Figure 1.
Experimental system for the proteomic identification of LMP1 interaction partners in EBV-infected primary human B-cells.
(A) Schematic representation of the LMP1 wildtype molecule (top) and the engineered HA-LMP1-liTEV-CT derivative (bottom). For proteomics, HA-LMP1-liTEV-CT was immunoprecipitated by anti-HA-antibodies. Subsequently, the LMP1 signaling domain together with its interaction partners was cleaved off by TEV protease, eluted from the beads, and subsequently analyzed by immunoblotting or mass spectrometry. The known LMP1-binding protein TRAF3 and hypothetical direct and indirect interaction partners x and y, respectively, are shown as examples to illustrate the experimental approach. (B) Lymphoblastoid B-cell clone LCL-TEV.5 growing out from an infection of primary human B-cells with the recombinant maxi-EBV 2089-HA-LMP1-liTEV-CT, which has been generated by replacing the endogenous LMP1 gene of wildtype maxi-EBV 2089 by HA-LMP1-liTEV-CT. Expression of the GFP marker gene (left panel) in LCL-TEV.5 cells verified successful infection with recombinant virus. (C) Immunoprecipitation and TEV cleavage experiment. As indicated, the experiment was performed with LCLs expressing LMP1 wildtype (LCL 721, left panel), HA-LMP1 (LCL3, middle panel), or HA-LMP1-liTEV-CT (LCL-TEV.5, right panel). Anti-HA immunoprecipitations were performed (HA-IP, middle lanes). Precipitates were treated with TEV protease and the released LMP1 C-terminus together with bound proteins was eluted and analyzed by immunoblotting (TEV eluate, right lanes) or mass spectrometry (for the identification of TNIK in the TEV eluate of LCL-TEV.5 cells, see Tables S1 and S2). As expected, the tracer molecule TRAF3 was only found in the TEV eluate of LCL-TEV.5 but not of LCL 721 or LCL3 cells. The following antibodies were used for immunoblotting: anti-LMP1 (CS1-4) directed against the LMP1 signaling domain and anti-TRAF3 (C-20). Apparent molecular masses are given in kilodaltons (kDa). IB, immunoblot.
Figure 2.
TNIK binds to the LMP1 signaling complex.
(A) Endogenous TNIK interacts with LMP1 in lymphoblastoid cells. TNIK was immunoprecipitated from LCL 721 cell lysates with the anti-TNIK antibody (left panel). Co-precipitated wildtype LMP1 was detected by the anti-LMP1 (1G6-3) antibody. An unrelated mouse isotype IgG (IsoG) served as a negative control for immunoprecipitation. Vice versa, endogenous LMP1 was immunoprecipitated using the anti-LMP1 (1G6-3) antibody (right panel). Co-precipitated TNIK was detected using the anti-TNIK antibody. Rat isotype IgG served as a negative control. IB, immunoblot; IP, immunoprecipitation. Shown is one representative of four independent experiments (n = 4). (B) The CTAR2 domain of LMP1 is essential for TNIK recruitment. HEK293 cells were transfected in 15 cm culture dishes with 20 µg of the indicated LMP1 constructs. The Δ371-386 deletion inactivates the CTAR2 domain. CTAR2 with Y384G mutation has no signaling capacity towards NF-κB and JNK. AAA harbors a mutated TRAF interaction motif within CTAR1. Endogenous TNIK was immunoprecipitated from cell lysates. LMP1 co-precipitation was analyzed using the anti-LMP1 (CS1-4) antibody. The anti-TNIK antibody was used to verify equal TNIK precipitation in all samples. Comparable expression of LMP1 constructs was confirmed in cell lysates. n = 3. (C) The 16 C-terminal amino acids of CTAR2 are sufficient for interaction with TNIK. HEK293 cells were transfected with pCMV-HA-LMP1-TNFR1ΔDD, a chimera of the LMP1 transmembrane domain and the signaling domain of TNFR1 lacking its death domain (DD), or pCMV-HA-LMP1-TNFR1-CTAR2 carrying amino acids 371–386 of LMP1 instead of DD. Endogenous TNIK was immunoprecipitated from cell lysates and co-precipitation of the HA-tagged chimeras was analyzed by immunoblotting with the anti-HA (3F10) antibody. The anti-TNIK antibody was used to confirm comparable TNIK precipitation. n = 3.
