JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation
2003; Springer Nature; Volume: 22; Issue: 17 Linguagem: Inglês
10.1093/emboj/cdg438
ISSN1460-2075
Autores Tópico(s)interferon and immune responses
ResumoArticle1 September 2003free access JNK-interacting protein 3 associates with Toll-like receptor 4 and is involved in LPS-mediated JNK activation Tetsuya Matsuguchi Corresponding Author Tetsuya Matsuguchi Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Akio Masuda Akio Masuda Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Kenji Sugimoto Kenji Sugimoto Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yoshiyuki Nagai Yoshiyuki Nagai Toyama Institute of Health, Toyama, Japan Search for more papers by this author Yasunobu Yoshikai Yasunobu Yoshikai Division of Host Defense, Research Center of Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Tetsuya Matsuguchi Corresponding Author Tetsuya Matsuguchi Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Akio Masuda Akio Masuda Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Kenji Sugimoto Kenji Sugimoto Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan Search for more papers by this author Yoshiyuki Nagai Yoshiyuki Nagai Toyama Institute of Health, Toyama, Japan Search for more papers by this author Yasunobu Yoshikai Yasunobu Yoshikai Division of Host Defense, Research Center of Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan Search for more papers by this author Author Information Tetsuya Matsuguchi 1, Akio Masuda1, Kenji Sugimoto1, Yoshiyuki Nagai2 and Yasunobu Yoshikai3 1Division of Host Defense, Center for Neural Disease and Cancer, Nagoya University Graduate School of Medicine, Nagoya, Japan 2Toyama Institute of Health, Toyama, Japan 3Division of Host Defense, Research Center of Prevention of Infectious Diseases, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan *Corresponding author. E-mail: [email protected] The EMBO Journal (2003)22:4455-4464https://doi.org/10.1093/emboj/cdg438 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Lipopolysaccharide (LPS) is recognized by Toll-like receptor (TLR) 4 and activates NF-κB and a set of MAP kinases. Here we have investigated proteins associated with the cytoplasmic domain of mouse TLR4 by yeast two-hybrid screening and identified JNK-interacting protein 3 (JIP3), a scaffold protein for JNK, as a TLR4-associated protein. In mammalian cells, JIP3, through its N-terminal region, constitutively associates with TLR4. The association is specific to JIP3, as the two other JIPs, JIP1 and JIP2, failed to bind TLR4. In HEK 293 cells exogenously expressing TLR4, MD2 and CD14, co-expression of JIP3 significantly increased the complex formation of TLR4–JNK and LPS-mediated JNK activation. In contrast, expression of C-terminally truncated forms of JIP3 impaired LPS-induced JNK activation in a mouse macrophage cell line, RAW264.7. Moreover, RNA interference of JIP3 inhibited LPS-mediated JNK activation. In RAW264.7 cells, JIP3 associates MEKK-1, but not with TAK-1. Finally, JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. Altogether, our data indicate the involvement of JIP3 in JNK activation in downstream signals of some TLRs. Introduction Toll-like receptors (TLRs) play important roles in host defense mechanisms by pathogen recognition. Ten members of the TLRs (TLR1–10) have so far been reported. TLR4 mediates lipopolysaccharide (LPS) signals in collaboration with other molecules, such as CD14, MD-2, myeloid differentiation factor 88 (MyD88) and Toll receptor–IL-1 receptor domain containing adapter protein (TIRAP)/MyD88-adapter-like (Mal). On the other hand, TLR2 is considered to be an essential receptor for lipoprotein, peptidoglycan, zymosans and lipoteichoic acid presumably by forming heteromers with TLR1 or TLR6. TLR3, TLR5 and TLR9 have recently been shown to mediate signals from double-stranded RNA, flagella and bacterial DNA, respectively (Akira, 2001). Stimulation of TLRs by specific ligands induces nuclear transport of NF-κB and the activation of a set of mitogen-activated protein kinases (MAPKs): extracellular signal-regulated