TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK–JAK interaction

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven... Abstract Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1–JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5−/− CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway. cytokine, Janus kinase, protein interaction, signal transduction, TNF receptor-associated factor Introduction Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are intracellular proteins that exhibit a variety of signaling functions in inflammation, apoptosis and immune responses. Six TRAF family proteins, TRAF1 to TRAF6, which share a conserved TRAF-C domain at the C-terminus, interact with TNFR family molecules, Toll-like receptors (TLRs), interleukin receptors, nucleotide binding-oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), interferon receptors, transforming growth factor-β (TGF-β) receptor, the T-cell receptor (TCR) and platelet receptors, and these interactions control various signaling pathways that are important in health and disease (1–6). IL-6 is a pro-inflammatory cytokine and exerts its action via the common signaling receptor subunit glycoprotein 130 kDa (gp130) (7). TRAF2 and TRAF5 constitutively bind to gp130 and inhibit IL-6-driven phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3). In naive CD4+ T cells, TRAF2 and TRAF5 work as negative regulators in the IL-6-receptor (IL-6R) signaling complex and limit the generation of pro-inflammatory IL-17-producing helper T cells (Th17 cells) that require activation of the IL-6–IL-6R–gp130–STAT3 signaling axis for their development (8, 9). In accordance with this, Traf5−/− mice displayed enhanced clinical signs of experimental autoimmune encephalomyelitis (EAE), and Traf5−/− donor CD4+ T cells induced exaggerated EAE in Traf5-sufficient recipient wild-type mice (8). Upon interaction of IL-6 with the IL-6R, the complex of IL-6 and IL-6R next binds to the gp130 receptor, which leads to dimerization of gp130. Janus kinases (JAKs) are constitutively bound to the intracellular domains of gp130, and thus this event brings JAKs into close proximity, inducing transphosphorylation of each JAK on a tyrosine residue that stimulates kinase activity of JAKs. The activated JAKs then phosphorylate the cytoplasmic tail of gp130 on specific tyrosine residues, generating binding sites for STAT transcription factors including STAT3. Recruitment of a STAT3 to the phosphorylated gp130 brings the STAT3 close to the activated JAK, which then phosphorylates a tyrosine residue of the STAT3 (10, 11). As described above, the expression of TRAF2 and TRAF5 inhibits phosphorylation of STAT3, and therefore an important question would be how TRAF2 and TRAF5 negatively control the activation process of the IL-6R complex. JAK activation is an essential molecular event for initiating the IL-6R signaling pathway and is regulated by its conformational change during IL-6R activation (12). Thus, it is possible that TRAF proteins that bind to gp130 could influence the reorientation of cytoplasmic tail of gp130 that might be required for JAK’s conformational changes or JAK–JAK interactions. In the present study, we examined the hypothesis that the binding of TRAF2 or TRAF5 to gp130 inhibits the process of JAK–JAK interactions. To examine this, we have established a protein fragment complementation assay based on firefly luciferase (13). In this assay, two N-terminal and C-terminal luciferase enzyme fragments with no enzymatic activity were fused to a JAK1 protein separately. These JAK1-luciferase fragments showed minimal background association when co-expressed in the same cells without stimulation. Upon cross-linking of gp130 with a complex of IL-6 and IL-6R, the interaction between two JAKs reconstituted active firefly luciferase activity in the IL-6R complex. Using this system, we found that TRAF2 and TRAF5 inhibited the induction of bioluminescence from active luciferase. Our results not only underscore how TRAF proteins control the IL-6R signaling, but also provide important insights for understanding the mechanism of a variety of biological responses that are regulated by the shared cytokine receptor gp130. Methods Mice and cell lines Naive (CD44lowCD62Lhigh) CD4+ T cells were purified from spleens of Traf5−/− or wild-type age-matched B6 mice with a naive CD4+ T cell isolation kit II (130-093-227, Miltenyi Biotech, Bergisch Gladbach, Germany) and an autoMACS Pro cell separator (Miltenyi Biotech). Experiments with mice were approved by the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine. IL-3-dependent BAF/B03 pro-B cell lines stably expressing mouse gp130 (BAF-gp130) were previously described (9). Antibodies and cytokines Anti-c-Myc (9E10), anti-V5 (AB3792) and anti-α-Tubulin (2G10) were from Millipore (Billerica, MA, USA). Anti-Flag (DYKDDDDK, 01822381) was from WAKO (Osaka, Japan). Purified Mouse IgG2a κ isotype control monoclonal antibody (mAb) was from BD Biosciences (Franklin Lakes, NJ, USA). Human IL-6 (200-06) and soluble human IL-6R (200-06R) were from PeproTech (Rocky Hill, NJ, USA). Anti-phospho-JAK1 (3331), anti-JAK1 (3344), anti-phospho-STAT3 (9145), anti-STAT3 (9139) and anti-phospho-tyrosine (9411) were from Cell Signaling Technology (Danvers, MA, USA). Anti-TRAF5 (C-19, sc-6195) and anti-gp130 (M-20, sc-656) were from Santa Cruz Biotechnology (Dallas, TX, USA). Plasmids and transfection Vectors containing cDNA encoding mouse gp130, TRAF2 and TRAF5 were previously described (8, 9). Based on cDNA sequence of mouse Jak1 (NM_146145.2), cDNA of the entire coding region was amplified with PCR using primers that added a 5′-EcoRV site and a 3′-NotI site and ligated into a pcDNA3.1/V5-His A vector (Invitrogen, Waltham, MA, USA). The N- and C-terminal fragments of firefly (Photinus pyralis) luciferase, LucN (amino acids 1-416) and LucC (amino acids 398-550), respectively, were derived from pGL3-Basic vector (Promega, Madison, WI, USA) (13). The LucN (1-416) fragment was amplified with primer pairs that added a 5′-XhoI site with a GGGGS linker and a 3′-XbaI site: forward 5′-AACTCGAGGGTGGTGGT GGTTCTGAAGACGCCAA AAACATAAAG-3′ and reverse 5′-AATCTAGATCCATCC TTGTCAATCAAGGC-3′. The LucC (398-550) fragment was amplified with primer pairs that added a 5′-XhoI site with a GGGGS linker and a 3′-XbaI site: forward 5′-AACTCGAGGGTGGTG GTGGTTCTATGTCCGG TTATGTAAACAATC-3′ and reverse 5′-AATCTAGACACGG CGATCTTTCCGCCC-3′. The purified fragments were digested by XhoI/XbaI and then cloned into the XhoI/XbaI of digested/dephosphorylated pcDNA3.1/V5-His A-JAK1 vector. HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing penicillin, streptomycin, 2-mercaptoethanol and 10% fetal calf serum (FCS). Polyethylenimine (408727, Sigma-Aldrich, St Louis, MO, USA) was used for transient transfection of HEK293T cells grown in 48-well plates. One day before transfection, HEK293T cells were seeded at a density of 2.5 × 104 cells per well in 48-well plates. For transient transfection, culture media were removed and replaced with 950 µl per well of fresh DMEM containing 10% FCS. For making the transfection mixture, 0.7 µl per well of 2 mg ml−1 of polyethylenimine in H2O was initially mixed with 38.8 µl per well of DMEM without serum and antibiotics. Then, plasmid DNA was added into the solution, followed by incubation of the polyethyleneimine-DNA mixture for 15 min at room temperature. Finally, the mixture was dropwise added into culture wells of HEK293T cells. Bioluminescence assay Forty-eight hours after transfection, cells were washed once with 1 ml per well of Dulbecco’s phosphate buffered saline without Ca2+ and Mg2+ and were cultured for an additional 4 h in 200 µl per well of 0.1% BSA-containing DMEM for serum starvation. Cells were stimulated with a complex of IL-6 and IL-6R, and whole-cell lysates were prepared by using passive lysis buffer (E194A, Promega) according to the manufacturer’s directions. Cell lysates were centrifuged at 12000 × g and at 4°C for 2 min. The supernatants were used for luciferase assay and immunoblot analysis. The samples were assayed by mixing 20 µl of supernatants and 100 µl of luciferase assay substrate (E151A, Promega), and luciferase activity was measured using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Immunoprecipitation and immunoblot analysis Transfected HEK293T cells were lysed for 30 min on ice in ice-cold 1% Nonidet P-40 (NP-40) buffer [20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM Na3VO4 and protease-inhibitor mixture (P8340, Sigma-Aldrich)]. Insoluble material was removed by centrifugation at 4°C and at 15000 × g for 10 min. Proteins were immunoprecipitated from the supernatants overnight at 4°C with anti-cMyc mAb immobilized on Dynabeads protein G (100-04D, Invitrogen). After being washed extensively with ice-cold 1% NP-40 buffer, beads were boiled at 100°C for 5 min in 4× lithium dodecyl sulfate sample buffer (NP0007, Invitrogen). Eluted samples were further reduced at 70°C for 10 min with 2-mercaptoethanol for immunoblot analysis. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) and analyzed by immunoblot with appropriate antibodies (identified above). All blots were developed with Immobilon HRP substrate (Millipore). Statistics Statistical significance was assessed with Student’s t-test with two-sided distributions. Results Inhibition of IL-6–IL-6R-mediated phosphorylation of JAK1, gp130 and STAT3 by TRAF5 The membrane-proximal cytoplasmic region of gp130 is constitutively associated with JAK proteins. Upon binding of a complex of IL-6 and IL-6R to the extracellular part of gp130, the dimerized gp130 brings the JAKs into close proximity, causing JAK–JAK transphosphorylation of tyrosine residues that promotes JAK activity. The activated JAKs phosphorylate on specific tyrosine residues in the membrane-distal cytoplasmic part of gp130. STAT proteins are recruited by their SH2 domain to specific phosphotyrosine motifs in gp130, and then JAKs phosphorylate STATs. TRAF5 binds to gp130 and inhibits phosphorylation of STAT3 mediated by IL-6–IL-6R (8). The question is how TRAF5 controls this molecular event in the IL-6R complex. We hypothesized that TRAF5 associated with gp130 would inhibit JAK–JAK protein interactions that are essential for following phosphorylation and activation of STAT3. To test this, we measured tyrosine phosphorylation of JAK1 (p-JAK1) in the presence or absence of TRAF5 in primary T cells. We found that Traf5−/− naive CD4+ T cells displayed higher amounts of phosphorylated JAK1 and STAT3 than did wild-type naive CD4+ T cells when cell surface gp130 was cross-linked with a complex of IL-6 and IL-6R (Fig. 1A). This result suggests that the interaction of two JAKs is negatively controlled by TRAF5. To test more carefully the role for TRAF5 in JAK1 activation, BAF-gp130 cells, which stably express gp130 in an IL-3-dependent cell line BAF/B03, were further transduced with a vector encoding TRAF5 to establish BAF-gp130 cell lines that stably over-express TRAF5 (9), and we measured the levels of p-JAK1 in this cell line. As expected, after stimulation with IL-6–IL-6R we observed a decreased p-JAK1 in TRAF5-overexpressed BAF-gp130 cells (Fig. 1B). TRAF5 expression had no major role for p-JAK1 mediated by IL-3, showing specificity (Fig. 1C). In addition, the tyrosine phosphorylation of gp130 was impaired in TRAF5-overexpressed cells (Fig. 1D). These results demonstrate that TRAF5 restrains activation of gp130-associated JAK kinases that are important upstream regulators of the IL-6R signaling pathway. Fig. 1. View largeDownload slide TRAF5 inhibits phosphorylation of JAK1, gp130 and STAT3 mediated by IL-6–IL-6R. (A) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1, tyrosine-phosphorylated STAT3 (p-STAT3), total STAT3 and TRAF5 in wild-type and Traf5−/− naive CD4+ T cells stimulated for 5 min with various concentrations of IL-6–IL-6R. (B and C) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1 and TRAF5 in BAF-gp130 cells stably transduced with control vector (pMX-IRES-GFP) or TRAF5 (pMX-TRAF5-IRES-GFP) stimulated for various times with 200 ng ml−1 IL-6–IL-6R (B) or for 5 min with various concentration of IL-3 (C). (D) Phosphorylation of gp130 in BAF-gp130 cells stimulated for various times with 200 ng ml−1 IL-6–IL-6R, followed by immunoprecipitation of lysates with anti-p-Tyr mAb or isotype-matched control mAb (iso) and immunoblot with anti-gp130. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 1. View largeDownload slide TRAF5 inhibits phosphorylation of JAK1, gp130 and STAT3 mediated by IL-6–IL-6R. (A) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1, tyrosine-phosphorylated STAT3 (p-STAT3), total STAT3 and TRAF5 in wild-type and Traf5−/− naive CD4+ T cells stimulated for 5 min with various concentrations of IL-6–IL-6R. (B and C) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1 and TRAF5 in BAF-gp130 cells stably transduced with control vector (pMX-IRES-GFP) or TRAF5 (pMX-TRAF5-IRES-GFP) stimulated for various times with 200 ng ml−1 IL-6–IL-6R (B) or for 5 min with various concentration of IL-3 (C). (D) Phosphorylation of gp130 in BAF-gp130 cells stimulated for various times with 200 ng ml−1 IL-6–IL-6R, followed by immunoprecipitation of lysates with anti-p-Tyr mAb or isotype-matched control mAb (iso) and immunoblot with anti-gp130. Data are from one experiment representative of at least two independent experiments with similar results. Split luciferase system for JAK–JAK interaction To investigate how TRAF5 inhibits the activation of JAK kinases, we established a system for assessment of JAK–JAK interactions based on luciferase enzyme fragment complementation (13) (Fig. 2A and B). Firefly luciferase can be split into two N-terminal and C-terminal fragments, LucN (1-416) and LucC (398-550), respectively. We combined each luciferase fragment with a JAK1 protein using a glycine-glycine-glycine-glycine-serine (GGGGS) linker to make JAK1-LucN (1-416) and JAK1-LucC (398-550). Active luciferase conformation must be restored when gp130-associated JAK1 proteins bring LucN (1-416) and LucC (398-550) fragments into close proximity. Indeed, we observed a significant increase in luciferase activity and p-JAK1 only when HEK293T cells expressing JAK1-LucN, JAK1-LucC and gp130 were stimulated with IL-6–IL-6R. Induction of luminescence and p-JAK1 peaked at 5–10 min after IL-6–IL-6R, and remained high at 30 min (Fig. 2C and D). Importantly, JAK1-LucC was co-immunoprecipitated with JAK1-LucN only after IL-6–IL-6R stimulation (Fig. 2E), indicating that IL-6–IL-6R-mediated proximal JAK1–JAK1 interactions restore the correct conformation of luciferase enzyme. Thus, using this system, we decided to examine the possible regulatory role of TRAF5 in JAK1–JAK1 interactions. Fig. 2. View largeDownload slide Firefly luciferase protein fragment complementation assay system for evaluation of the interaction between two JAK1 molecules in the IL-6R complex. (A) Schematic diagram of luciferase complementation induced by JAK1–JAK1 interaction in the activated IL-6R signaling complex. The N-terminal (1-416) or the C-terminal (398-550) fragment of firefly luciferase is fused at the end of the C-terminus of full-length mouse JAK1 via a linker peptide. Binding of a complex of IL-6 and IL-6R to transmembrane gp130 induces interaction between JAK1-LucN (1-416) and JAK1-LucC (398-550), which leads to reconstitution of firefly luciferase enzyme activity. (B) Immunoblot analysis of HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130. (C) Kinetics of induction of bioluminescence. Luciferase assay of mixed lysates from HEK cells transiently transduced to express V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation of triplicate wells. *P < 0.05, **P < 0.01 (Student’s t-test). (D) Immunoblot analysis of HEK293T cells as in C and immunoblot analysis with anti-p-JAK1, anti-V5, anti-c-Myc and anti-Tubulin. (E) IL-6–IL-6R-dependent binding between JAK1-LucN and JAK1-LucC. Pull-down assay of JAK1-LucN from lysates of HEK cells transiently transfected with plasmid vector encoding Flag-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-Flag or control IgG (iso) and immunoblot analysis with anti-Flag, anti-V5, anti-c-Myc and anti-JAK1. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 2. View largeDownload slide Firefly luciferase protein fragment complementation assay system for evaluation of the interaction between two JAK1 molecules in the IL-6R complex. (A) Schematic diagram of luciferase complementation induced by JAK1–JAK1 interaction in the activated IL-6R signaling complex. The N-terminal (1-416) or the C-terminal (398-550) fragment of firefly luciferase is fused at the end of the C-terminus of full-length mouse JAK1 via a linker peptide. Binding of a complex of IL-6 and IL-6R to transmembrane gp130 induces interaction between JAK1-LucN (1-416) and JAK1-LucC (398-550), which leads to reconstitution of firefly luciferase enzyme activity. (B) Immunoblot analysis of HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130. (C) Kinetics of induction of bioluminescence. Luciferase assay of mixed lysates from HEK cells transiently transduced to express V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation of triplicate wells. *P < 0.05, **P < 0.01 (Student’s t-test). (D) Immunoblot analysis of HEK293T cells as in C and immunoblot analysis with anti-p-JAK1, anti-V5, anti-c-Myc and anti-Tubulin. (E) IL-6–IL-6R-dependent binding between JAK1-LucN and JAK1-LucC. Pull-down assay of JAK1-LucN from lysates of HEK cells transiently transfected with plasmid vector encoding Flag-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-Flag or control IgG (iso) and immunoblot analysis with anti-Flag, anti-V5, anti-c-Myc and anti-JAK1. Data are from one experiment representative of at least two independent experiments with similar results. Inhibition of IL-6-driven JAK–JAK interaction by TRAF5 JAK1 is associated with gp130 via membrane-proximal intracellular regions called Box1 (649IWPNVDP657) and Box2 (689VSVVEIEANNKKP701), whereas the region in gp130 that interacts with TRAF5 has been mapped to the amino acid sequence 774VFSRSESTQPLLDSEERPEDLQLVD798 (8). To evaluate the inhibitory role of TRAF5 in JAK activation, we firstly tested whether TRAF5 could inhibit the binding between gp130 and JAK1. We transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC in the presence or absence of TRAF5 and immunoprecipitated gp130 from cell lysates. JAK1-LucN, JAK1-LucC and TRAF5 were co-immunoprecipitated with gp130, and TRAF5 expression did not affect the amount of JAK1 associated with gp130 (Fig. 3, lane 3 versus lane 4). In addition, triggering of gp130 with IL-6–IL-6R did not change the amount of JAK1 associated with gp130 (Fig. 3, lane 3 versus lane 5, lane 4 versus lane 6). In addition, JAK1 expression did not change the amount of TRAF5 associated with gp130 (Fig. 3, lane 1 versus lane 4). These results demonstrate that the interaction between TRAF5 and gp130 can occur independently of the