Dephosphorylating TRAM TRIF-Dependent TLR4 Pathway By

Phosphatase PTPN4 Preferentially Inhibits TRIF-Dependent TLR4 Pathway by Dephosphorylating TRAM

This information is current as Wanwan Huai, Hui Song, Lijuan Wang, Bingqing Li, Jing of September 29, 2021. Zhao, Lihui Han, Chengjiang Gao, Guosheng Jiang, Lining Zhang and Wei Zhao J Immunol 2015; 194:4458-4465; Prepublished online 30 March 2015;

doi: 10.4049/jimmunol.1402183 Downloaded from http://www.jimmunol.org/content/194/9/4458

References This article cites 32 articles, 11 of which you can access for free at: http://www.jimmunol.org/content/194/9/4458.full#ref-list-1 http://www.jimmunol.org/

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. The Journal of Immunology

Phosphatase PTPN4 Preferentially Inhibits TRIF-Dependent TLR4 Pathway by Dephosphorylating TRAM

Wanwan Huai,* Hui Song,* Lijuan Wang,† Bingqing Li,‡ Jing Zhao,* Lihui Han,* Chengjiang Gao,* Guosheng Jiang,‡ Lining Zhang,* and Wei Zhao*

TLR4 recruits TRIF-related adaptor molecule (TRAM, also known as TICAM2) as a sorting adaptor to facilitate the interaction between TLR4 and TRIF and then initiate TRIF-dependent IRF3 activation. However, the mechanisms by which TRAM links downstream molecules are not fully elucidated. In this study, we show that TRAM undergoes tyrosine phosphorylation upon TLR4 activation and that is required for TLR4-induced IRF3 activation. Protein tyrosine phosphatase nonreceptor type 4 (PTPN4), a protein tyrosine phosphatase, inhibits tyrosine phosphorylation and subsequent cytoplasm translocation of TRAM, resulting in the disturbance of TRAM–TRIF interaction. Consequently, PTPN4 specifically inhibits TRIF-dependent IRF3 activation and IFN-b production in TLR4 pathway. Therefore, our results provide new insight into the TLR4 pathway and identify PTPN4 as a specific Downloaded from inhibitor of TRIF-dependent TLR4 pathway. Targeting PTPN4 would be beneficial for the development of new strategy to control TLR4-associated diseases without unwanted side effects. The Journal of Immunology, 2015, 194: 4458–4465.

attern recognition receptors (PRRs) mediate the recogni- IRF3 and then induces the expression of type I IFN (IFN-a/b)(3). tion of pathogen-associated molecular patterns and trigger BesidesTLR4,TLR3alsousesTRIFtoactivatedownstream http://www.jimmunol.org/ P innate immune responses against pathogens invasion (1, 2). pathways, while it recruits TRIF directly, without the need for TLRs are the best characterized PRRs, which possess leucine- TRAM. rich repeats, transmembrane domains, and intracellular Toll-IL-1R Reversible phosphorylation of proteins, which is catalyzed by (TIR) domains (2, 3). Unique among the large family of TLRs, kinases and phosphatases, is a key regulatory mechanism for nu- TLR4 engages two distinct adaptor proteins: MyD88 and TRIF, merous important aspects of physiology. Phosphorylation status of which activates two separate pathways, the MyD88-dependent and receptors or adaptors involved in PRRs signaling directly affects TRIF-dependent pathway (also called MyD88-independent path- the transduction of activation signals and is crucial for optimal ways) (3). In the case of TLR4 signaling, TLR4 uses two adaptors: innate immune responses. For example, a dynamic balance be-

