The Journal of Immunology
Modulation of TLR4 Signaling by a Novel Adaptor Protein Signal-Transducing Adaptor Protein-2 in Macrophages1 Yuichi Sekine,* Taro Yumioka,* Tetsuya Yamamoto,* Ryuta Muromoto,* Seiyu Imoto,* Kenji Sugiyma,† Kenji Oritani,‡ Kazuya Shimoda,¶ Mayu Minoguchi,储 Shizuo Akira,§ Akihiko Yoshimura,储 and Tadashi Matsuda2* Signal-transducing adaptor protein-2 (STAP-2) is a recently identified adaptor protein that contains pleckstrin and Src homology 2-like domains as well as a YXXQ motif in its C-terminal region. Our previous studies have demonstrated that STAP-2 binds to STAT3 and STAT5, and regulates their signaling pathways. In the present study, STAP-2 was found to positively regulate LPS/TLR4-mediated signals in macrophages. Disruption of STAP-2 resulted in impaired LPS/TLR4-induced cytokine production and NF-B activation. Conversely, overexpression of STAP-2 enhanced these LPS/TLR4-induced biological activities. STAP-2, particularly its Src homology 2-like domain, bound to both MyD88 and IB kinase (IKK)-␣, but not TNFR-associated factor 6 or IL-1R-associated kinase 1, and formed a functional complex composed of MyD88-STAP-2-IKK-␣. These interactions augmented MyD88- and/or IKK-␣-dependent signals, leading to enhancement of the NF-B activity. These results demonstrate that STAP-2 may constitute an alternative LPS/TLR4 pathway for NF-B activation instead of the TNFR-associated factor 6-IL-1Rassociated kinase 1 pathway. The Journal of Immunology, 2006, 176: 380 –389.
T
oll-like receptor family proteins are characterized by the presence of extracellular domains with leucine-rich repeats and intracytoplasmic regions called Toll/IL-1R homology (TIR)3 domains. They are abundantly expressed on a variety of cells, including macrophages and dendritic cells, and serve as an important link between innate and adaptive immune responses (1– 6). At least 11 of the mammalian TLRs recognize distinct microbial pathogen-associated molecular patterns to evoke an inflammatory response. Ligand ligation of TLRs mediates signals such as the activation of NF-B and MAPKs, resulting in the production of inflammatory cytokines and expression of costimulatory molecules. Subsequently, the secreted inflammatory cytokines not only stimulate macrophages and NK cells that directly kill pathogens but also enhance clonal B and T lymphocyte responses. Recently, we cloned two novel adaptor molecules, signal-transducing adaptor protein (STAP)-1 and STAP-2 (7, 8). STAP-1 was *Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan; †Nippon Boehringer Ingelheim, Kawanishi Pharma Research Institute, Hyogo, Japan; ‡Department of Hematology and Oncology, Graduate School of Medicine, and §Department of Host Defense, Research Institute for Microbial Diseases, Osaka University, Osaka, Japan; and ¶First Department of Internal Medicine, Faculty of Medicine, and 储Division of Molecular and Cellular Immunology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Received for publication April 28, 2005. Accepted for publication October 24, 2005. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 This work was supported in part by grants from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government, the Osaka Foundation for Promotion of Clinical Immunology, the Akiyama Foundation, the Naito Foundation, and the Ichiro Kanehara Foundation. 2 Address correspondence and reprint requests to Dr. Tadashi Matsuda, Department of Immunology, Graduate School of Pharmaceutical Sciences, Hokkaido University, Kita-Ku Kita 12 Nishi 6, Sapporo 060-0812, Japan. E-mail address:
[email protected] 3 Abbreviations used in this paper: TIR, Toll/IL-1R homology; STAP, signal-transducing adaptor protein; PH, pleckstrin homology; SH2, Src homology 2; IKK, IB kinase; TRAF, TNFR-associated factor; IRAK, IL-1R-associated kinase; His, histine tagged; WT, wild type; ELAM, endothelial leukocyte adhesion molecule; TIRAP, TIR domain-containing adaptor protein; TRIF, TIR domain-containing molecule; TRAM, TRIF-related adaptor molecule; SOCS, suppressor of cytokine signaling.
Copyright © 2005 by The American Association of Immunologists, Inc.
