NFκB Signaling Is Induced by the Oncoprotein Tio through Direct Interaction with TRAF6*

The transcription factor NFκB is a major regulator of genes involved in inflammation and oncogenesis. NFκB is induced upon stimulation of cellular receptors coupled to different intracellular signaling molecules. Further downstream, TRAF6 links at least two receptor pathways to take control of IκB, the administrator of NFκB activity. Here we report on a strong NFκB activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NFκB induction by Tio is independent of Src-kinase interaction and tyrosine phosphorylation of Tio. Mutation of a glutamic acid-rich motif at the N terminus of Tio, corresponding to a TRAF6 consensus binding motif, completely abrogated NFκB activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NFκB activation. Furthermore, we provide evidence that TRAF6 directly binds to the Tio oncoprotein. Identification of TRAF6 as the direct target of Tio describes a novel mechanism for the constitutive activation of NFκB through an oncoprotein.

The transcription factor NFB is a major regulator of genes involved in inflammation and oncogenesis. NFB is induced upon stimulation of cellular receptors coupled to different intracellular signaling molecules. Further downstream, TRAF6 links at least two receptor pathways to take control of IB, the administrator of NFB activity. Here we report on a strong NFB activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NFB induction by Tio is independent of Src-kinase interaction and tyrosine phosphorylation of Tio. Mutation of a glutamic acid-rich motif at the N terminus of Tio, corresponding to a TRAF6 consensus binding motif, completely abrogated NFB activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NFB activation. Furthermore, we provide evidence that TRAF6 directly binds to the Tio oncoprotein. Identification of TRAF6 as the direct target of Tio describes a novel mechanism for the constitutive activation of NFB through an oncoprotein.
NFB plays an important role in oncogenesis by promoting genes that have an influence on cell proliferation, cell cycle, differentiation, and apoptosis. It also triggers both innate and adaptive immune responses by transcriptional activation of genes coding for cytokines, chemokines, and adhesion molecules (1,(1)(2)(3)(4)(5)(6). Three classical signaling pathways leading to the activation of NFB are initiated by the stimulation of different receptors. Antigen recognition by the T cell receptor induces NFB by recruiting protein tyrosine kinases Lck and Zap70 to the receptor complex. The signal is processed by several intermediate proteins, including protein kinase C (PKC) 2 and a multimolecular module containing CARMA, Bcl-10, MALT1, and TRAF6. Subsequently, the IB kinase (IKK) complex and NFB are activated (5,(7)(8)(9)(10). Upon engagement by their cognate ligands, members of the tumor necrosis factor receptor (TNFR) family activate NFB by recruiting TNFR-associated death domain protein (TRADD) and TNFR-associated factor 2 (TRAF2) (11,12). The induction of NFB by the Toll/ interleukin-1 receptor family is a prominent event after stimulation with pro-inflammatory cytokines or recognition of pathogen-associated molecular patterns (13). The signal derived from the prototypic Tolllike receptor-4 is dependent on a module containing myeloid differentiation primary response gene 88 (MYD88) and interleukin-1 receptorassociated kinase-1 (IRAK1). The ubiquitin ligase TRAF6 links this module to a trimolecular complex consisting of transforming growth factor-␤-activated kinase-1 (TAK1), TAK1-binding protein-1 (TAB1), and TAB2 leading to modification of the IKK complex (14,15).
The critical step common to these major NFB-inducing pathways is the activation of the IKK complex. This complex consists of two catalytic subunits, IKK-␣ (IKK1) and IKK-␤ (IKK2), and the regulatory subunit IKK-␥ (NEMO), which is devoid of kinase activity. In the absence of IKK activity, cytoplasmic IB binds NFB and thereby prevents nuclear translocation of this transcription factor. The activated IKK complex phosphorylates IB, which in turn gets ubiquitinated, dissociates from the NFB complex, and is degraded by the proteasome. Free NFB then enters the nucleus and activates transcription (1,4,16).