TIR domain–containing adapter protein (TIRAP, also known as tween phosphorylation and dephosphorylation of RIG-I and MDA5 by guest on September 29, 2021 Mal) and TRIF-related adaptor molecule (TRAM, also known as is essential for their signal transduction (9). Serine phosphorylation TICAM2) as sorting adaptors to facilitate signal transduction (3). of NLRC4 (a cytosolic member of the NOD-like receptor family) TIRAP recruits MyD88 to facilitate the activation of the MyD88- is critical for inflammasome activation (10). In the setting of TLR dependent pathway at the plasma membrane, resulting in the ac- pathways, TLR2, TLR3, TLR4, TLR5, TLR8, and TLR9 undergo tivation of NF-kB and MAPK pathways, and subsequent secretion tyrosine phosphorylation (11), and mutations of tyrosines within of proinflammatory cytokines, such as TNF-a and IL-6. As for the their TIR domains suppress TLRs activation. In addition to the TRIF-dependent pathway, TRAM is needed for the recruitment of receptors, other components of the PRRs signaling pathways (e.g., TRIF (4–6). Following LPS stimulation, TRAM dissociate from the adaptors, also undergo tyrosine phosphorylation upon ligand the membrane to endosomes, where the TRAM–TRIF pathway is engagement) (11). Tyrosine phosphorylation of TIRAP is required initiated (7, 8). This leads to the activation of transcription factor for the recruitment of MyD88 to TLR2 and TLR4 (12, 13). TRIF and MyD88 have been shown to be tyrosine phosphorylated *Department of Immunology, Shandong University School of Medicine, Jinan, Shan- causing downregulation of TLR signaling (14). However, the dong 250012, China; †Pathology Tissue Bank, Qilu Hospital, Shandong University, phosphorylation modification of key proteins in PRRs signaling Jinan, Shandong 250012, China; and ‡Institute of Basic Medicine, Shandong Acad- emy of Medical Sciences, Jinan, Shandong 250062, China has not been fully elucidated. Protein tyrosine phosphatases Received for publication August 25, 2014. Accepted for publication February 18, (PTPs), which dephosphorylate tyrosine residues of target sub- 2015. strates (15, 16), have been reported to be involved in the regulation This work was supported by grants from the National Natural Science Foundation of of innate immune responses, such as Src homology region 2 do- China (Grant 31370017), Shandong Provincial Nature Science Foundation for Dis- main–containing phosphatase-1 (17), Src homology region 2 do- tinguished Young Scholars (Grant JQ201420), and the National “973” Program of China (Grant 2011CB503906). main–containing phosphatase-2 (18), PTPN22 (19), PTP with Address correspondence and reprint requests to Dr. Wei Zhao, Department of Immu- proline-glutamine-serine-threonine-rich motifs (20), PTP1B (21), nology, Shandong University School of Medicine, 44 Wenhua Xi Road, Jinan, Shandong, and so on. However, the PTPs identified to be PRRs regulators 250012, China. E-mail address: [email protected]. possessed broadly regulatory effects and that limited their poten- Abbreviations used in this article: HA, hemagglutinin; HEK, human embryonic tial implication for immunotherapy. Thus, more specific regulator kidney; IP, immunoprecipitation; LTA, lipothechoic acid; PGN, peptidoglycan; poly(I:C), polyinosinic:polycytidylic acid; PRR, pattern recognition receptor; remains to be identified. PTPN4, protein tyrosine phosphatase nonreceptor type 4; SeV, Sendai virus; PTP nonreceptor type 4 (PTPN4, also known as PTP-MEG1) TAG, TRAM adaptor with GOLD; TIR, Toll-IL 1R; TIRAP, TIR domain–containing functions in TCR cell signaling and apoptosis (22, 23). How- adapter protein; TRAM, TRIF-related adaptor molecule; WT, wild-type. ever, to our knowledge, the roles of PTPN4 in innate immune Copyright Ó 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00 responses have never been investigated. In this study, we show that www.jimmunol.org/cgi/doi/10.4049/jimmunol.1402183 The Journal of Immunology 4459

PTPN4 specifically inhibits TRIF-dependent TLR4 pathway by GGAUUUGUUAAACCAUUAA-39 (siRNA 1) and 59-GGUUUGGAUUC- suppressing tyrosine phosphorylation of TRAM. TRAM under- AAUGUAAA-39 (siRNA 2) for PTPN4, and “scrambled” control sequences 9 9 goes tyrosine phosphorylation at Tyr167 upon TLR4 activation were 5 -UUCUCCGAACGUGUCACGU-3 . siRNA sequences for TRIM38 were described previously (25). and that is required for TLR4-induced IRF3 activation and IFN-b secretion. PTPN4 attenuates TRAM tyrosine phosphorylation and ELISA inhibits its translocation and subsequent TRAM–TRIF interaction. The concentration of IFN-b was measured with ELISA kits (BioLegend, These results indicate that tyrosine phosphorylation of TRAM is San Diego, CA). The concentrations of TNF-a and IL-6 were measured critical for TLR4 activation and also identify PTPN4 as a specific with ELISA kits (Dakewe Biotech, Shenzhen, China). inhibitor of TRIF-dependent pathway triggered by TLR4 en- RNA quantitation gagement. Total RNA was extracted with TRIzol reagent, according to the manu- facturer’s instructions (Invitrogen). A LightCycler (ABI PRISM 7000) and Materials and Methods a SYBR RT-PCR kit (Takara) were used for quantitative real-time RT-PCR 9 Mice and reagents analysis. Specific primers used for RT-PCR assays were 5 -ATGAGTG- GTGGTTGCAGGC-39 and 59-TGACCTTTCAAATGCAGTAGATTCA-39 Female C57BL/6 mice (5–6 wk) were obtained from Joint Ventures Sipper for IFN-b and 59-TGTTACCAACTGGGACGACA-39 and 59-CTGGGT- BK Experimental Animal (Shanghai, China). All animal experiments were CATCTTTTCACGGT-39 for b-actin. Data are normalized to b-actin ex- undertaken in accordance with the National Institutes of Health’s Guide for pression in each sample. the Care and Use of Laboratory Animals, with the approval of the Sci- entific Investigation Board of Medical School of Shandong University Immunoprecipitation and Western blot

