The Germinal Center Kinase TNIK Is Required for Canonical NF-κB and JNK Signaling in B-Cells by the EBV Oncoprotein LMP1 and the CD40 Receptor

TNIK has an important function in physiological activation and viral transformation of human B-cells by interacting with the TRAF6 adapter complex and mediating NF-κB and JNK signal transduction.


Introduction
TNIK was discovered in a yeast-two-hybrid screen for interaction partners of the adapter proteins TRAF2 and Nck [1]. The serine/threonine kinase TNIK is a member of the germinal center kinase (GCK) family, which belongs to the Ste20 group of kinases [2]. GCKs share high sequence homology in their N-terminal kinase and C-terminal germinal center kinase homology (GCKH) domains, while the intermediate domain is less conserved [2]. Current knowledge about the molecular and biological functions of TNIK is very limited. TNIK overexpression modulates the actin cytoskeleton and activates the JNK pathway, which is induced through the GCKH domain by a yet undefined mechanism [1,3]. The molecular function of TNIK's interaction with TRAF molecules is unclear. A recent study suggested that TRAF2 and TNIK might be located within one signaling pathway that leads to Wnt pathway induction in chronic myelogenous leukemia stem cells [4]. TNIK also mediates proliferative Wnt signals in crypts of the small intestine and colorectal cancer cells by nuclear translocation and subsequent phosphorylation of the transcription factor TCF4 [5,6]. In neurons, TNIK is involved in the regulation of neurite growth and neuronal structure [7,8]. However, a physiological role for TNIK in hematopoietic cells has not been described.
The latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV) serves as proto-type of a viral receptor-like oncoprotein that usurps cellular signal transduction pathways for cell transformation. The gamma-herpesvirus EBV, classified as a human DNA tumor virus by the WHO, establishes a chronic latent infection in B-cells and is associated with various malignancies, such as Hodgkin's and Burkitt's lymphoma, life-threatening post-transplant lymphoproliferative disorders, or nasopharyngeal carcinoma [9]. LMP1 is found expressed in most EBV-associated tumors and it is crucial for viral cell transformation and continued in vitro proliferation of latently EBV-infected B-cells, so-called lymphoblastoid cell lines (LCLs) [9]. LMP1 resembles a constitutively active cellular receptor whose ligand-independent signaling activity is attributable to spontaneous homo-oligomerization of LMP1 molecules within the membrane [10]. By the recruitment of TRAF molecules, LMP1 mimics molecular functions of the CD40 receptor in B-cell activation and proliferation. However, compared to CD40, LMP1 assembles a unique and more efficient signaling complex [11][12][13][14]. Constitutive expression of LMP1 in the B-cell compartment of transgenic mice induces lymphomas, whereas timely activation of LMP1 signaling largely substitutes for CD40 deficiency in B-cell development and function [15][16][17].
Apart from LMP1, TRAF6 also mediates canonical NF-kB and JNK signaling by cellular receptors such as CD40 or Toll-like receptors [43]. Current concepts of TAK1 and IKKb activation by TRAF6 have been reviewed [43][44][45]. In brief, activated and K63-autoubiquitinated TRAF6 binds TAB2, which then mediates the recruitment of the MAP3kinase TAK1 to TRAF6. TRAF6derived unanchored ubiquitin chains bind TAB2 and help to induce TAK1 [46]. Activated TAK1 phosphorylates MKK6 to upregulate the JNK pathway. TAK1 also phosphorylates IKKb within its activation loop and IKKb activation is further facilitated by interaction of its regulatory component IKKc with TRAF6. However, IKKb is also induced by a TAK1-independent mechanism [44,46]. IKKb phosphorylates IkB, which results in IkB degradation and the release of active p65/p50 NF-kB dimers to the nucleus. It is tempting to speculate that yet unknown factors might serve as additional organizers or scaffolding proteins for TRAF-TAK-IKK complexes within the cell to orchestrate NF-kB and JNK signaling.
It is still not fully understood how the signaling complex at CTAR2 of LMP1 is assembled and how activation of transforming downstream signals is conveyed. We hypothesized the existence of still undefined molecular players and set out to identify novel LMP1 interaction partners by a functional proteomics approach. We report the characterization of TNIK as a component of the LMP1 signaling complex in EBV-transformed human B-cells. TNIK has a critical role in LMP1-induced JNK and canonical NF-kB signaling by the formation of an activation-induced complex at LMP1 with TRAF6, TAK1/TAB2, and IKKb. Accordingly, TNIK is required for proliferation and survival of lymphoblastoid cells. TNIK is also of critical importance for physiological activation of the two pathways in B-cells by the CD40 receptor. Taken together, we identified TNIK as a novel key player in TRAF6-dependent JNK and NF-kB activation by two members of the TNF receptor family.

Functional Proteomics Identifies TNIK as LMP1 Interaction Partner
We set out to identify novel interaction partners of the LMP1 signaling complex in its native context, the EBV-transformed primary human B-cell. To this end, HA-LMP1-liTEV-CT, an LMP1 variant optimized for proteomics studies, was expressed from a recombinant maxi-EBV genome in lymphoblastoid cells. To generate HA-LMP1-liTEV-CT, an N-terminal hemagglutinin (HA)-tag was added and a tobacco etch virus protease cleavage site coupled to a flexible linker (liTEV) was inserted between the transmembrane domain and the C-terminal (CT) signaling domain of LMP1 ( Figure 1A). TEV protease cleavage after immunoprecipitation of the HA-LMP1-liTEV-CT complex allowed the release of the LMP1 signaling domain and its interaction partners from the beads for further analysis by mass spectrometry. By this approach the background of proteins was reduced which either interacted with the LMP1 N-terminus and/or the transmembrane domain or which unspecifically bound to the beads or antibodies.
Recombinant EBV expressing HA-LMP1-liTEV-CT from the viral LMP1 promoter was used to infect primary B-cells isolated

