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Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip

Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 9, Issue of March 1, pp. 7059 –7065, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip* Received for publication, October 2, 2001, and in revised form, December 12, 2001 Published, JBC Papers in Press, December 18, 2001, DOI 10.1074/jbc.M109537200 Guolong Zhang and Sankar Ghosh‡ From the Section of Immunobiology and the Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520 Toll-like receptor (TLR)-mediated recognition of nous stimuli, TLRs are also able to trigger cell activation in pathogens represents one of the most important mech- response to endogenous signals, such as heat shock protein 60 anisms of innate immunity and disease resistance. The (7), fibronectin (8), fibrinogen (9), and unknown factors from adaptor protein Tollip was identified initially as an in- the injured myocardium (10) and necrotic cells (11). Activation termediate in interleukin (IL)-1 signaling. Here we re- of TLRs by endogenous ligands presumably provides immune port that Tollip also associates directly with TLR2 and surveillance at sites of inflammation, but sustained activation TLR4 and plays an inhibitory role in TLR-mediated cell may lead to development of chronic inflammatory disorders activation. Inhibition by Tollip is mediated through its and autoimmune diseases. ability to potently suppress the activity of IL-1 receptor- All mammalian TLRs are type I transmembrane proteins associated kinase (IRAK) after TLR activation. In addi- consisting of multiple copies of leucine-rich repeats in the ex- tion, we show for the first time that Tollip is a bona fide tracellular domain and a conserved Toll/interleukin-1 receptor substrate for IRAK and is phosphorylated by IRAK upon (IL-1R) homology (TIR) domain in the cytoplasmic tail (1, 3). stimulation with lipopolysaccharide or IL-1. Negative Because TIR domains of TLRs, IL-1R, and IL-18R are highly regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory medi- homologous, it is believed that these receptors activate similar ators during inflammation and infection. signaling pathways when stimulated with their cognate li- gands (1). Consistent with this hypothesis, the downstream signaling components, myeloid differentiation protein 88 Toll-like receptors (TLRs) represent a family of phylogeneti- (MyD88), IL-1R-associated kinase (IRAK) and tumor necrosis cally conserved proteins that have been found in insects, factor receptor-associated factor 6 (TRAF6) are all utilized by plants, and mammals (1–3). In addition to playing a critical these three groups of receptors to activate both nuclear factor role in the dorso-ventral axis formation in insects, TLRs are (NF)-B and mitogen-activated protein kinase signaling cas- also involved in innate immunity and disease resistance (2– 4). cades (12–16). MyD88 functions as a crucial adaptor that links In mammals, 10 members of the TLR family have been identi- the receptors to IRAK after activation. IRAK in turn is phos- fied which are expressed by host immune cells most likely to phorylated, dissociates from the receptor, and associates with come into direct contact with pathogens from the environment, TRAF6 to signal activation of either NF-B or mitogen-acti- such as dendritic cells, peripheral phagocytes, and mucosal vated protein kinases. However, genetic studies revealed that epithelia (5). Recent studies have revealed that a striking fea- neither MyD88 (17), IRAK (18), nor TRAF6 (19) is absolutely ture of TLRs is their ability to discriminate among different essential for cell activation in response to stimulation with classes of pathogens (3, 4). For example, TLR4 detects lipopo- LPS, implying the existence of alternative signaling pathways. lysaccharide (LPS) from Gram-negative bacteria, whereas The recent description of TIR domain-containing adaptor pro- TLR2 recognizes peptidoglycan (PGN), lipoproteins, and zymo- tein (TIRAP) (20), also known as Mal (MyD88 adaptor-like) san from Gram-positive bacteria and yeast. TLRs alone or in (21), probably explains the residual signaling observed in the combination are believed to mediate host immune responses to absence of MyD88. Interestingly TIRAP/Mal also appears to be a large array of pathogens (6). In addition to detecting exoge- involved in signaling through TLR4 but not IL-1, thereby dem- onstrating a unique specificity in the adaptor molecules that participate in signaling. * This work was supported by the Howard Hughes Medical Institute In addition to MyD88, another adaptor protein named Toll- and National Institutes of Health Grant R37 AI33443. The costs of interacting protein (Tollip) was recently found to associate with publication of this article were defrayed in part by the payment of page the cytoplasmic TIR domain of IL-1Rs after IL-1 stimulation charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (22). Tollip forms a complex with IRAK in resting cells and ‡ To whom correspondence should be addressed: Section of Immuno- inhibits IL-1-induced signaling by blocking IRAK phosphoryl- biology, Yale University School of Medicine, 310 Cedar St., New Haven, ation. Because of the significant homology in the intracellular CT 06520. Tel.: 203-737-4419; Fax: 203-737-1764; E-mail: sankar. [email protected]. portion of TLRs, IL-1R, and IL-18R, we hypothesized that The abbreviations used are: TLR(s), Toll-like receptor(s); AP-1, ac- Tollip might also inhibit TLR-mediated signaling by interact- tivator protein-1; GST, glutathione S-transferase; HA, hemagglutinin; ing with TLRs through the TIR domain. Here we report that IL-1R, interleukin-1 receptor; IL-18R, interleukin-18 receptor; IRAK, Tollip associates directly with TLR2 and TLR4 and inhibits IL-1R-associated kinase; LPS, lipopolysaccharide; Luc, luciferase; Mal, MyD88-adaptor-like; MyD88, myeloid differentiation protein 88; NF- TLR-mediated cellular responses by suppressing phosphoryla- B, nuclear factor-B; PGN, peptidoglycan; TIR, Toll/IL-1R homology tion and kinase activity of IRAK. Furthermore, we find that domain; TIRAP, TIR domain-containing adaptor protein; Tollip, Toll- Tollip is phosphorylated by activated IRAK, making it the first interacting protein; TRAF6, tumor necrosis factor receptor-associated factor 6; UBA, ubiquitin-associated. direct substrate for IRAK to be characterized. This paper is available on line at http://www.jbc.org 7059 This is an Open Access article under the CC BY license. 7060 Tollip Inhibits TLR-mediated Signaling MATERIALS AND METHODS GST pull-down assays were performed essentially as described previ- ously (24). Briefly, 5 lof S-labeled lysates was incubated for1hat Cells and Reagents—Human embryonic kidney epithelial 293 cells 4 °C with 5 g of purified GST or GST-Tollip and 20 l of glutathione stably expressing the NF-B-dependent luciferase reporter pBIIX-Luc beads in 1 ml of TNT buffer. The beads were washed three times with (293-Luc) were developed as described previously (23). Mouse macro- TNT buffer, boiled in SDS sample buffer, and fractionated by 12.5% phage RAW264.7 cells were obtained from ATCC (Manassas, VA). Both SDS-PAGE. The radiolabeled proteins were visualized by cell lines were maintained in high glucose Dulbecco’s modified Eagle’s autoradiography. medium supplemented with 7% heat-inactivated fetal calf serum, 2 mM In Vitro Kinase Assays—For the detection of endogenous IRAK ac- L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. LPS tivity, 3  10 293/TLR2 or 293/TLR4 cells were plated into 10-cm from Escherichia coli 0111:B4, PGN from Staphylococcus aureus, anti- dishes and transfected the following day with 5 g of HA-Tollip and/or FLAG M2 antibody, and M2 agarose beads were purchased from Sigma. 5 g of MD-2 plasmid using FuGene 6. 