(Jinan, Shandong, China). LPS (Escherichia coli, 055:B5) and lipothechoic For IP,whole-cell extracts were lysed in IP buffer containing 1% (v/v) Nonidet Downloaded from acid (LTA) were from Sigma-Aldrich (St. Louis, MO); peptidoglycan P-40, 50 mM Tris-HCl (pH 7.4), 50 mM EDTA, 150 mM NaCl, and a protease (PGN), polyinosinic:polycytidylic acid (poly[I:C]), and imidazoquinoline inhibitor “mixture” (Merck). After centrifugation for 10 min at 14,000 3 g, compound R848 were purchased from InvivoGen (San Diego, CA). The supernatants were collected and incubated with protein G Plus–Agarose IP concentration of agonists were used as below: 100 ng/ml LPS, 10 mg/ml m m m reagent together with specific Ab. After 6 h of incubation, beads were poly(I:C), 10 g/ml PGN, 1 g/ml R848, and 5 g/ml LTA. The Abs washed five times with IP buffer. Immunoprecipitates were eluted by boiling specific to p-tyr (9416), anti-Myc (2272), IRF3 (4302), STAT1 (9175), 701 with 1% (w/v) SDS sample buffer. For Western blot, cells were lysed with anti-(p)-IRF3(Ser396) (4947), anti–p-STAT1(Tyr ) (7649), anti–p-JNK M-PER Protein Extraction Reagent (Pierce, Rockford, IL) supplemented (4668), anti–p-p38 (4511) anti–p-ERK (4370), anti-JNK (9258), anti-p38 http://www.jimmunol.org/ k a with a protease inhibitor “mixture,” and then protein concentrations in the (8690), anti-ERK1/2 (4695), and anti–p-I B (2859) were from Cell extracts were measured with a bicinchoninic acid assay (Pierce). Equal Signaling Technology (Beverly, MA). Anti-Flag (F3165) was from Sigma- amounts of extracts were separated by SDS-PAGE and then were trans- Aldrich. Anti-PTPN4 (PAB4081) was from Abnova. Anti-TRAM (sc- ferred onto nitrocellulose membranes for immunoblot analysis (24, 25). 34748), anti–b-actin (sc-81178), and protein G–agarose (sc-2002) used for immunoprecipitation (IP) and HRP-conjugated secondary Abs were from Membrane fractionation Santa Cruz Biotechnology (Santa Cruz, CA). Sendai virus (SeV) was purchased from China Center for Type Culture Collection (Wuhan Uni- Membrane and cytoplasmic protein extraction was performed using a versity, Wuhan, China). Membrane and Cytosol Protein Extraction kit (Beyotime, Shanghai, China). Briefly, 4 3 106 cells were resuspended in 200 ml membrane Cell culture protein isolation solution A and homogenized on ice. The homogenate was centrifuged at 700 3 g for 10 min at 4˚C. The supernatant was then by guest on September 29, 2021 To obtain mouse primary peritoneal macrophages, C57BL/6J mice were centrifuged at 14,000 3 g for 30 min at 4˚C. The resulting supernatant was injected i.p. with 3% Brewer’s thioglycollate broth. Three days later, the cytoplasmic protein fraction. The pellet was resuspended in 40 ml peritoneal exudate cells were harvested and incubated. Two hours later, membrane protein isolation solution B. The homogenate was incubated on nonadherent cells were removed, and the adherent monolayer cells were ice for 10 min, vortexed for 5 s, and then centrifuged at 14,000 3 g for used as peritoneal macrophages. Mouse macrophage cell line RAW264.7 5 min. The resulting supernatant was the membrane protein fraction. and human embryonic kidney (HEK)293 cells were obtained from American Type Culture Collection (Manassas, VA). HEK293–TLR3 and Luciferase assay HEK293–TLR4 cell lines were obtained from InvivoGen. The cells were cultured at 37˚C under 5% CO2 in DMEM supplemented with 10% FCS Luciferase activities were measured with Dual-Luciferase Reporter Assay (Invitrogen Life Technologies), 100 U/ml penicillin, and 100 mg/ml System (Promega), according to the manufacturer’s instructions (24, 25). streptomycin (24, 25). Data are normalized for transfection efficiency by subtracting Firefly lu- ciferase activity with that of Renilla luciferase. Plasmid constructs Production of a phosphospecific TRAM Ab Flag-tagged PTPN4 wild-type (WT) and DA plasmids were provided by Dr. N. S. van Oers (University of Texas Southwestern Medical Center, Dallas, ChinaPeptides (Shanghai, China) immunized a rabbit with a synthetic peptide TX) and subcloned in pCMV-Myc plasmids (Promega). PTPN4 DA plasmid corresponding to aa 163–174 of TRAM (RQHKYNSVIPMR) with a phos- is a phosphatase activity–disrupted mutant in which an Asp to Ala point photyrosine incorporated instead of the tyrosine. The phosphospecific Abs mutation was introduced in the PTPase domain (22). PTP1B plasmid were then affinity purified from the blood by using the phosphopeptide. was provided by Dr. N. K. Tonks (Cold Spring Harbor Laboratory, Cold Spring, NY) (26). Flag-TRAM expression plasmid was provided by Structural analysis Dr. L. A. O’Neill (Trinity College Dublin, Dublin, Ireland). The TRAM The Protein Data Bank file (PDB code: 2M1W) of TIR domain of human Y154F and Y167F mutations were generated using the KOD-Plus-Mutagenesis TRAM is downloaded from Protein Data Bank (http://www.rcsb.org). k kit (Toyobo, Osaka, Japan). NF- B reporter plasmid was purchased from Structural figures were generated using PyMol (http://www.pymol.org). Stratagene. IFN-b and IRF3 reporter plasmids were gifts from Dr. X. Cao (Second Military Medical University, Shanghai, China). Expression plas- Statistical analysis mids for RIG-I, MAVS, TRIF, and TBK1 were described previously (27). All experiments were independently performed three times. Data are Transfection presented as means 6 SD of three or four experiments. Analysis was performed using a Student t test or ANOVA. The p values , 0.05 were For transient transfection of plasmids into RAW264.7 cells, jetPEI considered to be statistically significant. reagents were used (Polyplus-transfection). For stable selection of RAW264.7 cell lines overexpressing PTPN4, transfected RAW264.7 macrophages were selected with G418 (800 mg/ml) and pooled for further Results experiments (24, 25). For transient silencing, duplexes of small inter- PTPN4 negatively regulates TLR4-induced IFN-b production fering RNA (siRNA) were transfected into cells with the Geneporter 2 Transfection Reagent (GTS, San Diego, CA), according to the standard To identify possible roles for PTPN4 in innate immune responses, protocol (23, 24). Target sequences for transient silencing were 59- we transfected HEK293-TLR4, HEK293-TLR3, or HEK293 cells 4460 PTPN4 INHIBITS TRAM TYROSINE PHOSPHORYLATION with an IFN-b or NF-kB luciferase reporter, as well as PTPN4 R848, or SeV was enhanced by TRIM38 knockdown, which was expression plasmid, and then treated the cells with LPS (TLR4 consistent with previous report (25). The effect of PTPN4 ligand) or poly(I:C) (TLR3 ligand) or infected with SeV (a type of knockdown was also analyzed by quantitative RT-PCR. PTPN4 ssRNA virus recognized by RIG-I). PTPN4 specifically inhibited siRNA treatment significantly increased LPS-induced IFN-b LPS-induced IFN-b activation and had no effects on IFN-b acti- mRNA expression (Fig. 1G) and had no effects on LPS-induced vation induced by poly(I:C) or SeV (Fig. 1A). In addition, PTPN4 TNF-a or IL-6 mRNA expression (Fig. 1G), demonstrating that had no effects on NF-kB activation induced by TLR4, TLR3, or PTPN4 inhibits LPS-induced IFN-b expression at both mRNA RIG-I (Fig. 1B). As a parallel control, TLR4/3- and RIG-I–in- and protein levels in macrophages. Collectively, these data indi- duced NF-kB activation was greatly inhibited by PTP1B, which cate that PTPN4 specifically inhibits TLR4-induced IFN-b pro- was consistent with a previous report (21). duction. To further confirm that PTPN4 specifically attenuates TLR4- induced IFN-b activation, knockdown experiments were per- PTPN4 inhibits TLR4-induced IRF3 activation formed. The expression of PTPN4 was greatly decreased with IRF3 is the key transcription factor that mediates the expression of transfection of PTPN4-specific siRNA in mouse peritoneal mac- IFN-b in TLR3-, TLR4-, and RIG-I–mediated signal transduction. rophages (Fig. 1C). PTPN4 siRNA 1, which has a higher effi- We then observed the effect of PTPN4 on IRF3 activation. IRF3 ciency to knockdown PTPN4 protein expression (Fig. 1C), has phosphorylation was greatly increased by PTPN4 knockdown in a greater potential to increase the LPS-induced IFN-b production LPS-stimulated macrophages (Fig. 2A) but not in poly(I:C)- or (Fig. 1D). Therefore, PTPN4 siRNA 1 was used in the following SeV-treated macrophages (Fig. 2B, 2C). LPS induces phosphor- experiments. Although PTPN4 knockdown significantly increased ylation of STAT1 at Tyr701, which is IFN-b dependent (28). To Downloaded from LPS-induced IFN-b production, it had no effects on poly(I:C), investigate whether the effect of PTPN4 is consistent for PGN (a TLR2 ligand), R848 (TLR7/8 ligand), or SeV-induced IFN-mediated signaling, we examined the effects of PTPN4 IFN-b production (Fig. 1E). In addition, PTPN4 knockdown on STAT1 phosphorylation. PTPN4 knockdown substantially could not promote TNF-a or IL-6 production induced by LPS, increased LPS-induced phosphorylation of STAT1 at Tyr701 poly(I:C), PGN, R848, or SeV (Fig. 1F). As a parallel control, (Fig. 2A). We next observed the effect of PTPN4 on IFR3 acti-