Author Summary
The germinal center kinase family member TNIK was discovered in a yeast-two-hybrid screen for interaction partners of the adapter proteins TRAF2 and Nck, and here we show it is one of the missing molecular players in two key signaling pathways in B-lymphocytes. We found that TNIK is crucial for the activities of the CD40 receptor on Bcells and its viral mimic, the latent membrane protein 1 (LMP1) of Epstein-Barr virus (EBV). EBV is a human DNA tumor virus that is associated with various malignancies. It targets and transforms B-cells by hijacking the cellular signaling machinery via its oncogene LMP1. In normal Bcell physiology, the CD40 receptor is central to the immune response by mediating B-cell activation and proliferation. TNIK turns out to be an organizer of the LMP1-and CD40-induced signaling complexes by interacting with the TRAF6 adapter protein, well known for its role in linking distinct signaling pathways. Through this mechanism the two receptors depend on TNIK to activate the canonical NF-kB and JNK signal transduction pathways, which are important for the physiological activation of B-cells (a process that enables antibody production), as well as for their transformation into tumor cells. TNIK thus constitutes a key player in the transmission of physiological and pathological signals in human B-cells that might serve as a future therapeutic target against B-cell malignancies.
from human adenoids. The recombinant virus efficiently transformed B-cells into lymphoblastoid cells, which showed typical clumpy LCL growth and green fluorescence due to the expression of a green fluorescence protein (GFP) marker gene located on the recombinant virus genome ( Figure 1B). The clone LCL-TEV.5 was used for proteomics studies. The outgrowth of LCL-TEV cells further proved that HA-LMP1-liTEV-CT was fully functional because an intact LMP1 is mandatory for B-cell transformation by EBV [11]. Moreover, HA-LMP1-liTEV-CT was able to induce signaling as wildtype LMP1 in HEK293 cells ( Figure S1).
HA-LMP1-liTEV-CT was immunoprecipitated from lysates of LCL-TEV.5 cells ( Figure 1C). Parallel precipitations were performed with the lymphoblastoid cell lines LCL 721 expressing wildtype LMP1 and LCL3 expressing HA-tagged LMP1 [35]. Expression levels of the LMP1 proteins were comparable in all three cell types ( Figure 1C). HA-LMP1-liTEV-CT and HA-LMP1 were efficiently immunoprecipitated by anti-HA antibodies. TEV protease cleavage released the signaling domain of HA-LMP1-liTEV-CT but not that of HA-LMP1 ( Figure 1C). The known CTAR1 interaction partner TRAF3 verified functionality of the experimental system. As expected, TRAF3 specifically co-precipitated with both HA-tagged LMP1 variants but was only detected in the TEV eluate of LCL-TEV.5 immunoprecipitations ( Figure 1C). TEV eluates of LCL-TEV.5 immunoprecipitations were analyzed by mass spectrometry as described in Materials and Methods. The identified candidate LMP1 interaction partners included signaling proteins, proteins involved in ubiquitinylation processes, cytoskeletal proteins, and proteins with other or unknown functions. Two peptides identifying the TRAF2-and Nck-interacting kinase (TNIK) were detected in the TEV eluate of LCL-TEV.5, but not of control cells, which indicated that TNIK interacts with the signaling domain of HA-LMP1-liTEV-CT and is thus a novel component of the LMP1 signaling complex (Tables S1 and S2).

LMP1 Recruits TNIK via the CTAR2 Domain
To confirm the interaction between TNIK and LMP1 in lymhoblastoid cells, endogenous TNIK was immunoprecipitated from lysates of LCL 721 cells and analyzed for LMP1 binding 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. doi:10.1371/journal.pbio.1001376.g001 ( Figure 2A). Indeed, endogenous LMP1 specifically co-precipitated with TNIK. Vice versa, immunoprecipitation of LMP1 brought down TNIK (Figure 2A). These experiments verified the results that were previously obtained in the functional proteomics experiment and showed that TNIK is in fact part of the LMP1 signalosome in EBV-transformed B-cells.
Next we asked whether one of the two signaling-active subdomains of LMP1, CTAR1 or CTAR2, mediates the interaction between TNIK and LMP1. Wildtype LMP1 as well as LMP1(AAA) harboring a mutated PxQxT motif within CTAR1, the CTAR2 deletion mutant LMP1D371-386, and the CTAR1/CTAR2 double mutants LMP1(AAA, D371-386) and LMP1(AAA, Y384G) were transiently expressed in HEK293 cells and endogenous TNIK was immunoprecipitated from cell lysates. Immunoblot analysis of the precipitations revealed that wildtype LMP1 and the LMP1(AAA) mutant bound to TNIK equally well, excluding a critical role of CTAR1 for TNIK binding. In contrast, mutation of CTAR2 completely abolished interaction of LMP1 and TNIK, the exchange of tyrosine 384 to glycine being equally effective as the deletion of the 16 C-terminal amino acids of CTAR2 ( Figure 2B). These experiments indicated but did not definitely prove that CTAR2 is the critical domain for LMP1's interaction with TNIK. Therefore, we repeated the experiment with the HA-LMP1-TNFR1-CTAR2 chimera, which consists of the LMP1 transmembrane domain fused to the TNFR1 signaling domain that carries amino acids 371 to 386 of CTAR2 replacing the TNFR1 death domain [35]. Except for CTAR2 residues 371-386, no other sequences of the LMP1 signaling domain are present within the chimera. TNIK readily bound to HA-LMP1-TNFR1-CTAR2 but not the control construct lacking the CTAR2 sequences ( Figure 2C). In summary, these experiments demonstrated that CTAR2 is both critical and sufficient for TNIK recruitment to LMP1, whereas CTAR1 has no apparent role in mediating this interaction.

TNIK Is Essential for JNK Activation by LMP1
Having identified TNIK as a novel CTAR2 interaction partner, we asked whether TNIK has a role in LMP1 signal transduction. The JNK pathway initiates at CTAR2 and TNIK was shown to induce JNK signaling upon overexpression [1,30]. Therefore, we 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 mg of the indicated LMP1 constructs. The D371-386 deletion inactivates the CTAR2 domain. CTAR2 with Y384G mutation has no signaling capacity towards NF-kB 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-TNFR1DDD, 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. doi:10.1371/journal.pbio.1001376.g002 investigated a potential role for TNIK as mediator of LMP1induced JNK signal transduction. HEK293 cells were transfected with TNIK-specific siRNA or non-targeting control siRNA. Subsequently, wildtype LMP1 or the null control LMP1D194-386 were expressed, and HA-JNK kinase assays were performed to monitor LMP1-induced JNK1 activity. The knockdown of TNIK caused a drastic reduction of JNK activation by LMP1 ( Figure 3A). We confirmed this result in the human lymphoblastoid cell line EREB2-5. Upon knockdown of TNIK with siRNA a robust reduction of endogenous JNK phosphorylation, a measure of JNK activity, was detected in EREB2-5 cells ( Figure 3B). Notably, JNK activity in LCLs depends on LMP1 [11,30,47]. We have thus identified TNIK as a novel signaling mediator of LMP1 that is crucial for the induction of the JNK pathway.