identified as a c-kit-interacting protein, while STAP-2 is a c-fmsinteracting protein. Human STAP-2 is identical to a recently cloned adaptor molecule, BKS, which is a substrate of breast tumor kinase (BRK) tyrosine kinase (9). Both STAP-1 and STAP-2 contain an N-terminal pleckstrin homology (PH) domain and a region distantly related to the Src homology 2 (SH2) domain (overall amino acid identity: 33%). The N-terminal PH domains of STAP-2 and STAP-1 share 36% identity and 58% similarity. The central region of STAP-2 is distantly related to the SH2 domain. This region of STAP-2 shares 40% sequence identity with that of STAP-1 and 29% sequence identity with the SH2 domain of human phospholipase C-␥2. However, STAP-2 has a C-terminal proline-rich region and a YXXQ motif, neither of which are present in STAP-1. We previously reported that STAP-2 interacts with STAT3 through its YXXQ motif and enhances STAT3 transcriptional activity. STAP-2 also interacts with STAT5 through its PH and SH2-like domains (10). Although STAP-2-knockout mice did not show severe abnormalities, LPS- or IL-6-stimulated acute phase protein gene induction was significantly decreased in STAP2-deficient hepatocytes (8). The induction of STAT3 phosphorylation by LPS, particularly the responses at the later phase after stimulation, was also decreased in these cells. These results suggest that STAP-2 may play a role in LPS-induced signaling. LPS binds to a specific receptor, TLR4, whose signals are mediated in either a MyD88-dependent or -independent manner. In this study, we analyzed the role of STAP-2 in LPS/TLR4 signaling. Our data indicate that STAP-2 enhances cytokine production and NF-B activation after LPS stimulation, and that it binds directly to both MyD88 and IB kinase ␣ (IKK-␣) but not to TNFR-associated factor 6 (TRAF6) or IL-1R-associated kinase 1 (IRAK1). We further discuss the possible mechanisms for its modification of LPS/ TLR4 signaling.
Materials and Methods Reagents, Abs, and mice LPS from Escherichia coli (O55:B5) and poly(I:C) were purchased from Sigma-Aldrich. Phosphorothioate-stabilized CpG DNA was purchased 0022-1767/05/$02.00
The Journal of Immunology from Hokkaido System Science (11). The NF-B-responsive reporter plasmid (NF-B-LUC), a gift from Dr. T. Fujita (Tokyo Metropolitan Institute of Medical Science, Tokyo, Japan), contains three copies of an NF-Bbinding site from the L chain Ig enhancer (12). Recombinant plasmids for expression of TRAF6, IKK-␣, IKK- K44M were provided by Dr. J. Inoue (Waseda University, Tokyo, Japan) (13), and Drs. T. Sugita and H. Sakurai (Tanabe Seiyaku, Osaka, Japan) (14). GST-IB was a gift from Dr. M. Hibi (RIKEN, Kobe, Japan) (15). Myc-tagged STAP-2 and MyD88 constructs were described previously (8, 16). To obtain mammalian expression vectors for GST-fusion STAP-2 mutants, STAP-2 mutant cDNAs were generated by PCR methods and sequenced (primer sequences are available upon request) and inserted into pEBgs vector (10). Myc-tagged IKK- mutants were also generated by PCR methods and sequenced (primer sequences are available upon request). Anti-Myc, anti-GST, antiHis, and anti-IKK-␣ Abs were purchased from Santa Cruz Biotechnology. Anti-FLAG M2 Ab and rabbit polyclonal anti-FLAG Ab were purchased from Sigma-Aldrich. Abs against IB, phospho-IB, phospho-JNK, JNK, phospho-p38 MAPK, and p38 MAPK were purchased from Cell Signaling. Anti-human STAP-2 Ab was described previously (17). The generation of STAP-2-deficient mice was described previously (8). STAP2-deficient mice were backcrossed for more than seven generations onto C57BL/6 mice, and littermate wild-type (WT) mice were used as controls. Mice were housed and bred in the Pharmaceutical Sciences Animal Center of Hokkaido University.
Cell culture, transfection, luciferase assays, and RT-PCR analysis The murine macrophage cell line, Raw264.7, was cultured in DMEM supplemented with 10% FBS. Raw264.7 cells were transfected with the pcDNA3-Myc-STAP-2 and its mutant constructs (8) using Fugene 6 (Roche) according to the manufacturer’s instructions. Stable Raw264.7 cell transformants were selected with 1 mg/ml G418 (Sigma-Aldrich). Mouse peritoneal macrophages were collected by peritoneal lavage with HBSS at 3 days after i.p. injection of 2 ml of 3% sterile thioglycolate (BD Biosciences) into 8- to 12-wk-old mice. Peritoneal macrophages were cultured in RPMI 1640 medium with 10% FBS. The human T cell leukemia cell line, Jurkat, was maintained in RPMI 1640 medium containing 10% FBS and transfected with pcDNA3-Myc-STAP-2 using electroporation. Stable Jurkat transformants were selected with 1 mg/ml G418. The human acute monocytic leukemia cell line THP-1 was cultured in RPMI 1640 medium supplemented with 10% FBS. THP-1 differentiation was induced by treatment with 50 ng/ml PMA (Sigma-Aldrich) for 3 days (18). The human embryonic kidney carcinoma cell line, 293T, was maintained in DMEM containing 10% FBS and transfected by the standard calcium precipitation protocol (19). The cells were harvested 48 h after transfection and lysed in 100 l of PicaGene Reporter Lysis Buffer (Toyo Ink) and assayed for luciferase and -galactosidase activities according to the manufacturer’s instructions. Luciferase activities were normalized to the -galactosidase activities. Three or more independent experiments were conducted for each assay. The stable transformant of Raw264.7 cells expressing human endothelial leukocyte adhesion molecule (ELAM) promoter luciferase was described previously (20). Raw264.7 cells were transfected using Metafectene (Biontex Laboratories) according to the manufacturer’s instructions. RT-PCR analysis was performed using RT-PCR high -Plus- Kit (Toyobo). The primers were used as described previously (21).