TNF-a or IL-6 production induced by LPS, poly(I:C), PGN, vation by detecting IRF3 luciferase reporter gene expression. http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. PTPN4 inhibits TLR4-induced IFN-b production. (A) HEK293-TLR4, HEK293-TLR3 or HEK293 cells were transfected with IFN-b plasmid together with PTPN4 expression plasmid or control plasmid, analyzed luciferase activity after treatment with indicated agonists for 6 h. (B) HEK293-TLR4, HEK293-TLR3, or HEK293 cells were transfected with NF-kB reporter plasmid together with PTPN4, PTP1B, or control plasmid and analyzed luciferase activity after treatment with indicated agonists for 6 h. PTPN4 expression was detected by Western blot analysis. (C) Western blot analysis of PTPN4 expression in mouse peritoneal macrophages transfected with control siRNA or PTPN4 siRNA 1 or 2 for 36 h. (D) ELISA of IFN-b in the supernatants of peritoneal macrophages as in (C) stimulated with LPS. (E) ELISA of IFN-b in the supernatants of peritoneal macrophages transfected with control siRNA or PTPN4 siRNA 1 for 36 h and then stimulated with LPS, poly(I:C), PGN, R848, or SeV for 12 h. (F) ELISA of TNF-a and IL-6 in the supernatants of peritoneal macrophages transfected with control siRNA, PTPN4 siRNA 1, or TRIM38 siRNA for 36 h and then stimulated with LPS, poly(I:C), PGN, R848, or SeV for 12 h. PTPN4 and TRIM38 expression were detected by Western blot. (G) Quantitative real-time RT-PCR analysis IFN-b,TNF-a,and IL-6 mRNA expression in peritoneal macrophages transfected with control siRNA or PTPN4 siRNA 1 and then stimulated with LPS for indicated time periods. Data are representative of three independent experiments with similar results (C) or are from three independent experiments [(A, B,andD–G) mean 6 SD, n =6].**p , 0.01. The Journal of Immunology 4461