LMP1-Induced Canonical NF-kB Signaling Requires TNIK
Canonical NF-kB constitutes the second important signaling pathway that is initiated at the CTAR2 domain of LMP1. CTAR2, but not CTAR1, induces IKKb activity, which is essential for CTAR2-mediated NF-kB signaling [25,35,42]. To test if TNIK is involved in this pathway as well, Flag-IKKb kinase assays were performed in HEK293 cells ( Figure 4A). Endogenous TNIK was depleted by TNIK siRNA, and LMP1 wildtype or the inactive null mutant LMP1(AAA, D371-386) was expressed and tested for their ability to activate IKKb. LMP1 expression in cells treated with control siRNA caused a 2.6-fold induction of IKKb activity, monitored as in vitro GST-IkBa substrate phosphorylation by the immunoprecipitated Flag-IKKb. The knockdown of TNIK almost entirely abolished the activation of IKKb by LMP1, demonstrating the critical importance of TNIK in the canonical NF-kB pathway ( Figure 4A). In order to exclude a role for TNIK in non-canonical NF-kB signaling by LMP1, the effect of a TNIK knockdown on NF-kB p52 was examined. NF-kB p52 activation is a hallmark for CTAR1 signaling [24][25][26][27]. Downregulation of TNIK by a shRNA vector in HEK293 cells did not affect the LMP1-induced p52 translocation to the nucleus, whereas the nuclear shift of canonical p65 was largely inhibited ( Figure 4B).
NF-kB reporter assays were performed in HEK293 cells to test the role of TNIK also at the level of NF-kB-dependent transcription. The siRNA-mediated knockdown of TNIK caused a nearly 50% reduction in NF-kB activation by LMP1 as compared to cells treated with control siRNA ( Figure 4C). Given that a substantial proportion of total LMP1-induced NF-kB activity detected in reporter assays constitutes CTAR1-induced non-canonical NF-kB [28,29], we concluded that knockdown of TNIK largely blocked CTAR2 signaling in the reporter assay. This conclusion was later corroborated by the use of a dominantnegative TNIK mutant that inhibited CTAR2, but not CTAR1, activation of the NF-kB reporter (see Figure 6F). We confirmed our findings by siRNA experiments in EBVtransformed EREB2-5 cells. Knockdown of TNIK in these cells resulted in a marked reduction of phosphorylated IkBa and a concomitant stabilization of IkBa showing that canonical NF-kB signaling is defective upon depletion of TNIK in LCLs ( Figure 4D). TNIK is thus an important signaling mediator of the canonical NF-kB pathway.

TNIK Mediates Proliferation and Survival of EBV-Transformed B-Cells
The LMP1-induced IkB-dependent NF-kB pathway and the JNK pathway are essential for lymphoblastoid cell survival and proliferation [31,32]. Given the important role of TNIK in both pathways, its knockdown should interfere with LCL physiology. To test this hypothesis, TNIK expression was downregulated in EREB2-5 lymphoblastoid cells by siRNA and proliferation was monitored. In fact, TNIK deficiency strongly retarded proliferation of the cells and apoptosis was induced concomitantly ( Figure 5A and 5B, respectively). The spontaneous apoptosis rate in EREB2-5 cells increased by a factor of 3.8 on average after the knockdown of TNIK ( Figure 5B). Accordingly, many dead cells were visible in disintegrating LCL clumps in the siTNIK-treated EREB2-5 culture, whereas the siCTR-treated cells displayed normal LCL morphology ( Figure S2). Thus, TNIK has a critical The JNK and Canonical NF-kB Pathways Bifurcate at the Level of TNIK As TNIK is critically involved in both JNK and canonical NF-kB signal transduction downstream of LMP1, we next asked whether these two pathways might bifurcate at the level of TNIK by dissecting the contribution of individual TNIK domains to the activation of JNK and NF-kB signaling. A set of HA-tagged TNIK constructs was generated that comprise full-length TNIK, the kinase domain (KD), the germinal center kinase homology domain (GCKH), as well as the DKD and DGCKH deletion mutants ( Figure 6A). Additionally, a kinase-negative mutant (KM) of TNIK was used, which carries a mutation of the conserved lysine 54 HEK293 cells were transfected in 6-well plates with TNIK siRNA or non-targeting siRNA. Subsequently, the cells were co-transfected with 1 mg each of HA-LMP1 wildtype or inactive HA-LMP1(AAA, D371-386) together with 1 mg of Flag-IKKb, as indicated. Flag-IKKb was immunoprecipitated and its activity was monitored in in vitro kinase assays using purified GST-IkBa as a substrate. Immunoprecipitated Flag-IKKb, expression of LMP1 constructs, and downregulation of TNIK were detected in lysates using the anti-IKKb, anti-LMP1 ( As indicated, 4 mg 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-kB 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-kB 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 mg of expression vectors for HA-LMP1(AAA, D371-386) or HA-LMP1 wildtype together with 5 ng of the NF-kB reporter 3xkBLuc and Renilla control vector. 24 h posttransfection cells were lysed and NF-kB 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 6 standard deviations; statistics, two-tailed Student's t test. *p = 0.006. (D) The downregulation of TNIK in lymphoblastoid cells impairs the canonical NF-kB pathway. Identical samples of siTNIK or siCTR-treated EREB2-5 LCLs as shown in Figure 3B were blotted for phospho-IkBa and IkBa. Control immunoblots for TNIK and LMP1 expression are displayed again for better comparison. n = 3. doi:10.1371/journal.pbio.1001376.g004 residue in the ATP-binding pocket of the kinase domain [1]. We then tested for the ability of the individual TNIK constructs to induce canonical NF-kB signaling in IKKb kinase activity assays. Wildtype TNIK activated IKKb-dependent phosphorylation of GST-IkBa by a factor of 4.7-fold ( Figure 6B). Notably, expression of the TNIK kinase domain alone was sufficient to fully induce IKKb as TNIK-KD caused an 11-fold activation of IKKb. Vice versa, mutation or deletion of the kinase domain completely abolished TNIK's potential to activate IKKb. In contrast, neither deletion of the GCKH domain nor its overexpression had any effect on IKKb activation. In line with these results, the exogenous expression of TNIK wildtype or TNIK-KD was sufficient to also induce the nuclear translocation of canonical NF-kB p65, whereas non-canonical NF-kB p52 remained unaffected ( Figure 6C). This finding further corroborated our previous observations that TNIK has no function in non-canonical NF-kB signaling (see above). As expected, TNIK-KM was unable to shift any of the two NF-kB proteins to the nucleus (unpublished data). In summary, we concluded that the TNIK kinase domain and in particular its kinase activity is critical for canonical NF-kB induction by TNIK, while the GCKH domain is dispensable for this pathway.
Notably, JNK activation maps to a region of TNIK different from the NF-kB-activating kinase domain. The GCKH domain alone activates JNK as efficiently as full-length TNIK, whereas mutation of the kinase domain had no effect on TNIK's ability to induce JNK as determined by kinase assays in HEK293 cells ( Figure 6D). This finding is consistent with previous results showing that the GCKH domain alone can induce the JNK pathway whereas the TNIK kinase domain is dispensable [1]. Taken together, JNK and IKKb induction map to different TNIK domains, suggesting that TNIK constitutes the point of bifurcation of these two pathways.
Next we asked about the functional role of the TNIK kinase domain in IKKb/NF-kB activation. One straightforward scenario would be that TNIK phosphorylates IKKb for its activation. However, we did not detect direct IKKb phosphorylation by TNIK in our experimental systems, for instance in TNIK kinase assays using IKKb as a substrate (unpublished data). Previous studies demonstrated that TNIK phosphorylates itself [1,3]. Therefore, we investigated if LMP1 expression affects TNIK autophosphorylation. In fact, LMP1 enhanced the phosphorylating activity of TNIK versus itself by a factor of 4.2-fold, demonstrating a role for TNIK autophosphorylation in LMP1 signaling ( Figure 6E). The vast majority of the about 40 Ser/Thr phosphorylation sites of TNIK detected so far in vivo by mass spectrometry are located within the intermediate domain (databank: www.phosphosite.org; search term: TNIK). If the TNIK kinase domain phosphorylates TNIK within its intermediate domain and TNIK autophosphorylation is critical for NF-kB signaling, the exogenously expressed TNIK kinase domain alone would be non-functional but depend on endogenous wildtype TNIK to activate IKKb. To test this possibility, HEK293 cells were depleted of endogenous wildtype TNIK by siRNA. Subsequently, the construct HA-TNIK-KDwob was transfected, which expresses the wildtype TNIK kinase domain, and IKKb kinase assays were performed. As the HA-TNIK-KDwob construct carries silent wobble mutations at the nucleotide level, it is not targeted by TNIK-specific siRNA. The knockdown of endogenous TNIK abolished the potential of the exogenous TNIK kinase domain to activate IKKb ( Figure S3). A similar mechanism for JNK activation can be excluded because the kinase domain is dispensable for JNK signaling (see Figure 6D) and TNIK-KD overexpression does not induce JNK in HEK293 cells [1]. Taken together, these findings are in line with the concept of a role for TNIK autophosphorylation in NF-kB signaling by LMP1.
To further validate the importance of the TNIK kinase domain for canonical NF-kB signaling, we tested if overexpression of the kinase-negative mutant TNIK-KM had a dominant-negative effect on LMP1-induced NF-kB signaling in reporter assays ( Figure 6F). In fact, TNIK-KM expression reduced NF-kB activation by wildtype LMP1 to almost 50%, a factor that was comparable to the effect of TNIK knockdown on LMP1-induced  NF-kB (see Figure 4C). Moreover, NF-kB signaling of LMP1(AAA), which only harbors functional CTAR2, was affected by TNIK-KM but not that of LMP1D371-386, which solely signals via CTAR1 ( Figure 6F). Thus, TNIK-KM exerted its dominant-negative effect on CTAR2-induced NF-kB signaling, confirming that the kinase activity of TNIK is critical for activation of canonical NF-kB by LMP1-CTAR2.