24 h after transfection, cells Purified polyclonal rabbit antibodies against IRAK and hemagglutinin were either left untreated or stimulated with 1 g/ml LPS for 10 or 30 (HA) epitope were from Santa Cruz Biotechnology (Santa Cruz, CA). min and lysed in 1.2 ml of TNT buffer containing 2 mM NaF, 1 mM Protein A-Sepharose and glutathione-conjugated agarose beads were dithiothreitol, 1 mM EDTA, and a mixture of protease inhibitors. Cell from Amersham Biosciences, Inc. lysates were precipitated at 4 °C for 4 h with 1 g of anti-IRAK antibody Plasmids—Mammalian expression vectors containing N-terminal and 20 l of 50% (v/v) slurry of protein A-Sepharose beads. IRAK FLAG-tagged human TLR2 and TLR4 that were made by inserting autophosphorylation was measured essentially as described previously PCR-generated cDNA fragments lacking the signal sequence into using protocols developed for detecting IKK autophosphorylation (24). pFLAG-CMV-1 (Sigma) were kindly provided by R. Medzhitov (Yale After washing twice with TNT buffer and twice with kinase buffer (20 Medical School). The intracellular domains of TLR2 and TLR4 were mM HEPES, pH 7.5, 200 mM NaCl, 20 mM MgCl ,1mM EDTA, 2 mM amplified by PCR and cloned into pGBKT7 (Clontech, Palo Alto, CA). NaF, 1 mM dithiothreitol, 10 M ATP), the beads were suspended in 20 Plasmids for human CD14, FLAG-tagged human Tollip, HA-tagged l of kinase buffer containing 10 Ci of [- P]ATP (Amersham Bio- Tollip, and its deletion mutants were constructed in-frame by inserting sciences, Inc.). The reactions were allowed to proceed at 30 °C for 20 PCR-generated cDNA fragments into pCDNA3.1 (Invitrogen). FLAG- min and terminated with 20 l of SDS sample buffer. The beads were IRAK and HA-IRAK were generated by inserting cDNA into boiled, 20-l reaction aliquots were separated by 10% SDS-PAGE, and pCDNA3.1. Kinase-inactive FLAG-IRAK (K239A) and HA-IRAK visualized by autoradiography. For measuring phosphorylation of (K239A) were made using the Quick site-directed mutagenesis kit from Tollip by IRAK, IRAK was first precipitated, and immune complexes Stratagene (La Jolla, CA). All FLAG and HA epitope tags were gener- were washed with TNT and kinase buffers, then incubated in 25 lof ated by incorporating tag sequences into primers at the N terminus of kinase buffer with 10 Ci of [- P]ATP and 2 g of purified GST, respective cDNA fragments. Correct inserts were confirmed by direct GST-Tollip, or its truncation mutants. Phosphorylated proteins were sequencing. The expression vector pEFBOS containing human MD-2 precipitated further with 20 l of glutathione-agarose beads, separated was kindly provided by K. Miyake (Saga Medical School, Japan). by SDS-PAGE, and visualized by autoradiography. Development of 293/TLR2 and 293/TLR4 Stable Cell Lines—293- Luc cells were seeded into 10-cm dishes and transfected using FuGene RESULTS 6 (Roche Molecular Biochemicals) with 5 g of pFLAG-TLR2 or pFLAG- TLR4 together with 0.5 g of a plasmid encoding CD14 containing a Tollip Physically Associates with TLR2 and TLR4 via Its hygromycin resistance gene. Cells were selected in Dulbecco’s modified C-Terminal Domain—To determine whether Tollip interacts Eagle’s medium with 200 g/ml hygromycin B (Calbiochem). Individual with TLRs via the TIR domain, we developed two cell lines colonies were picked, expanded, and confirmed for the surface expres- (293/TLR2 and 293/TLR4) that stably express human CD14, as sion of TLRs by flow cytometry and immunoblotting using anti-FLAG well as FLAG-tagged human TLR2 or TLR4. Flow cytometry M2. Stable cell lines were propagated and maintained in complete Dulbecco’s modified Eagle’s medium for regular 293-Luc cells. and immunoblotting revealed significant amounts of TLR ex- Transfection and Luciferase Reporter Assays—RAW264.7 cells were pression on the cell surface (data not shown). Furthermore, plated at a density of 3  10 cells/well in 24-well plates 1 day before expression of the TLRs rendered the 293 cells highly responsive transfection. Cells were transfected in triplicate using FuGene 6 with to various bacterial products, which do not otherwise activate 0.2 g of pBIIX-Luc or pAP-1-Luc and the indicated amounts of HA- these cells (data not shown). Tollip plasmid. The total amount of transfected plasmids was equalized Interactions between Tollip and TLRs were examined by by supplementing with the empty vector pCDNA3.1. After 24 h, cells transfecting these cell lines with plasmids expressing HA- were either left untreated or stimulated with 100 ng/ml LPS or 10 g/ml PGN for 6 h. Cells were then lysed and assayed for luciferase activity Tollip and/or MD-2, which is a secreted molecule that binds to (Promega, Madison, WI). The data were normalized to total protein TLR4 and is required for signaling (25). The FLAG-tagged concentrations of each sample measured using a protein assay kit from TLRs were immunoprecipitated using anti-FLAG antibody fol- Bio-Rad. lowed by immunoblotting with anti-HA antibody. HA-Tollip Immunoprecipitation and Immunoblotting—For coimmunoprecipita- coprecipitated efficiently with TLR2 (Fig. 1A, lane 3 of the top tion of transfected proteins, 7  10 293, 293/TLR2, or 293/TLR4 cells panel). Association between Tollip and TLR4 was also detect- were plated into six-well plates and transfected the following day with the indicated plasmids using FuGene 6. 24 –36 h after transfection, cells able but was enhanced significantly in the presence of MD-2 were lysed in 0.5 ml of TNT buffer (20 mM Tris, pH 7.5, 200 mM NaCl, (Fig. 1A, lanes 5 and 6 of the top panel), suggesting that 1% Triton X-100) containing a protease inhibitor mixture (Roche Mo- conformational changes in the cytoplasmic tail of TLR4 induced lecular Biochemicals). A portion of the lysates (50 l) was saved for by MD-2 may be necessary for efficient binding to Tollip. To immunoblotting, and FLAG-tagged proteins were precipitated from the determine whether Tollip directly interacts with the TIR do- remainder for 2–4hat4 °C with 20 l of 50% (v/v) slurry of anti-FLAG main of TLRs, in vitro GST pull-down assays were performed. M2 beads. After extensive washing with TNT buffer, the beads were boiled in SDS sample buffer. Proteins were separated by 10% SDS- [ S]Methionine-labeled intracellular portions of TLR2 and PAGE, transferred to polyvinylidene difluoride membranes, and blotted TLR4 were produced by in vitro translation and incubated with with the indicated antibodies. The reactive bands were visualized by either GST or GST-Tollip. As shown in Fig. 1B, both TLR2 and enhanced chemiluminescence (Amersham Biosciences, Inc.). TLR4 bound to GST-Tollip, but not GST alone, demonstrating Generation of Glutathione S-Transferase (GST)-Tollip and in Vitro that Tollip associates directly with the TIR domain of TLRs. Pull-down Assays—GST-Tollip and its N-terminal (1–52), central (53– To map the region of Tollip responsible for association with 178), and C-terminal (179 –273) truncation mutants were made by cloning respective human Tollip cDNA fragments in-frame into pGEX- the TLRs, we analyzed the interaction of TLR2 and TLR4 with 4T-1 (Amersham Biosciences, Inc.). GST fusion proteins were induced truncation mutants of Tollip as indicated in Fig. 2A. TLR4 with 0.4 mM isopropyl--D-thiogalactopyranoside for 4 –6hat37 °C and bound efficiently to either full-length Tollip (1–273) or mutants incubated with glutathione beads. Purified GST proteins were obtained with an intact C-terminal sequence, Tollip (53–178) and by elution from the beads with 10 mM reduced glutathione followed by 35 Tollip (53–273) (Fig. 2B, lanes 1, 4 and 5 of the top panel). Also, dialysis in phosphate-buffered saline. [ S]Methionine-labeled proteins whereas Tollip (1–229) could interact with TLR4, no associa- were generated with TNT-T7 Quick-coupled transcription/translation system (Promega) according to the manufacturer’s instructions. In vitro tion was found between TLR4 and Tollip (1–178) or Tollip Tollip Inhibits TLR-mediated Signaling 7061 FIG.1. Tollip associates directly with TLR2 and TLR4. A, coim- munoprecipitation of Tollip with TLR2 and TLR4. 293, 293/FLAG- TLR2, or 293/FLAG-TLR4 cells were transfected with expression plas- mids as indicated (1 g of each); after 24 h, proteins in the cell lysates FIG.2. Analysis of domain interactions of Tollip with TLR4. A, were immuoprecipitated (IP) using anti-FLAG M2 beads. Coprecipi- coimmunoprecipitation of Tollip mutants with TLR4. 