FIGURE 2. PTPN4 inhibits TLR4-induced IRF3 activation. (A) Western blot analysis of p-IRF3, p-STAT1, and total IRF3, STAT1 in mouse peritoneal macrophages transfected with control siRNA or PTPN4 siRNA and stimulated with LPS. (B and C) Western blot analysis of p-IRF3 and total IRF3 in mouse peritoneal macrophages transfected with control siRNA or PTPN4 siRNA and stimulated with poly(I:C) or SeV. (D) HEK293-TLR4, HEK293-TLR3, or

HEK293 cells were transfected with IRF3 reporter plasmid together with PTPN4 expression plasmid or control plasmid and analyzed luciferase activity Downloaded from after treatment with indicated agonists for 6 h. (E) Western blot analysis of p-IRF3 and total IRF3 in HEK293-TLR4 cells transfected with Myc-PTPN4 expression plasmid or control empty vector (Ctrl) stimulated with LPS. (F) Western blot analysis of phosphorylated and total signaling proteins in mouse peritoneal macrophages was transfected with control siRNA or PTPN4 siRNA and stimulated with LPS. Data are representative of three independent experiments with similar results (A–C, E, and F) or are from three independent experiments [(D) mean 6 SD, n = 6]. **p , 0.01.

PTPN4 attenuated TLR4-induced IRF3 activation and had no effects (Fig. 3C). The phosphatase inactive mutant PTPN4 DA http://www.jimmunol.org/ effects on TLR3- or RIG-I–induced IRF3 activation (Fig. 2D). bears no effects on TLR4-induced IFN-b activation, suggesting Accordingly, PTPN4 greatly inhibited LPS-induced IRF3 that PTPN4 inhibited TRIF-dependent TLR4 signaling via its phosphorylation in HEK293–TLR4 cells (Fig. 2E). However, phosphatase activity. PTPN4 knockdown had no effects on LPS-induced phosphor- PTPN4 targets TRAM ylation of ERK, JNK, p38, and IkBa (Fig. 2F), indicating that PTPN4 bears no regulatory effects on MAPK and NF-kBac- To determine the molecular targets of PTPN4 in TLR4 pathway, the tivation induced by TLR4 engagement. Taken together, these effects of PTPN4 on IFN-b or IRF3 promoter activation mediated data suggested that PTPN4 specifically attenuated TLR4- by various molecules were examined in reporter assays. PTPN4 induced IRF3 activation. significantly inhibited TRAM-induced IFN-b or IRF3 activation by guest on September 29, 2021 (Fig. 4A). However, TRIF-, RIG-I–, MAVS-, and TBK1-induced PTPN4 inhibits TLR4 signaling via its phosphatase activity IFN-b or IRF3 activation remain unchanged by PTPN4 over- PTPN4 is an intracellular protein tyrosine phosphatase, and its expression (Fig. 4A). These data indicate that PTPN4 may target phosphatase activity is important for its role in dephosphorylation TRAM or its upstream receptors. Our previous data indicated that of downstream substrates. We then investigated the role of phos- PTPN4 specifically inhibited TRIF-dependent IRF3 activation phatase activity in its inhibitory effects. PTPN4 WT greatly (Fig. 2A, 2E) and had no regulatory effects on MAPK or NF-kB inhibited TLR4-induced IFN-b and IRF3 activation, whereas activation in TLR4 pathway (Figs. 1B, 2F). Furthermore, TLR3 phosphatase inactive mutant (PTPN4 DA) lost the inhibitory and RIG-I pathways, whose signals do not use TRAM, were not effects on TLR4-induced IFN-b and IRF3 activation in HEK293– affected by PTPN4 (Figs. 1A, 1D, 2B–D). Therefore, we specu- TLR4 cells (Fig. 3A). Next, we established RAW264.7 cell lines lated that PTPN4 targeted TRAM to inhibit IFN-b production. that stably expressed Myc-tagged PTPN4 WT plasmid, PTPN4 Consistent with our speculation, PTPN4 inhibited TRAM-induced DA plasmid, or empty vector. Overexpression of PTPN4 was IFN-b activation in a dose-dependent manner, and the phosphatase confirmed by Western blotting with both PTPN4 and Myc Abs mutant PTPN4 DA lost the inhibitory effects (Fig. 4B). TRAM– (Fig. 3B). PTPN4 WT greatly inhibited IFN-b production in LPS- TRIF pathway could trigger late NF-kB activation in TLR4 induced RAW264.7 cells, whereas PTPN4 DA lost the inhibitory pathway. We then investigated whether PTPN4 could affect the