interaction between JAK1 and gp130 and that TRAF5 does not inhibit the association between JAK1 and gp130. Fig. 3. View largeDownload slide Binding between TRAF5 and gp130 occurs independently of binding between JAK1 and gp130. Immunoassay of gp130 from lysates of HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 or control vector, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-c-Myc or control IgG (iso) and immunoblot analysis with anti-c-Myc, anti-V5 and anti-Flag. Data are from one experiment representative of three independent experiments with similar results. Fig. 3. View largeDownload slide Binding between TRAF5 and gp130 occurs independently of binding between JAK1 and gp130. Immunoassay of gp130 from lysates of HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 or control vector, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-c-Myc or control IgG (iso) and immunoblot analysis with anti-c-Myc, anti-V5 and anti-Flag. Data are from one experiment representative of three independent experiments with similar results. Secondly, we tested the possibility that TRAF5 prevents the interaction between two JAK1 proteins using the split luciferase system (Fig. 2). We additionally transfected HEK293T cells with titrated amounts of plasmid vector encoding TRAF5 to examine how the expression levels of TRAF5 affect luciferase activity that is dependent on JAK–JAK interactions. Under stimulation with IL-6–IL-6R, TRAF5 dose-dependently inhibited the induction of luminescence (Fig. 4A and B). In addition, IL-6–IL-6R stimulation produced a dose-dependent increase in luminescence, and this response was significantly suppressed by the expression of TRAF5, at doses of 50 and 200 ng ml−1 of IL-6–IL-6R (Fig. 4C and D), although at higher concentrations of IL-6–IL-6R, TRAF5 could not inhibit the luminescence (Fig. 4C). Fig. 4. View largeDownload slide TRAF5 inhibits the interaction between two JAK1 proteins in the IL-6R complex. (A and B) Dose-dependent inhibitory effect of TRAF5 on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with various amounts of TRAF5 () or control vector (), left unstimulated (None) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (A). Immunoblot analysis of HEK293T cells as in A (B). (C and D) Dose-dependent effect of IL-6–IL-6R on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with 500 ng of TRAF5 () or control vector (), stimulated for 5 min with various concentration of IL-6–IL-6R (C). Immunoblot analysis of HEK293T cells as in C (D). Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). One representative experimental result is shown. Fig. 4. View largeDownload slide TRAF5 inhibits the interaction between two JAK1 proteins in the IL-6R complex. (A and B) Dose-dependent inhibitory effect of TRAF5 on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with various amounts of TRAF5 () or control vector (), left unstimulated (None) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (A). Immunoblot analysis of HEK293T cells as in A (B). (C and D) Dose-dependent effect of IL-6–IL-6R on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with 500 ng of TRAF5 () or control vector (), stimulated for 5 min with various concentration of IL-6–IL-6R (C). Immunoblot analysis of HEK293T cells as in C (D). Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). One representative experimental result is shown. Thirdly, we examined if the inability of TRAF5 to bind to gp130 would affect the JAK–JAK interaction. In our previous study, amino acid residues 774-798 in gp130 were essential for its recognition by TRAF5, and a gp130 mutant lacking this region, gp130 (∆774-798), exhibited considerably diminished binding to TRAF5 relative to that of full-length gp130 (8). As expected, the reduction in luminescence mediated by TRAF5 was abolished in gp130 (∆774-798) expressing cells (Fig. 5A–D). Thus, TRAF5 requires the TRAF5-binding region in gp130 to inhibit JAK1–JAK1 interactions. Fig. 5. View largeDownload slide TRAF5 binding to gp130 is required for the inhibition against JAK1–JAK1 interactions. (A and C) Impaired TRAF5-mediated inhibition against JAK1–JAK1 interactions in cells expressing gp130 lacking the cytoplasmic region necessary for TRAF binding. Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN and V5-tagged JAK1-LucC together with c-Myc-tagged full-length gp130 (1-917) (A) or c-Myc-tagged deletion mutant of gp130 (∆774-798) (C) in the presence () or absence () of TRAF5, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B and D) Immunoblot analysis of HEK293T cells transfected with plasmid vectors encoding c-Myc-tagged full-length gp130 (1-917) (B) and c-Myc-tagged deletion mutant of gp130 (∆774-798) (D) as in A and C, respectively. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Fig. 5. View largeDownload slide TRAF5 binding to gp130 is required for the inhibition against JAK1–JAK1 interactions. (A and C) Impaired TRAF5-mediated inhibition against JAK1–JAK1 interactions in cells expressing gp130 lacking the cytoplasmic region necessary for TRAF binding. Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN and V5-tagged JAK1-LucC together with c-Myc-tagged full-length gp130 (1-917) (A) or c-Myc-tagged deletion mutant of gp130 (∆774-798) (C) in the presence () or absence () of TRAF5, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B and D) Immunoblot analysis of HEK293T cells transfected with plasmid vectors encoding c-Myc-tagged full-length gp130 (1-917) (B) and c-Myc-tagged deletion mutant of gp130 (∆774-798) (D) as in A and C, respectively. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Collectively, these results clearly show that gp130-associated TRAF5 is inhibitory for the interaction between two JAK molecules associated with gp130. Comparable inhibitory activity of TRAF2 toward JAK–JAK interaction We previously reported that TRAF2 shared the binding region in gp130 with TRAF5 and inhibited STAT3 activation mediated by IL-6–IL-6R (9). Thus, it is reasonable to speculate that TRAF2 has a similar inhibitory function against JAK1. To investigate this, we transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC together with TRAF2. As expected, TRAF2 did prevent luminescence derived from JAK1–JAK1 associations induced by IL-6–IL-6R, and TRAF2 could decrease the peak response of luminescence similarly to TRAF5 (Fig. 6A and B). Thus, both TRAF2 and TRAF5 inhibit JAK–JAK interactions in the IL-6R complex. Fig. 6. View largeDownload slide TRAF2 also inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 () or Flag-tagged TRAF2 () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation. **P < 0.01 (Student’s t-test). (B) Immunoblot analysis of HEK293T cells as in A. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 6. View largeDownload slide TRAF2 also inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 () or Flag-tagged TRAF2 () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation. **P < 0.01 (Student’s t-test). (B) Immunoblot analysis of HEK293T cells as in A. Data are from one experiment representative of at least two independent experiments with similar results. Inhibitory function of C-terminal domains of TRAF2 and TRAF5 for JAK–JAK interaction The inhibitory activity of TRAF2 and TRAF5 for STAT3 relies entirely on the TRAF-C domain (8, 9), suggesting that the TRAF-C domain also plays a critical inhibitory role for JAK1. To investigate which domains of TRAF are responsible for the inhibition, we transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC together with the N-terminus of TRAF2 (amino acids 1-271), which contains really interesting new gene (RING) and zinc-finger domains [TRAF2 (1-271)], or the C-terminus of TRAF2 (amino acids 272-501), which contains leucine-zipper and TRAF-C domains [TRAF2 (272-501)], or the N-terminus of TRAF5 (amino acids 1-241), which contains RING and zinc-finger domains [TRAF5 (1-241)], or the C-terminus of TRAF5 (amino acids 242-558), which contains leucine-zipper and TRAF-C domains [TRAF5 (242-558)]. As expected, both TRAF2 (272-501) and TRAF5 (242-558) significantly inhibited luminescence derived from JAK1–JAK1 interactions, whereas TRAF2 (1-271) and TRAF5 (1-241) did not (Fig. 7A–D). These results demonstrate that the binding activity of TRAF-C domain toward gp130 is responsible for the inhibitory activity of TRAF2 and TRAF5 against JAK1 activation. Fig. 7. View largeDownload slide The C-terminal TRAF domain inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF2 (1-271) () or Flag-tagged TRAF2 (272-501) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B) Immunoblot analysis of HEK293T cells as in A. (C) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 (1-241) () or Flag-tagged TRAF5 (242-558) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (D) Immunoblot analysis of HEK293T cells as in C. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Fig. 7. View largeDownload slide The C-terminal TRAF domain inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF2 (1-271) () or Flag-tagged TRAF2 (272-501) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B) Immunoblot analysis of HEK293T cells as in A. (C) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 (1-241) () or Flag-tagged TRAF5 (242-558) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (D) Immunoblot analysis of HEK293T cells as in C. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. In conclusion, we have identified a novel mechanism by which TRAF2 and TRAF5 limit the IL-6R signaling. Our results demonstrate that the binding activity of TRAF2 and TRAF5 to gp130 is responsible for the inhibition toward JAK1–JAK1 interactions in the IL-6R complex, and that the E3 ubiquitin ligase activity in TRAF2 and TRAF5 may be dispensable for the inhibition. Discussion In this study, we show that the expression of TRAF5 was inhibitory for phosphorylations in JAK1, gp130 and STAT3 that occur in the IL-6R signaling complex. Although TRAF5 binding to gp130 did not alter the constitutive interaction between JAK1 and gp130, TRAF5–gp130 interactions decreased transphosphorylation and corresponding activation of JAK1 that are dependent on JAK1–JAK1 interactions. TRAF2 also suppressed JAK1–JAK1 interactions. Thus, we identify a novel inhibitory mechanism of TRAF adaptor proteins for JAK activation and demonstrate that this inhibitory activity of TRAF2 and TRAF5 limits IL-6-driven STAT3 activation that is essential for the lineage commitment of inflammatory and pathogenic Th17 cells. Although our results support the conclusion that the elevated IL-6-driven STAT3 activation in Traf5−/− CD4+ T cells was due to increased activation of JAK1, detailed mechanisms by which TRAF proteins regulate JAK activation in the IL-6R signaling complex are still unclear. The JAK1 FERM domain constitutively binds to the membrane-proximal Box1 and Box2 in gp130, whereas the TRAF-binding region is located in the middle part of the cytoplasmic tail of gp130 and does not contain tyrosine residues. Indeed, the expression of TRAF5 did not inhibit the interaction between JAK1 and gp130, showing that the binding between gp130 and JAK1 occurs independently of the interaction between gp130 and TRAF5. Based on some of the mechanisms that are proposed for JAK activation within the receptor complexes, formation of gp130 dimer and repositioning of the associated JAKs would be important steps for inducing the catalytic activity of JAKs (12, 14). We speculate that TRAF2 and TRAF5 associated with gp130 might sterically interfere with the repositioning process of inter domain communication that is required for JAK activation. According to a useful characteristic of the enzyme fragment complementation assay (13, 15, 16), we have decided to address the hypothesis that TRAF proteins inhibit the activation process of JAK–JAK interactions in the IL-6R complex. The N-terminus and C-terminus fragments of firefly luciferase were separately fused to the C-terminus of JAK1 using a flexible linker composed of GGGGS to promote reorganization of active luciferase enzyme structure. We examined the ability of reconstitution of split luciferase fragments in transiently co-transfected HEK293T cells with JAK1-LucN, JAK1-LucC and gp130 vectors in the presence or absence of TRAF vectors. We detected a quick and significant increase in luciferase activity, which is dependent on interactions between LucN and LucC, in transfected HEK293T cells after treatment of cells with IL-6–IL-6R. In this system, we have identified a novel inhibitory function of TRAF2 and TRAF5 for JAK1 activation. However, it is a matter of concern how gp130-associated TRAF proteins can impede the proximal JAK1 interactions in the IL-6R complex, as discussed above. Additional work is required for understanding of the role for gp130-associated TRAFs in JAK activation. Although TRAF3 inhibited IL-6R signaling by facilitating the association between phosphatase PTPN22 and JAK1 leading to blockade of STAT3 phosphorylation in B cells (17), our present results support the notion that TRAF2 and TRAF5 sterically inhibit the interaction between two JAKs associated with gp130. We previously reported that TRAF2 and TRAF5 did not require their RING and zinc-finger domains to interact with gp130 and that STAT3 binding to gp130 was suppressed by the expression of a TRAF5 mutant lacking RING and zinc-finger domains (8, 9). In addition, this TRAF5 mutant without RING and zinc-finger domains served an inhibitory role in IL-6–IL-6R-mediated Th17 development. In addition, over-expression of TRAF2 or TRAF5 but not TRAF3 inhibited IL-6–IL-6R-mediated phosphorylation of STAT3 and Th17 responses (8, 9). In this study, we demonstrated that TRAF2 and TRAF5 mutants lacking RING and zinc-finger domains inhibited JAK–JAK protein interactions. We also confirmed that TRAF5 expression suppressed IL-6–IL-6R-mediated phosphorylation of JAK1 even in the presence of tyrosine phosphatase inhibitor Na3VO4 in BAF-gp130 cells (data not shown). Thus, we think that a steric hindrance caused by TRAF binding to gp130 is responsible for the inhibition toward JAK activation in the IL-6R complex. In conclusion, using a firefly luciferase enzyme fragment complementation assay, we have discovered a novel inhibitory role for TRAF2 and TRAF5 in JAK activation in the IL-6R signaling complex. However, we still do not know how gp130-associated TRAF and JAK proteins organize active IL-6R complexes on the T-cell membrane. In this study, to cross-link membrane bound gp130, cells were treated with a complex of IL-6 and IL-6R instead of IL-6 alone. We think that the elevated phosphorylation of JAK1 in Traf5−/− T cells may be induced by IL-6 alone, but this is a matter of future study. It will be also important in the future to understand whether the inhibitory mechanism regulated by TRAF2 and TRAF5 is also applicable to other IL-6 family cytokines that use the common signaling receptor subunit gp130. Funding This work was supported by JSPS KAKENHI grant numbers JP15H04640 and JP18H02572 (T.S.) and the Yamaguchi Educational and Scholarship Foundation (to T.S.), the Daiichi-Sankyo Foundation of Life Science (T.S.), and the Tamura Science and Technology Foundation (T.S.). Acknowledgements We thank members of Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine for their assistance and help. We thank the Biomedical Research Core and the Institute for Animal Experimentation (Tohoku University Graduate School of Medicine) for technical support. Conflicts of interest statement: the authors declared no conflicts of interest. References 1 Inoue , J. I. , Ishida , T. , Tsukamoto , N. , et al. 2000 . Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling . Exp. Cell Res . 254 : 14 . Google Scholar CrossRef Search ADS PubMed 2 Chung , J. Y. , Park , Y. C. , Ye , H. and Wu , H . 2002 . All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction . J. Cell Sci . 115 : 679 . Google Scholar PubMed 3 Ha , H. , Han , D. and Choi , Y . 2009 . TRAF-mediated TNFR-family signaling . Curr. Protoc. Immunol . Chapter 11 : Unit11.9D . Google Scholar PubMed 4 Xie , P . 2013 . TRAF molecules in cell signaling and in human diseases . J. Mol. Signal . 8 : 7 . Google Scholar CrossRef Search ADS PubMed 5 So , T. , Nagashima , H. and Ishii , N . 2015 . TNF receptor-associated factor (TRAF) signaling network in CD4(+) T-lymphocytes . Tohoku J. Exp. Med . 236 : 139 . Google Scholar CrossRef Search ADS PubMed 6 Bishop , G. A . 2016 . TRAF3 as a powerful and multitalented regulator of lymphocyte functions . J. Leukoc. Biol . 100 : 919 . Google Scholar CrossRef Search ADS PubMed 7 Taga , T. and Kishimoto , T . 1997 . Gp130 and the interleukin-6 family of cytokines . Annu. Rev. Immunol . 15 : 797 . Google Scholar CrossRef Search ADS PubMed 8 Nagashima , H. , Okuyama , Y. , Asao , A. , et al. 2014 . The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6 . Nat. Immunol . 15 : 449 . Google Scholar CrossRef Search ADS PubMed 9 Nagashima , H. , Okuyama , Y. , Hayashi , T. , Ishii , N. and So , T . 2016 . TNFR-associated factors 2 and 5 differentially regulate the instructive IL-6 receptor signaling required for Th17 development . J. Immunol . 196 : 4082 . Google Scholar CrossRef Search ADS PubMed 10 Kishimoto , T. , Akira , S. , Narazaki , M. and Taga , T . 1995 . Interleukin-6 family of cytokines and gp130 . Blood 86 : 1243 . Google Scholar PubMed 11 Heinrich , P. C. , Behrmann , I. , Haan , S. , Hermanns , H. M. , Müller-Newen , G. and Schaper , F . 2003 . Principles of interleukin (IL)-6-type cytokine signalling and its regulation . Biochem. J . 374 : 1 . Google Scholar CrossRef Search ADS PubMed 12 Babon , J. J. , Lucet , I. S. , Murphy , J. M. , Nicola , N. A. and Varghese , L. N . 2014 . The molecular regulation of Janus kinase (JAK) activation . Biochem. J . 462 : 1 . Google Scholar CrossRef Search ADS PubMed 13 Luker , K. E. , Smith , M. C. , Luker , G. D. , Gammon , S. T. , Piwnica-Worms , H. and Piwnica-Worms , D . 2004 . Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals . Proc. Natl Acad. Sci. USA 101 : 12288 . Google Scholar CrossRef Search ADS 14 Waters , M. J. and Brooks , A. J . 2015 . JAK2 activation by growth hormone and other cytokines . Biochem. J . 466 : 1 . Google Scholar CrossRef Search ADS PubMed 15 Remy , I. , Wilson , I. A. and Michnick , S. W . 1999 . Erythropoietin receptor activation by a ligand-induced conformation change . Science 283 : 990 . Google Scholar CrossRef Search ADS PubMed 16 Liu , Y. , Berry , P. A. , Zhang , Y. , et al. 2014 . Dynamic analysis of GH receptor conformational changes by split luciferase complementation . Mol. Endocrinol . 28 : 1807 . Google Scholar CrossRef Search ADS PubMed 17 Lin , W. W. , Yi , Z. , Stunz , L. L. , Maine , C. J. , Sherman , L. A. and Bishop , G. A . 2015 . The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development . Sci. Signal . 8 : ra88 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices) http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png International Immunology Oxford University Press

