K. Kim, C. H. Lin, B. S. Parekh, M. G. Wathelet. 1998. Structure and function of the interferon-{beta} enhanceosome. Cold Spring Harbor Symp. Quant. Biol. 63: 609-620. [Medline]
  10. Wathelet, M. G., C. H. Lin, B. S. Parekh, L. V. Ronco, P. M. Howley, T. Maniatis. 1998. Virus infection induces the assembly of coordinately activated transcription factors on the IFN-{beta} enhancer in vivo. Mol. Cell 1: 507-518. [Medline]
  11. Fitzgerald, K. A., S. M. McWhirter, K. L. Faia, D. C. Rowe, E. Latz, D. T. Golenbock, A. J. Coyle, S. M. Liao, T. Maniatis. 2003. IKK{epsilon} and TBK1 are essential components of the IRF3 signaling pathway. Nat. Immunol. 4: 491-496. [Medline]
  12. Sharma, S., B. R. tenOever, N. Grandvaux, G. P. Zhou, R. Lin, J. Hiscott. 2003. Triggering the interferon antiviral response through an IKK-related pathway. Science 300: 1148-1151. [Abstract/Free Full Text]
  13. Perry, A. K., E. K. Chow, J. B. Goodnough, W. C. Yeh, G. Cheng. 2004. Differential requirement for TANK-binding kinase-1 in type I interferon responses to Toll-like receptor activation and viral infection. J. Exp. Med. 199: 1651-1658. [Abstract/Free Full Text]
  14. Hemmi, H., O. Takeuchi, S. Sato, M. Yamamoto, T. Kaisho, H. Sanjo, T. Kawai, K. Hoshino, K. Takeda, S. Akira. 2004. The roles of two I{kappa}B kinase-related kinases in lipopolysaccharide and double stranded RNA signaling and viral infection. J. Exp. Med. 199: 1641-1650. [Abstract/Free Full Text]
  15. McWhirter, S. M., K. A. Fitzgerald, J. Rosains, D. C. Rowe, D. T. Golenbock, T. Maniatis. 2004. IFN-regulatory factor 3-dependent gene expression is defective in Tbk1-deficient mouse embryonic fibroblasts. Proc. Natl. Acad. Sci. USA 101: 233-238. [Abstract/Free Full Text]
  16. Yamamoto, M., S. Sato, K. Mori, K. Hoshino, O. Takeuchi, K. Takeda, S. Akira. 2002. Cutting edge: a novel Toll/IL-1 receptor domain-containing adapter that preferentially activates the IFN-{beta} promoter in the Toll-like receptor signaling. J. Immunol. 169: 6668-6672. [Abstract/Free Full Text]
  17. Yamamoto, M., S. Sato, H. Hemmi, K. Hoshino, T. Kaisho, H. Sanjo, O. Takeuchi, M. Sugiyama, M. Okabe, K. Takeda, et al 2003. Role of adaptor TRIF in the MyD88-independent Toll-like receptor signaling pathway. Science 301: 640-643. [Abstract/Free Full Text]
  18. Oshiumi, H., M. Matsumoto, K. Funami, T. Akazawa, T. Seya. 2003. TICAM-1, an adaptor molecule that participates in Toll-like receptor 3-mediated interferon-{beta} induction. Nat. Immunol. 4: 161-167. [Medline]
  19. Han, K. J., X. Su, L. G. Xu, L. H. Bin, J. Zhang, H. B. Shu. 2004. Mechanisms of the TRIF-induced interferon-stimulated response element and NF-{kappa}B activation and apoptosis pathways. J. Biol. Chem. 279: 15652-15661. [Abstract/Free Full Text]
  20. Jiang, Z., T. W. Mak, G. Sen, X. Li. 2004. Toll-like receptor 3-mediated activation of NF-{kappa}B and IRF3 diverges at Toll-IL-1 receptor domain-containing adapter inducing IFN-{beta}. Proc. Natl. Acad. Sci. USA 101: 3533-3538. [Abstract/Free Full Text]
  21. Karin, M., Y. Ben-Neriah. 2000. Phosphorylation meets ubiquitination: the control of NF-{kappa}B activity. Annu. Rev. Immunol. 18: 621-663. [Medline]