FIGURE 3. PTPN4 inhibits TLR4 signaling via its phosphatase activity. (A) HEK293-TLR4 cells were transfected with IFN-b or IRF3 reporter plasmid together with PTPN4 WT and DA. 24 h later, luciferase activity was measured. (B) Western blot analysis of PTPN4 expression in RAW264.7 cells stably transfected with Myc-PTPN4 WT, Myc-PTPN4 DA or control empty plasmid (Ctrl). (C) ELISA of IFN-b in the supernatants of RAW264.7 cells as in (B) stimulated with LPS or poly(I:C) for 12 h. Data are representative of three independent experiments with similar results (B) or are from three independent experiments [(A and C) mean 6 SD, n = 6]. **p , 0.01. 4462 PTPN4 INHIBITS TRAM TYROSINE PHOSPHORYLATION

FIGURE 4. PTPN4 targets TRAM. (A) HEK293 cells were transfected with TRAM, TRIF, RIG-I, MAVS, or TBK1, along with IFN-b or IRF3 reporter B b plasmid and PTPN4 plasmid, and analyzed luciferase activity. ( ) HEK293 cells were transfected with TRAM, along with IFN- reporter plasmid and Downloaded from increasing amount (50, 100, and 200 ng) of PTPN4 WT or PTPN4 DA plasmid, and analyzed luciferase activity. (C) Membrane fractionation was carried out on mouse peritoneal macrophages following LPS stimulation for indicated time periods. Western blot analysis of PTPN4 and TRAM expression in membrane (M) and cytoplasm (C). (D) Lysates from mouse peritoneal macrophages stimulated with LPS for indicated time periods were subjected to IP with anti-TRAM Ab, followed by Western blot analysis with anti-PTPN4 Ab. Proteins in whole-cell lysate were used as positive control (input). (E) Lysates from HEK293 cells transiently transfected with Flag-TRAM or Flag-TRIF and Myc-PTPN4 were subjected to IP with anti-Myc Ab, followed by Western blot analysis with anti-Flag Ab. Data are representative of three independent experiments with similar results (C–E) or are from three independent experiments [(A and B) mean 6 SD, n = 6]. **p , 0.01. http://www.jimmunol.org/

TRAM-dependent NF-kB activation. PTPN4 inhibited TRAM- also bears conserved TIR domain and function as a sorting adaptor induced NF-kB activation, whereas PTPN4 DA lost the inhibi- for bridging TLR4 and TRIF in TLR4 activation (4–6). PTPN4 tory effects (Fig. 4B). Taken together, these data indicated that inhibited TRAM-induced signal transduction via its phosphatase PTPN4 inhibited TRIF-dependent TLR4 signaling by targeting activity, and that promoted us to investigate whether TRAM TRAM. could undergo tyrosine phosphorylation during TLR4 activation. TRAM predominately locates in the plasma membrane and We then detected the tyrosine phosphorylation of endogenous translocates to cytoplasm upon TLR4 activation. To investigate TRAM in macrophages following TLRs activation. LPS stimulation by guest on September 29, 2021 whether PTPN4 possess the potential to target TRAM, we first prompted an increase in tyrosine phosphorylation of TRAM, detected the subcellular localization of PTPN4. PTPN4 was pre- whereas poly(I:C) and LTA could not induce it (Fig. 5A). Next, we sented predominantly at the plasma membrane and translocated to transfected with Flag-tagged TRAM plasmid into HEK293–TLR4 cytoplasm following TLR4 activation in macrophages (Fig. 4C). cells and examined the tyrosine phosphorylation of the ectopically Similarly, TRAM was presented at the plasma membrane, and the expressed TRAM. Flag-tagged TRAM was tyrosine phosphory- amount of TRAM in the membrane fraction was decreased upon lated with LPS stimulation (Fig. 5B). These data indicate that LPS stimulation (Fig. 4C), suggesting that TRAM is disappearing TRAM undergoes tyrosine phosphorylation in TLR4 signaling. from the membrane, which was consistent with previous report To determine the tyrosine phosphorylation sites of TRAM, we (7). We then investigated whether endogenous PTPN4 interacted first performed alignment of TRAM sequences from human, with TRAM. An association between PTPN4 and TRAM could mouse, bovine, and rat. Only two tyrosine (Y) residues, which both be detected in resting macrophages and was enhanced by LPS located in the TIR domain, are conserved in the entire TRAM stimulation (Fig. 4D). To further confirm the interaction between sequences from the four species (Fig. 5C), attesting to their pos- PTPN4 and TRAM, Myc-tagged PTPN4 together with Flag- sible importance in the functioning of TRAM. In humans, the two tagged TRAM were transfected into HEK293 cells, and TRAM tyrosine residues are located at 154 aa (Y-154) and 167 aa (Y-167), was precipitated with specific Flag Ab. PTPN4 coimmunopreci- respectively (Fig. 5D). The structure of TIR domain of TRAM pitated with Flag-tagged TRAM (Fig. 4E). As a negative control, from human has been determined (30). We analyzed this struc- TRIF could not interact with PTPN4 (Fig. 4E). Collectively, these ture and noted that both Y-154 and Y-167 were surface exposed data indicated that PTPN4 could interact with TRAM. (Fig. 5E) and possessed the potential to be phosphorylated. No- tably, Y-167 was located in the middle of a mobile randon coil TRAM is tyrosine phosphorylated upon LPS stimulation (from 161 to 181 aa), which made it easier to be phosphorylated. Several posttranslational modifications are reported to be critical To examine the role of these tyrosine residues in TRAM phos- for the optimal function of TRAM, including myristoylation and phorylation, we mutated the two tyrosine residues conservatively serine phosphorylation (7, 29). The myristoylation of TRAM to phenylalanine (F) by using site-directed mutagenesis. The localizes it on the plasma membrane (29), where it undergoes phosphorylation of TRAM in response to LPS was completely phosphorylation at Ser16 and then translocates to the cytoplasm abolished when Y-167 was mutated (Fig. 5F). Mutation of Y-154 (7). TIRAP, the sorting adaptor for MyD88-dependent pathway, had no effect on TRAM phosphorylation (Fig. 5F), suggesting that undergoes tyrosine phosphorylation at Tyr86,Tyr106, and Tyr159 Y-154 is not involved in LPS-induced phosphorylation of TRAM. residues within its TIR domain following LPS stimulation, and It is therefore likely that TRAM is phosphorylated on Y-167. We that is required for the activation of MyD88-dependent pathway then raised an Ab to a synthetic peptide comprising aa 163–174 of in TLR2 and TLR4 signaling (12, 13). Similar to TIRAP, TRAM TRAM with a phosphotyrosine inserted instead of a tyrosine at The Journal of Immunology 4463 Downloaded from http://www.jimmunol.org/