TRAF2 and TRAF5 associated with the signal transducing receptor gp130 limit IL-6-driven transphosphorylation of JAK1 through the inhibition of proximal JAK–JAK interaction

International Immunology , Volume Advance Article (7) – Apr 13, 2018

Loading next page...
 
/lp/ou_press/traf2-and-traf5-associated-with-the-signal-transducing-receptor-gp130-01gqmkPiCL
Publisher
Oxford University Press
Copyright
© The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com
ISSN
0953-8178
eISSN
1460-2377
D.O.I.
10.1093/intimm/dxy029
Publisher site
See Article on Publisher Site

Abstract

Abstract Tumor necrosis factor receptor-associated factor 2 (TRAF2) and TRAF5 constitutively bind to glycoprotein 130 kDa (gp130) and inhibit IL-6-driven activation of signal transducer and activator of transcription 3 (STAT3) in CD4+ T cells, which limits the differentiation of pro-inflammatory IL-17-producing helper T cells that require IL-6-receptor (IL-6R) signals for their development. However, it is not known how the interaction between TRAF and gp130 negatively regulates STAT3 activity in the IL-6R complex. We hypothesized that TRAF proteins associated with gp130 might limit the activation of Janus kinase that is needed for the activation of STAT3. To test this, we transfected HEK293T cells to express gp130 and TRAF2 or TRAF5 together with two chimeric JAK1 proteins combined with either the N-terminal or the C-terminal protein fragment of firefly luciferase. Using this luciferase fragment complementation system, we found that the recovery of luciferase enzyme activity was coincident with proximal JAK1–JAK1 interaction and phosphorylation of JAK1 in the IL-6R complex and that the expression of TRAF protein significantly inhibited the recovery of luciferase activity. The binding of TRAF to gp130 via the C-terminal TRAF domain was essential for the inhibition. In accordance with this, upon stimulation of endogenous gp130 with a complex of IL-6 and IL-6R, Traf5−/− CD4+ T cells displayed significantly higher amounts of phosphorylated JAK1 than did their wild-type counterparts. Therefore, our results demonstrate that gp130-associated TRAF2 and TRAF5 inhibit the interaction between two JAK proteins in the IL-6R complex that is essential for initiating the JAK-STAT signaling pathway. cytokine, Janus kinase, protein interaction, signal transduction, TNF receptor-associated factor Introduction Tumor necrosis factor receptor (TNFR)-associated factors (TRAFs) are intracellular proteins that exhibit a variety of signaling functions in inflammation, apoptosis and immune responses. Six TRAF family proteins, TRAF1 to TRAF6, which share a conserved TRAF-C domain at the C-terminus, interact with TNFR family molecules, Toll-like receptors (TLRs), interleukin receptors, nucleotide binding-oligomerization domain (NOD)-like receptors (NLRs), retinoic acid-inducible gene (RIG)-I-like receptors (RLRs), interferon receptors, transforming growth factor-β (TGF-β) receptor, the T-cell receptor (TCR) and platelet receptors, and these interactions control various signaling pathways that are important in health and disease (1–6). IL-6 is a pro-inflammatory cytokine and exerts its action via the common signaling receptor subunit glycoprotein 130 kDa (gp130) (7). TRAF2 and TRAF5 constitutively bind to gp130 and inhibit IL-6-driven phosphorylation and activation of signal transducer and activator of transcription 3 (STAT3). In naive CD4+ T cells, TRAF2 and TRAF5 work as negative regulators in the IL-6-receptor (IL-6R) signaling complex and limit the generation of pro-inflammatory IL-17-producing helper T cells (Th17 cells) that require activation of the IL-6–IL-6R–gp130–STAT3 signaling axis for their development (8, 9). In accordance with this, Traf5−/− mice displayed enhanced clinical signs of experimental autoimmune encephalomyelitis (EAE), and Traf5−/− donor CD4+ T cells induced exaggerated EAE in Traf5-sufficient recipient wild-type mice (8). Upon interaction of IL-6 with the IL-6R, the complex of IL-6 and IL-6R next binds to the gp130 receptor, which leads to dimerization of gp130. Janus kinases (JAKs) are constitutively bound to the intracellular domains of gp130, and thus this event brings JAKs into close proximity, inducing transphosphorylation of each JAK on a tyrosine residue that stimulates kinase activity of JAKs. The activated JAKs then phosphorylate the cytoplasmic tail of gp130 on specific tyrosine residues, generating binding sites for STAT transcription factors including STAT3. Recruitment of a STAT3 to the phosphorylated gp130 brings the STAT3 close to the activated JAK, which then phosphorylates a tyrosine residue of the STAT3 (10, 11). As described above, the expression of TRAF2 and TRAF5 inhibits phosphorylation of STAT3, and therefore an important question would be how TRAF2 and TRAF5 negatively control the activation process of the IL-6R complex. JAK activation is an essential molecular event for initiating the IL-6R signaling pathway and is regulated by its conformational change during IL-6R activation (12). Thus, it is possible that TRAF proteins that bind to gp130 could influence the reorientation of cytoplasmic tail of gp130 that might be required for JAK’s conformational changes or JAK–JAK interactions. In the present study, we examined the hypothesis that the binding of TRAF2 or TRAF5 to gp130 inhibits the process of JAK–JAK interactions. To examine this, we have established a protein fragment complementation assay based on firefly luciferase (13). In this assay, two N-terminal and C-terminal luciferase enzyme fragments with no enzymatic activity were fused to a JAK1 protein separately. These JAK1-luciferase fragments showed minimal background association when co-expressed in the same cells without stimulation. Upon cross-linking of gp130 with a complex of IL-6 and IL-6R, the interaction between two JAKs reconstituted active firefly luciferase activity in the IL-6R complex. Using this system, we found that TRAF2 and TRAF5 inhibited the induction of bioluminescence from active luciferase. Our results not only underscore how TRAF proteins control the IL-6R signaling, but also provide important insights for understanding the mechanism of a variety of biological responses that are regulated by the shared cytokine receptor gp130. Methods Mice and cell lines Naive (CD44lowCD62Lhigh) CD4+ T cells were purified from spleens of Traf5−/− or wild-type age-matched B6 mice with a naive CD4+ T cell isolation kit II (130-093-227, Miltenyi Biotech, Bergisch Gladbach, Germany) and an autoMACS Pro cell separator (Miltenyi Biotech). Experiments with mice were approved by the Institute for Animal Experimentation, Tohoku University Graduate School of Medicine. IL-3-dependent BAF/B03 pro-B cell lines stably expressing mouse gp130 (BAF-gp130) were previously described (9). Antibodies and cytokines Anti-c-Myc (9E10), anti-V5 (AB3792) and anti-α-Tubulin (2G10) were from Millipore (Billerica, MA, USA). Anti-Flag (DYKDDDDK, 01822381) was from WAKO (Osaka, Japan). Purified Mouse IgG2a κ isotype control monoclonal antibody (mAb) was from BD Biosciences (Franklin Lakes, NJ, USA). Human IL-6 (200-06) and soluble human IL-6R (200-06R) were from PeproTech (Rocky Hill, NJ, USA). Anti-phospho-JAK1 (3331), anti-JAK1 (3344), anti-phospho-STAT3 (9145), anti-STAT3 (9139) and anti-phospho-tyrosine (9411) were from Cell Signaling Technology (Danvers, MA, USA). Anti-TRAF5 (C-19, sc-6195) and anti-gp130 (M-20, sc-656) were from Santa Cruz Biotechnology (Dallas, TX, USA). Plasmids and transfection Vectors containing cDNA encoding mouse gp130, TRAF2 and TRAF5 were previously described (8, 9). Based on cDNA sequence of mouse Jak1 (NM_146145.2), cDNA of the entire coding region was amplified with PCR using primers that added a 5′-EcoRV site and a 3′-NotI site and ligated into a pcDNA3.1/V5-His A vector (Invitrogen, Waltham, MA, USA). The N- and C-terminal fragments of firefly (Photinus pyralis) luciferase, LucN (amino acids 1-416) and LucC (amino acids 398-550), respectively, were derived from pGL3-Basic vector (Promega, Madison, WI, USA) (13). The LucN (1-416) fragment was amplified with primer pairs that added a 5′-XhoI site with a GGGGS linker and a 3′-XbaI site: forward 5′-AACTCGAGGGTGGTGGT GGTTCTGAAGACGCCAA AAACATAAAG-3′ and reverse 5′-AATCTAGATCCATCC TTGTCAATCAAGGC-3′. The LucC (398-550) fragment was amplified with primer pairs that added a 5′-XhoI site with a GGGGS linker and a 3′-XbaI site: forward 5′-AACTCGAGGGTGGTG GTGGTTCTATGTCCGG TTATGTAAACAATC-3′ and reverse 5′-AATCTAGACACGG CGATCTTTCCGCCC-3′. The purified fragments were digested by XhoI/XbaI and then cloned into the XhoI/XbaI of digested/dephosphorylated pcDNA3.1/V5-His A-JAK1 vector. HEK293T cells were maintained in Dulbecco’s modified Eagle medium (DMEM) containing penicillin, streptomycin, 2-mercaptoethanol and 10% fetal calf serum (FCS). Polyethylenimine (408727, Sigma-Aldrich, St Louis, MO, USA) was used for transient transfection of HEK293T cells grown in 48-well plates. One day before transfection, HEK293T cells were seeded at a density of 2.5 × 104 cells per well in 48-well plates. For transient transfection, culture media were removed and replaced with 950 µl per well of fresh DMEM containing 10% FCS. For making the transfection mixture, 0.7 µl per well of 2 mg ml−1 of polyethylenimine in H2O was initially mixed with 38.8 µl per well of DMEM without serum and antibiotics. Then, plasmid DNA was added into the solution, followed by incubation of the polyethyleneimine-DNA mixture for 15 min at room temperature. Finally, the mixture was dropwise added into culture wells of HEK293T cells. Bioluminescence assay Forty-eight hours after transfection, cells were washed once with 1 ml per well of Dulbecco’s phosphate buffered saline without Ca2+ and Mg2+ and were cultured for an additional 4 h in 200 µl per well of 0.1% BSA-containing DMEM for serum starvation. Cells were stimulated with a complex of IL-6 and IL-6R, and whole-cell lysates were prepared by using passive lysis buffer (E194A, Promega) according to the manufacturer’s directions. Cell lysates were centrifuged at 12000 × g and at 4°C for 2 min. The supernatants were used for luciferase assay and immunoblot analysis. The samples were assayed by mixing 20 µl of supernatants and 100 µl of luciferase assay substrate (E151A, Promega), and luciferase activity was measured using a Lumat LB 9507 luminometer (Berthold Technologies, Bad Wildbad, Germany). Immunoprecipitation and immunoblot analysis Transfected HEK293T cells were lysed for 30 min on ice in ice-cold 1% Nonidet P-40 (NP-40) buffer [20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% NP-40, 50 