kinases (ERKs), c-Jun N-terminal kinases (JNKs) and p38 kinases. Activation mechanisms of MAPKs are generally through phosphorylation of threonine and tyrosine residues within the signature sequence of T-X-Y by dual specificity MAPK kinases (MKKs). MKKs are in turn phosphorylated and activated by a family of serine/threonine MKK kinases (MKKKs) (Robinson and Cobb, 1997). MKKKs integrate signals mediated by upstream signaling molecules. The distinct classes of MAPKs play important roles in various cellular events including cell proliferation, differentiation and apoptosis. JNKs are activated by diverse stimuli including DNA damage, heat-shock, bacterial components, inflammatory cytokines and Fas (Leppa and Bohmann, 1999). Activated JNKs play an essential role in the activation of transcriptional factors, such as c-Jun, ATF-2, Elk-1 and ets-2 (Smith et al., 2000). In macrophages, activated JNKs mediate the expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), chemokines and cytokines, all of which potently activate host defense mechanisms. The critical roles of JNK signaling in immuno-regulatory cells are evident as jnk1−/− and jnk2−/− mice exhibit various defective immune responses (Dong et al., 1998; Yang et al., 1998). MAPK activation may be facilitated by the formation of signaling modules, and it has been well established in both yeast and mammalian cells that 'scaffold proteins' play important roles by interacting with MAPKs and their upstream kinases (Whitmarsh and Davis, 1998). For example, in yeast, a scaffold protein, Ste5p, mediates the mating Fus3 MAPK pathway (Choi et al., 1994) and Pbs2p plays a part in the osmoregulatory MAPK pathway (Posas and Saito, 1997). Mammalian scaffold proteins, MP1 and KSR were found to function in the ERK activation pathway (Schaeffer et al., 1998; Cacace et al., 1999). β-arrestin2, which binds to β2-adrenergic receptors, has recently been reported to function as a scaffold protein for JNK activation (McDonald et al., 2000). Also in the JNK signaling cascade, three other putative scaffold proteins have been reported: JNK-interacting protein 1 (JIP1; Whitmarsh et al., 1998), JIP2 (Yasuda et al., 1999) and JIP3 (also termed JSAP1) (Ito et al., 1999; Kelkar et al., 2000). They promote JNK activation by interacting with JNKs and the upstream kinases in vitro. A recent study of jip1 gene-disrupted mice has indicated that JIP1 functions as an JNK activator in vivo (Whitmarsh et al., 2001). The role of JIP2 is somewhat controversial, as it has recently been reported to bind and activate p38 kinase, but not JNK (Buchsbaum et al., 2002). JIPs may also participate in vesicular transport by binding a motor protein, kinesin-1 (Verhey et al., 2001). JIP3 is ubiquitously expressed in mouse tissues, with notably higher expression detected in brain, heart and lung (Kelkar et al., 2000). The homologue of JIP3 has been reported in Drosophila and Caenorhabiditis elegans (Bowman et al., 2000; Byrd et al., 2001), indicating JIP3 is evolutionally well conserved. Although TLRs activate three groups of MAPKs, the possible involvement of scaffold proteins in the activating processes is not known. LPS, which signals through TLR4, activates two MKKKs, TGFβ-activated kinase (TAK) 1 and MEK kinase (MEKK) 1, presumably through TRAF6 (Kopp et al., 1999; Ninomiya-Tsuji et al., 1999). Activation of these two kinases has been indicated to involve interacting proteins, TAK1-binding protein (TAB) 1/2 and ECSIT (evolutionarily conserved signaling intermediate in Toll pathways), respectively. Here we report that TLR4 constitutively associates with JIP3, but not with JIP1 or JIP2. JIP3 also associates with TLR2 and TLR9, but not with TLR1 or TLR6. In a mouse macrophage cell line, JIP3 associates with both full-length and processed forms of MEKK1. Moreover, expression of C-terminally deleted JIP3 mutants and RNA interference of JIP3 inhibited LPS-mediated JNK activation. These data indicate that JIP3, as a scaffold protein, plays a role in mediating JNK activation by LPS through the association with TLR4. Results Identification of JIP3 as a TLR4-interacting