  22. Ghosh, S., M. Karin. 2002. Missing pieces in the NF-{kappa}B puzzle. Cell 109: (Suppl.):S81-S96.
  23. Chen, D., X. Li, Z. Zhai, H. B. Shu. 2002. A novel zinc finger protein interacts with receptor-interacting protein (RIP) and inhibits tumor necrosis factor (TNF)- and IL1-induced NF-{kappa}B activation. J. Biol. Chem. 277: 15985-15991. [Abstract/Free Full Text]
  24. Bin, L. H., L. G. Xu, H. B. Shu. 2003. TIRP, a novel Toll/interleukin-1 receptor (TIR) domain-containing adapter protein involved in TIR signaling. J. Biol. Chem. 278: 24526-24532. [Abstract/Free Full Text]
  25. Hu, W. H., H. Johnson, H. B. Shu. 1999. Tumor necrosis factor-related apoptosis-inducing ligand receptors signal NF-{kappa}B and JNK activation and apoptosis through distinct pathways. J. Biol. Chem. 274: 30603-30610. [Abstract/Free Full Text]
  26. Cao, T., K. L. Borden, P. S. Freemont, L. D. Etkin. 1997. Involvement of the rfp tripartite motif in protein-protein interactions and subcellular distribution. J. Cell Sci. 110: 1563-1571. [Abstract]
  27. Cao, T., E. Duprez, K. L. Borden, P. S. Freemont, L. D. Etkin. 1998. Ret finger protein is a normal component of PML nuclear bodies and interacts directly with PML. J. Cell Sci. 111: 1319-1329. [Abstract]
  28. Tezel, G., T. Nagasaka, N. Iwahashi, N. Asai, T. Iwashita, K. Sakata, M. Takahashi. 1999. Different nuclear/cytoplasmic distributions of RET finger protein in different cell types. Pathol. Int. 49: 881-886. [Medline]
  29. Harbers, M., T. Nomura, S. Ohno, S. Ishii. 2001. Intracellular localization of the Ret finger protein depends on a functional nuclear export signal and protein kinase C activation. J. Biol. Chem. 276: 48596-48607. [Abstract/Free Full Text]
  30. Saurin, A. J., K. L. Borden, M. N. Boddy, P. S. Freemont. 1996. Does this have a familiar RING?. Trends Biochem. Sci. 21: 208-214. [Medline]
  31. Borden, K. L.. 1998. RING fingers and B-boxes: zinc-binding protein-protein interaction domains. Biochem. Cell Biol. 76: 351-358. [Medline]
  32. Servant, M. J., B. Tenoever, R. Lin. 2002. Overlapping and distinct mechanisms regulating IRF-3 and IRF-7 function. J. Interferon Cytokine Res. 22: 49-58. [Medline]
  33. Takahashi, M., G. M. Cooper. 1987. ret transforming gene encodes a fusion protein homologous to tyrosine kinases. Mol. Cell. Biol. 7: 1378-1385. [Abstract/Free Full Text]
  34. Dho, S. H., K. S. Kwon. 2003. The Ret finger protein induces apoptosis via its RING finger-B box-coiled-coil motif. J. Biol. Chem. 278: 31902-31908. [Abstract/Free Full Text]
  35. Trockenbacher, A., V. Suckow, J. Foerster, J. Winter, S. Krauss, H. H. Ropers, R. Schneider, S. Schweiger. 2001. MID1, mutated in Opitz syndrome, encodes an ubiquitin ligase that targets phosphatase 2A for degradation. Nat. Genet. 29: 287-294. [Medline]
  36. Ancient missense mutations in a new member of the RoRet gene family are likely to cause familial Mediterranean fever: the International FMF Consortium. Cell 90: 1997797-807. [Medline]

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