Figure 3.
TNIK is essential for JNK activation by LMP1.
(A) HEK293 cells were transfected in 6-well plates with siRNA against human TNIK (siTNIK) or non-targeting control siRNA (siCTR). Subsequently, the cells were co-transfected with 1 µg of HA-JNK1 and 1 µg of HA-LMP1 wildtype or HA-LMP1Δ194–386 lacking the LMP1 signaling domain, as indicated. HA-JNK1 was immunoprecipitated from cell lysates and immunocomplex kinase assays were performed using recombinant GST-c-Jun as a substrate. Equal HA-JNK1 immunoprecipitation was confirmed by the anti-JNK1 antibody. TNIK downregulation and LMP1 expression was monitored in cell lysates using anti-TNIK and anti-LMP1 (CS1-4) antibodies. The null control construct LMP1Δ194–386 cannot be detected by the CS1-4 antibody because all CS1-4 epitopes are located within the LMP1 signaling domain. Quantification of four independent experiments ± standard deviations, given as x-fold inductions: lane 1, 1.15±0.32; lane 2, 1.48±0.6; lane 3, 1.0±0.0; lane 4, 3.23±1.03. (B) The knockdown of TNIK in lymphoblastoid cells blocks the JNK pathway. EREB2-5 B-cells were treated with Accell siRNA against human TNIK or non-targeting siRNA for 72 h. Cell lysates were analyzed by immunoblotting using anti-TNIK, anti-phospho-JNK, anti-JNK1, and anti-LMP1 (CS1-4) antibodies. n = 3.
Figure 4.
TNIK has a critical role in LMP1-induced canonical NF-κB signaling.
(A) The knockdown of TNIK inhibits LMP1 activation of IKKβ. HEK293 cells were transfected in 6-well plates with TNIK siRNA or non-targeting siRNA. Subsequently, the cells were co-transfected with 1 µg each of HA-LMP1 wildtype or inactive HA-LMP1(AAA, Δ371–386) together with 1 µg of Flag-IKKβ, as indicated. Flag-IKKβ was immunoprecipitated and its activity was monitored in in vitro kinase assays using purified GST-IκBα as a substrate. Immunoprecipitated Flag-IKKβ, expression of LMP1 constructs, and downregulation of TNIK were detected in lysates using the anti-IKKβ, anti-LMP1 (1G6-3), and anti-TNIK antibodies, respectively. Quantification of three independent experiments: lane 1, 1.0±0.0; lane 2, 2.6±0.17; lane 3, 0.5±0.0; lane 4, 0.97±0.47. (B) TNIK mediates LMP1-induced canonical NF-κB signaling without affecting the non-canonical pathway. HEK293 cells were transfected in 10 cm culture dishes with 4 µg each of HA-LMP1 wildtype or HA-LMP1(AAA, Δ371–386). As indicated, 4 µg of pSM2-shTNIK expressing a short hairpin RNA targeting TNIK or non-targeting vector were co-transfected. 48 h post-transfection the cells were lysed, and cytoplasmic and nuclear fractions were prepared and analyzed by immunoblotting using the indicated antibodies for the NF-κB proteins p65 and p52. Tubulin and SAM68 were used as markers for the cytoplasmic and nuclear fractions, respectively. TNIK knockdown and LMP1 expression was monitored in total cell lysates (right). n = 2. (C) NF-κB reporter assays in HEK293 cells. TNIK was depleted from HEK293 cells by transfection in 6-well plates with TNIK siRNA. Cells were co-transfected with 2 µg of expression vectors for HA-LMP1(AAA, Δ371–386) or HA-LMP1 wildtype together with 5 ng of the NF-κB reporter 3xκBLuc and Renilla control vector. 24 h post-transfection cells were lysed and NF-κB reporter assays were performed. Luciferase activities were corrected for transfection efficiencies as described in Materials and Methods. Given data are mean values of three independent experiments ± standard deviations; statistics, two-tailed Student's t test. *p = 0.006. (D) The downregulation of TNIK in lymphoblastoid cells impairs the canonical NF-κB pathway. Identical samples of siTNIK or siCTR-treated EREB2-5 LCLs as shown in Figure 3B were blotted for phospho-IκBα and IκBα. Control immunoblots for TNIK and LMP1 expression are displayed again for better comparison. n = 3.