protein by yeast two-hybrid screening In an attempt to identify novel proteins interacting with the cytoplasmic domain of TLR4, we screened a mouse embryo cDNA library by yeast two-hybrid method using the mouse TLR4 cytoplasmic domain as the bait. We isolated 34 independent clones, two of which encode MyD88, a previously known TLR4-interacting protein, confirming validation of the screening. By nucleotide sequencing, we found that two of the isolated clones encode JIP3. These two clones contained different lengths of JIP3 cDNA (encoding amino acids 154–1337 and 172–1337), and were confirmed to associate with TLR4 cytoplasmic domain by re-transfection in yeast with bait plasmid (Figure 1A). Figure 1.Interaction of JIP3 and TLR4. (A) The cytoplasmic portion of mouse TLR4 cloned in frame with GAL4 DNA binding domain in pDBLeu vector was cotransformed with pPC86 empty vector containing GAL4 activating domain, pPC86-MyD88 or pPC86-JIP3 into MaV203 yeast strain carrying the three reporter genes: HIS3, URA3 and lacZ. The transformed colonies were grown on either SC/-Leu or SC/-His-Trp-His+3AT selection. (B) HEK 293 cells were transiently transfected with pcDNA3.1(+)-CD14, pcDNA3.1(+)-MD2 and p3XFlagCMV14-TLR4 in combination with either pEFBOSMyc (EV: empty vector) or pEFBOSMyc-JIP3. At 48 h after transfection, cells were either left untreated or treated with 1 μg/ml LPS for 20 min. Anti-Flag and anti-Myc immunoprecipitates were separated by SDS–PAGE and immunoblotting was performed with anti-Flag (upper) and anti-Myc (lower) antibodies. (C) HEK 293 cells were transiently transfected with pEFBOSFlag-JIP3 in combination with either pcDNA3.1(+)-GCSFR/TLR2 or pcDNA3.1(+)-GCSFR/TLR4. At 48 h after transfection, cell lysates were prepared. Anti-Flag and control antibody immunoprecipitates were separated by SDS–PAGE, and immunoblotting was performed with anti-GCSFR antibody (top panel). Cell lysates were also probed with anti-Flag or anti-GCSFR antibody. (D) RAW264.7 cells were stimulated with 1 μg/ml LPS for 20 min or left untreated. Cell lysates were prepared and anti-TLR4 and control antibody immunoprecipitates were separated by SDS–PAGE, followed by the analysis with anti-JIP3 antibody. All the experiments in this paper were repeated at least twice, typically 3–5 times, with reproducible results. Download figure Download PowerPoint In order to test the JIP3–TLR4 association in mammalian cells, we expressed JIP3 N-terminally tagged with Myc and TLR4 C-terminally tagged with Flag in HEK 293 cells in combination with mouse CD14 and MD-2. We found that JIP3 was coprecipitated with TLR4 in the absence of LPS stimulation of the cells (Figure 1B). No increase of binding was detected when the cells were stimulated with LPS. JIP3–TLR4 interaction was also detected in the absence of CD14 or MD-2 (data not shown), which were co-expressed in order to increase the LPS-responsiveness of the cells. We next examined the association by using a chimera protein of the extracellular domain of human G-CSF receptor fused to the cytoplasmic domain of TLR4 (Figure 1C). The G-CSFR–TLR4 fusion protein was transiently expressed with Flag-tagged JIP3 in HEK 293 cells. The fusion protein was coprecipitated with JIP3, indicating that the extracellular domain of TLR4 does not contribute to the TLR4–JIP3 association. We further examined the association of endogenous TLR4 and JIP3. Using a mouse macrophage cell line, RAW264.7, we could detect endogenous JIP3 in the TLR4 immunoprecipitates with or without LPS stimulation, but not in the control immunoprecipitates (Figure 1D). The C-terminal domain of TLR4 is necessary for binding JIP3 In an attempt to identify the region of TLR4 required for JIP3 binding, we utilized two C-terminal deletion mutants: TLR4del13 and TLR4 del130, deleting C-terminal 13 and 130 amino acids of TLR4, respectively. Unlike the full-length TLR4, neither mutant was coprecipitated with JIP3 (Figure 2A). Thus the C-terminal 13 amino acids of TLR4 are essential for binding JIP3. Figure 2.Identification of TLR4 region essential for interaction with JIP3. (A) Schematic presentation of the mouse