TRAF6 Directly Binds TNIK and Mediates TNIK Interaction with LMP1
To better understand TNIK's molecular functions in JNK and NF-kB activation and its role as bifurcation point of the two pathways, it was necessary to identify TNIK interaction partners in LMP1 signaling. The first step was to investigate how TNIK interacts with LMP1 and to characterize potential mediators of this interaction. TNIK has been shown to bind TRAF2 via its intermediate domain [1]. This finding suggested that TRAF molecules might physically couple TNIK to upstream inducers/ receptors. CTAR2 signaling to JNK and IKKb/NF-kB essentially requires TRAF6 but not TRAF2 [18,33,34,38,48]. Despite the fact that an interaction of TRAF6 with TNIK has not been described so far, we tested if TRAF6 binds to TNIK in LMP1 signaling by immunoprecipitation experiments in HEK293 cells ( Figure 7A). In the absence of LMP1 a weak co-precipitation of HA-TNIK and Flag-TRAF6 was detected. Strikingly, LMP1 induced a very strong interaction of both proteins, demonstrating (i) that TRAF6 is a novel binding partner of TNIK and (ii) that interaction of both proteins is greatly enhanced upon activation ( Figure 7A). The effects of LMP1 on TNIK-TRAF interaction were, however, not restricted to TRAF6. CTAR2, but not CTAR1, induced a weak but detectable interaction of TNIK with TRAF2 ( Figure S4). Because studies in TRAF2-deficient cells have clearly excluded a critical function for TRAF2 in CTAR2 signaling [18,33,48], we concentrated our further studies on the newly identified and CTAR2-critical TNIK interaction partner TRAF6.
The TNIK intermediate domain directly binds TRAF2, as has been shown by yeast-two-hybrid assays and immunoprecipitations [1]. To determine whether TRAF6 and TNIK are also direct interaction partners, in vitro binding assays using recombinant proteins purified from bacteria were performed ( Figure 7B). Indeed, the C-terminal TRAF domain of TRAF6 (amino acids 310-522) specifically bound to the immobilized GST-tagged TNIK intermediate domain. Purified TRAF2 (amino acids 311-501) was included into the experiment as a control, which also interacted with the intermediate domain of TNIK. No interaction of the two TRAFs with the TNIK kinase domain, the GCKH domain, or the GST control beads was observed. Thus, the C-terminal TRAF domain of TRAF6 directly binds to the TNIK intermediate domain.
In order to investigate whether TRAF6 acts as mediator of the interaction between TNIK and LMP1 we analyzed the subcellular localization of transiently expressed HA-TNIK and LMP1 in TRAF6-deficient and wildtype mouse embryonic fibroblasts. Confocal immunofluorescence microscopy revealed a high degree of co-localization of TNIK and LMP1 in the TRAF6+/+ cells ( Figure 7C). LMP1 did not induce translocation of TNIK into the nucleus as it has been shown for Wnt signaling in intestinal cells [5]. There was no significant co-localization of LMP1 and TNIK in TRAF62/2 cells. This finding was substantiated by a grey scale line scan analysis of the microscopic images confirming that the distribution of TNIK and LMP1 displays a high degree of colocalization in wildtype cells. In contrast, the absence of TRAF6 caused a more dispersed localization of TNIK and prevented its recruitment to LMP1 ( Figure 7C). This result showed that TNIK and LMP1 interact in an indirect manner and that TRAF6 is crucial for this interaction. To verify this finding by a biochemical approach we performed a rescue experiment in TRAF62/2 cells. LMP1 and Flag-TNIK were expressed in TRAF62/2 cells in the absence or presence of exogenously expressed TRAF6 ( Figure 7D). LMP1 co-precipitated with Flag-TNIK only when TRAF6 was transfected. Exogenous TRAF6 expression was thus able to rescue the interaction between TNIK and LMP1 in TRAF6-deficient cells. Taken together we revealed TRAF6 as a novel direct interaction partner of the TNIK intermediate domain and as critical mediator of the interaction between TNIK and LMP1.
TNIK Forms a Dynamic Signaling Complex with TAK1, TAB2, and IKKb TAK1 interacts via TAB2 with TRAF6 to activate JNK and IKKb/NF-kB signaling (see Introduction). Previous studies have shown that TAK1 mediates JNK signaling by LMP1, whereas the role of TAK1 in NF-kB activation is controversial [37,40,42]. Having defined a role for TNIK as an interaction partner of TRAF6 in JNK and canonical NF-kB signaling by LMP1, we asked whether TAK1 and TAB2 interact with TNIK as well. Indeed, TNIK and TAK1 readily interacted in HEK293 cells ( Figure 8A). As the presence or absence of LMP1 had no striking effect on the affinity of both proteins we concluded that TNIK and TAK1 bind to each other constitutively. We next analyzed this interaction with regard to the TNIK domains that mediate TAK1 binding by using TNIK deletion constructs for immunoprecipitations ( Figure 8B). Whereas the GCKH domain alone bound to TAK1, no interaction was detectable with the TNIK kinase domain. Deletion of the GCKH domain (HA-TNIK-DGCKH construct) strongly diminished the interaction with TAK1. The main TAK1 interaction site of TNIK is thus the GCKH domain and the intermediate domain contributes some binding activity as well, possibly by an indirect mechanism. It is important to note at this point that the GCKH domain of the MAP4K TNIK induces JNK signaling (see Figure 6D) and at the same time binds the critical MAP3K for this pathway, TAK1.
Co-immunoprecipitation experiments in HEK293 cells showed that TAB2 also specifically co-precipitates with TNIK ( Figure 8C). However, this interaction is activation-dependent, as TAB2 did only very weakly bind to TNIK unless LMP1 was present. LMP1 co-expression induced a strong interaction of TNIK with TAB2.  Notably, TNIK is required for the interaction of TAK1/TAB2 with the LMP1 complex. The knockdown of endogenous TNIK by expression of shRNA abolished binding of TAK1 and TAB2 to LMP1 in co-immunoprecpitation experiments ( Figure 8D and 8E, respectively). Thus, TNIK has an important role in the assembly of the LMP1 signalosome by acting as an interaction mediator of critical components of the complex.
We have shown that LMP1 activates IKKb via TNIK. Therefore, we asked whether IKKb is also a component of the TNIK signaling complex. Indeed, IKKb also bound to TNIK, albeit only in the presence of LMP1 ( Figure 8F). The interaction of TNIK with IKKb appeared to be weaker as compared to TRAF6, TAK1, or TAB2, potentially indicating an indirect recruitment of IKKb to TNIK. In summary, we found that TNIK forms a dynamic complex incorporating critical components of TRAF6dependent JNK and NF-kB signaling, namely TRAF6, TAK1/ TAB2, and IKKb. TAK1 seems to be constitutively associated with TNIK, whereas the other components enter the complex after activation.