293/FLAG-TLR4 tated proteins were detected by immunoblotting (IB) with anti-HA and cells were cultured in six-well plates and transfected with 1 g of MD-2 anti-FLAG antibodies. Cell lysates (20 l) were also immunoblotted plasmid together with 0.5 g of the indicated HA-Tollip mutants. Im- using anti-HA antibody to monitor the expression of transfected Tollip. munoprecipitation and immunoblotting were performed as described in B, direct interactions of Tollip with TLR2 and TLR4. Intracellular the legend to Fig. 1A. B, analysis of interactions of Tollip mutants with domains of TLR2 and TLR4 were S labeled by in vitro translation and TLR4 and Tollip. Diagrammatic representations of Tollip mutants are incubated with purified GST or GST-Tollip bound to glutathione beads. shown; black segments represent the C2 domain, and striped segments The beads were then washed, boiled in SDS sample buffer, and frac- are the UBA domain. , very strong; , strong; , weak; ,no tionated by 12.5% SDS-PAGE. The radiolabeled proteins were visual- interaction. ized by exposure to x-ray film. (53–178), indicating that the interaction domain lies in the 178) (Figs. 3B and 2A). Therefore, self-association of Tollip is C-terminal region of Tollip between residues 179 and 229 (Fig. mediated by both the N- and C-terminal regions of the protein 2B, lanes 2, 3 and 6 of the top panel). Similar results were and may result in formation of a stable protein complex. observed with TLR2 (data not shown). Interestingly, binding of Although both Tollip and MyD88 function as adaptors to link Tollip to IL-1R is also mediated through a similar C-terminal IL-1R or TLRs to downstream molecules, neither in vitro GST region (22). pull-down nor in vivo mammalian coimmunoprecipitation as- Tollip Interacts with Itself to Form Oligomers—TLRs, IL-1R, says revealed a stable complex between these two molecules and IL-18R are all known to form either homo- or heterodimers (data not shown). These results suggested that MyD88 and upon stimulation with their cognate ligands. Therefore, it is Tollip exist in distinct protein complexes, which is consistent possible that downstream proteins might also oligomerize to with previous findings that both molecules are recruited inde- allow signals to be transduced efficiently. To test whether pendently to the IL-1R after stimulation (22). Tollip forms homo-oligomers, we in vitro translated full-length Tollip Inhibits Cell Activation Induced by Bacterial Prod- S-labeled Tollip (1–273) and used it in GST pull-down assays. ucts—To assess the functional role of Tollip in TLR-mediated We found that Tollip bound to GST-Tollip but not to GST alone signaling, we transfected mouse macrophage RAW264.7 cells (Fig. 3A), suggesting that Tollip is capable of self-association. with increasing amounts of Tollip together with either NF-B To identify further the domains involved in Tollip homo-oli- or AP-1 luciferase reporter vectors. Luciferase activity was gomerization, 293-Luc cells were cotransfected with FLAG- measured after stimulation of the transfected cells with vari- tagged full-length Tollip and the HA-tagged Tollip deletion ous bacterial products. Both NF-B and AP-1 were activated mutants described in Fig. 2A, and the interaction between the readily in response to LPS (Fig. 4) and PGN (data not shown), proteins was analyzed by coimmunoprecipitation (Fig. 3B). All and both were inhibited dose-dependently by Tollip, suggesting Tollip mutants associated efficiently with full-length Tollip that Tollip is a negative regulator of TLR2 and TLR4 signaling. except for the construct containing only the C2 domain (53– Overexpression of Tollip also impaired NF-B activation in 7062 Tollip Inhibits TLR-mediated Signaling FIG.3. Tollip forms oligomers. A, self-association of Tollip. Tollip was translated in vitro and incubated with purified GST or GST-Tollip bound to glutathione beads. The pull-down assay was performed as described in the legend to Fig. 1B. B, analysis of domains involved in self-association of Tollip. 293-Luc cells were cultured in six-well plates and transfected with 0.5 g of FLAG-Tollip plasmid together with 0.5 g of the indicated HA-Tollip deletion mutants. Immunoprecipitation and immunoblotting were performed as described in the legend to Fig. 1A. FIG.5. Tollip suppresses phosphorylation and kinase activity of IRAK. A, inhibition of the recruitment of phosphorylated IRAK to the TLR4 receptor complex. 293/FLAG-TLR4 cells were cultured in six-well plates and transfected with 1 g of each of the indicated plasmids. Immunoprecipitation and immunoblotting were performed as described in the legend to Fig. 1A. B, suppression of LPS-induced kinase activity of IRAK by Tollip. 293/FLAG-TLR2 and 293/FLAG-TLR4 cells were cultured in 10-cm dishes and transfected with the plasmids indi- cated (5 g of each). 24 h after transfection, cells were either left untreated or stimulated with 1 g/ml LPS for 10 or 30 min. Proteins in cell lysates were precipitated using anti-IRAK antibody and protein A-Sepharose beads. Immune complexes were washed, and an aliquot of the immunoprecipitate was saved for immunoblotting to confirm equal loading. The remainder of the precipitated proteins was incubated in FIG.4. Tollip impairs LPS-induced cell activation. RAW264.7 kinase buffer at 30 °C for 20 min, separated by SDS-PAGE, and phos- cells were plated in 24-well plates and transfected in triplicate with 0.2 phorylated IRAK was visualized by autoradiography. g of luciferase reporter vector pBIIX-Luc or pAP-1-Luc together with the indicated amounts of HA-Tollip plasmid. After 24 h, cells were FLAG-TLR2 and 293/FLAG-TLR4 cells were transfected with either left untreated (white bars) or stimulated (black bars) with 100 Tollip together with either wild type or kinase-inactive ng/ml LPS for a further 6 h. Cells were then lysed and assayed for luciferase activity. The data were normalized to total protein concen- (K239A) IRAK. Consistent with previous findings, transfection trations and are represented as the means  S.D. Similar results were of wild type IRAK resulted in significant autophosphorylation obtained from three independent experiments. These data demonstrate of IRAK; however, the level of phosphorylation was diminished that Tollip inhibits activation of both NF-B(A) and AP-1 (B)ina dramatically in the presence of Tollip (Ref. 22 and Fig. 5A, dose-dependent manner. lanes 7 and 9 versus 8 and 10 of the second panel from the 293/TLR2 and 293/TLR4 cells after stimulation with LPS or bottom). Immunoprecipitation of TLR4 with anti-FLAG anti- PGN (data not shown). body revealed that Tollip did not appear to influence the Tollip Suppresses the Phosphorylation and Kinase Activity of amount of IRAK (K239A) recruited to the TLR4 receptor com- IRAK—To understand better the molecular mechanism by plex in the presence or absence of MD-2 (Fig. 5A, lanes 3 and 4 which Tollip inhibits TLR-mediated signaling, we analyzed its versus 5 and 6 of the top panel). In contrast, recruitment of effect on the recruitment of IRAK to the TLR complex. 293/ phosphorylated IRAK was completely abolished by Tollip re- Tollip Inhibits TLR-mediated Signaling 7063 FIG.6. Tollip is phosphorylated by activated IRAK. A, phosphorylation of Tollip by IRAK. 293/FLAG-TLR4 cells were cultured in 10-cm dishes and transfected with 5 g of MD-2 plasmid with or without 5 g of HA-Tollip. 24 h after transfection, cells were either left untreated or stimulated with 1 g/ml LPS for 10 –30 min. Cell lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads, and kinase assays were performed as described in the legend to Fig. 5B. The lysates (20 l) were also blotted with anti-IRAK and anti-HA antibodies to monitor the expression of transfected Tollip and phosphorylation of endogenous IRAK upon stimulation. B, phosphorylation of GST-Tollip by IRAK. 293/FLAG-TLR4 cells were cultured in six-well plates and transfected with 1 g/well MD-2 plasmid. Cells were then left untreated or stimulated with 1 g/ml LPS for 10 –30 min, and lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads. Kinase assays were performed in the presence of 2 g of purified GST or GST-Tollip. Phosphorylated proteins were precipitated further with glutathione beads, separated by SDS-PAGE, and visualized by autoradiography. Approximately equal amounts of GST protein input are shown on the bottom panel. C, Tollip is phosphorylated by IRAK at the C terminus. 293/FLAG-TLR4 cells were seeded in six-well plates and transfected with 1 g/well MD-2 plasmid. After 24 h, cells were stimulated with 1 g/ml LPS for 20 min, and lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads. Kinase assays were performed in the presence of 2 g of purified GST, GST-Tollip, or its truncation mutants. The bottom panel shows approximately equal amounts of GST proteins used in the kinase assay. 