Immunoprecipitation, immunoblotting, and in vitro kinase assays The immunoprecipitation and Western blotting assays were performed as described previously (22). Cells were harvested and lysed in lysis buffer (50 mM Tris-HCl (pH 7.4), 0.15 M NaCl, containing 1% Nonidet P-40, 1 M sodium orthovanadate, 1 M PMSF, and 10 g/ml each of aprotinin, pepstatin, and leupeptin). The immunoprecipitates from the cell lysates were resolved on SDS-PAGE and transferred to an Immobilon filter (Millipore). The filters were then immunoblotted with the respective Ab. IKK- kinase activity was measured by in vitro kinase assay as described previously (15, 23). Briefly, the immune complex of IKK- was washed in kinase buffer (10 mM HEPES (pH 7.4), 50 mM NaCl, 0.1 mM sodium orthovanadate, 5 mM MnCl2, 5 mM MgCl2) and mixed with 5 Ci/l [␥-32P]ATP with or without GST-IB at 25°C for 30 min. The products of these reactions were separated by SDS-PAGE.
Measurement of cytokines in the culture supernatants Peritoneal macrophages or Raw264.7 cells were cultured with the indicated concentration of LPS for 24 h. Concentrations of TNF-␣ or IL-6 in culture supernatants were measured by ELISA according to the manufacturer’s instructions (BioSource International).
381 EMSA Raw264.7 or Raw/STAP-2 cells were stimulated with 100 ng/ml LPS for the indicated periods. Nuclear extracts were purified from cells and incubated with a specific probe for the NF-B DNA-binding site, electrophoresed, and visualized by autoradiography as described previously (24).
Statistical methods The significance of differences between group means was determined by Student’s t test.
Results Disruption of STAP-2 results in reduced cytokine production after LPS stimulation Our previous studies involving STAP-2-deficient hepatocytes suggested that STAP-2 participates in LPS/TLR4-mediated signaling, and prompted us to examine LPS-induced cytokine production in STAP-2-deficient macrophages in more detail. We first examined the production of TNF-␣ by WT or STAP-2-deficient macrophages after LPS stimulation. A time-course study revealed that TNF-␣ was detected in the culture supernatant within 3 h, and that its concentration was ⬃30% lower in STAP-2-deficient macrophages than in WT macrophages during the observation period (Fig. 1A). Production of TNF-␣ and IL-6 after LPS stimulation was evident at 0.1 g/ml LPS, and increased in a dose-dependent manner. The production of TNF-␣ and IL-6 was significantly lower in STAP-2-deficient macrophages than in WT macrophages at all examined concentrations of LPS (Fig. 1A). When the gene expression of TNF-␣ and IL-6 was analyzed by RT-PCR, PCR products for TNF-␣ and IL-6 were detected in RNA samples obtained from WT macrophages stimulated with LPS for 60 min (Fig. 1B). However, significant reductions in the TNF-␣ and IL-6 mRNA levels were observed in RNA samples from STAP-2deficient macrophages. Therefore, STAP-2 disruption reduces cytokine production by macrophages after LPS stimulation. In addition, the observation that STAP-2-deficient macrophages expressed as much TLR4 mRNA as WT macrophages (data not shown) may suggest that STAP-2 participates in LPS/TLR4signaling. To evaluate the physiological role of STAP-2 in TNF-␣ production in vivo, the serum concentrations of TNF-␣ in WT and STAP-2-deficient mice after LPS injection were measured by ELISA. The serum TNF-␣ levels in WT and STAP-2-deficient mice in the absence of LPS were below the detectable level (6.0 pg/ml). As shown in Fig. 1C, the serum concentration of TNF-␣ was lower in STAP-2-deficient mice than in WT mice at 30 min after LPS injection, but showed no difference at 1 h after LPS injection. Therefore, STAP-2 modulates LPS-induced TNF-␣ production at an early stage after LPS exposure in vivo. STAP-2 enhances LPS-induced activation of NF-B In LPS/TLR4-signaling, NF-B is a major molecule that induces the promoter activity of cytokine genes. To assess the NF-B activation, the phosphorylation of IB after LPS stimulation was evaluated using a phospho-IB-specific Ab. As shown in Fig. 2A, phosphorylation of IB was detected within 30 min, and a higher level of phosphorylation was observed in WT macrophages at 60 min after LPS stimulation. The phosphorylation of IB after LPS stimulation was much lower in STAP-2-deficient macrophages. However, similar levels of p38 MAPK or JNK activation were observed after LPS stimulation in STAP-2-deficient and WT macrophages (Fig. 2A). To further delineate the role of STAP-2 in LPS/TLR4-mediated NF-B activation in macrophages, we established Raw/STAP-2 cells overexpressing human STAP-2. As