Induction of the transcription factor NFB is also considered to be important for transformation and immortalization of lymphoid cells by human T cell leukemia virus type-1 or Epstein-Barr virus (2,(17)(18)(19). In this context, activation of NFB is attributed to the viral oncoproteins, the 40-kDa transactivator protein of the pX region (Tax) of human T cell leukemia virus type-1, and the latent membrane protein (LMP-1) of Epstein-Barr virus, respectively (20,21). According to the most widely accepted concept, Tax deregulates the IKK complex by linking the scaffold protein IKK-␥ to the upstream kinases MEKK1 and NIK, which in turn phosphorylate IKK-␣ and IKK-␤. In addition, Tax supports the oligomerization of IKK-␥ to enhance the activity of the IKK complex (22). LMP-1 induces NFB activity 20 -40-fold (19,23,24). The signal is transduced by a TNFR-like pathway, where TRADD and TRAF2, which directly interact with LMP-1, are essential (25)(26)(27). TRAF6 also affects NFB activation by LMP-1 through its action downstream of TRADD and TRAF2 (28,29). An interaction of TRAF6 with TRADD or TRAF2 has not been described for TNFR signaling so far. Thus, Tax activates NFB by direct modification of the IKK complex, whereas LMP-1 mimics a constitutively active receptor.
Herpesvirus saimiri and Herpesvirus ateles are members of the ␥2-herpesviruses or rhadinoviruses. Both induce malignant lymphoproliferation in New World monkeys, except for their natural hosts, the squirrel monkey and the spider monkey, respectively (30 -33). Furthermore, H. saimiri strain C488 is able to transform human T lymphocytes to permanent growth in culture (34). The saimiri transformation-associated protein of subgroup C (StpC) and the tyrosine kinase-interacting protein (Tip) are the two oncoproteins essential for the T cell-transforming phenotype of strain C488 (35)(36)(37). In contrast, the related H. ateles encodes only one oncoprotein, the "two-in-one" (Tio) protein.
Tio is a membrane-bound protein, forming homodimers or multimers, which exhibits sequence homologies to both StpC and Tip (38). Recombinant H. saimiri carrying tio in place of stpC and tip demonstrated that Tio can substitute for StpC and Tip in T cell transformation (39). In previous studies, different analogous functions for Tip and Tio have been reported. They both interact with cellular Src family kinases by binding to their Src homology 3 (SH3) domain (38,40,41) and are phosphorylated on distinct tyrosine residues by the bound kinase (42,43). Tip is phosphorylated on two tyrosine residues, creating binding sites for the SH2 domains of STAT3 and Lck, respectively (43)(44)(45). The sole phosphorylation site of Tio, tyrosine residue 136, is essential for the immortalization of primary human T cells (42). A function of Tio related to StpC could not be demonstrated so far. StpC raises the transcriptional activity of NFB 3.5-6-fold. A TRAF2 binding site in the N-terminal region of StpC is essential, not only for TRAF2 binding, but also for NFB induction and transformation of primary human T lymphocytes (46).
In this report, we describe a novel mode of constitutive NFB activation by demonstrating a direct interaction between the viral oncoprotein Tio and TRAF6, a key modification enzyme and adapter molecule of multiple NFB-inducing signaling cascades.

MATERIALS AND METHODS
Cell Culture and Expression Plasmids-Cultures of transformed human cord blood lymphocytes (CBL-Tio; CBL-M124) were generated as described previously (39). Long term cultures and Jurkat T cells (E6.1; American Type Culture Collection, TIB-152) were maintained in RPMI 1640 medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen), glutamine, and antibiotics. For transfection purposes, Jurkat T cells were cultivated at a maximum concentration of 10 6 cells/ml.
Transfection-Ten million Jurkat T cells per sample were electroporated with 25-100 g of DNA. Vector plasmid (pcDNA3) was used to equalize promoter copy numbers in all transfections. Electroporation was carried out with an Easyject Plus apparatus (Equibio, Boughton, UK) at 250 V, 1500 microfarads. The cells were transfected in complete medium without antibiotics, harvested 48 h post-transfection and washed in phosphate-buffered saline. Two-thirds of the cells were used for Western blot analysis, and one-third was used for luciferase reporter assays.