7064 Tollip Inhibits TLR-mediated Signaling gardless of MD-2 expression (Fig. 5A, lanes 7 and 9 versus 8 initially as a protein involved in IL-1 signaling, we now show and 10 of the top panel). Similar results were observed in that Tollip can associate with TLR2 and TLR4 and play an 293/FLAG-TLR2 cells (data not shown). inhibitory role in TLR-mediated cell activation. Consistent In vitro kinase assays were also performed to confirm that with our findings, during the preparation of the manuscript, suppression of IRAK autophosphorylation by Tollip was the Bulut et al. (31) reported independently that Tollip coprecipi- result of decreased IRAK kinase activity. 293/FLAG-TLR2 and tates with TLR2 and TLR4, and overexpression of Tollip inhib- 293/FLAG-TLR4 cells were transfected with Tollip and/or its TLR2- and TLR4-induced NF-B activation in human der- MD-2, stimulated with LPS, and then IRAK was immunopre- mal microvessel endothelial cells. cipitated and assayed for its kinase activity. As expected, IRAK One striking feature of Tollip is its ability to suppress the was heavily phosphorylated after LPS stimulation (Fig. 5B, kinase activity of IRAK. Several lines of evidence suggest that lanes 2 and 8 of the top panel), but this autophosphorylation the mechanism by which Tollip inhibits TLR signaling is by was decreased dramatically in the presence of Tollip (Fig. 5B, preassociating with IRAK, thus preventing it from being phos- lanes 4, 5, 10, and 11 of the top panel). These findings suggest phorylated and activated on the TLR receptor complex. In fact, a possible mechanism by which Tollip might impair TLR sig- studies using IRAK-deficient cells demonstrated that phospho- naling, namely by blocking the autophosphorylation of IRAK, rylation of IRAK is required for it to dissociate from the recep- which is necessary for the dissociation of IRAK from the recep- tor and transduce signals to downstream molecules leading to tor complex (26, 27). NF-B activation (26, 27). The ability of Tollip to interact Tollip Is Phosphorylated by IRAK—IRAK is a serine/threo- directly with both the death (1–208) and kinase (181–545) nine kinase with a centrally located catalytic domain (13). domains of IRAK may explain the inhibitory role of Tollip (22, Upon stimulation with either IL-1 or bacterial products, IRAK and data not shown). is activated and heavily phosphorylated (Ref. 13 and Fig. 6A, Upon activation, IRAK undergoes rapid autophosphorylation lanes 2 and 3 of both top and bottom panels). However, the and dissociates from the receptor. Our studies reveal that after identity of potential IRAK substrate(s) has remained unknown. stimulation IRAK also phosphorylates Tollip. The physiological In addition, recent studies have suggested that the kinase significance of this phosphorylation is still unclear, although it activity of IRAK is dispensable for its role as an intermediate in is possible that phosphorylation of Tollip may facilitate its IL-1 signaling (28, 29). However, we were surprised to find that dissociation from IRAK and subsequent degradation by ubiq- after LPS stimulation, activation of IRAK is accompanied by a uitination. This is consistent with the fact that Tollip contains concomitant phosphorylation of Tollip in 293/FLAG-TLR4 (Fig. a highly conserved UBA (ubiquitin-associated) or CUE (cou- 6A, lanes 5 and 6 of the top panel) and 293/FLAG-TLR2 cells pling of ubiquitin conjugation to endoplasmic reticulum degra- (data not shown). Phosphorylation of Tollip did not occur in dation) domain at the C terminus (230 –270). Similar domains resting cells without activated IRAK (Fig. 6A, lane 4 of the top in other proteins have been shown to recruit ubiquitin-conju- panel). gating enzymes, leading to proteosome-dependent degradation To confirm this observation, the 293/FLAG-TLR2 and 293/ (32, 33). Interestingly, the region around the UBA domain of FLAG-TLR4 cells were either left untreated or stimulated with Tollip binds to unphosphorylated IRAK under resting condi- LPS. IRAK was immunoprecipitated from these cells and incu- tions and also associates transiently with the TLRs and IL-R bated with purified GST-Tollip in in vitro kinase assays. We upon stimulation (22). The same region of Tollip can also be found that upon stimulation with LPS, concordant with the phosphorylated by IRAK (Fig. 6C). Therefore, once activated on phosphorylation of IRAK, GST-Tollip (Fig. 6B, lanes 4 – 6 of the the receptor, IRAK phosphorylates Tollip, which may lead to top panel), but not GST alone (Fig. 6B, lanes 1–3 of the top the dissociation of Tollip from IRAK and the receptor complex. panel), was heavily phosphorylated. Similar results were also Unmasking the UBA domain on Tollip may lead to its subse- observed in 293-Luc cells stimulated with IL-1 (data not quent degradation by a ubiquitination-dependent process. The shown). Therefore these experiments strongly suggest that in removal of Tollip would allow signaling to continue by freeing addition to undergoing autophosphorylation, IRAK also phos- activated IRAK to bind to downstream TRAF6. Because IRAK phorylates Tollip. Phosphorylation of Tollip by activated IRAK also undergoes rapid ubiquitination and degradation after ac- may facilitate the dissociation of IRAK from Tollip, thereby tivation (13, 34), it is tempting to speculate that phosphoryl- allowing IRAK to escape the inhibitory effect of Tollip and ated Tollip facilitates the ubiquitination of IRAK by recruiting subsequently modify downstream signaling components. ubiquitin-conjugating enzymes. Further studies will be needed To determine further the domain of Tollip which is phospho- to explore fully the biological ramifications of Tollip phospho- rylated by IRAK, we produced N-terminal (1–52), central (53– rylation by IRAK. 178), and C-terminal (179 –273) truncation mutants of Tollip Although a more complete understanding of biological func- fused with GST. However, the central region of Tollip, which tions of Tollip awaits the generation and analysis of mice lack- encompasses only the C2 domain, is completely insoluble when ing Tollip, it is possible that the primary role of Tollip-mediated expressed in bacteria. Therefore, kinase assays were performed pathway may be to maintain immune cells in a quiescent state with purified N- and C-terminal Tollip domains together with in the absence of infection and facilitate the termination of wild type GST-Tollip. As shown in Fig. 6C, the C-terminal TLR-induced cell signaling during inflammation and infection. (179 –273) but not N-terminal (1–52) region of Tollip is phos- It is conceivable that this evolutionarily conserved inhibitory phorylated by IRAK. Interestingly, the same region is also system benefits the host by limiting the production of proin- responsible for the interactions of Tollip with TLRs and IRAK flammatory mediators and subsequent tissue damage under (Fig. 2 and Ref. 22). However, we cannot rule out the possibility infectious conditions. Therefore, strategies to up-regulate that the central C2 domain of Tollip might also be phosphoryl- Tollip expression may prove effective in treating both chronic ated by IRAK upon activation because the intensity of phos- and acute inflammatory diseases, such as inflammatory bowel phorylation at the C terminus is considerably weaker than that disease and septic shock. for wild type Tollip (Fig. 6C, lane 3 versus 4ofthe top panel). Acknowledgments—We thank Ruslan Medzhitov, Elizabeth Kopp, and Kensuke Miyake for providing the various plasmids, and members DISCUSSION of the Ghosh laboratory for helpful discussions. We thank Michael May In this paper we demonstrate that the adaptor protein Tollip for careful reading of the manuscript. We are also grateful to the can negatively regulate TLR signaling pathways. Described anonymous reviewers’ constructive comments on the manuscript. 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Immunol. 34. Yamin, T. T., and Miller, D. K. (1997) J. Biol. Chem. 272, 21540 –21547 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip

Zhang, Guolong; Ghosh, Sankar
Journal of Biological Chemistry – Mar 1, 2002