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FIGURE 1. Cytokine production and activation of signaling cascades in peritoneal macrophages in response to LPS. A, Upper panel, Peritoneal macrophages (105 cells/well) from WT (⫹/⫹) and STAP-2-deficient (⫺/⫺) mice were cultured with 100 ng/ml LPS for the indicated periods. Concentrations of TNF-␣ in the culture supernatants were measured with ELISA. Lower panels, Peritoneal macrophages (105 cells/well) were also stimulated with the indicated concentrations of LPS for 12 h, and the supernatants were analyzed for TNF-␣ or IL-6 with ELISA. All values are the mean ⫾ SEM of triplicate cultures from three different experiments. ⴱ, p ⬍ 0.05. B, Peritoneal macrophages were stimulated with 10 ng/ml LPS for the indicated periods. Total RNAs were extracted and analyzed for expression of TNF-␣ and IL-6 mRNA by PT-PCR. C, Age-matched WT (n ⫽ 5) and STAP-2-deficient (n ⫽ 5) mice were injected i.p. with 1.0 mg of LPS. Sera were taken after the indicated period of time, and serum concentrations of TNF-␣ were determined with ELISA. Data are the means SD of samples from five mice. ⴱ, p ⬍ 0.05
shown in Fig. 2B, TNF-␣ and IL-6 production after LPS stimulation was significantly higher in Raw/STAP-2 cells than in Raw/ pcDNA3 cells transfected with the pcDNA3 vector alone. Using these transfected Raw264.7 cells, the activation of NF-B was evaluated with EMSAs. As shown in Fig. 2C, the EMSA time course revealed enhanced DNA-binding activity of NF-B in nuclear extracts from LPS-stimulated Raw/STAP-2 cells. We also tested whether STAP-2 enhances LPS/TLR4-induced transcriptional activation of NF-B using Raw264.7 cells expressing human ELAM promoter-LUC. As shown in Fig. 2D, LPS/TLR4induced ELAM-LUC activity was significantly enhanced by the overexpressed STAP-2. Therefore, STAP-2 positively regulates NF-B activation in LPS/TLR4-signaling.
Physical interactions of STAP-2 with MyD88 and IKK-␣ One of the mechanisms consistent with the above-described data is direct interactions between STAP-2 and some signaling molecules that participate in NF-B activation through LPS/TLR4. To clarify this possibility, coimmunoprecipitation experiments were performed using 293T cells. Following transfection of expression vectors for FLAG-tagged TRAF6 or IRAK1 together with Myctagged STAP-2 into 293T cells, the cells were lysed, immunoprecipitated with an anti-Myc Ab, and immunoblotted with an anti-FLAG Ab. As shown in Fig. 3A, no significant interaction of STAP-2 with TRAF6 or IRAK1 was observed. When expression vectors for FLAG-tagged MyD88 or His-tagged IKK-␣ together with Myc-tagged STAP-2 were transfected into
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FIGURE 2. Enhancement of LPS response by STAP-2 in Raw264.7 cells. A, Peritoneal macrophages (106 cells/ml) were stimulated with 100 ng/ml LPS for the indicated periods. Their whole cell lysates were prepared and subjected to Western blot analysis using Abs specific for the indicated molecules. B, Parental Raw264.7 or Raw/STAP-2 cells (5 ⫻ 104 cells/well) were stimulated with 100 ng/ml LPS for the indicated periods, and their supernatants were analyzed for TNF-␣ or IL-6 with ELISA. All values are the mean ⫾ SEM of triplicate cultures from three different experiments. ⴱ, p ⬍ 0.05. C, Raw264.7 and Raw/STAP-2 cells (5 ⫻ 106 cells) were stimulated with 100 ng/ml LPS for the indicated periods. NF-B activity was determined with EMSA. Arrow indicates the inducible NF-B complex. D, RAW264.7 cells, stably transfected with human ELAM promoter-LUC (Raw/ELAM-LUC), was used to measure the NF-B promoter activities. Raw/ELAMLUC cells were transfected with empty vector or STAP-2. After 36 h of transfection, cells were treated with or without 5 ng/ml LPS for 8 h. The luciferase activities were normalized to protein concentration.