FIGURE 5. TRAM is tyrosine phosphorylated upon LPS stimulation. (A) Lysates from mouse peritoneal macrophages stimulated with indicated agonists were subjected to IP with anti-TRAM Ab, followed by Western blot analysis with anti–p-tyr Ab. (B) Lysates from HEK293–TLR4 cells transfected with Flag–TRAM expression plasmid stimulated with LPS were subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti–p-tyr Ab. (C) Alignment of TRAM. Two tyrosine (Y) residues conserved in four species are underlined. (D) Schematic diagram of human TRAM. G2, myristoylation site; S16, serine phosphorylation site; Y154 and Y167, potential tyrosine phosphorylation sites. (E) Structural surface analysis of TIR domain of hTRAM. The potential phospho-accepting tyrosines Y154 and Y167 are highlighted in red. (F) Lysates from HEK293–TLR4 cells transfected with Flag-TRAM WT, Y154F, or Y167F expression plasmid stimulated with LPS were subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti–p-tyr Ab. (G) Western blot analysis of phosphorylated and total signaling proteins in mouse peritoneal macrophages stimulated with indicated agonists. (H) HEK293 cells were transfected with TRAM, TRAM Y154F, or TRAM Y167F, along with IFN-b or IRF3 reporter plasmid, and analyzed luciferase activity. Data are by guest on September 29, 2021 representative of three independent experiments with similar results (A, B, F, and G) or are from three independent experiments [(H) mean 6 SD, n = 6]. **p , 0.01.