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Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 9, Issue of March 1, pp. 7059 –7065, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Negative Regulation of Toll-like Receptor-mediated Signaling by Tollip* Received for publication, October 2, 2001, and in revised form, December 12, 2001 Published, JBC Papers in Press, December 18, 2001, DOI 10.1074/jbc.M109537200 Guolong Zhang and Sankar Ghosh‡ From the Section of Immunobiology and the Department of Molecular Biophysics and Biochemistry, Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, Connecticut 06520 Toll-like receptor (TLR)-mediated recognition of nous stimuli, TLRs are also able to trigger cell activation in pathogens represents one of the most important mech- response to endogenous signals, such as heat shock protein 60 anisms of innate immunity and disease resistance. The (7), fibronectin (8), fibrinogen (9), and unknown factors from adaptor protein Tollip was identified initially as an in- the injured myocardium (10) and necrotic cells (11). Activation termediate in interleukin (IL)-1 signaling. Here we re- of TLRs by endogenous ligands presumably provides immune port that Tollip also associates directly with TLR2 and surveillance at sites of inflammation, but sustained activation TLR4 and plays an inhibitory role in TLR-mediated cell may lead to development of chronic inflammatory disorders activation. Inhibition by Tollip is mediated through its and autoimmune diseases. ability to potently suppress the activity of IL-1 receptor- All mammalian TLRs are type I transmembrane proteins associated kinase (IRAK) after TLR activation. In addi- consisting of multiple copies of leucine-rich repeats in the ex- tion, we show for the first time that Tollip is a bona fide tracellular domain and a conserved Toll/interleukin-1 receptor substrate for IRAK and is phosphorylated by IRAK upon (IL-1R) homology (TIR) domain in the cytoplasmic tail (1, 3). stimulation with lipopolysaccharide or IL-1. Negative Because TIR domains of TLRs, IL-1R, and IL-18R are highly regulation of TLR signaling by Tollip may therefore serve to limit the production of proinflammatory medi- homologous, it is believed that these receptors activate similar ators during inflammation and infection. signaling pathways when stimulated with their cognate li- gands (1). Consistent with this hypothesis, the downstream signaling components, myeloid differentiation protein 88 Toll-like receptors (TLRs) represent a family of phylogeneti- (MyD88), IL-1R-associated kinase (IRAK) and tumor necrosis cally conserved proteins that have been found in insects, factor receptor-associated factor 6 (TRAF6) are all utilized by plants, and mammals (1–3). In addition to playing a critical these three groups of receptors to activate both nuclear factor role in the dorso-ventral axis formation in insects, TLRs are (NF)-B and mitogen-activated protein kinase signaling cas- also involved in innate immunity and disease resistance (2– 4). cades (12–16). MyD88 functions as a crucial adaptor that links In mammals, 10 members of the TLR family have been identi- the receptors to IRAK after activation. IRAK in turn is phos- fied which are expressed by host immune cells most likely to phorylated, dissociates from the receptor, and associates with come into direct contact with pathogens from the environment, TRAF6 to signal activation of either NF-B or mitogen-acti- such as dendritic cells, peripheral phagocytes, and mucosal vated protein kinases. However, genetic studies revealed that epithelia (5). Recent studies have revealed that a striking fea- neither MyD88 (17), IRAK (18), nor TRAF6 (19) is absolutely ture of TLRs is their ability to discriminate among different essential for cell activation in response to stimulation with classes of pathogens (3, 4). For example, TLR4 detects lipopo- LPS, implying the existence of alternative signaling pathways. lysaccharide (LPS) from Gram-negative bacteria, whereas The recent description of TIR domain-containing adaptor pro- TLR2 recognizes peptidoglycan (PGN), lipoproteins, and zymo- tein (TIRAP) (20), also known as Mal (MyD88 adaptor-like) san from Gram-positive bacteria and yeast. TLRs alone or in (21), probably explains the residual signaling observed in the combination are believed to mediate host immune responses to absence of MyD88. Interestingly TIRAP/Mal also appears to be a large array of pathogens (6). In addition to detecting exoge- involved in signaling through TLR4 but not IL-1, thereby dem- onstrating a unique specificity in the adaptor molecules that participate in signaling. * This work was supported by the Howard Hughes Medical Institute In addition to MyD88, another adaptor protein named Toll- and National Institutes of Health Grant R37 AI33443. The costs of interacting protein (Tollip) was recently found to associate with publication of this article were defrayed in part by the payment of page the cytoplasmic TIR domain of IL-1Rs after IL-1 stimulation charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. (22). Tollip forms a complex with IRAK in resting cells and ‡ To whom correspondence should be addressed: Section of Immuno- inhibits IL-1-induced signaling by blocking IRAK phosphoryl- biology, Yale University School of Medicine, 310 Cedar St., New Haven, ation. Because of the significant homology in the intracellular CT 06520. Tel.: 203-737-4419; Fax: 203-737-1764; E-mail: sankar. [email protected]. portion of TLRs, IL-1R, and IL-18R, we hypothesized that The abbreviations used are: TLR(s), Toll-like receptor(s); AP-1, ac- Tollip might also inhibit TLR-mediated signaling by interact- tivator protein-1; GST, glutathione S-transferase; HA, hemagglutinin; ing with TLRs through the TIR domain. Here we report that IL-1R, interleukin-1 receptor; IL-18R, interleukin-18 receptor; IRAK, Tollip associates directly with TLR2 and TLR4 and inhibits IL-1R-associated kinase; LPS, lipopolysaccharide; Luc, luciferase; Mal, MyD88-adaptor-like; MyD88, myeloid differentiation protein 88; NF- TLR-mediated cellular responses by suppressing phosphoryla- B, nuclear factor-B; PGN, peptidoglycan; TIR, Toll/IL-1R homology tion and kinase activity of IRAK. Furthermore, we find that domain; TIRAP, TIR domain-containing adaptor protein; Tollip, Toll- Tollip is phosphorylated by activated IRAK, making it the first interacting protein; TRAF6, tumor necrosis factor receptor-associated factor 6; UBA, ubiquitin-associated. direct substrate for IRAK to be characterized. This paper is available on line at http://www.jbc.org 7059 This is an Open Access article under the CC BY license. 7060 Tollip Inhibits TLR-mediated Signaling MATERIALS AND METHODS GST pull-down assays were performed essentially as described previ- ously (24). Briefly, 5 lof S-labeled lysates was incubated for1hat Cells and Reagents—Human embryonic kidney epithelial 293 cells 4 °C with 5 g of purified GST or GST-Tollip and 20 l of glutathione stably expressing the NF-B-dependent luciferase reporter pBIIX-Luc beads in 1 ml of TNT buffer. The beads were washed three times with (293-Luc) were developed as described previously (23). Mouse macro- TNT buffer, boiled in SDS sample buffer, and fractionated by 12.5% phage RAW264.7 cells were obtained from ATCC (Manassas, VA). Both SDS-PAGE. The radiolabeled proteins were visualized by cell lines were maintained in high glucose Dulbecco’s modified Eagle’s autoradiography. medium supplemented with 7% heat-inactivated fetal calf serum, 2 mM In Vitro Kinase Assays—For the detection of endogenous IRAK ac- L-glutamine, 100 units/ml penicillin, and 100 g/ml streptomycin. LPS tivity, 3  10 293/TLR2 or 293/TLR4 cells were plated into 10-cm from Escherichia coli 0111:B4, PGN from Staphylococcus aureus, anti- dishes and transfected the following day with 5 g of HA-Tollip and/or FLAG M2 antibody, and M2 agarose beads were purchased from Sigma. 5 g of MD-2 plasmid using FuGene 6. 