293T cells and the cells were lysed, immunoprecipitated with an anti-Myc Ab, and immunoblotted with an anti-FLAG or anti-His Ab, the immunoprecipitates contained MyD88 and IKK-␣ (Fig. 3, B and C), indicating that STAP-2 associated with MyD88 and IKK-␣ in 293T cells. A similar interaction between STAP-2 and IKK-␣ was also observed in Jurkat cells stably transfected with Myc-tagged STAP-2. The Jurkat/STAP-2 cells were lysed, immunoprecipitated with an anti-Myc Ab and immunoblotted with an anti-IKK-␣ Ab. As shown in Fig. 3D, the immunoprecipitates contained endogenous IKK-␣ proteins. To examine the physiological interactions between STAP-2 and IKK- in more detail, the human acute monocytic leukemia cell line THP-1, which differentiates into macrophages after treatment with PMA, was used. After treatment of THP-1 cells with PMA for 3 days, the cells were lysed and immunoprecipitated with a control Ab or anti-STAP-2 Ab. The immunoprecipitate with the anti-STAP-2 Ab contained IKK- (Fig. 3E). To further examine whether MyD88 and IKK-␣ are bridged by STAP-2 to form a complex, we assessed the association of MyD88 with IKK-␣ in the absence or presence of STAP-2. Expression vectors for FLAG-tagged MyD88 and Histagged IKK-␣ together with or without Myc-tagged STAP-2 were transfected into 293T cells. The cells were lysed, immunoprecipitated with an anti-His Ab, and immunoblotted with an antiFLAG or anti-His Ab. As shown in Fig. 3F, the association between MyD88 and IKK- was only detected in the presence of
STAP-2. Therefore, STAP-2 associates with MyD88 and IKK-, and plays a role in forming a complex of these three molecules. Physiological roles of the interaction of STAP-2 with MyD88 and IKK- in the enhancement of NF-B activity When MyD88 or IKK- was overexpressed in 293T cells, NF-B activation was observed (25). Using this experimental system, we analyzed the molecular mechanisms for the STAP-2-mediated enhancement of NF-B activation. First, we examined the effects of STAP-2 on MyD88-induced activation of NF-B-LUC in 293T cells. As shown in Fig. 4A, NF-B-LUC activity was induced by the overexpressed MyD88 (⬃5-fold induction compared with the control cells). In addition, the MyD88-induced NF-B-LUC activity was significantly enhanced in parallel with the expression of STAP-2. Similarly, ectopic expression of STAP-2 also enhanced IKK-␣-induced NF-B activity (Fig. 4B). Next, we tested the possibility that the enhanced NF-B activation mediated by STAP-2 was due to activation of the kinase activity of IKK-␣. To this end, we performed in vitro kinase assays for IKK-. Following transfection of expression vectors for His-IKK- together with increasing amounts of Myc-STAP-2 into 293T cells, the cells were lysed and immunoprecipitated with an anti-His Ab. Next, the immunoprecipitates were subjected to in vitro kinase assays in the presence of 32P and GST-IB, resolved by SDS-PAGE and
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FIGURE 3. Physical interactions of STAP-2 with IKK-␣ and MyD88. A, 293T cells (1 ⫻ 107) were transfected with FLAG-tagged IRAK1 (10 g) or FLAG-tagged TRAF6 (10 g) and/or Myc-tagged STAP-2 (5 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-Myc Ab and immunoblotted with anti-FLAG (upper panel) or anti-Myc Ab (middle panel). Total cell lysates (1%) were blotted with anti-FLAG Ab (bottom panel) to monitor the expression of IRAK1 or TRAF6. B, 293T cells (1 ⫻ 107) were transfected with Myc-tagged STAP-2 (5 g) and/or FLAG-tagged MyD88 (10 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-Myc Ab and immunoblotted with anti-FLAG (upper panel) or anti-Myc Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-FLAG (bottom panel) to monitor the expression of MyD88. C, 293T cells (1 ⫻ 107) were transfected with His-tagged IKK-␣ (10 g) or IKK- (10 g) and/or Myc-tagged STAP-2 (5 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-Myc Ab and immunoblotted with anti-His (upper panel) or anti-Myc Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-His Ab (bottom panel) to monitor the expression of IKK-␣ or IKK-. D, Jurkat cells stably transfected with Myc-tagged STAP-2 (1 ⫻ 107) were lysed, and immunoprecipitated with anti-Myc Ab and immunoblotted with anti-IKK-␣ Ab (upper panel) or anti-Myc Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-IKK-␣ Ab (bottom panel) to monitor the expression of IKK-␣ or IKK-. E, PMA-treated THP-1 cells (5 ⫻ 107) were lysed, and immunoprecipitated with control or anti-STAP-2 Ab and immunoblotted with anti-IKK- Ab (upper panels) or anti-STAP-2 Ab (lower panels). Total cell lysates (1%) were blotted with anti-IKK- or anti-STAP-2 Ab (left panels) to monitor the expression of IKK- or STAP-2. F, 293T cells (1 ⫻ 107) were transfected with FLAG-tagged MyD88 (10 g) and His-tagged IKK- (10 g) and/or Myc-tagged STAP-2 (5 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-His Ab and immunoblotted with anti-FLAG, anti-Myc, or anti-His Ab (upper panel). Total cell lysates (1%) were immunoblotted with anti-FLAG or anti-Myc Ab (bottom panel) to monitor the expression of MyD88 and STAP-2.
assessed by autoradiography. As shown in Fig. 4C (upper panels), autophosphorylation of IKK- was markedly enhanced in the presence of higher amounts of STAP-2. Furthermore, IKK--mediated incorporation of 32P into GST-IB as a substrate was also stimulated in the presence of STAP-2. The specificity of these experi-
ments was confirmed by the absence of 32P incorporation into IKK- and IB when a critical amino acid residue for the kinase activity of IKK- was mutated (IKK- K44M). Therefore, expression of STAP-2 enhances the kinase activity of IKK-, thereby resulting in the induction of NF-B activity.