TLR4 mutants used in the assay. Black boxes represent transmembrane domains (TM). (B) C-terminal region of TLR4 is essential for the interaction with JIP3. HEK 293 cells were transiently transfected with expression plasmids of a Myc-tagged JIP3 (wild type or JNK-binding mutant) and an indicated Flag-tagged TLR4. At 48 h after transfection, JIP3 was precipitated with anti-Myc antibody and coprecipitation of TLR4 was detected with anti-Flag antibody. Lysates were also run as controls for the input. (C) Pro712 of TLR4 is not essential for JIP3 binding. HEK 293 cells were transiently transfected with expression plasmids of a Myc-tagged wild-type JIP3 construct and the TLR4 wild-type or Pro712His point mutant. At 48 h after transfection, lysates were prepared and anti-Myc and control immunoprecipitates were analyzed for the coprecipitation of TLR4 with anti-Flag antibody. Download figure Download PowerPoint Although not included in the C-terminal 13 amino acids, Pro712 is considered essential for the TLR4 signaling. Mutation of the proline to histidine, which is found in the C3H/HeJ mouse strain, severely impaired LPS-stimulated NF-κB and JNK activation presumably by inhibiting TLR4–MyD88 binding (data not shown) (Rhee and Hwang, 2000). We examined if this Pro712His mutation affects TLR4–JIP3 binding and found that the TLR4 Pro712His mutant is capable of binding JIP3 to the same extent as the wild-type TLR4 (Figure 2B), indicating that the TLR4–JIP3 interaction alone is not sufficient to induce JNK activation. JIP3 increases LPS-stimulated JNK activity As TLR4 associates with JIP3, which is a known scaffold for a JNK signaling module, we speculated that the expression of JIP3 may enhance JNK activation by LPS. To test this hypothesis, we performed transfection assays using COS7 cells, which do not express endogenous JIP3 protein (Kelkar et al., 2000) (Figure 3). Expression of JIP3 in combination with TLR4, CD14 and MD-2 did not cause the activation of the cotransfected haemagglutinin (HA)-JNK1. However, JIP3 expression significantly increased LPS-induced JNK activity. In contrast, JIP3 expression did not affect JNK activation by anisomycin, a potent activator of MKK4. Thus JIP3 is specifically involved in LPS-mediated JNK activation. Also, the effect of JIP3 is specific to JNK, as activation of p38 kinase by LPS was not affected by JIP3 expression (Figure 3). Figure 3.JIP3 overexpression enhances LPS-mediated JNK activation. COS7 cells were transiently transfected with p3XFlag-CMV14-TLR4, pcDNA3.1(+)-mCD14, p3XFlag-CMV10-mMD-2 and pcDNA3.1(+)-HA-JNK1. At 48 h after the transfection, cells were left untreated or stimulated with 1 μg/ml LPS or 10 μg/ml anisomycin for 20 min, and lysed. Anti-HA immunoprecipitates were tested for in vitro kinase assay on GST–cJun5-89 as the substrate. Cell lysates were also analyzed for p38 kinase activity by anti-p38 immunoprecipitation using GST–ATF-2 as the substrate. Download figure Download PowerPoint Identification of TLR4 binding domain of JIP3 Two independent partial cDNA clones of JIP3 isolated by the two-hybrid screening both contain parts of the N-terminal region of JIP3. We did not isolate any short cDNA clones encoding only the C-terminal regions of JIP3, hinting that the N-terminal region of JIP3 may be responsible for TLR4 binding. In order to define the TLR4 binding domain of JIP3, we expressed several C-terminal deletion mutants of JIP3 in combination with the wild-type TLR4 for coprecipitation assays. As shown in Figure 4, Flag-tagged TLR4 was coprecipitated with JIP3 C-terminal deletion mutants: 1–221, 1–355, 1–677, 1–1015, at similar levels to the full JIP3 protein. However, no coprecipitation was detected for the JIP3 1–163 mutant, suggesting that the residues 163 to 221 are essential for JIP3 to bind TLR4. Figure 4.JIP3 associates with TLR4 through its N-terminal region and induces TLR4–JIP3–JNK complex formation. (A) JIP3 N-terminal region is sufficient for the interaction with TLR4. HEK 293 cells were transiently transfected with p3XFlag-CMV14-mTLR4 in combination with a series of Myc-tagged C-terminal deletion mutants