24 h after transfection, cells Purified polyclonal rabbit antibodies against IRAK and hemagglutinin were either left untreated or stimulated with 1 g/ml LPS for 10 or 30 (HA) epitope were from Santa Cruz Biotechnology (Santa Cruz, CA). min and lysed in 1.2 ml of TNT buffer containing 2 mM NaF, 1 mM Protein A-Sepharose and glutathione-conjugated agarose beads were dithiothreitol, 1 mM EDTA, and a mixture of protease inhibitors. Cell from Amersham Biosciences, Inc. lysates were precipitated at 4 °C for 4 h with 1 g of anti-IRAK antibody Plasmids—Mammalian expression vectors containing N-terminal and 20 l of 50% (v/v) slurry of protein A-Sepharose beads. IRAK FLAG-tagged human TLR2 and TLR4 that were made by inserting autophosphorylation was measured essentially as described previously PCR-generated cDNA fragments lacking the signal sequence into using protocols developed for detecting IKK autophosphorylation (24). pFLAG-CMV-1 (Sigma) were kindly provided by R. Medzhitov (Yale After washing twice with TNT buffer and twice with kinase buffer (20 Medical School). The intracellular domains of TLR2 and TLR4 were mM HEPES, pH 7.5, 200 mM NaCl, 20 mM MgCl ,1mM EDTA, 2 mM amplified by PCR and cloned into pGBKT7 (Clontech, Palo Alto, CA). NaF, 1 mM dithiothreitol, 10 M ATP), the beads were suspended in 20 Plasmids for human CD14, FLAG-tagged human Tollip, HA-tagged l of kinase buffer containing 10 Ci of [- P]ATP (Amersham Bio- Tollip, and its deletion mutants were constructed in-frame by inserting sciences, Inc.). The reactions were allowed to proceed at 30 °C for 20 PCR-generated cDNA fragments into pCDNA3.1 (Invitrogen). FLAG- min and terminated with 20 l of SDS sample buffer. The beads were IRAK and HA-IRAK were generated by inserting cDNA into boiled, 20-l reaction aliquots were separated by 10% SDS-PAGE, and pCDNA3.1. Kinase-inactive FLAG-IRAK (K239A) and HA-IRAK visualized by autoradiography. For measuring phosphorylation of (K239A) were made using the Quick site-directed mutagenesis kit from Tollip by IRAK, IRAK was first precipitated, and immune complexes Stratagene (La Jolla, CA). All FLAG and HA epitope tags were gener- were washed with TNT and kinase buffers, then incubated in 25 lof ated by incorporating tag sequences into primers at the N terminus of kinase buffer with 10 Ci of [- P]ATP and 2 g of purified GST, respective cDNA fragments. Correct inserts were confirmed by direct GST-Tollip, or its truncation mutants. Phosphorylated proteins were sequencing. The expression vector pEFBOS containing human MD-2 precipitated further with 20 l of glutathione-agarose beads, separated was kindly provided by K. Miyake (Saga Medical School, Japan). by SDS-PAGE, and visualized by autoradiography. Development of 293/TLR2 and 293/TLR4 Stable Cell Lines—293- Luc cells were seeded into 10-cm dishes and transfected using FuGene RESULTS 6 (Roche Molecular Biochemicals) with 5 g of pFLAG-TLR2 or pFLAG- TLR4 together with 0.5 g of a plasmid encoding CD14 containing a Tollip Physically Associates with TLR2 and TLR4 via Its hygromycin resistance gene. Cells were selected in Dulbecco’s modified C-Terminal Domain—To determine whether Tollip interacts Eagle’s medium with 200 g/ml hygromycin B (Calbiochem). Individual with TLRs via the TIR domain, we developed two cell lines colonies were picked, expanded, and confirmed for the surface expres- (293/TLR2 and 293/TLR4) that stably express human CD14, as sion of TLRs by flow cytometry and immunoblotting using anti-FLAG well as FLAG-tagged human TLR2 or TLR4. Flow cytometry M2. Stable cell lines were propagated and maintained in complete Dulbecco’s modified Eagle’s medium for regular 293-Luc cells. and immunoblotting revealed significant amounts of TLR ex- Transfection and Luciferase Reporter Assays—RAW264.7 cells were pression on the cell surface (data not shown). Furthermore, plated at a density of 3  10 cells/well in 24-well plates 1 day before expression of the TLRs rendered the 293 cells highly responsive transfection. Cells were transfected in triplicate using FuGene 6 with to various bacterial products, which do not otherwise activate 0.2 g of pBIIX-Luc or pAP-1-Luc and the indicated amounts of HA- these cells (data not shown). Tollip plasmid. The total amount of transfected plasmids was equalized Interactions between Tollip and TLRs were examined by by supplementing with the empty vector pCDNA3.1. After 24 h, cells transfecting these cell lines with plasmids expressing HA- were either left untreated or stimulated with 100 ng/ml LPS or 10 g/ml PGN for 6 h. Cells were then lysed and assayed for luciferase activity Tollip and/or MD-2, which is a secreted molecule that binds to (Promega, Madison, WI). The data were normalized to total protein TLR4 and is required for signaling (25). The FLAG-tagged concentrations of each sample measured using a protein assay kit from TLRs were immunoprecipitated using anti-FLAG antibody fol- Bio-Rad. lowed by immunoblotting with anti-HA antibody. HA-Tollip Immunoprecipitation and Immunoblotting—For coimmunoprecipita- coprecipitated efficiently with TLR2 (Fig. 1A, lane 3 of the top tion of transfected proteins, 7  10 293, 293/TLR2, or 293/TLR4 cells panel). Association between Tollip and TLR4 was also detect- were plated into six-well plates and transfected the following day with the indicated plasmids using FuGene 6. 24 –36 h after transfection, cells able but was enhanced significantly in the presence of MD-2 were lysed in 0.5 ml of TNT buffer (20 mM Tris, pH 7.5, 200 mM NaCl, (Fig. 1A, lanes 5 and 6 of the top panel), suggesting that 1% Triton X-100) containing a protease inhibitor mixture (Roche Mo- conformational changes in the cytoplasmic tail of TLR4 induced lecular Biochemicals). A portion of the lysates (50 l) was saved for by MD-2 may be necessary for efficient binding to Tollip. To immunoblotting, and FLAG-tagged proteins were precipitated from the determine whether Tollip directly interacts with the TIR do- remainder for 2–4hat4 °C with 20 l of 50% (v/v) slurry of anti-FLAG main of TLRs, in vitro GST pull-down assays were performed. M2 beads. After extensive washing with TNT buffer, the beads were boiled in SDS sample buffer. Proteins were separated by 10% SDS- [ S]Methionine-labeled intracellular portions of TLR2 and PAGE, transferred to polyvinylidene difluoride membranes, and blotted TLR4 were produced by in vitro translation and incubated with with the indicated antibodies. The reactive bands were visualized by either GST or GST-Tollip. As shown in Fig. 1B, both TLR2 and enhanced chemiluminescence (Amersham Biosciences, Inc.). TLR4 bound to GST-Tollip, but not GST alone, demonstrating Generation of Glutathione S-Transferase (GST)-Tollip and in Vitro that Tollip associates directly with the TIR domain of TLRs. Pull-down Assays—GST-Tollip and its N-terminal (1–52), central (53– To map the region of Tollip responsible for association with 178), and C-terminal (179 –273) truncation mutants were made by cloning respective human Tollip cDNA fragments in-frame into pGEX- the TLRs, we analyzed the interaction of TLR2 and TLR4 with 4T-1 (Amersham Biosciences, Inc.). GST fusion proteins were induced truncation mutants of Tollip as indicated in Fig. 2A. TLR4 with 0.4 mM isopropyl--D-thiogalactopyranoside for 4 –6hat37 °C and bound efficiently to either full-length Tollip (1–273) or mutants incubated with glutathione beads. Purified GST proteins were obtained with an intact C-terminal sequence, Tollip (53–178) and by elution from the beads with 10 mM reduced glutathione followed by 35 Tollip (53–273) (Fig. 2B, lanes 1, 4 and 5 of the top panel). Also, dialysis in phosphate-buffered saline. [ S]Methionine-labeled proteins whereas Tollip (1–229) could interact with TLR4, no associa- were generated with TNT-T7 Quick-coupled transcription/translation system (Promega) according to the manufacturer’s instructions. In vitro tion was found between TLR4 and Tollip (1–178) or Tollip Tollip Inhibits TLR-mediated Signaling 7061 FIG.1. Tollip associates directly with TLR2 and TLR4. A, coim- munoprecipitation of Tollip with TLR2 and TLR4. 293, 293/FLAG- TLR2, or 293/FLAG-TLR4 cells were transfected with expression plas- mids as indicated (1 g of each); after 24 h, proteins in the cell lysates FIG.2. Analysis of domain interactions of Tollip with TLR4. A, were immuoprecipitated (IP) using anti-FLAG M2 beads. Coprecipi- coimmunoprecipitation of Tollip mutants with TLR4. 