A Complex of TNIK with LMP1, TRAF6, TAK1/TAB2, and IKKb Exists in EBV-Transformed Human B-Cells
We sought to verify the existence of an endogenous TNIK signaling complex in lymphoblastoid cells that endogenously express LMP1. TNIK was immunoprecipitated from LCL 721 cell lysates and components of the signaling complex were analyzed by immunoblotting ( Figure 8G). We found that LMP1, TRAF6, TAK1, TAB2, and IKKb specifically bind to TNIK in LCLs, thus proving the existence of the LMP1-induced TNIK signaling complex in its native context. Taken together, our results show that the TNIK complex, which is composed of TRAF6 and LMP1 as upstream components and of TAK1/TAB2 and IKKb as downstream mediators, is essential for JNK and canonical NF-kB activation by LMP1 in EBV-transformed human B-cells.

TNIK Is Critical for JNK and Canonical NF-kB Signaling by CD40
Having characterized TNIK as a mediator of signal transduction by the viral pseudoreceptor LMP1, we tested a general requirement for TNIK in JNK and canonical NF-kB activation by a cellular receptor in B-cells. Because LMP1 is a functional mimic of CD40 and TRAF6 plays a pivotal role as an adapter protein for both LMP1 and CD40, we tested whether CD40 engages TNIK for signal transduction.
First we analyzed the effect of TNIK knockdown on JNK1 and IKKb activation by CD40 in HEK293 cells. Overexpression of CD40 was sufficient to activate CD40 signaling in HEK293 cells without the need to further stimulate with CD40L (CD40 ligand). TNIK was downregulated by siRNA and cells were co-transfected with either HA-JNK or Flag-IKKb and CD40 expression vectors. HA-JNK and Flag-IKKb kinase assays proved that the downregulation of TNIK in fact blocked CD40-induced JNK and IKKb activation ( Figure 9A and 9B, respectively).
In order to confirm these results in human B-cells, BL41 cells were depleted of endogenous TNIK by siRNA and stimulated with recombinant soluble CD40L ( Figure 9C). The knockdown of TNIK resulted in a nearly complete inhibition of CD40-induced JNK phosphorylation, demonstrating an important role of TNIK in JNK activation by CD40 also in B-cells. IkBa degradation after 10 to 20 min of CD40 stimulation indicated activation of the NF-kB pathway when cells were treated with non-targeting control siRNA. In contrast, after TNIK downregulation by siRNA the NF-kB pathway did not respond to CD40 stimulation as IkBa levels did not decrease over time ( Figure 9C). These data demonstrated that TNIK is a novel and critical intermediate of endogenous CD40 signaling in human B-cells on the JNK and NF-kB axes.
CD40 stimulation activates BL41 cells, detectable as upregulation of activation markers at the cell surface such as CD54, an adhesion molecule also known as ICAM-1 and hallmark of B-cell activation [49]. CD54 upregulation by CD40 is dependent on canonical NF-kB in BL cells [50]. We tested if the knockdown of TNIK affected CD54 surface upregulation by CD40 ligand stimulation of BL41 cells. TNIK dowregulation resulted in a marked decrease of CD40induced CD54 surface levels, demonstrating an important role for TNIK also in B-cell activation ( Figure 9D).
TRAF6 is an essential signaling mediator of both LMP1 and CD40, and we have demonstrated recruitment of TRAF6 to TNIK in the context of LMP1 signaling. Therefore we asked whether CD40 stimulation can also induce an interaction between TNIK and TRAF6 in B-cells. BL41 cells were stimulated with CD40L for 0, 5, and 15 min and TNIK was immunoprecipitated and tested for TRAF6 co-precipitation. We observed that CD40 induced an interaction between endogenous TNIK and endogenous TRAF6 already 5 min after stimulation ( Figure 9E). Ten minutes later the majority of TRAF6 had already dissociated from TNIK. The prompt interaction between TNIK and TRAF6 in response to CD40 stimulation demonstrates a role for the TNIK-TRAF6 complex in the context of CD40 signaling, suggesting that interaction of both molecules is a key step in signaling by LMP1 and CD40. Taken together we have identified TNIK as an important mediator of JNK and also canonical NF-kB in physiological CD40 stimulation.