of JIP3 (see the scheme). Cell lysates were prepared at 48 h after transfection, and the anti-Myc immunoprecipitates were tested for the coprecipitation of TLR4 using anti-Flag antibody. The results are summarized in the scheme. The immunoblots for some constructs are shown. (B) JIP3 induces the complex formation by TLR4 and JNK. HEK 293 cells were transiently transfected with pEFBOSMyc-mTLR4 in combination with either pEFBOSFlag (empty vector) or pEFBOSFlag-mJIP3. Cell lysates were prepared at 48 h after transfection, and the protein expression of endogenous JNK1 and Flag-JIP3 was confirmed by immunoblot (top and middle panel). The control or anti-JNK1 antibody immunoprecipitates were separated by SDS–PAGE, and the coprecipitation of TLR4 was tested using anti-Myc antibody (bottom panel). Download figure Download PowerPoint It has previously been reported that residues 207 to 216 are required for JIP3–JNK binding (Kelkar et al., 2000). To rule out the possibility that TLR4 and JNK compete for the same binding site in JIP3, we examined if JIP3 induces TLR4–JIP3–JNK complex formation. We transiently transfected HEK 293 cells with Myc-tagged TLR4 with or without JIP3, immunoprecipitated endogenous JNK1 and detected coprecipitated TLR4 by anti-Myc antibodies. As shown in Figure 4B, JIP3 expression significantly increased TLR4 coprecipitating with JNK1, indicating that there is TLR4–JIP3–JNK1 complex formation. We also introduced two amino acid substitutions into the JNK-binding motif of JIP3 (Kelkar et al., 2000). This mutant (JIP3-GG) was significantly less potent for JNK binding (data not shown), but was capable of binding TLR4 with similar affinity to the wild-type JIP3 (Figure 2). Thus, JNK- and TLR4-binding is not mutually exclusive for JIP3. Expression of JIP3 C-terminal deletion mutants and JIP3 RNA interference inhibit LPS-mediated JNK activation It has previously been reported that JIP3 binds, in addition to JNK, upstream kinases: MKK7 and MLK3, through the central region of the JIP3 molecule (Kelkar et al., 2000). In another report, JIP3 has been reported to bind MKK4 and MEKK1 through the C-terminal and the central region, respectively (Ito et al., 1999). These reports indicated that the expression of N-terminal region of JIP3 alone may function in a dominant negative fashion. We tested this hypothesis with RAW264.7 cells using both transient and stable expression systems. First we confirmed that RAW264.7 cells constitutively express endogenous JIP3 protein, using two antibodies recognizing the N- and C-terminal domains of JIP3 (Figure 5A). We also easily detected JIP3 protein in mouse peritoneal macrophages induced by thioglycollate (data not shown), indicating that JIP3 protein is generally expressed in mouse macrophages. We then transiently transfected RAW264.7 cells with the expression plasmid for C-terminal deletion mutants of JIP3, in combination with the expression plasmid for HA-tagged JNK1, and compared JNK activity of the anti-HA immunoprecipitates after LPS stimulation. We found that transient expression of either JIP3 1–221 or 1–355 significantly decreased JNK activity in response to LPS but not to TNF-α (Figure 5B). Figure 5.JIP3 is involved in JNK activation by LPS in a mouse macrophage cell line. (A) RAW264.7 cells were stimulated with 1 μg/ml LPS for the indicated times. Cell lysates were prepared and JIP3 protein contents were analyzed by antibodies specific to the N- and C-terminal domains of JIP3. (B) RAW264.7 cells were transiently transfected with the empty vector, the expression plasmid of the wild-type JIP3 or a JIP3 C-terminal mutant, in combination with the expression plasmid for HA-tagged JNK1. Cells were untreated, treated with 1 μg/ml LPS or 10 ng/ml TNF-α for 20 min, and anti-HA antibody immunoprecipitates were examined for their kinase activity on GST–cJun5-89. (C) RAW264.7 cells stably transfected with the empty vector, the expression plasmid of the wild-type JIP3, or a JIP3 C-terminal mutant were either untreated or treated with 1 μg/ml LPS for 20 min, and anti-JNK1 immunoprecipitates were examined for their kinase activity on