Figure 5.
TNIK mediates proliferation and survival in EBV-transformed human B-cells.
(A) The knockdown of TNIK inhibits proliferation of LCLs. EREB2-5 cells were seeded in the presence of Accell siRNA targeting TNIK or non-targeting siRNA. Cell proliferation was monitored at the indicated times by MTT conversion. Shown are the results of one representative experiment in triplicates of three independent experiments. (B) Apoptosis induction by TNIK knockdown in LCLs. EREB2-5 cells were incubated with Accell siTNIK or siCTR, as indicated. Apoptosis was monitored after 3 d in the presence of siRNA by co-staining of the cells with Cy5-labeled annexin V (AnV-Cy5) and propidium iodide (PI). The population of intact cells within the lymphocyte gate in the forward scatter (FSC)/sideward scatter (SSC) plot (left panels) was strongly reduced after the knockdown of TNIK. The gated cells were then analyzed for apoptosis rates, indicated by PI−/AnV-Cy5+ staining (right panels). The graph shows mean values of PI−/AnV-Cy5+ percentages of three independent experiments ± standard deviations; two-tailed Student's t test. *p = 0.033.
Figure 6.
The JNK and canonical NF-κB pathways bifurcate at the level of TNIK.
(A) Schematic diagram of TNIK mutants cloned with an N-terminal HA-tag. Amino acid positions are given in numbers. The asterisk marks the position of the K54R mutation that inactivates the kinase. GCKH, germinal center kinase homology domain; KD, kinase domain; KM, kinase mutant. (B) The intact TNIK kinase domain is required for IKKβ activation. HEK293 cells were transfected in 6-well plates with 2 µg of the indicated TNIK constructs or pRK5 empty vector and 1 µg of Flag-IKKβ. Flag-IKKβ activity was monitored in immunocomplex kinase assays. Immunoprecipitated Flag-IKKβ and expressed TNIK-constructs were detected using the anti-IKKβ and anti-HA (3F10) antibodies, respectively. Asterisks indicate the TNIK constructs. Apparent molecular masses are given in kDa. Quantification of three independent experiments: lane 1, 1.0±0.0; lane 2, 4.73±1.0; lane 3, 0.93±0.46; lane 4, 11.0±5.86; lane 5, 1.3±0.56; lane 6, 1.4±0.62; lane 7, 5.13±1.72. (C) The TNIK kinase domain induces the nuclear shift of canonical p65 NF-κB but not of non-canonical p52 NF-κB. HEK293 cells were transfected in 10 cm dishes with 4 µg each of the indicated constructs. LMP1 served as positive control. Cytoplasmic and nuclear fractions were prepared and analyzed by immunoblotting using the indicated antibodies. Expression of the constructs was verified in total lysates by the anti-HA (3F10) antibody. n = 4. (D) The GCKH domain of TNIK is sufficient for JNK activation and TNIK kinase activity is dispensable. HEK293 cells were transfected with 0.5 µg of the indicated HA-tagged TNIK constructs or pRK5 empty vector and 1 µg of HA-JNK1. Subsequently, immunocomplex kinase assays were performed. Immunoprecipitated HA-JNK1 and the expressed TNIK-constructs were detected using the anti-JNK1 and anti-HA (3F10) antibodies. Quantification of three independent experiments: lane 1, 1.0±0.0; lane 2, 3.2±1.66; lane 3, 4.57±1.35; lane 4, 6.0±0.1. (E) LMP1 enhances autophosphorylation of TNIK. HEK293 cells were transfected in 6-well plates with 1 µg of HA-TNIK vector together with or without 1 µg of pSV-LMP1, as indicated. Subsequently, HA-TNIK was immunoprecipitated