Y-167 and applied to specifically detect the Y-167 phosphorylation Next, we investigated whether tyrosine phosphorylation of of TRAM. LPS could induce TRAM Y-167 phosphorylation, and TRAM was also required for the association between TRAM and the phosphorylation peaks at 30 min in peritoneal macrophages TRIF. Hemagglutinin (HA)-tagged TRIF and Flag-tagged TRAM (Fig. 5G), whereas poly(I:C), LTA, or SeV could not induce ty- WT, TRAM Y154F, or TRAM Y167F were transfected into rosine phosphorylation of TRAM (Fig. 5G). We next tested the HEK293 cells. TRAM Y167F significantly reduced the ability of ability of the TRAM mutants to activate IFN-b, IRF3, and NF-kB TRAM to interact with TRIF (Fig. 6D), indicating that Y-167 reporter genes in HEK293 cells. Y-167 mutant TRAM (TRAM phosphorylation of TRAM is critical for TRAM–TRIF complex Y167F) greatly lost the ability to activate IFN-b, IRF3, or NF-kB formation. To determine whether PTPN4 could disrupt the (Fig. 5H). Taken together, these data indicate that TRAM under- TRAM–TRIF complex formation, HA-tagged TRIF and Flag- goes tyrosine phosphorylation following TLR4 activation, and tagged WT TRAM were transfected in HEK293 cells with or the Y-167 phosphorylation is critical for TRIF-dependent TLR4 without Myc-tagged PTPN4. PTPN4 overexpression resulted in pathway. a reduction in the interaction between TRIF and TRAM (Fig. 6E). Altogether, these data indicated that Y167 phosphorylation of PTPN4 inhibits TRAM tyrosine phosphorylation and TRAM is required for the TRAM–TRIF complex formation and subsequent TRAM–TRIF interaction PTPN4 could suppress TRAM–TRIF interaction by inhibiting We then investigated whether PTPN4 could inhibit tyrosine Y167 phosphorylation of TRAM. phosphorylation of TRAM using specific Ab for Y-167 phos- phorylation of TRAM. PTPN4 overexpression inhibited Y-167 Discussion phosphorylation of TRAM in HEK293–TLR4 cells (Fig. 6A), In the current study, we reported that TRAM underwent tyrosine whereas PTPN4 knockdown had the opposite effects in mouse phosphorylation, and this modification was required for optimal peritoneal macrophages (Fig. 6B). TRAM cytoplasm translocation activation of TRAM. Moreover, we identified that the Y167 residue is a key step for the activation of downstream pathway. We next located in TIR domain of TRAM was the phospho-accepting ty- investigated whether PTPN4 could regulate TRAM translocation. rosine, and Y167 mutation of TRAM lost the activity of triggering TRAM translocated from plasma membrane to cytoplasm fol- downstream signals. It has been reported that TIRAP underwent lowing LPS stimulation and PTPN4 inhibited the dissociation of tyrosine phosphorylation at the residues within its TIR domain TRAM from the membrane in HEK293–TLR4 cells (Fig. 6C). following LPS stimulation, and this modification initiated a con- 4464 PTPN4 INHIBITS TRAM TYROSINE PHOSPHORYLATION Downloaded from FIGURE 6. PTPN4 inhibits TRAM tyrosine phosphorylation and TRAM–TRIF interaction. (A) Western blot analysis of p-TRAM and total TRAM in HEK293-TLR4 cells transfected with Myc-PTPN4 expression plasmid or control empty vector (Ctrl) stimulated with LPS. (B) Western blot analysis of p-TRAM and total TRAM in mouse peritoneal macrophages transfected with control siRNA or PTPN4 siRNA and stimulated with LPS. (C) Membrane fractionation was carried out on HEK293–TLR4 cells overexpressing Flag-tagged TRAM along with or without Myc-PTPN4 and stimulated with LPS for 30 min. Western blot analysis of TRAM expression in membrane. (D) Lysates from HEK293 cells transiently transfected with HA-TRIF and Flag-TRAM WT, and TRAM mutants Y154F or Y167F were subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti-HA Ab. (E) Lysates from HEK293 cells transfected with Flag-TRAM and HA-TRIF in the presence or absence of Myc-PTPN4 were http://www.jimmunol.org/ subjected to IP with anti-Flag Ab, followed by Western blot analysis with anti-HA Ab. Data are representative of three independent experiments with similar results. formational change in the TIR domain of TIRAP (12, 13), thereby sclerosis, arthritis, asthma, and ischemia–reperfusion injury, were leading to activation of downstream signals. Tyrosine phosphor- documented to be linked with TLR4. Inappropriate TLR4 acti- ylation results in the recruitment of the phosphorylase kinase, such vation and unbalanced production of type I IFNs and proin- as Ser/Thr kinase and PI3K, to the signaling complex, a step that flammatory cytokines can promote the development of multiple is essential for the activation of downstream transcription factors diseases. Thus, elucidating the mechanisms by which differen- by guest on September 29, 2021 (11). Tyrosine phosphorylation then provides docking sites for tially regulates the production of type I IFNs and proinflammatory other proteins (11). Structural analysis indicates that both TRIF cytokines in TLR4 signaling would be beneficial for the devel- and TRAM TIR domains form a BB-loop–mediated homodimer opment of new strategy to control TLR4-associated diseases. Al- (30). The dimerization of TRAM TIR presents an interaction though multiple molecules have been identified as regulators of surface for TRIF (30). Tyrosine phosphorylation within the TIR TLR4 signaling up to now, few of them were specific for TRIF- domain of TRAM may initiate the conformational change and dependent TLR4 pathway. In the current study, we identified then provide docking sites to facilitate the interaction between PTPN4 as a specific inhibitor of TRIF-dependent TLR4 pathway. TRAM and TRIF. Targeting PTPN4 would be beneficial for the development of new Modulating the TRAM–TRIF interaction is an important strategy to control TLR4-associated diseases without unwanted mechanism for the specific regulation of TRIF-dependent pathway side effects. in TLR4 signaling. TRAM adaptor with GOLD (TAG) domain and transmembrane emp24 domain-containing protein 7 (TMED7) Acknowledgments have been reported to be involved in the regulation of TRIF- We thank Drs. Nicolai S. van Oers, Nicholas K. Tonks, Luke A. O’Neill, and dependent TLR4 pathway. Transmembrane emp24 domain- Xuetao Cao for providing plasmids. containing protein 7interacts with TRAM and displaces the TRIF from TRAM in endosomes (31). TMED7 colocalizes with Disclosures TRAM and TLR4 in endosomes, where it encounters TAG and The authors have no financial conflicts of interest. mediates the inhibitory effects of TAG on TRAM–TRIF associa- tion (32). Here we identified PTPN4 as another specific inhibitor by directly targeting TRAM via its phosphatase activity. PTPN4 References attenuates TRAM tyrosine phosphorylation, inhibits its cytoplasm 1. Janeway, C. A., Jr., and R. Medzhitov. 2002. Innate immune recognition. Annu. Rev. Immunol. 20: 197–216. translocation and subsequent TRAM–TRIF interaction, resulting 2. Kawai, T., and S. Akira. 2010. The role of pattern-recognition receptors in innate in the decrease of TLR4-mediated IRF3 activation and IFN-b immunity: update on Toll-like receptors. Nat. Immunol. 11: 373–384. production. 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