mM NaF, 1 mM Na3VO4 and protease-inhibitor mixture (P8340, Sigma-Aldrich)]. Insoluble material was removed by centrifugation at 4°C and at 15000 × g for 10 min. Proteins were immunoprecipitated from the supernatants overnight at 4°C with anti-cMyc mAb immobilized on Dynabeads protein G (100-04D, Invitrogen). After being washed extensively with ice-cold 1% NP-40 buffer, beads were boiled at 100°C for 5 min in 4× lithium dodecyl sulfate sample buffer (NP0007, Invitrogen). Eluted samples were further reduced at 70°C for 10 min with 2-mercaptoethanol for immunoblot analysis. Samples were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) and analyzed by immunoblot with appropriate antibodies (identified above). All blots were developed with Immobilon HRP substrate (Millipore). Statistics Statistical significance was assessed with Student’s t-test with two-sided distributions. Results Inhibition of IL-6–IL-6R-mediated phosphorylation of JAK1, gp130 and STAT3 by TRAF5 The membrane-proximal cytoplasmic region of gp130 is constitutively associated with JAK proteins. Upon binding of a complex of IL-6 and IL-6R to the extracellular part of gp130, the dimerized gp130 brings the JAKs into close proximity, causing JAK–JAK transphosphorylation of tyrosine residues that promotes JAK activity. The activated JAKs phosphorylate on specific tyrosine residues in the membrane-distal cytoplasmic part of gp130. STAT proteins are recruited by their SH2 domain to specific phosphotyrosine motifs in gp130, and then JAKs phosphorylate STATs. TRAF5 binds to gp130 and inhibits phosphorylation of STAT3 mediated by IL-6–IL-6R (8). The question is how TRAF5 controls this molecular event in the IL-6R complex. We hypothesized that TRAF5 associated with gp130 would inhibit JAK–JAK protein interactions that are essential for following phosphorylation and activation of STAT3. To test this, we measured tyrosine phosphorylation of JAK1 (p-JAK1) in the presence or absence of TRAF5 in primary T cells. We found that Traf5−/− naive CD4+ T cells displayed higher amounts of phosphorylated JAK1 and STAT3 than did wild-type naive CD4+ T cells when cell surface gp130 was cross-linked with a complex of IL-6 and IL-6R (Fig. 1A). This result suggests that the interaction of two JAKs is negatively controlled by TRAF5. To test more carefully the role for TRAF5 in JAK1 activation, BAF-gp130 cells, which stably express gp130 in an IL-3-dependent cell line BAF/B03, were further transduced with a vector encoding TRAF5 to establish BAF-gp130 cell lines that stably over-express TRAF5 (9), and we measured the levels of p-JAK1 in this cell line. As expected, after stimulation with IL-6–IL-6R we observed a decreased p-JAK1 in TRAF5-overexpressed BAF-gp130 cells (Fig. 1B). TRAF5 expression had no major role for p-JAK1 mediated by IL-3, showing specificity (Fig. 1C). In addition, the tyrosine phosphorylation of gp130 was impaired in TRAF5-overexpressed cells (Fig. 1D). These results demonstrate that TRAF5 restrains activation of gp130-associated JAK kinases that are important upstream regulators of the IL-6R signaling pathway. Fig. 1. View largeDownload slide TRAF5 inhibits phosphorylation of JAK1, gp130 and STAT3 mediated by IL-6–IL-6R. (A) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1, tyrosine-phosphorylated STAT3 (p-STAT3), total STAT3 and TRAF5 in wild-type and Traf5−/− naive CD4+ T cells stimulated for 5 min with various concentrations of IL-6–IL-6R. (B and C) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1 and TRAF5 in BAF-gp130 cells stably transduced with control vector (pMX-IRES-GFP) or TRAF5 (pMX-TRAF5-IRES-GFP) stimulated for various times with 200 ng ml−1 IL-6–IL-6R (B) or for 5 min with various concentration of IL-3 (C). (D) Phosphorylation of gp130 in BAF-gp130 cells stimulated for various times with 200 ng ml−1 IL-6–IL-6R, followed by immunoprecipitation of lysates with anti-p-Tyr mAb or isotype-matched control mAb (iso) and immunoblot with anti-gp130. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 1. View largeDownload slide TRAF5 inhibits phosphorylation of JAK1, gp130 and STAT3 mediated by IL-6–IL-6R. (A) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1, tyrosine-phosphorylated STAT3 (p-STAT3), total STAT3 and TRAF5 in wild-type and Traf5−/− naive CD4+ T cells stimulated for 5 min with various concentrations of IL-6–IL-6R. (B and C) Immunoblot analysis of tyrosine-phosphorylated JAK1 (p-JAK1), total JAK1 and TRAF5 in BAF-gp130 cells stably transduced with control vector (pMX-IRES-GFP) or TRAF5 (pMX-TRAF5-IRES-GFP) stimulated for various times with 200 ng ml−1 IL-6–IL-6R (B) or for 5 min with various concentration of IL-3 (C). (D) Phosphorylation of gp130 in BAF-gp130 cells stimulated for various times with 200 ng ml−1 IL-6–IL-6R, followed by immunoprecipitation of lysates with anti-p-Tyr mAb or isotype-matched control mAb (iso) and immunoblot with anti-gp130. Data are from one experiment representative of at least two independent experiments with similar results. Split luciferase system for JAK–JAK interaction To investigate how TRAF5 inhibits the activation of JAK kinases, we established a system for assessment of JAK–JAK interactions based on luciferase enzyme fragment complementation (13) (Fig. 2A and B). Firefly luciferase can be split into two N-terminal and C-terminal fragments, LucN (1-416) and LucC (398-550), respectively. We combined each luciferase fragment with a JAK1 protein using a glycine-glycine-glycine-glycine-serine (GGGGS) linker to make JAK1-LucN (1-416) and JAK1-LucC (398-550). Active luciferase conformation must be restored when gp130-associated JAK1 proteins bring LucN (1-416) and LucC (398-550) fragments into close proximity. Indeed, we observed a significant increase in luciferase activity and p-JAK1 only when HEK293T cells expressing JAK1-LucN, JAK1-LucC and gp130 were stimulated with IL-6–IL-6R. Induction of luminescence and p-JAK1 peaked at 5–10 min after IL-6–IL-6R, and remained high at 30 min (Fig. 2C and D). Importantly, JAK1-LucC was co-immunoprecipitated with JAK1-LucN only after IL-6–IL-6R stimulation (Fig. 2E), indicating that IL-6–IL-6R-mediated proximal JAK1–JAK1 interactions restore the correct conformation of luciferase enzyme. Thus, using this system, we decided to examine the possible regulatory role of TRAF5 in JAK1–JAK1 interactions. Fig. 2. View largeDownload slide Firefly luciferase protein fragment complementation assay system for evaluation of the interaction between two JAK1 molecules in the IL-6R complex. (A) Schematic diagram of luciferase complementation induced by JAK1–JAK1 interaction in the activated IL-6R signaling complex. The N-terminal (1-416) or the C-terminal (398-550) fragment of firefly luciferase is fused at the end of the C-terminus of full-length mouse JAK1 via a linker peptide. Binding of a complex of IL-6 and IL-6R to transmembrane gp130 induces interaction between JAK1-LucN (1-416) and JAK1-LucC (398-550), which leads to reconstitution of firefly luciferase enzyme activity. (B) Immunoblot analysis of HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130. (C) Kinetics of induction of bioluminescence. Luciferase assay of mixed lysates from HEK cells transiently transduced to express V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation of triplicate wells. *P < 0.05, **P < 0.01 (Student’s t-test). (D) Immunoblot analysis of HEK293T cells as in C and immunoblot analysis with anti-p-JAK1, anti-V5, anti-c-Myc and anti-Tubulin. (E) IL-6–IL-6R-dependent binding between JAK1-LucN and JAK1-LucC. Pull-down assay of JAK1-LucN from lysates of HEK cells transiently transfected with plasmid vector encoding Flag-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-Flag or control IgG (iso) and immunoblot analysis with anti-Flag, anti-V5, anti-c-Myc and anti-JAK1. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 2. View largeDownload slide Firefly luciferase protein fragment complementation assay system for evaluation of the interaction between two JAK1 molecules in the IL-6R complex. (A) Schematic diagram of luciferase complementation induced by JAK1–JAK1 interaction in the activated IL-6R signaling complex. The N-terminal (1-416) or the C-terminal (398-550) fragment of firefly luciferase is fused at the end of the C-terminus of full-length mouse JAK1 via a linker peptide. Binding of a complex of IL-6 and IL-6R to transmembrane gp130 induces interaction between JAK1-LucN (1-416) and JAK1-LucC (398-550), which leads to reconstitution of firefly luciferase enzyme activity. (B) Immunoblot analysis of HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130. (C) Kinetics of induction of bioluminescence. Luciferase assay of mixed lysates from HEK cells transiently transduced to express V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation of triplicate wells. *P < 0.05, **P < 0.01 (Student’s t-test). (D) Immunoblot analysis of HEK293T cells as in C and immunoblot analysis with anti-p-JAK1, anti-V5, anti-c-Myc and anti-Tubulin. (E) IL-6–IL-6R-dependent binding between JAK1-LucN and JAK1-LucC. Pull-down assay of JAK1-LucN from lysates of HEK cells transiently transfected with plasmid vector encoding Flag-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-Flag or control IgG (iso) and immunoblot analysis with anti-Flag, anti-V5, anti-c-Myc and anti-JAK1. Data are from one experiment representative of at least two independent experiments with similar results. Inhibition of IL-6-driven JAK–JAK interaction by TRAF5 JAK1 is associated with gp130 via membrane-proximal intracellular regions called Box1 (649IWPNVDP657) and Box2 (689VSVVEIEANNKKP701), whereas the region in gp130 that interacts with TRAF5 has been mapped to the amino acid sequence 774VFSRSESTQPLLDSEERPEDLQLVD798 (8). To evaluate the inhibitory role of TRAF5 in JAK activation, we firstly tested whether TRAF5 could inhibit the binding between gp130 and JAK1. We transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC in the presence or absence of TRAF5 and immunoprecipitated gp130 from cell lysates. JAK1-LucN, JAK1-LucC and TRAF5 were co-immunoprecipitated with gp130, and TRAF5 expression did not affect the amount of JAK1 associated with gp130 (Fig. 3, lane 3 versus lane 4). In addition, triggering of gp130 with IL-6–IL-6R did not change the amount of JAK1 associated with gp130 (Fig. 3, lane 3 versus lane 5, lane 4 versus lane 6). In addition, JAK1 expression did not change the amount of TRAF5 associated with gp130 (Fig. 3, lane 1 versus lane 4). These results demonstrate that the interaction between TRAF5 and gp130 can occur independently of the interaction between JAK1 and gp130 and that TRAF5 