GST–cJun5-89. Cell lysates were also examined for the exogenous JIP3 expression with anti-Flag antibody. (D) RAW264.7 cells were stably transfected with either the empty vector or the pSilencer-JIP3 plasmid. Cells were untreated, treated with 1 μg/ml LPS or 10 ng/ml TNF-α for 20 min. JIP3 and β-actin protein contents were examined by their specific antibodies. Subsequently, JNK1 was immunoprecipitated and in vitro kinase assay was performed on GST–cJun5-89. Download figure Download PowerPoint We next isolated multiple RAW264.7 clones stably expressing full-length or C-terminally deleted JIP3s and analyzed their JNK activity of anti-JNK1 immunoprecipitates after LPS stimulation (Figure 5C). Consistently with the transient transfection experiment described above, more than three independent clones for each of JIP3 1–221 and JIP3 1–355 showed decreased JNK1 activity compared with the empty vector clones (results from two typical clones are shown for each deletion construct in Figure 5B). These results indicate that both JIP3 1–221 and 1–355 function as dominant negative mutants and suggest that JIP3 is physiologically involved in LPS-mediated JNK activation. Furthermore, we introduced small inhibitory RNA (siRNA) of JIP3 into RAW264.7 cells. It reduced the endogenous protein level of JIP3, but not that of β-actin, which was analyzed as a control (Figure 5D). Introduction of JIP3 siRNA efficiently decreased JNK activation by LPS but not that by TNF-α (Figure 5D), further confirming the specific involvement of JIP3 in LPS signaling. JIP3 associates with MEKK1, but not TAK1, in RAW264.7 cells LPS induces activation of several MKKKs. In particular, both MEKK1 and TAK1 have been indicated as essential upstream kinases for JNK activation by LPS. JIP3 has been reported to bind MEKK1 (Ito et al., 1999), whereas the association of TAK1 and JIP3 has not been reported. To examine the possible interaction between JIP3 and the two MKKKs, we used RAW264.7 cells stably expressing Flag-tagged JIP3, and MKKKs coprecipitated with JIP3 were detected with their specific antibodies in the anti-Flag immunoprecipitates. As shown in Figure 6, although no coprecipitation of TAK1 and JIP3 was detected, we found that the full-length form of MEKK1 constitutively associated with JIP3. Furthermore, LPS stimulation rapidly induced the temporary coprecipitation of the processed 72 kDa form of MEKK1 with JIP3. As the ratio of processed/full-length MEKK1 did not change in response to LPS in this cell line (Figure 6A, left panel), it apparently indicated that only a portion of MEKK1 protein that interacts with JIP3 becomes processed on the scaffold protein in response to LPS stimulation. Figure 6.Complex formation of JIP3 and MEKK1. RAW264.7 cells stably expressing Flag-tagged JIP3 were stimulated with LPS for the indicated times. (A) The anti-MEKK1 immunoblot of the cell lysates is shown in the left panel. For the immunoprecipitation experiments, anti-Flag or control antibody was cross-linked to protein A beads and incubated with the cell lysates. The immunoprecipitates were separated by SDS–PAGE and probed with anti-MEKK1 antibody (two right panels). (B) Anti-Flag and control antibody immunoprecipitates were separated by SDS–PAGE and probed with anti-TAK1 antibody. Download figure Download PowerPoint Interaction of other JIP and TLR proteins Two other JIP family proteins (JIP1 and JIP2), both of which function as JNK scaffold proteins, have been reported (Whitmarsh et al., 1998; Yasuda et al., 1999). Although JIP1 and JIP2 are homologous, JIP3 does not share significant amino acid sequence homology with JIP1 or JIP2. In order to test the possible association of JIP1 and JIP2 with TLR4, we transiently transfected Flag-tagged TLR4 in HEK 293 cells, and analyzed coprecipitated JIP proteins in the anti-Flag immunoprecipitates. HEK 293 cells constitutively express endogenous JIP1, 2 and 3 as shown by the immunoblot using their specific antibodies (Figure 7A). In contrast to JIP3, neither JIP1 nor JIP2 was coprecipitated with TLR4, indicating TLR4 specifically interacts with JIP3. Consistently, expression of