from cell lysates using the anti-HA (12CA5) antibody and TNIK autophosphorylation reaction was performed with the precipitated TNIK in the presence of radioactive ATP. Equal TNIK precipitation and LMP1 expression was confirmed on immunoblots. Quantification of six independent experiments: lane 1, 1.0±0.0; lane 2, 4.28±2.76; two-tailed Student's t test, p = 0.015. (F) Kinase-negative TNIK(KM) exerts a dominant-negative effect specifically on the NF-κB pathway induced by CTAR2. HEK293 cells were transfected in 6-well plates with 1 µg of the indicated LMP1 constructs, 2 µg of kinase-negative HA-TNIK(KM) or empty vector, and 5 ng of the NF-κB reporter 3xκBLuc and Renilla control reporter. Luciferase activities were corrected for transfection efficiencies. Given data are mean values of three independent experiments ± standard deviations; two-tailed Student's t test. *p = 0.03, **p = 0.003, #p = 0.88.
Figure 7.
TRAF6 directly binds to TNIK and mediates TNIK recruitment to LMP1.
(A) LMP1 induces interaction of TNIK and TRAF6. HEK293 cells were transfected in 10 cm cell culture dishes with 0.5 µg each of Flag-TRAF6 and HA-TNIK expression vectors, both in the presence or absence of 4 µg pSV-LMP1, as indicated. Cells were lysed and HA-TNIK was immunoprecipitated using the anti-HA (12CA5) antibody. As indicated, the following antibodies were used for immunoblot analysis of immunoprecipitations and lysates: anti-TRAF6 (H-274), anti-TNIK, and anti-LMP1 (1G6-3). IB, immunoblot; IP, immunoprecipitation. n = 3. (B) TRAF6 and TRAF2 bind directly to the intermediate domain of TNIK. Protein binding assays with purified proteins. Interaction of His-tagged TRAF2(311–501) and TRAF6(310–522) with GST-tagged kinase-negative kinase domain, intermediate domain, and GCKH domain of TNIK was analyzed on immunoblots with the anti-TRAF2 (C-20) and anti-TRAF6 (C-20) antibodies (upper panels). Plain GST was incorporated as a control. For immunoblots only 25 ng of the TRAF input was loaded to avoid overexposure. Input of TRAF and GST-TNIK proteins is shown in a coomassie-stained gel (lower panel). Asterisks indicate GST and GST-TNIK proteins. Apparent molecular masses are given in kDa. n = 2. (C) TRAF6 mediates the interaction between TNIK and LMP1. Wildtype and TRAF6−/− MEF cells were electroporated with Flag-TNIK and LMP1 vectors. Cells were immunostained for TNIK (red) and LMP1 (green) as described in Materials and Methods. Nuclei were counterstained with DAPI (blue). The graph gives the percentage of cells with co-localization of TNIK and LMP1. A line scan analysis (right panel) shows intensities of TNIK (red) and LMP1 (green) signals along the indicated arrows. LMP1 and TNIK localization peaks overlap only in wildtype but not in TRAF6−/− cells. n = 3. (D) Exogenous TRAF6 rescues the interaction between TNIK and LMP1 in TRAF6−/− cells. TRAF6−/− MEFs were electroporated with 5 µg each of Flag-TNIK, HA-TRAF6, and HA-LMP1 vectors as indicated. Total transfected DNA was adjusted to 20 µg with empty vector. TNIK was immunoprecipitated from cell lysates using the anti-TNIK antibody. Complex formation of LMP1 and TNIK was analyzed by immunoblotting of the IPs using the anti-TNIK and anti-LMP1 (1G6-3) antibodies. Lysates were probed with anti-LMP1 (1G6-3), anti-TRAF6 (H-274), and anti-TNIK. The arrow indicates the position of LMP1. n = 2.