does not inhibit the association between JAK1 and gp130. Fig. 3. View largeDownload slide Binding between TRAF5 and gp130 occurs independently of binding between JAK1 and gp130. Immunoassay of gp130 from lysates of HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 or control vector, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-c-Myc or control IgG (iso) and immunoblot analysis with anti-c-Myc, anti-V5 and anti-Flag. Data are from one experiment representative of three independent experiments with similar results. Fig. 3. View largeDownload slide Binding between TRAF5 and gp130 occurs independently of binding between JAK1 and gp130. Immunoassay of gp130 from lysates of HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 or control vector, then left unstimulated (−) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (+), followed by immunoprecipitation of proteins from lysates with anti-c-Myc or control IgG (iso) and immunoblot analysis with anti-c-Myc, anti-V5 and anti-Flag. Data are from one experiment representative of three independent experiments with similar results. Secondly, we tested the possibility that TRAF5 prevents the interaction between two JAK1 proteins using the split luciferase system (Fig. 2). We additionally transfected HEK293T cells with titrated amounts of plasmid vector encoding TRAF5 to examine how the expression levels of TRAF5 affect luciferase activity that is dependent on JAK–JAK interactions. Under stimulation with IL-6–IL-6R, TRAF5 dose-dependently inhibited the induction of luminescence (Fig. 4A and B). In addition, IL-6–IL-6R stimulation produced a dose-dependent increase in luminescence, and this response was significantly suppressed by the expression of TRAF5, at doses of 50 and 200 ng ml−1 of IL-6–IL-6R (Fig. 4C and D), although at higher concentrations of IL-6–IL-6R, TRAF5 could not inhibit the luminescence (Fig. 4C). Fig. 4. View largeDownload slide TRAF5 inhibits the interaction between two JAK1 proteins in the IL-6R complex. (A and B) Dose-dependent inhibitory effect of TRAF5 on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with various amounts of TRAF5 () or control vector (), left unstimulated (None) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (A). Immunoblot analysis of HEK293T cells as in A (B). (C and D) Dose-dependent effect of IL-6–IL-6R on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with 500 ng of TRAF5 () or control vector (), stimulated for 5 min with various concentration of IL-6–IL-6R (C). Immunoblot analysis of HEK293T cells as in C (D). Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). One representative experimental result is shown. Fig. 4. View largeDownload slide TRAF5 inhibits the interaction between two JAK1 proteins in the IL-6R complex. (A and B) Dose-dependent inhibitory effect of TRAF5 on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with various amounts of TRAF5 () or control vector (), left unstimulated (None) or stimulated for 5 min with 200 ng ml−1 IL-6–IL-6R (A). Immunoblot analysis of HEK293T cells as in A (B). (C and D) Dose-dependent effect of IL-6–IL-6R on JAK1–JAK1 interactions. Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with 500 ng of TRAF5 () or control vector (), stimulated for 5 min with various concentration of IL-6–IL-6R (C). Immunoblot analysis of HEK293T cells as in C (D). Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). One representative experimental result is shown. Thirdly, we examined if the inability of TRAF5 to bind to gp130 would affect the JAK–JAK interaction. In our previous study, amino acid residues 774-798 in gp130 were essential for its recognition by TRAF5, and a gp130 mutant lacking this region, gp130 (∆774-798), exhibited considerably diminished binding to TRAF5 relative to that of full-length gp130 (8). As expected, the reduction in luminescence mediated by TRAF5 was abolished in gp130 (∆774-798) expressing cells (Fig. 5A–D). Thus, TRAF5 requires the TRAF5-binding region in gp130 to inhibit JAK1–JAK1 interactions. Fig. 5. View largeDownload slide TRAF5 binding to gp130 is required for the inhibition against JAK1–JAK1 interactions. (A and C) Impaired TRAF5-mediated inhibition against JAK1–JAK1 interactions in cells expressing gp130 lacking the cytoplasmic region necessary for TRAF binding. Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN and V5-tagged JAK1-LucC together with c-Myc-tagged full-length gp130 (1-917) (A) or c-Myc-tagged deletion mutant of gp130 (∆774-798) (C) in the presence () or absence () of TRAF5, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B and D) Immunoblot analysis of HEK293T cells transfected with plasmid vectors encoding c-Myc-tagged full-length gp130 (1-917) (B) and c-Myc-tagged deletion mutant of gp130 (∆774-798) (D) as in A and C, respectively. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Fig. 5. View largeDownload slide TRAF5 binding to gp130 is required for the inhibition against JAK1–JAK1 interactions. (A and C) Impaired TRAF5-mediated inhibition against JAK1–JAK1 interactions in cells expressing gp130 lacking the cytoplasmic region necessary for TRAF binding. Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN and V5-tagged JAK1-LucC together with c-Myc-tagged full-length gp130 (1-917) (A) or c-Myc-tagged deletion mutant of gp130 (∆774-798) (C) in the presence () or absence () of TRAF5, stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B and D) Immunoblot analysis of HEK293T cells transfected with plasmid vectors encoding c-Myc-tagged full-length gp130 (1-917) (B) and c-Myc-tagged deletion mutant of gp130 (∆774-798) (D) as in A and C, respectively. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Collectively, these results clearly show that gp130-associated TRAF5 is inhibitory for the interaction between two JAK molecules associated with gp130. Comparable inhibitory activity of TRAF2 toward JAK–JAK interaction We previously reported that TRAF2 shared the binding region in gp130 with TRAF5 and inhibited STAT3 activation mediated by IL-6–IL-6R (9). Thus, it is reasonable to speculate that TRAF2 has a similar inhibitory function against JAK1. To investigate this, we transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC together with TRAF2. As expected, TRAF2 did prevent luminescence derived from JAK1–JAK1 associations induced by IL-6–IL-6R, and TRAF2 could decrease the peak response of luminescence similarly to TRAF5 (Fig. 6A and B). Thus, both TRAF2 and TRAF5 inhibit JAK–JAK interactions in the IL-6R complex. Fig. 6. View largeDownload slide TRAF2 also inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 () or Flag-tagged TRAF2 () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation. **P < 0.01 (Student’s t-test). (B) Immunoblot analysis of HEK293T cells as in A. Data are from one experiment representative of at least two independent experiments with similar results. Fig. 6. View largeDownload slide TRAF2 also inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 () or Flag-tagged TRAF2 () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. Data are average with standard deviation. **P < 0.01 (Student’s t-test). (B) Immunoblot analysis of HEK293T cells as in A. Data are from one experiment representative of at least two independent experiments with similar results. Inhibitory function of C-terminal domains of TRAF2 and TRAF5 for JAK–JAK interaction The inhibitory activity of TRAF2 and TRAF5 for STAT3 relies entirely on the TRAF-C domain (8, 9), suggesting that the TRAF-C domain also plays a critical inhibitory role for JAK1. To investigate which domains of TRAF are responsible for the inhibition, we transfected HEK293T cells to express gp130, JAK1-LucN and JAK1-LucC together with the N-terminus of TRAF2 (amino acids 1-271), which contains really interesting new gene (RING) and zinc-finger domains [TRAF2 (1-271)], or the C-terminus of TRAF2 (amino acids 272-501), which contains leucine-zipper and TRAF-C domains [TRAF2 (272-501)], or the N-terminus of TRAF5 (amino acids 1-241), which contains RING and zinc-finger domains [TRAF5 (1-241)], or the C-terminus of TRAF5 (amino acids 242-558), which contains leucine-zipper and TRAF-C domains [TRAF5 (242-558)]. As expected, both TRAF2 (272-501) and TRAF5 (242-558) significantly inhibited luminescence derived from JAK1–JAK1 interactions, whereas TRAF2 (1-271) and TRAF5 (1-241) did not (Fig. 7A–D). These results demonstrate that the binding activity of TRAF-C domain toward gp130 is responsible for the inhibitory activity of TRAF2 and TRAF5 against JAK1 activation. Fig. 7. View largeDownload slide The C-terminal TRAF domain inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF2 (1-271) () or Flag-tagged TRAF2 (272-501) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B) Immunoblot analysis of HEK293T cells as in A. (C) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 (1-241) () or Flag-tagged TRAF5 (242-558) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (D) Immunoblot analysis of HEK293T cells as in C. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. Fig. 7. View largeDownload slide The C-terminal TRAF domain inhibits the interaction between two JAK1 proteins. (A) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF2 (1-271) () or Flag-tagged TRAF2 (272-501) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (B) Immunoblot analysis of HEK293T cells as in A. (C) Luciferase assay of mixed lysates from HEK293T cells transiently transfected with plasmid vectors encoding V5-tagged JAK1-LucN, V5-tagged JAK1-LucC and c-Myc-tagged gp130 together with Flag-tagged TRAF5 (1-241) () or Flag-tagged TRAF5 (242-558) () or control vector (), stimulated for various times with 200 ng ml−1 IL-6–IL-6R. (D) Immunoblot analysis of HEK293T cells as in C. Data are average with standard deviation. *P < 0.05, **P < 0.01 (Student’s t-test). Data are from one experiment representative of at least two independent experiments with similar results. In conclusion, we have identified a novel mechanism by which TRAF2 and TRAF5 limit the IL-6R signaling. Our results demonstrate that the binding activity of TRAF2 and TRAF5 to gp130 is responsible for the inhibition toward JAK1–JAK1 interactions in the IL-6R complex, and that the E3 ubiquitin ligase activity in TRAF2 and TRAF5 may be dispensable for the inhibition. Discussion In this study, we show that the expression of TRAF5 was inhibitory for phosphorylations in JAK1, gp130 and STAT3 that occur in the IL-6R signaling complex. Although TRAF5 binding to gp130 did not alter the constitutive interaction between JAK1 and gp130, TRAF5–gp130 interactions decreased transphosphorylation and corresponding