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FIGURE 4. Physiological roles of the interaction of STAP-2 with MyD88 and IKK- in the enhancement of NF-B activity. A, 293T cells in a 12-well plate were transfected with NF-B-LUC (0.4 g) and/or MyD88 (0.4 g) together with the increasing amounts of STAP-2 as indicated. Forty-eight hours after transfection, the cells were harvested, and luciferase activities were measured. The results are indicated as fold induction of luciferase activity from triplicate experiments, and the error bars represent the SD. B, 293T cells in a 12-well plate were transfected with NF-B-LUC (0.4 g) and/or IKK-␣ or IKK- (100 ng) together with the increasing amounts of STAP-2 as indicated. Forty-eight hours after transfection, the cells were harvested, and luciferase activities were measured. The results are indicated as fold induction of luciferase activity, and all values are the mean ⫾ SEM of triplicate cultures from three different experiments. ⴱ, p ⬍ 0.05. C, 293T cells (1 ⫻ 107) were transfected with His-tagged IKK- (5 g) and/or the increasing amounts of Myc-tagged STAP-2 as indicated. Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-His Ab. The kinase activity of IKK- in the immunoprecipitates was measured by in vitro kinase assay in the presence of GST-IB (1 g). Total cell lysates (1%) were immunoblotted with anti-His and anti-Myc Abs to monitor the expression of IKK- and STAP-2. IKK- K44M (lower panels), A kinase-negative form of IKK-
Involvement of the SH2-like domain of STAP-2 in its associations with MyD88 and IKK-␣ To determine the domains of STAP-2 involved in its associations with MyD88 and IKK-, a series of deletion mutants of STAP-2 fused with GST (GST-STAP-2 PH, GST-STAP-2 SH2, and GSTSTAP-2 C) were constructed (Fig. 5A). The respective mutants together with His-tagged IKK- or FLAG-tagged MyD88 were transiently expressed in 293T cells. The binding potential of these proteins with FLAG-tagged MyD88 or His-tagged IKK- was examined by immunoprecipitation with an anti-FLAG or anti-His Ab followed by Western blotting with an anti-GST Ab. As shown in Fig. 5, B and C, the precipitate for GST-STAP-2 SH2 contained both MyD88 and IKK-. However, no association of GSTSTAP-2 PH or GST-STAP-2 C with MyD88 or IKK- was detected. To further confirm this involvement of the SH2-like domain of STAP-2, we performed another set of coimmunoprecipitation experiments using a different series of STAP-2 deletion mutants (Fig. 5A). As shown in Fig. 5D, a deletion mutant of the SH2-like domain of STAP-2 failed to interact with IKK-, while full-length
and deletion mutants of the PH or C-terminal domains of STAP-2 retained strong binding to IKK-. Therefore, the SH2-like domain of STAP-2 can bind to both MyD88 and IKK-. To determine the domains of IKK- involved in the association with the SH2-like domain of STAP-2, 293T cells were transfected with GST-STAP-2 SH2 and/or a series of Myc-tagged IKK- deletion mutants (Fig. 5E). The transfectants were lysed, pulled down with glutathione-Sepharose beads (GSH bound), and immunoblotted with an anti-Myc Ab. As shown in Fig. 5F, the LZ domain of IKK- alone failed to interact with the SH2-like domain of STAP-2, while both the kinase and HLH domains of IKK- retained strong binding to STAP-2. Therefore, the SH2-like domain of STAP-2 interacts with the N-terminal and C-terminal domains of IKK-. To determine the domains of MyD88 involved in the association with the SH2-like domain of STAP-2, 293T cells were transfected with GST-STAP-2 SH2 and/or a series of Myc-tagged MyD88 deletion mutants (Fig. 5G). The transfectants were lysed, pulled down with glutathione-Sepharose beads (GSH bound), and blotted
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FIGURE 5. Involvement of the SH2-like domain of STAP-2 in the association with MyD88 and IKK-. A, Domain structure of STAP-2, GST-fused mutant fragments are schematically shown. B, 293T cells (1 ⫻ 107) were transfected with His-tagged IKK- (10 g) or FLAG-tagged MyD88 (10 g) and/or GST or a series of GST-fused STAP-2 mutants (10 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-FLAG Ab and immunoblotted with anti-GST Ab (upper panel) or anti-FLAG Ab (middle panel). Total cell lysates (1%) were blotted with anti-GST Ab (lower panel) to monitor the expression of GST or its fusion proteins. C, 293T cells (1 ⫻ 107) were transfected with His-tagged IKK- (10 g) and/or a series of GST-tagged STAP-2 mutants (5 mg). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-His Ab and immunoblotted with anti-GST Ab (upper panel) or anti-IKK- Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-GST Ab (lower panel) to monitor the expression of WT STAP-2 or its mutants. D, 293T cells (1 ⫻ 107) were transfected with His-tagged IKK- (10 g) and/or a series of Myc-tagged STAP-2 mutants (5 g). Forty-eight hours after transfection, the cells were lysed, and immunoprecipitated with anti-His Ab and immunoblotted with anti-Myc Ab (upper panel) or anti-IKK- Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-Myc Ab (lower panel) to monitor the expression of WT STAP-2 or its mutants. E, Domain structure of IKK- and its mutant fragments are schematically (Figure legend continues)
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FIGURE 6. Physiological role of the SH2-like domain of STAP-2 in LPS/TLR4-signaling. A, Stable Raw264.7 transformants with a series of STAP-2 mutants were established. Total extracts (1%) of each transformant were examined by immunoblotting using anti-Myc Ab to monitor the expression of WT STAP-2 or its mutants. B, Raw264.7 transformants (2 ⫻ 103) were cultured in a 96-well plate with the indicated concentrations of LPS for 24 h, and the supernatants were analyzed for TNF-␣ by ELISA. Data are values are the mean ⫾ SEM of triplicates.
with an anti-Myc Ab. As shown in Fig. 5H, both the death domain and TIR domains of MyD88 interacted with the SH2-like domain of STAP-2. Therefore, the SH2-like domain of STAP-2 interacts with at least two regions of MyD88.