293/FLAG-TLR4 tated proteins were detected by immunoblotting (IB) with anti-HA and cells were cultured in six-well plates and transfected with 1 g of MD-2 anti-FLAG antibodies. Cell lysates (20 l) were also immunoblotted plasmid together with 0.5 g of the indicated HA-Tollip mutants. Im- using anti-HA antibody to monitor the expression of transfected Tollip. munoprecipitation and immunoblotting were performed as described in B, direct interactions of Tollip with TLR2 and TLR4. Intracellular the legend to Fig. 1A. B, analysis of interactions of Tollip mutants with domains of TLR2 and TLR4 were S labeled by in vitro translation and TLR4 and Tollip. Diagrammatic representations of Tollip mutants are incubated with purified GST or GST-Tollip bound to glutathione beads. shown; black segments represent the C2 domain, and striped segments The beads were then washed, boiled in SDS sample buffer, and frac- are the UBA domain. , very strong; , strong; , weak; ,no tionated by 12.5% SDS-PAGE. The radiolabeled proteins were visual- interaction. ized by exposure to x-ray film. (53–178), indicating that the interaction domain lies in the 178) (Figs. 3B and 2A). Therefore, self-association of Tollip is C-terminal region of Tollip between residues 179 and 229 (Fig. mediated by both the N- and C-terminal regions of the protein 2B, lanes 2, 3 and 6 of the top panel). Similar results were and may result in formation of a stable protein complex. observed with TLR2 (data not shown). Interestingly, binding of Although both Tollip and MyD88 function as adaptors to link Tollip to IL-1R is also mediated through a similar C-terminal IL-1R or TLRs to downstream molecules, neither in vitro GST region (22). pull-down nor in vivo mammalian coimmunoprecipitation as- Tollip Interacts with Itself to Form Oligomers—TLRs, IL-1R, says revealed a stable complex between these two molecules and IL-18R are all known to form either homo- or heterodimers (data not shown). These results suggested that MyD88 and upon stimulation with their cognate ligands. Therefore, it is Tollip exist in distinct protein complexes, which is consistent possible that downstream proteins might also oligomerize to with previous findings that both molecules are recruited inde- allow signals to be transduced efficiently. To test whether pendently to the IL-1R after stimulation (22). Tollip forms homo-oligomers, we in vitro translated full-length Tollip Inhibits Cell Activation Induced by Bacterial Prod- S-labeled Tollip (1–273) and used it in GST pull-down assays. ucts—To assess the functional role of Tollip in TLR-mediated We found that Tollip bound to GST-Tollip but not to GST alone signaling, we transfected mouse macrophage RAW264.7 cells (Fig. 3A), suggesting that Tollip is capable of self-association. with increasing amounts of Tollip together with either NF-B To identify further the domains involved in Tollip homo-oli- or AP-1 luciferase reporter vectors. Luciferase activity was gomerization, 293-Luc cells were cotransfected with FLAG- measured after stimulation of the transfected cells with vari- tagged full-length Tollip and the HA-tagged Tollip deletion ous bacterial products. Both NF-B and AP-1 were activated mutants described in Fig. 2A, and the interaction between the readily in response to LPS (Fig. 4) and PGN (data not shown), proteins was analyzed by coimmunoprecipitation (Fig. 3B). All and both were inhibited dose-dependently by Tollip, suggesting Tollip mutants associated efficiently with full-length Tollip that Tollip is a negative regulator of TLR2 and TLR4 signaling. except for the construct containing only the C2 domain (53– Overexpression of Tollip also impaired NF-B activation in 7062 Tollip Inhibits TLR-mediated Signaling FIG.3. Tollip forms oligomers. A, self-association of Tollip. Tollip was translated in vitro and incubated with purified GST or GST-Tollip bound to glutathione beads. The pull-down assay was performed as described in the legend to Fig. 1B. B, analysis of domains involved in self-association of Tollip. 293-Luc cells were cultured in six-well plates and transfected with 0.5 g of FLAG-Tollip plasmid together with 0.5 g of the indicated HA-Tollip deletion mutants. Immunoprecipitation and immunoblotting were performed as described in the legend to Fig. 1A. FIG.5. Tollip suppresses phosphorylation and kinase activity of IRAK. A, inhibition of the recruitment of phosphorylated IRAK to the TLR4 receptor complex. 293/FLAG-TLR4 cells were cultured in six-well plates and transfected with 1 g of each of the indicated plasmids. Immunoprecipitation and immunoblotting were performed as described in the legend to Fig. 1A. B, suppression of LPS-induced kinase activity of IRAK by Tollip. 293/FLAG-TLR2 and 293/FLAG-TLR4 cells were cultured in 10-cm dishes and transfected with the plasmids indi- cated (5 g of each). 24 h after transfection, cells were either left untreated or stimulated with 1 g/ml LPS for 10 or 30 min. Proteins in cell lysates were precipitated using anti-IRAK antibody and protein A-Sepharose beads. Immune complexes were washed, and an aliquot of the immunoprecipitate was saved for immunoblotting to confirm equal loading. The remainder of the precipitated proteins was incubated in FIG.4. Tollip impairs LPS-induced cell activation. RAW264.7 kinase buffer at 30 °C for 20 min, separated by SDS-PAGE, and phos- cells were plated in 24-well plates and transfected in triplicate with 0.2 phorylated IRAK was visualized by autoradiography. g of luciferase reporter vector pBIIX-Luc or pAP-1-Luc together with the indicated amounts of HA-Tollip plasmid. After 24 h, cells were FLAG-TLR2 and 293/FLAG-TLR4 cells were transfected with either left untreated (white bars) or stimulated (black bars) with 100 Tollip together with either wild type or kinase-inactive ng/ml LPS for a further 6 h. Cells were then lysed and assayed for luciferase activity. The data were normalized to total protein concen- (K239A) IRAK. Consistent with previous findings, transfection trations and are represented as the means  S.D. Similar results were of wild type IRAK resulted in significant autophosphorylation obtained from three independent experiments. These data demonstrate of IRAK; however, the level of phosphorylation was diminished that Tollip inhibits activation of both NF-B(A) and AP-1 (B)ina dramatically in the presence of Tollip (Ref. 22 and Fig. 5A, dose-dependent manner. lanes 7 and 9 versus 8 and 10 of the second panel from the 293/TLR2 and 293/TLR4 cells after stimulation with LPS or bottom). Immunoprecipitation of TLR4 with anti-FLAG anti- PGN (data not shown). body revealed that Tollip did not appear to influence the Tollip Suppresses the Phosphorylation and Kinase Activity of amount of IRAK (K239A) recruited to the TLR4 receptor com- IRAK—To understand better the molecular mechanism by plex in the presence or absence of MD-2 (Fig. 5A, lanes 3 and 4 which Tollip inhibits TLR-mediated signaling, we analyzed its versus 5 and 6 of the top panel). In contrast, recruitment of effect on the recruitment of IRAK to the TLR complex. 293/ phosphorylated IRAK was completely abolished by Tollip re- Tollip Inhibits TLR-mediated Signaling 7063 FIG.6. Tollip is phosphorylated by activated IRAK. A, phosphorylation of Tollip by IRAK. 293/FLAG-TLR4 cells were cultured in 10-cm dishes and transfected with 5 g of MD-2 plasmid with or without 5 g of HA-Tollip. 24 h after transfection, cells were either left untreated or stimulated with 1 g/ml LPS for 10 –30 min. Cell lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads, and kinase assays were performed as described in the legend to Fig. 5B. The lysates (20 l) were also blotted with anti-IRAK and anti-HA antibodies to monitor the expression of transfected Tollip and phosphorylation of endogenous IRAK upon stimulation. B, phosphorylation of GST-Tollip by IRAK. 293/FLAG-TLR4 cells were cultured in six-well plates and transfected with 1 g/well MD-2 plasmid. Cells were then left untreated or stimulated with 1 g/ml LPS for 10 –30 min, and lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads. Kinase assays were performed in the presence of 2 g of purified GST or GST-Tollip. Phosphorylated proteins were precipitated further with glutathione beads, separated by SDS-PAGE, and visualized by autoradiography. Approximately equal amounts of GST protein input are shown on the bottom panel. C, Tollip is phosphorylated by IRAK at the C terminus. 