the functionally defective mutants of JIP1 (JIP1 281) (Whitmarsh et al., 1998) and JIP2 (JIP2 232) (Yasuda et al., 1999) did not show any significant effects on LPS-induced JNK activation (Figure 7B). Figure 7.Involvement of JIP3 in other TLR signals. (A) HEK 293 cells were transiently transfected with p3XFlag-CMV14-TLR4, pcDNA3.1(+)-mCD14, and pcDNA3.1(+)-mMD-2. At 48 h after the transfection, cells were left untreated or stimulated with 1 μg/ml LPS for 20 min and lysed. At 48 h after transfection, cells were lysed and immunoprecipitates of anti-Flag and control antibodies were separated by SDS–PAGE. Immunoblotting was performed with anti-JIP1, anti-JIP2, anti-JIP3 or anti-Flag antibody. (B) HEK 293 cells were transiently transfected with pEEBOS-Flag-JIP3 221, pEFBOS-Flag-JIP1 281 or pEFBOS-Flag-JIP2 232, along with p3XFlag-CMV14-TLR4, pcDNA3.1(+)-mCD14, p3cDNA3.1(+)-mMD-2 and pcDNA3.1(+)-HA-JNK1. At 48 h after the transfection, cells were left untreated or stimulated with 1 μg/ml LPS for 20 min, and lysed. Immunoblotting results using anti-Flag antibody are shown in the upper panel. Anti-HA immunoprecipitates were tested for in vitro kinase assay on GST–cJun5-89 as the substrate. (C) 293T cells were transiently transfected with an expression plasmid of Myc-tagged JIP3 in combination with the indicated expression plasmid of Flag-tagged TLR. At 48 h after transfection, cells were lysed and protein expression was examined using anti-Flag and anti-Myc antibodies (upper two panels). JIP3 was immunoprecipitated with anti-Myc antibody. Coprecipitated TLRs were detected by anti-Flag antibody. (D) RAW264.7 cells stably transfected with the empty vector or the expression plasmid of JIP3 1–355 were treated with 1 μg/ml LPS, 10 μg/ml synthetic lipoprotein, or 1 μM CpG ODN for 20 min and anti-JNK1 antibody immunoprecipitates were examined for their kinase activity on GST–cJun5-89. Download figure Download PowerPoint We next examined the association of JIP3 with several members of TLR proteins. Flag-tagged TLR1, 2, 4, 6 and 9 were transiently expressed with Myc-JIP3 in HEK 293 cells, and the interaction was analyzed by co-immunoprecipitation assays. As shown in Figure 7C, JIP3 associated with TLR2, 4 and 9, but not with TLR1 or 6. The association was also detected between JIP3 and G-CSFR–TLR2 fusion protein, indicating the TLR2 cytoplasmic domain is sufficient to bind JIP3 (Figure 1C). Furthermore, we stimulated RAW264.7 cells stably expressing a C-terminal deletion mutant of JIP3, JIP3 1–355, with synthetic lipoprotein, which signals through TLR2/TLR6, and CpG bacterial DNA, which signals through TLR9, in addition to LPS (Figure 7D). Consistently, JNK activation was reduced in this cell line after lipoprotein and CpG stimulation as well as LPS treatment compared with that in the control cell line, confirming that JIP3 is also involved in TLR2 and TLR9 signaling. Discussion We report that a scaffold protein, JIP3, interacts with TLR4 and appears to enhance JNK activation by LPS. The efficiency of MAPK activation is generally enhanced by the presence of scaffold proteins in both mammals and yeasts. A scaffold protein provides a site for the formation of a signaling module, which contains the elements of an MKKK, an MKK and a MAPK. In yeast, for example, a scaffold protein, Ste5p, is required for the mating Fus3 MAPK activation (Choi et al., 1994). In response to pheromone, a heterotrimeric G protein associates with the N-terminus of Ste5 and transmits the pheromone response pathway (Whiteway et al., 1995). Also, Pbs2p bound to the Sho1p osmosensor, the MKKK Ste11p and the MAPK Hog1p mediate Hog1p activation in response to high extracellular osmolarity (Posas and Saito, 1997). In mammals, the β-arrestin-2 scaffold protein has been reported to be engaged by G protein-coupled receptors and activate the JNK pathway when these receptors bind ligands (McDonald et al., 2000). These examples clearly indicate that at least some scaffold proteins participate in MAPK activation downstream of signaling receptors. However, for most of the m
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