Discussion
In this study we have identified and characterized the germinal center kinase family member TNIK as a novel component of the TRAF6/TAK1/TAB2/IKKb complex. TNIK is required for JNK and canonical NF-kB signaling by the EBV oncoprotein LMP1 and its cellular counterpart CD40. According to this critical function in signaling, TNIK has an important role in mediating proliferation and survival of EBV-transformed B-cells and in physiological B-cell activation by CD40. In an unbiased functional proteomics screen TNIK was isolated as an interaction partner of the LMP1 complex in EBV-infected primary human B-cells. TNIK binding to the CTAR2 domain of LMP1 is mediated by TRAF6, a newly described direct interaction partner of TNIK. We thus report the first molecular function for TNIK's interaction with TRAF molecules. The existence of a CTAR2-induced signaling complex was revealed involving activation-dependent binding of TRAF6, TAB2, and IKKb to TNIK. Importantly, CD40 stimulation also induces association of TNIK with TRAF6. Because TNIK's activities in JNK1 and NF-kB signaling map to different TNIK domains, we propose a model in which TNIK orchestrates bifurcation and signal transmission of both pathways at the level of the TRAF6/TAK1/TAB2/IKKb complex ( Figure 10). Our discovery that TNIK is a new key player in TRAF6-dependent   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 IKKb to the TNIK complex. HEK293 cells were transfected in 10 cm culture dishes with 2 mg HA-TNIK, 2 mg Flag-IKKb, and 3 mg 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-IKKb, 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 IKKb 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-IKKb antibodies. n = 2. doi:10.1371/journal.pbio.1001376.g008 JNK and canonical NF-kB signaling significantly extends the current concept of molecular regulation of these pathways. LMP1 is constitutively active and closely mimics the TNFR family member CD40 in B-cell activation [11]. Despite differences in the molecular composition and efficiency of their signaling complexes, LMP1 and CD40 share similarities with regard to the engagement of TRAF molecules and the pattern of activated signal transduction pathways [14]. TRAF6 plays a pivotal role in canonical NF-kB and JNK signaling by both receptors [33,37,38,51]. Both LMP1 and CD40 induce association of TNIK with TRAF6, whose interaction is direct and involves the TRAF domain of TRAF6 and the intermediate domain of TNIK. This interaction couples TNIK to the upstream receptor. Apart from TRAF6, TNIK can also interact with TRAF2. However, TRAF2 is dispensable for JNK activation, IkB-dependent NF-kB signaling, and p65 nuclear translocation by LMP1 and can thus be excluded as an essential mediator of TNIK interaction with CTAR2 [33,34,48]. The CTAR2-induced association of TNIK and TRAF2 detected here might have a non-essential accessory role in CTAR2 signaling. Also the CTAR2-interacting factor TRADD is unlikely to play a central role in TNIK recruitment because it is exclusively involved in IKKb/NF-kB activation by CTAR2 but not in JNK signaling, whereas TNIK is required for both signaling pathways [35]. Upon activation by LMP1, TAB2 and IKKb are recruited to TNIK. This observation of activation-induced complex formation is in line with findings that the dynamic association of TAB2 with TRAF6 and TAK1 also occurs in other pathways, for instance in interleukin-1 signaling [52]. TAK1, in contrast, interacts constitutively with TNIK. We therefore propose that upon activation the TNIK-TAK1 complex is recruited to the CTAR2 domain via TRAF6 and recruits additional downstream signaling mediators such as TAB2 and IKKb. The signaling complex is likely further stabilized by TRADD, which is involved in the recruitment of IKKb to the LMP1 complex [35]. The TRADD-dependent stabilization of the complex at CTAR2 might in part explain the more efficient signaling complex of LMP1 as compared to CD40. In contrast to LMP1, CD40 induction of JNK and IkB phosphorylation involves TRAF2 in B-cells [53,54]. Moreover, TRAF2 is involved in TRAF6 recruitment to the distal TRAF binding site of CD40 that induces JNK and canonical NF-kB signaling [45]. For these reasons, a more pronounced role of TRAF2 in TNIK interaction with CD40 seems feasible. Future studies will have to dissect the precise role of TRAF family members in coupling TNIK to CD40. However, because CD40 induces a rapid interaction of TNIK with TRAF6, we suggest a critical role of TRAF6 in this process, which could involve additional members of the TRAF family.
The TNIK-TRAF6-TAK1/TAB2-IKKb complex mediates activation of the canonical NF-kB and JNK pathways. TNIK is, to our knowledge, the only known protein within this complex whose activities on the NF-kB and JNK axes are clearly allocated to separate domains of the same protein. NF-kB activation depends on the kinase and intermediate domains of TNIK, whereas signaling to JNK only involves the GCKH domain. Thus, TNIK seems to constitute the molecular organizer of JNK and NF-kB bifurcation. It has been shown that purified TAK1 together with TAB1/TAB2 is sufficient to phosphorylate and thus activate IKKb in a test tube. This reaction further depends on TRAF6 and Ubc13/Uev1A [46,55]. Our results demonstrate that TNIK is additionally required to assemble, organize, and activate the holocomplex in vivo and to recruit the complex to the receptor (here: LMP1) by acting as an adapter and scaffolding protein. Due to its interaction with TRAF molecules TNIK is likely involved in the specific coupling of the TAK1-TAB2 and IKK modules to distinct receptors.
The GCKH domain of the MAP4kinase TNIK mediates JNK activation and is also the main interaction site of the JNK-inducing MAP3kinase TAK1, suggesting that TNIK acts directly upstream of TAK1 in the signaling cascade. However, TNIK's kinase activity is dispensable for JNK activation. Similar to other germinal center kinases, TNIK may facilitate MAP3kinase activation by inducing conformational changes that induce MAP3K autophosphorylation and thus activation of the MAP3K [2,56].
In contrast to the JNK pathway, activation of IKKb by TNIK critically depends on the kinase activity of TNIK. IKKb itself does not appear to be a TNIK substrate because we could not detect direct IKKb phosphorylation by TNIK (unpublished data). However, LMP1 induces TNIK's activity to phosphorylate itself, supporting a role for TNIK autophosphorylation in signaling. The fact that exogenous TNIK-KD is incapable of activating IKKb in the absence of endogenous TNIK suggests that phosphorylation is important for NF-kB activation but localizes to a domain other Figure 10. Model of TNIK's role in JNK and canonical NF-kB signaling. LMP1 induces the formation of the TNIK complex at its JNK1 and NF-kB-inducing CTAR2 domain. Interaction of TNIK with LMP1 is mediated by TRAF6, which forms an induced complex with TNIK. Also TAB2 and IKKb are recruited upon activation, whereas TAK1 is permanently complexed with TNIK. In LMP1 signaling, IKKb recruitment is further enhanced or stabilized by a TRADD-dependent mechanism. TNIK organizes bifurcation of the JNK and NF-kB pathways downstream of TRAF6. Auto-phosphorylation of TNIK likely constitutes an important step in transmitting signals on the NF-kB 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 IKKb/NF-kB. For more details, see the text. doi:10.1371/journal.pbio.1001376.g010 than the kinase domain of TNIK. A similar mechanism for JNK signaling is not likely because TNIK-KD is dispensable for JNK activation and TNIK-KD overexpression does not induce JNK (see Figure 6D) [1]. Multiple phosphorylation sites cluster within TNIK's intermediate domain (www.phosphosite.org) [1,3], which is the likely target of TNIK's autophosphorylation. Phosphorylation of TNIK seems to have different effects. One study reported that autophosphorylated TNIK is found in the cytoskeletal fraction, where it mediates disassembly of F-actin [3]. Wnt/bcatenin signaling leads to the phosphorylation of TNIK at serine 764 and translocation of TNIK into the nucleus, where it interacts with TCF4 to mediate activation of Wnt target genes [5,6]. Using a phophosite-specific antibody we observed that LMP1 does not induce phosphorylation of TNIK at serine 764 (unpublished data), which is consistent with the fact that LMP1 does not induce Wnt signaling [57]. We consider the relevance of a different phosphorylation site within TNIK in the context of canonical NF-kB signaling. It will be the focus of future studies to identify TNIK autophosphorylation sites as well as possible other TNIK substrates in the NF-kB pathway, for instance by phosphoproteomics.
Due to its universal expression pattern we envision that TNIK functions as a mediator of TRAF-dependent JNK and NF-kB activation in various tissues and cell types. So far, TNIK has been described to regulate neurite growth and neuronal morphology in the brain and to be involved in the activation of Wnt target genes in intestinal crypt cells [5,6,8,58]. Here we extend the functions of TNIK to lymphocytes. Our data indicate important roles for this kinase in B-cell function, immunity, and cancer. JNK and NF-kB have pivotal roles in physiological activation and oncogenic transformation of B-cells [45,59,60]. LMP1 and CD40 are involved in various malignant diseases of the hematopoietic system, such as Hodgkin's and non-Hodgkin's lymphoma, posttransplant lymphoproliferative disease, or chronic lymphocytic leukemia, as well as in non-hematopoietic cancers such as nasopharyngeal carcinoma or renal carcinoma [60][61][62]. Notably, the LMP1-induced canonical NF-kB and JNK pathways are known to be essential for LMP1-mediated B-cell transformation by activation of anti-apoptotic and cell cycle-promoting signals [31,32,63]. Accordingly, we showed that TNIK is essential for lymphoblastoid proliferation and survival. CD40-induced NF-kB, which also involves TNIK, protects cells from apoptosis in some low-grade B-cell malignancies and promotes cell transformation of epithelial cells, for instance in breast cancer [62,64]. Thus, our data implicate TNIK in LMP1-and CD40-induced cancer and indicate the potential of TNIK as a future target for therapy of EBV and CD40-associated malignancies.