CpG DNA was observed in STAP-2-deficient macrophages (Fig. 7B). These results suggest that STAP-2 plays a role in the MyD88dependent CpG DNA/TLR9 pathway as well as in LPS/TLR4 signaling.
Physiological role of the SH2-like domain of STAP-2 in LPS/TLR4-signaling
Discussion
To further assess the functional relevance between STAP-2 and LPS/TLR4 signaling, we established stable transformants expressing WT STAP-2 or its deletion mutants in Raw264.7 cells (Fig. 6A), and used them to evaluate the induction of cytokine production by LPS. As shown in Fig. 6B, the production of TNF-␣ after LPS stimulation was strongly enhanced in Raw/STAP-2 Full, Raw/STAP-2 ⌬PH, and Raw/STAP-2 ⌬C cells compared with Raw/pcDNA3 cells. However, Raw/STAP-2 ⌬SH2 cells did not show any enhanced TNF-␣ production after LPS stimulation. Therefore, the SH2-like domain of STAP-2 plays an important role in modifying LPS/TLR4-signaling. STAP-2 deficiency affects TLR9- but not TLR3-mediated cytokine production Finally, we examined whether STAP-2 is involved in other TLRmediated signaling pathways. To this end, we tested cytokine production by WT or STAP-2-deficient macrophages after stimulation with poly(I:C)/TLR3 or CpG DNA/TLR9. As shown in Fig. 7A, poly(I:C)/TLR3 stimulation induced TNF-␣ and IL-6 production equally in WT or STAP-2-deficient macrophages. However, a significant reduction in TNF-␣ and IL-6 production in response to
During the process of inflammation, macrophages play a crucial role by producing inflammatory cytokines including IL-1, IL-6, and TNF-␣, which initiate and spread inflammatory responses to eliminate invading foreign pathogens. TLR4 on macrophages recognizes LPS and mediates signals for cytokine production. In the present study, we found a new function for the adaptor protein STAP-2 in macrophages. LPS/TLR4-mediated cytokine production and NF-B activation were significantly impaired in STAP2-deficient macrophages, while overexpression of STAP-2 in the murine macrophage cell line Raw264.7 resulted in enhanced cytokine production and NF-B activation after LPS stimulation. These findings suggest that STAP-2 participates in the production of inflammatory cytokines in activated macrophages. Interestingly, STAP-2 is constitutively expressed in macrophages, and the 5⬘ region of the STAP-2 genomic sequence contains several potential binding sites for c-Rel, AP-1, p65/NF-B, and STATs (8). In the murine myeloid leukemia cell line M1, STAP-2 mRNA expression is strongly induced by LIF in parallel with its differentiation into macrophages (8). These expression patterns of STAP-2 strengthen the possibility that STAP-2 in macrophages may mediate signals for acute-phase responses after infection. Indeed, STAP-2 modulated TNF-␣ production at an early stage after LPS exposure in vivo.
shown. KD, kinase domain; LZ, leucine zipper; HLH, helix-loop-helix. F, 293T cells (1 ⫻ 107) were transfected with GST-fused STAP-2 SH2 (10 g) and/or a series of Myc-tagged IKK- (10 g). Forty-eight hours after transfection, the cells were lysed, and pulled down with glutathione-Sepharose beads (GSH bound) and immunoblotted with anti-Myc Ab (upper panel) or anti-GST Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-Myc Ab (lower panel) to monitor the expression of IKK- mutant proteins. G, Domain structure of MyD88 and its mutant fragments are schematically shown. DD, death domain; TIR, Toll-IL-1R. H, 293T cells (1 ⫻ 107) were transfected with GST-fused STAP-2 SH2 (10 g) and/or a series of Myc-tagged MyD88 mutants (10 g). Forty-eight hours after transfection, the cells were lysed, and pulled down with glutathione-Sepharose beads (GSH bound) and immunoblotted with anti-Myc Ab (upper panel) or anti-GST Ab (middle panel). Total cell lysates (1%) were immunoblotted with anti-Myc Ab (lower panel) to monitor the expression of MyD88 mutant proteins. ⴱ, Migration positions of the respective mutants.
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INTERACTIONS BETWEEN STAP-2 AND TLR4 SIGNALING
FIGURE 7. Cytokine production in peritoneal macrophages in response to poly(I:C) or CpG DNA. Peritoneal macrophages (105 cells/well) from WT (⫹/⫹) and STAP-2-deficient (⫺/⫺) mice were cultured with the indicated concentrations of poly(I:C) (A) or CpG DNA (B) for 12 h. Concentrations of TNF-␣ or IL-6 in the culture supernatants were measured with ELISA. All values are the mean ⫾ SEM of triplicate cultures from three different experiments. ⴱ, p ⬍ 0.05.