293/FLAG-TLR4 cells were seeded in six-well plates and transfected with 1 g/well MD-2 plasmid. After 24 h, cells were stimulated with 1 g/ml LPS for 20 min, and lysates were precipitated with anti-IRAK antibody and protein A-Sepharose beads. Kinase assays were performed in the presence of 2 g of purified GST, GST-Tollip, or its truncation mutants. The bottom panel shows approximately equal amounts of GST proteins used in the kinase assay. 7064 Tollip Inhibits TLR-mediated Signaling gardless of MD-2 expression (Fig. 5A, lanes 7 and 9 versus 8 initially as a protein involved in IL-1 signaling, we now show and 10 of the top panel). Similar results were observed in that Tollip can associate with TLR2 and TLR4 and play an 293/FLAG-TLR2 cells (data not shown). inhibitory role in TLR-mediated cell activation. Consistent In vitro kinase assays were also performed to confirm that with our findings, during the preparation of the manuscript, suppression of IRAK autophosphorylation by Tollip was the Bulut et al. (31) reported independently that Tollip coprecipi- result of decreased IRAK kinase activity. 293/FLAG-TLR2 and tates with TLR2 and TLR4, and overexpression of Tollip inhib- 293/FLAG-TLR4 cells were transfected with Tollip and/or its TLR2- and TLR4-induced NF-B activation in human der- MD-2, stimulated with LPS, and then IRAK was immunopre- mal microvessel endothelial cells. cipitated and assayed for its kinase activity. As expected, IRAK One striking feature of Tollip is its ability to suppress the was heavily phosphorylated after LPS stimulation (Fig. 5B, kinase activity of IRAK. Several lines of evidence suggest that lanes 2 and 8 of the top panel), but this autophosphorylation the mechanism by which Tollip inhibits TLR signaling is by was decreased dramatically in the presence of Tollip (Fig. 5B, preassociating with IRAK, thus preventing it from being phos- lanes 4, 5, 10, and 11 of the top panel). These findings suggest phorylated and activated on the TLR receptor complex. In fact, a possible mechanism by which Tollip might impair TLR sig- studies using IRAK-deficient cells demonstrated that phospho- naling, namely by blocking the autophosphorylation of IRAK, rylation of IRAK is required for it to dissociate from the recep- which is necessary for the dissociation of IRAK from the recep- tor and transduce signals to downstream molecules leading to tor complex (26, 27). NF-B activation (26, 27). The ability of Tollip to interact Tollip Is Phosphorylated by IRAK—IRAK is a serine/threo- directly with both the death (1–208) and kinase (181–545) nine kinase with a centrally located catalytic domain (13). domains of IRAK may explain the inhibitory role of Tollip (22, Upon stimulation with either IL-1 or bacterial products, IRAK and data not shown). is activated and heavily phosphorylated (Ref. 13 and Fig. 6A, Upon activation, IRAK undergoes rapid autophosphorylation lanes 2 and 3 of both top and bottom panels). However, the and dissociates from the receptor. Our studies reveal that after identity of potential IRAK substrate(s) has remained unknown. stimulation IRAK also phosphorylates Tollip. The physiological In addition, recent studies have suggested that the kinase significance of this phosphorylation is still unclear, although it activity of IRAK is dispensable for its role as an intermediate in is possible that phosphorylation of Tollip may facilitate its IL-1 signaling (28, 29). However, we were surprised to find that dissociation from IRAK and subsequent degradation by ubiq- after LPS stimulation, activation of IRAK is accompanied by a uitination. This is consistent with the fact that Tollip contains concomitant phosphorylation of Tollip in 293/FLAG-TLR4 (Fig. a highly conserved UBA (ubiquitin-associated) or CUE (cou- 6A, lanes 5 and 6 of the top panel) and 293/FLAG-TLR2 cells pling of ubiquitin conjugation to endoplasmic reticulum degra- (data not shown). Phosphorylation of Tollip did not occur in dation) domain at the C terminus (230 –270). Similar domains resting cells without activated IRAK (Fig. 6A, lane 4 of the top in other proteins have been shown to recruit ubiquitin-conju- panel). gating enzymes, leading to proteosome-dependent degradation To confirm this observation, the 293/FLAG-TLR2 and 293/ (32, 33). Interestingly, the region around the UBA domain of FLAG-TLR4 cells were either left untreated or stimulated with Tollip binds to unphosphorylated IRAK under resting condi- LPS. IRAK was immunoprecipitated from these cells and incu- tions and also associates transiently with the TLRs and IL-R bated with purified GST-Tollip in in vitro kinase assays. We upon stimulation (22). The same region of Tollip can also be found that upon stimulation with LPS, concordant with the phosphorylated by IRAK (Fig. 6C). Therefore, once activated on phosphorylation of IRAK, GST-Tollip (Fig. 6B, lanes 4 – 6 of the the receptor, IRAK phosphorylates Tollip, which may lead to top panel), but not GST alone (Fig. 6B, lanes 1–3 of the top the dissociation of Tollip from IRAK and the receptor complex. panel), was heavily phosphorylated. Similar results were also Unmasking the UBA domain on Tollip may lead to its subse- observed in 293-Luc cells stimulated with IL-1 (data not quent degradation by a ubiquitination-dependent process. The shown). Therefore these experiments strongly suggest that in removal of Tollip would allow signaling to continue by freeing addition to undergoing autophosphorylation, IRAK also phos- activated IRAK to bind to downstream TRAF6. Because IRAK phorylates Tollip. Phosphorylation of Tollip by activated IRAK also undergoes rapid ubiquitination and degradation after ac- may facilitate the dissociation of IRAK from Tollip, thereby tivation (13, 34), it is tempting to speculate that phosphoryl- allowing IRAK to escape the inhibitory effect of Tollip and ated Tollip facilitates the ubiquitination of IRAK by recruiting subsequently modify downstream signaling components. ubiquitin-conjugating enzymes. Further studies will be needed To determine further the domain of Tollip which is phospho- to explore fully the biological ramifications of Tollip phospho- rylated by IRAK, we produced N-terminal (1–52), central (53– rylation by IRAK. 178), and C-terminal (179 –273) truncation mutants of Tollip Although a more complete understanding of biological func- fused with GST. However, the central region of Tollip, which tions of Tollip awaits the generation and analysis of mice lack- encompasses only the C2 domain, is completely insoluble when ing Tollip, it is possible that the primary role of Tollip-mediated expressed in bacteria. Therefore, kinase assays were performed pathway may be to maintain immune cells in a quiescent state with purified N- and C-terminal Tollip domains together with in the absence of infection and facilitate the termination of wild type GST-Tollip. As shown in Fig. 6C, the C-terminal TLR-induced cell signaling during inflammation and infection. (179 –273) but not N-terminal (1–52) region of Tollip is phos- It is conceivable that this evolutionarily conserved inhibitory phorylated by IRAK. Interestingly, the same region is also system benefits the host by limiting the production of proin- responsible for the interactions of Tollip with TLRs and IRAK flammatory mediators and subsequent tissue damage under (Fig. 2 and Ref. 22). However, we cannot rule out the possibility infectious conditions. Therefore, strategies to up-regulate that the central C2 domain of Tollip might also be phosphoryl- Tollip expression may prove effective in treating both chronic ated by IRAK upon activation because the intensity of phos- and acute inflammatory diseases, such as inflammatory bowel phorylation at the C terminus is considerably weaker than that disease and septic shock. for wild type Tollip (Fig. 6C, lane 3 versus 4ofthe top panel). Acknowledgments—We thank Ruslan Medzhitov, Elizabeth Kopp, and Kensuke Miyake for providing the various plasmids, and members DISCUSSION of the Ghosh laboratory for helpful discussions. We thank Michael May In this paper we demonstrate that the adaptor protein Tollip for careful reading of the manuscript. We are also grateful to the can negatively regulate TLR signaling pathways. Described anonymous reviewers’ constructive comments on the manuscript. 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Published: Mar 1, 2002

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