Cell Culture Methods
The EBV-positive lymphoblastoid B-cell lines LCL 721, EREB2-5, and LCL3 have been described [35,65,66]. The generation of LCL-TEV.5 cells is described herein. HEK293 human embryonic kidney cells, the human EBV-negative Burkitt's lymphoma B-cell line BL41 [67], and all lymphoblastoid cell lines were grown in RPMI full medium (Invitrogen) supplemented with 10% fetal calf serum (Biochrom AG). EREB2-5 cells were additionally kept in the presence of 1 mM b-estradiol to activate the conditional ER-EBNA2 transcription factor that drives EBV latent genes required for proliferation of EREB2-5 cells [66]. Wildtype and TRAF62/2 mouse embryonic fibroblasts [51] were grown in DMEM (Invitrogen) supplemented with 10% fetal calf serum. BL41 cells were stimulated with the indicated amounts of human recombinant soluble CD40 ligand (Source BioScience).

RNA Interference
HEK293 cells were seeded in 6-well plates and transfected twice within 24 h with 100 nM of human ON TARGETplus SMARTpool TNIK siRNA (pool of four siRNAs J-004542-10 to 13, Dharmacon) or corresponding ON TARGETplus non-targeting control siRNA using the Dharmafect transfection reagent according to the manufacturer's protocol. The cells were transfected 24 h later with the indicated plasmids using Polyfect transfection (Qiagen) and analyzed 24 h after the last transfection. To achieve TNIK knockdown in larger cell culture dishes, HEK293 cells were co-transfected with pSM2-shTNIK (RHS1764-949310, Open Biosystems), an expression vector for short hairpin RNA targeting TNIK, or the non-targeting control vector pSM2-shControl (RHS1707-OB, Open Biosystems) as indicated in the figure legends. LCL 721, EREB2-5, and BL41 cells were incubated with 5 mM Accell SMARTpool TNIK siRNA (pool of four Accell siRNAs J-004542-18 to 21, Dharmacon) or Accell non-targeting pool siRNA for 72-96 h in serum-free Accell delivery medium tively, for 25 min at 26uC. Substrates were included in the reaction buffer mix and their concentrations were not rate limiting for the phosphorylation reaction at the given reaction conditions [70]. For TNIK autophosphorylation assays, HA-TNIK was immunoprecipitated using the anti-HA (12CA5) antibody, beads with precipitated HA-TNIK were washed thoroughly, and the kinase reaction was performed in kinase reaction buffer lacking any other substrate. Kinase reactions were terminated by denaturating samples in Laemmli-DTT buffer. Subsequently samples were subjected to SDS-PAGE and autoradiography. Radioactive signals were quantified using the Fuji FLA-5100 phosphoimager.