The currently available information enables us to discuss the molecular mechanisms of LPS/TLR4 signaling. TLR4 mediates signals responding to LPS in collaboration with CD14 and MD-2 (26 –28). The cytoplasmic tail of TLR4 contains a TIR domain whose activation results in recruitment of TIR domain-containing adaptor proteins, such as MyD88 (1, 29), TIR domain-containing adaptor protein (TIRAP; also called MAL) (30, 31), TIR domaincontaining molecule (TRIF; also called TICAM-1) (32, 33) and TRIF-related adaptor molecule (TRAM; also called TIRP) (34). Experiments using MyD88-deficient mice have revealed that TLR4 signaling is composed of at least two pathways: a MyD88dependent pathway leading to the production of proinflammatory cytokines and a MyD88-independent pathway associated with the induction of IFN--inducible genes and maturation of dendritic cells. In the MyD88-dependent pathway, the death domain of MyD88 recruits downstream IRAK to the receptor complex (29, 30). IRAK then becomes autophosphorylated, dissociates from the receptor complex, and recruits TRAF6, which in turn activates downstream kinases (1, 5) including IKKs. TIRAP/MAL is also important for activation of the MyD88-dependent signaling pathway through TLR2 and TLR4. In the MyD88-independent pathway, TRIF/TICAM-1 is crucial for signals shared by TLR3 and TLR4. TRAM/TIRP provides specificity for the MyD88-independent component of TLR4 signaling. In addition, Toll-interacting protein interacts with IRAK1 and inhibits its kinase activity (6, 35). The evolutionarily conserved signaling intermediate in Toll pathways transduces TLR signaling by bridging both TRAF6 and MEKK1 to activate the IKK complex (36, 37). IRAK-M lacks kinase activity and prevents dissociation of the IRAK1-IRAK4 complex from MyD88, thereby blocking the complex formation between IRAK1 and TRAF6 (38, 39). Suppressor of cytokine signaling 1 (SOCS1) contains an SH2 domain and a SOCS box (40). The SOCS-box recruits the Elongin B, C-Rbx1-Cul2 ubiquitination system (41), suggesting that ubiquitination may be involved in SOCS1-mediated suppression of TRAF6-dependent IKK activation (42). The SH2 domain of SOCS1 has also been shown to interact directly with IRAK1 (43). In the case of STAP-2, its SH2like domain acts as a positive regulator of LPS/TLR4-signaling.
The SH2-like domain of STAP-2 associates with MyD88 and IKK-␣, but not IRAK1 or TRAF6. Our data also suggest that the direct interactions of STAP-2 with MyD88 and IKK-␣ form a complex, which transmits signals for NF-B activation. Indeed, Raw264.7 cells expressing STAP-2 lacking the SH2-like domain failed to enhance LPS/TLR4-mediated cytokine production. Activation of the classical NF-B pathway converges on the IKK signalosome, a protein complex composed of two kinase subunits (IKK-␣ and IKK-) and the noncatalytic subunit NF-B essential modulator. Activated IKK phosphorylates IB proteins that sequester NF-B in the cytoplasm (44). The main NF-B signaling pathway through TLR4 in macrophages is MyD88-IRAK1TRAF6-IKK-mediated phosphorylation of IB and TRAF6-mediated JNK and p38 MAPK activation. Our results suggest a possible alternative LPS/TLR4-signaling pathway mediated by STAP-2. From the results of the present study, we propose that LPS/TLR4 signaling has a direct link from MyD88 to the IKK complex by STAP-2. This possible model has been established on the basis of the following data: 1) STAP-2 bound to MyD88 and IKK-␣, but not IRAK1 or TRAF6; 2) the association between MyD88 and IKK- was only detected in the presence of STAP-2; 3) STAP-2 enhanced MyD88-induced and IKK-␣-induced NF-B activity; and 4) STAP-2 enhanced the kinase activity of IKK-. Further investigations will clarify the new signaling pathway, and experiments are in progress in this regard. Recently, Syk kinase was shown to be critical for tyrosine phosphorylation of IB and NF-B activation in various T cell lines and myeloid cells (45, 46). Arndt et al. (47) further reported that LPS activated Syk kinase in neutrophils. Because Syk kinase directly associates with and tyrosine-phosphorylates STAP-2 (17), it is possible that LPS-activated Syk kinase mediates the link between MyD88 and IKKs by STAP-2. This possibility should also be clarified in future studies. In the present paper, we have described evidence that STAP-2 can directly enhance LPS induction of the NF-B signaling pathway through binding to MyD88 and IKK-␣. Thus, STAP-2 functions as a novel adaptor protein, which acts as a link between MyD88 and IKK-␣ in TLR4 signaling. Our proposed signaling
The Journal of Immunology pathway concerning STAP-2 will provide new insights toward understanding the molecular mechanisms of TLR4-mediated signaling pathways. In addition, our data suggest the possibility that STAP-2 can be considered as a novel candidate for antiinflammatory drug development to regulate the expression of NFB-dependent genes.
Acknowledgments We thank Drs. T. Fujita, J. Inoue, T. Sugita, H. Sakurai, M. Hibi, and M. Matsumoto for their gifts for reagents. We also thank Dr. J. Akiyama for encouraging our work.
Disclosures The authors have no financial conflict of interest.
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