activation of JAK1 that are dependent on JAK1–JAK1 interactions. TRAF2 also suppressed JAK1–JAK1 interactions. Thus, we identify a novel inhibitory mechanism of TRAF adaptor proteins for JAK activation and demonstrate that this inhibitory activity of TRAF2 and TRAF5 limits IL-6-driven STAT3 activation that is essential for the lineage commitment of inflammatory and pathogenic Th17 cells. Although our results support the conclusion that the elevated IL-6-driven STAT3 activation in Traf5−/− CD4+ T cells was due to increased activation of JAK1, detailed mechanisms by which TRAF proteins regulate JAK activation in the IL-6R signaling complex are still unclear. The JAK1 FERM domain constitutively binds to the membrane-proximal Box1 and Box2 in gp130, whereas the TRAF-binding region is located in the middle part of the cytoplasmic tail of gp130 and does not contain tyrosine residues. Indeed, the expression of TRAF5 did not inhibit the interaction between JAK1 and gp130, showing that the binding between gp130 and JAK1 occurs independently of the interaction between gp130 and TRAF5. Based on some of the mechanisms that are proposed for JAK activation within the receptor complexes, formation of gp130 dimer and repositioning of the associated JAKs would be important steps for inducing the catalytic activity of JAKs (12, 14). We speculate that TRAF2 and TRAF5 associated with gp130 might sterically interfere with the repositioning process of inter domain communication that is required for JAK activation. According to a useful characteristic of the enzyme fragment complementation assay (13, 15, 16), we have decided to address the hypothesis that TRAF proteins inhibit the activation process of JAK–JAK interactions in the IL-6R complex. The N-terminus and C-terminus fragments of firefly luciferase were separately fused to the C-terminus of JAK1 using a flexible linker composed of GGGGS to promote reorganization of active luciferase enzyme structure. We examined the ability of reconstitution of split luciferase fragments in transiently co-transfected HEK293T cells with JAK1-LucN, JAK1-LucC and gp130 vectors in the presence or absence of TRAF vectors. We detected a quick and significant increase in luciferase activity, which is dependent on interactions between LucN and LucC, in transfected HEK293T cells after treatment of cells with IL-6–IL-6R. In this system, we have identified a novel inhibitory function of TRAF2 and TRAF5 for JAK1 activation. However, it is a matter of concern how gp130-associated TRAF proteins can impede the proximal JAK1 interactions in the IL-6R complex, as discussed above. Additional work is required for understanding of the role for gp130-associated TRAFs in JAK activation. Although TRAF3 inhibited IL-6R signaling by facilitating the association between phosphatase PTPN22 and JAK1 leading to blockade of STAT3 phosphorylation in B cells (17), our present results support the notion that TRAF2 and TRAF5 sterically inhibit the interaction between two JAKs associated with gp130. We previously reported that TRAF2 and TRAF5 did not require their RING and zinc-finger domains to interact with gp130 and that STAT3 binding to gp130 was suppressed by the expression of a TRAF5 mutant lacking RING and zinc-finger domains (8, 9). In addition, this TRAF5 mutant without RING and zinc-finger domains served an inhibitory role in IL-6–IL-6R-mediated Th17 development. In addition, over-expression of TRAF2 or TRAF5 but not TRAF3 inhibited IL-6–IL-6R-mediated phosphorylation of STAT3 and Th17 responses (8, 9). In this study, we demonstrated that TRAF2 and TRAF5 mutants lacking RING and zinc-finger domains inhibited JAK–JAK protein interactions. We also confirmed that TRAF5 expression suppressed IL-6–IL-6R-mediated phosphorylation of JAK1 even in the presence of tyrosine phosphatase inhibitor Na3VO4 in BAF-gp130 cells (data not shown). Thus, we think that a steric hindrance caused by TRAF binding to gp130 is responsible for the inhibition toward JAK activation in the IL-6R complex. In conclusion, using a firefly luciferase enzyme fragment complementation assay, we have discovered a novel inhibitory role for TRAF2 and TRAF5 in JAK activation in the IL-6R signaling complex. However, we still do not know how gp130-associated TRAF and JAK proteins organize active IL-6R complexes on the T-cell membrane. In this study, to cross-link membrane bound gp130, cells were treated with a complex of IL-6 and IL-6R instead of IL-6 alone. We think that the elevated phosphorylation of JAK1 in Traf5−/− T cells may be induced by IL-6 alone, but this is a matter of future study. It will be also important in the future to understand whether the inhibitory mechanism regulated by TRAF2 and TRAF5 is also applicable to other IL-6 family cytokines that use the common signaling receptor subunit gp130. Funding This work was supported by JSPS KAKENHI grant numbers JP15H04640 and JP18H02572 (T.S.) and the Yamaguchi Educational and Scholarship Foundation (to T.S.), the Daiichi-Sankyo Foundation of Life Science (T.S.), and the Tamura Science and Technology Foundation (T.S.). Acknowledgements We thank members of Department of Microbiology and Immunology, Tohoku University Graduate School of Medicine for their assistance and help. We thank the Biomedical Research Core and the Institute for Animal Experimentation (Tohoku University Graduate School of Medicine) for technical support. Conflicts of interest statement: the authors declared no conflicts of interest. References 1 Inoue , J. I. , Ishida , T. , Tsukamoto , N. , et al. 2000 . Tumor necrosis factor receptor-associated factor (TRAF) family: adapter proteins that mediate cytokine signaling . Exp. Cell Res . 254 : 14 . Google Scholar CrossRef Search ADS PubMed 2 Chung , J. Y. , Park , Y. C. , Ye , H. and Wu , H . 2002 . All TRAFs are not created equal: common and distinct molecular mechanisms of TRAF-mediated signal transduction . J. Cell Sci . 115 : 679 . Google Scholar PubMed 3 Ha , H. , Han , D. and Choi , Y . 2009 . TRAF-mediated TNFR-family signaling . Curr. Protoc. Immunol . Chapter 11 : Unit11.9D . Google Scholar PubMed 4 Xie , P . 2013 . TRAF molecules in cell signaling and in human diseases . J. Mol. Signal . 8 : 7 . Google Scholar CrossRef Search ADS PubMed 5 So , T. , Nagashima , H. and Ishii , N . 2015 . TNF receptor-associated factor (TRAF) signaling network in CD4(+) T-lymphocytes . Tohoku J. Exp. Med . 236 : 139 . Google Scholar CrossRef Search ADS PubMed 6 Bishop , G. A . 2016 . TRAF3 as a powerful and multitalented regulator of lymphocyte functions . J. Leukoc. Biol . 100 : 919 . Google Scholar CrossRef Search ADS PubMed 7 Taga , T. and Kishimoto , T . 1997 . Gp130 and the interleukin-6 family of cytokines . Annu. Rev. Immunol . 15 : 797 . Google Scholar CrossRef Search ADS PubMed 8 Nagashima , H. , Okuyama , Y. , Asao , A. , et al. 2014 . The adaptor TRAF5 limits the differentiation of inflammatory CD4(+) T cells by antagonizing signaling via the receptor for IL-6 . Nat. Immunol . 15 : 449 . Google Scholar CrossRef Search ADS PubMed 9 Nagashima , H. , Okuyama , Y. , Hayashi , T. , Ishii , N. and So , T . 2016 . TNFR-associated factors 2 and 5 differentially regulate the instructive IL-6 receptor signaling required for Th17 development . J. Immunol . 196 : 4082 . Google Scholar CrossRef Search ADS PubMed 10 Kishimoto , T. , Akira , S. , Narazaki , M. and Taga , T . 1995 . Interleukin-6 family of cytokines and gp130 . Blood 86 : 1243 . Google Scholar PubMed 11 Heinrich , P. C. , Behrmann , I. , Haan , S. , Hermanns , H. M. , Müller-Newen , G. and Schaper , F . 2003 . Principles of interleukin (IL)-6-type cytokine signalling and its regulation . Biochem. J . 374 : 1 . Google Scholar CrossRef Search ADS PubMed 12 Babon , J. J. , Lucet , I. S. , Murphy , J. M. , Nicola , N. A. and Varghese , L. N . 2014 . The molecular regulation of Janus kinase (JAK) activation . Biochem. J . 462 : 1 . Google Scholar CrossRef Search ADS PubMed 13 Luker , K. E. , Smith , M. C. , Luker , G. D. , Gammon , S. T. , Piwnica-Worms , H. and Piwnica-Worms , D . 2004 . Kinetics of regulated protein-protein interactions revealed with firefly luciferase complementation imaging in cells and living animals . Proc. Natl Acad. Sci. USA 101 : 12288 . Google Scholar CrossRef Search ADS 14 Waters , M. J. and Brooks , A. J . 2015 . JAK2 activation by growth hormone and other cytokines . Biochem. J . 466 : 1 . Google Scholar CrossRef Search ADS PubMed 15 Remy , I. , Wilson , I. A. and Michnick , S. W . 1999 . Erythropoietin receptor activation by a ligand-induced conformation change . Science 283 : 990 . Google Scholar CrossRef Search ADS PubMed 16 Liu , Y. , Berry , P. A. , Zhang , Y. , et al. 2014 . Dynamic analysis of GH receptor conformational changes by split luciferase complementation . Mol. Endocrinol . 28 : 1807 . Google Scholar CrossRef Search ADS PubMed 17 Lin , W. W. , Yi , Z. , Stunz , L. L. , Maine , C. J. , Sherman , L. A. and Bishop , G. A . 2015 . The adaptor protein TRAF3 inhibits interleukin-6 receptor signaling in B cells to limit plasma cell development . Sci. Signal . 8 : ra88 . Google Scholar CrossRef Search ADS PubMed © The Japanese Society for Immunology. 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/about_us/legal/notices)

Journal

International ImmunologyOxford University Press

Published: Apr 13, 2018

There are no references for this article.

You’re reading a free preview. Subscribe to read the entire article.


DeepDyve is your
personal research library

It’s your single place to instantly
discover and read the research
that matters to you.

Enjoy affordable access to
over 18 million articles from more than
15,000 peer-reviewed journals.

All for just $49/month

Explore the DeepDyve Library

Search

Query the DeepDyve database, plus search all of PubMed and Google Scholar seamlessly

Organize

Save any article or search result from DeepDyve, PubMed, and Google Scholar... all in one place.

Access

Get unlimited, online access to over 18 million full-text articles from more than 15,000 scientific journals.

Your journals are on DeepDyve

Read from thousands of the leading scholarly journals from SpringerNature, Elsevier, Wiley-Blackwell, Oxford University Press and more.

All the latest content is available, no embargo periods.

See the journals in your area

DeepDyve

Freelancer

DeepDyve

Pro

Price

FREE

$49/month
$360/year

Save searches from
Google Scholar,
PubMed

Create lists to
organize your research

Export lists, citations

Read DeepDyve articles

Abstract access only

Unlimited access to over
18 million full-text articles

Print

20 pages / month

PDF Discount

20% off