Figure 8.
TNIK forms a dynamic signaling complex with TAK1, TAB2, and IKKβ upon LMP1 activation.
(A) TAK1 interacts with TNIK independent of LMP1. HEK293 cells were transfected in 15 cm culture dishes with 5 µg of HA-TNIK and 2 µg of Flag-TAK1 vectors, both in the presence and absence of 5 µg pSV-LMP1, as indicated. HA-TNIK was immunoprecipitated with the anti-HA (12CA5) antibody. Immunoprecipitations were analyzed using anti-TNIK and anti-TAK1 antibodies. Cell lysates were stained with anti-TNIK, anti-TAK1, and anti-LMP1 (CS1-4) antibodies. n = 3. (B) TAK1 binds to the GCKH and intermediate domains of TNIK. HEK293 cells were transfected in 10 cm cell culture dishes with 2 µg of the indicated HA-TNIK constructs and 1 µg of Flag-TAK1 vector. HA-TNIK constructs were immunoprecipitated with the anti-HA (12CA5) antibody. Immunoprecipitations and lysates were analyzed with anti-HA (3F10) and anti-TAK1 antibodies, as indicated. Asterisks indicate TNIK proteins. Apparent molecular masses are given in kDa. n = 4. (C) LMP1 induces the interaction between TNIK and TAB2. HEK293 cells were transiently transfected in 15 cm culture dishes with 5 µg of HA-TAB2 and Flag-TNIK vectors in the presence or absence of 5 µg pSV-LMP1 as indicated. Flag-TNIK was immunoprecipitated using the anti-Flag (6F7) antibody. The following antibodies were used for immunoblotting: anti-Flag (6F7), anti-TAB2, anti-LMP1 (1G6-3). n = 3. (D) TNIK is essential for TAK1 interaction with LMP1. HEK293 cells were transfected in 15 cm culture dishes with 5 µg Flag-TAK1, 3 µg LMP1, and 7 µg each of shTNIK or shCTR vectors, as indicated. Cells were lysed 48 h post-transfection, LMP1 was immunoprecipitated using the anti-LMP1 (1G6-3) antibody, and co-precipitation of Flag-TAK1 was analyzed by immunoblotting for TAK1. TNIK knockdown and Flag-TAK1 expression was verified in total cell lysates. n = 3. (E) TNIK mediates the interaction between TAB2 and LMP1. HEK293 cells were transfected with the indicated vectors as described in (D). LMP1 was precipitated from cell lysates using the anti-LMP1(1G6-3) antibody. Immunoprecipitations and lysates were analyzed with the indicated antibodies. n = 2. (F) LMP1 induces the recruitment of IKKβ to the TNIK complex. HEK293 cells were transfected in 10 cm culture dishes with 2 µg HA-TNIK, 2 µg Flag-IKKβ, and 3 µg pSV-LMP1 as indicated. HA-TNIK was immunoprecipitated using the anti-HA (12CA5) antibody and immunocomplexes were analyzed by immunoblotting using anti-HA (12CA5) and anti-Flag (M2) antibodies. Expression of HA-TNIK, Flag-IKKβ, and LMP1 was detected in cell lysates with the anti-HA (12CA5), anti-Flag (M2), and anti-LMP1 (1G6-3) antibodies. n = 3. (G) TNIK signaling complex containing LMP1, TRAF6, TAK1, TAB2, and IKKβ in EBV-transformed lymphoblastoid cells. Endogenous TNIK was immunoprecipitated from LCL 721 cells with the anti-TNIK antibody. An unrelated mouse isotype IgG was used for control precipitation (IsoG). As indicated, immunoprecipitations and lysates were analyzed by immunoblotting using the anti-TNIK, anti-TRAF6 (H-274), anti-LMP1 (1G6-3), anti-TAK1, anti-TAB2, and anti-IKKβ antibodies. n = 2.