Cell Proliferation, Apoptosis, and Flow Cytometry
For cell proliferation assays, 2610 4 EREB2-5 cells per well of a 96-well plate were seeded at day zero in triplicates in Accell delivery medium supplemented with 1 mM b-estradiol and 5 mM Accell SMARTpool TNIK or Accell non-targeting siRNA. Proliferation was monitored at the indicated times by 3-(4,5-dimethylthiazol-2yl)-2,2,5-diphenyl tetrazolium bromide (MTT) conversion as described [11]. Apoptosis was assayed by staining of the cells with propidium iodide (PI) and Cy5-labeled Annexin V using the Apoptosis Detection Kit (Biocat) and subsequent flow cytometry analysis with the Becton Dickinson FACSCalibur flow cytometer. For detection of CD40-induced CD54 surface expression, 5610 4 BL41 cells were seeded per well of a 24-well plate in Accell delivery medium supplemented with 5 mM Accell siRNA. At day 1 and 2, the cells were stimulated with 1 mg/ml CD40 ligand or left untreated in the presence of 2% fetal calf serum. At day 3, surface CD54 was stained with an APC-conjugated anti-CD54 antibody (ImmunoTools) and detected by flow cytometry. Flow cytometry data were analyzed with the FlowJo software (TreeStar).
Immunofluorescence Confocal Microscopy 1.5610 6 mouse embryonic fibroblasts were electroporated using a BioRad Gene Pulser II at 240 V and 950 mF with 2 mg each of pSV-LMP1 and pRK5-Flag-TNIK. Total transfected DNA was adjusted to 20 mg with empty vector. After transfection cells were seeded onto glass coverslips and cultivated overnight. Cells were subsequently fixed with 2% paraformaldehyde (Merck) for 15 min, permeabilized with 0.15% Trtion X-100 (Sigma) in PBS three times for 5 min, and then blocked three times for 10 min with blocking solution (PBS, 1% bovine serum albumin, 0.15% glycine). Cells were then incubated with the primary antibody in blocking solution for 2 h at room temperature. After washing once with PBS and twice with PBS containing 0.15% Triton X-100 cells were blocked for 7 min in blocking solution. Subsequently cells were incubated with secondary antibody diluted 1:200 in blocking solution for 45 min at room temperature. The following primary antibodies were used: TNIK (mouse, BD Biosciences) and LMP1 (rat, 1G6-3). The following secondary antibodies were used: CY3-conjugated goat-anti-mouse IgG (H+L) and FITC-conjugated goat-anti-rat IgG (H+L) (both: Dianova). Images were acquired with a Leica TCS SP2 confocal laser scanning microscope fitted with a 6361.4 HCX Plan Apo blue objective. The acquired digital images were deconvoluted and evaluated with Huygens Essential Suite 3.2 software (Scientific Volume Imaging). Colocalization events were further analyzed with grey scale signal intensity line scans.

Reporter Assays
HEK293 cells were transfected in 6-well plates with the indicated constructs and 5 ng of the NF-kB luciferase reporter 3xkBLuc [28] together with 50 ng of a pPGK-Renilla housekeeping gene reporter construct using Polyfect transfection (Quiagen). Twenty-four hours post-transfection, cells were lysed in reporter lysis buffer and firefly and renilla luciferase activities were measured using the Dual-Luciferase reporter assay kit (Promega). Luciferase activities were normalized for renilla activities to standardize for transfection efficiency.

Protein Purification and In Vitro Binding Assays
His-tagged TRAF domains of TRAF2 (amino acids 311-501) and TRAF6 (amino acids 310-522) were expressed in E. coli BL21 Codon Plus RIPL cells (Stratagene) from pET17b vectors. Protein expression was induced by induction at an OD 600 of 0.8 with 0.1 mM isopropyl-b-D-1-thiogalactopyranoside at 20uC overnight. Bacteria were lysed by sonication in 50 mM phosphate buffer, pH 8.0, supplemented with 10 mM imidazole, 300 mM NaCl, 1 mg/ml lysozyme, and Roche complete proteinase inhibitor cocktail. Cleared lysates were incubated with Ni 2+ -NTA agarose (Qiagen) to bind His-tagged TRAF proteins. Subsequently, His-tagged proteins were washed with 50 mM phosphate buffer, pH 8.0, 300 mM NaCl and increasing imidazole concentrations (20 to 100 mM), and eluted from Ni 2+ -NTA agarose with 50 mM phosphate buffer, pH 7.4, 300 mM NaCl, and 500 mM imidazole. Eluted TRAF proteins were further purified by gel filtration on a DextraSEC PRO10 column (Applichem) and the buffer was exchanged to TBS, pH 7.4, 20% glycerol. Proteins were either directly used for experiments or stored at 220uC for up to 4 wk. For in vitro protein binding assays, 1 mg of His-TRAF2(311-501) or His-TRAF6(310-522) were incubated for 1 h at 4uC in 500 ml TBS, pH 7.4, 0.1% (w/v) BSA, 0.5% Tween 20, with immobilized GST-TNIK-KD(KM), GST-TNIK-IMD, GST-TNIK-GCKH or GST, purified from E. coli and coupled to glutathione sepharose (GE Healthcare). Beads were washed 3 times with TBS containing 0.1% Tween 20 and bound His-TRAF proteins were analyzed by immunoblotting. Figure S1 HA-LMP1-liTEV-CT induces signaling. HEK293 cells were transfected with expression vectors for HA-LMP1 and HA-LMP1-li-TEV-CT. HA-LMP1(AAA, Y384G) harboring mutated CTAR1 and CTAR2 domains served as null control. Co-transfected HA-JNK1 was immunoprecipitated and in vitro JNK1 assays were performed using GST-c-Jun as a substrate. Immunoprecipitated HA-JNK1 was detected using the anti-JNK1(C-17) antibody, and the expressed LMP1 constructs were detected by the anti-LMP1(CS1-4) antibody. (TIF) Figure S2 Effects of TNIK knockdown on EREB2-5 morphology. Microscopic images of the experiment shown in Figure 5B taken at day 3 after TNIK knockdown. Two representative images are shown each for Accell siTNIK and Accell siCTR treatment. The siCTR samples show typical LCL clusters containing mostly healthy and rounded cells. In contrast, the knockdown of TNIK caused the appearance of many apoptotic or dead cells and the disintegration of LCL clusters.
(DOC) Table S2 TNIK identification by mass spectrometry. The significance threshold for Mascot search (MOWSE score p value 0.05) was 28 and corresponds to a protein score confidence interval (C.I.) of 95%. (DOC)