Figure 9.
TNIK is required for JNK and canonical NF-κB signaling by CD40.
(A) The knockdown of TNIK impairs CD40-induced JNK signaling in HEK293 cells. HEK293 cells were transfected in 6-well plates with TNIK or non-targeting siRNA. Subsequently, the cells were transfected with 2 µg of the CD40 expression vector pESBOS-CD40 where indicated. 1 µg of pRK5-HA-JNK1 was co-transfected. HA-JNK activity was monitored in immunocomplex kinase assays. Immunoprecipitated HA-JNK1, expression of CD40, and downregulation of TNIK was detected using the anti-JNK1, anti-CD40, and anti-TNIK antibodies. Quantification of four independent experiments: lane 1, 1.0±0.0; lane 2, 7.18±3.15; lane 3, 0.8±0.59; lane 4, 3.28±1.68. (B) IKKβ activation by CD40 is blocked by TNIK downregulation. HEK293 cells were transfected as in (A) except that Flag-IKKβ was transfected instead of HA-JNK1. Flag-IKKβ immunocomplex kinase assays were performed. Flag-IKKβ, CD40, and TNIK were detected using the indicated antibodies. Quantification of three independent experiments: lane 1, 1.0±0.0; lane 2, 8.17±1.81; lane 3, 0.6±0.16; lane 4, 3.2±1.36. (C) The knockdown of TNIK in B-cells inhibits JNK and canonical NF-κB signaling after stimulation of the endogenous CD40 receptor. BL41 cells were treated with 5 µM Accell TNIK or non-targeting siRNA for 72 h. Cells were stimulated with 500 ng/ml recombinant human CD40 ligand for the indicated times. Cell lysates were subjected to immunoblot analysis using anti-phospho-JNK, anti-JNK1, anti-IκBα, and anti-TNIK antibodies. Equal loading was verified by staining with anti-tubulin antibodies. n = 3. (D) TNIK mediates CD54 upregulation by CD40. BL41 B-cells were incubated with TNIK or non-targeting Accell siRNA and stimulated with 1 µg/ml CD40 ligand as indicated and described in Materials and Methods. CD54 surface levels were analyzed by flow cytometry. n = 2. (E) CD40 stimulation induces the interaction of TNIK and TRAF6 in B-cells. BL41 cells were stimulated with 500 ng/ml CD40 ligand. At the indicated times, cells were lysed and endogenous TNIK was immunoprecipitated by the anti-TNIK antibody. TRAF6 co-precipitation was detected by the anti-TRAF6 (H-274) antibody. Lysate controls were stained with the indicated antibodies. n = 3.
Figure 10.
Model of TNIK's role in JNK and canonical NF-κB signaling.
LMP1 induces the formation of the TNIK complex at its JNK1 and NF-κB-inducing CTAR2 domain. Interaction of TNIK with LMP1 is mediated by TRAF6, which forms an induced complex with TNIK. Also TAB2 and IKKβ are recruited upon activation, whereas TAK1 is permanently complexed with TNIK. In LMP1 signaling, IKKβ recruitment is further enhanced or stabilized by a TRADD-dependent mechanism. TNIK organizes bifurcation of the JNK and NF-κB pathways downstream of TRAF6. Auto-phosphorylation of TNIK likely constitutes an important step in transmitting signals on the NF-κB axis. JNK signaling is triggered at the GCKH domain of TNIK through TAK1/TAB2. Activation of TAK1/TAB2 further involves K63 ubiquitinylation of TRAF6, which has been demonstrated earlier. Also CD40 signals through the TRAF6/TNIK complex to JNK and IKKβ/NF-κB. For more details, see the text.