Reporter Plasmids and Assays-The reporter construct pNFB-Luc contains five NFB binding sites in front of a luciferase gene (Stratagene, Heidelberg, Germany). Experimental integrity was confirmed with alternative NFB reporter constructs, pBIIxLuc with four NFB binding sites (49) and its mutant pBII-SK (48). In addition, AP-1 or NFAT luciferase reporter constructs (50) were tested. Jurkat T cells were transfected with expression plasmids and 8 g of reporter DNA. Cell pellets were resuspended in 300 l of lysis buffer (25 mM Tris-H 3 PO 4 , pH 7.8, 2 mM dithiothreitol, 1% Triton X-100, 10% glycerol, 2 mM DCTA (trans-1,2-diaminocyclohexane-N,N,NЈ,NЈ-tetraacetic acid monohydrate; Sigma) and incubated at room temperature for 30 min. Lysates were cleared by centrifugation for 5 min at 13,000 revolutions/min. Assay buffer (100 l; 100 mM K 3 PO 4 , 15 mM MgSO 4 , 5 mM ATP) was mixed with lysates (25 or 50 l) in a 96-well plate. Assay buffer (100 l) supplemented with D-luciferin (1 mM) (Roche Applied Science) was added, and luciferase activity was determined with a luminometer (Orion, Berthold, Germany). Relative luciferase activity was calculated by dividing the sample values by the value of vector DNA (pcDNA3) cotransfected with the reporter construct.
Drug Treatment of Transfected Cells-Transfected Jurkat T cells were dispensed into 12 wells 24 h post-electroporation and treated for 24 h with PMA (Sigma), ionomycin (Sigma), PKC inhibitors Gö6983 (51), or Ro-31-8220 (Calbiochem, Merck; Schwalbach, Germany), respectively (52), TNF␣ (ImmunoTools, Friesoythe, Germany), or the TNF␣ inhibitor Remicade (Infliximab, Essex Pharma, München, Germany), as indicated in the legend to Fig. 4. The volume of lysis buffer was reduced to 100 l/sample. Subcellular Fractionation of Proteins-Transformed human cord blood lymphocytes were harvested by low speed centrifugation at 500 ϫ g for 5 min. The pellet was resuspended in hypotonic lysis buffer (20 mM Tris, pH 7.5, 5 mM EDTA, protease inhibitors), and 30 strokes were delivered to the cell suspension in a tight-fitting Dounce homogenizer. Cell nuclei were removed by centrifugation at 4,000 ϫ g for 15 min. Cellular membranes were sedimented from the remaining supernatant at 20,000 ϫ g for 30 min. The final supernatant containing cytosol and free lipid was supplemented by adding an equal volume of 2-fold-concentrated TNE buffer including protease inhibitors. Pellets containing cellular membranes were dissolved in TNE buffer, and insoluble constituents were removed by centrifugation at 20,000 ϫ g for 10 min. Protein concentrations were determined and equal amounts were analyzed by Western blot or immunoprecipitation.

NFB Activity Is Induced by the Tio Oncoprotein in T Cells-NFB
activation is an important function of distinct viral oncoproteins, such as LMP-1, Tax, or StpC (21,23,24,46,53,54). Here we have investigated whether Tio also affects the activation of NFB. After cotransfection of Tio expression constructs with NFB-specific reporter plasmids into Jurkat T cells, relative luciferase activity was measured. The expression of the Tio oncoprotein was confirmed by Western blot analysis. The strong induction of NFB activity observed was directly proportional to the amount of Tio-DNA transfected (Fig. 1). Significant NFB activa-  tion could be measured for amounts of Tio far below the detection limit of the immunoblot (Fig. 1). NFB induction by Tio was confirmed with an alternative NFB luciferase reporter construct. In contrast, Tio did not stimulate luciferase expression from reporter plasmids with either mutated NFB binding sites or consensus AP-1 or NFAT response elements, respectively (data not shown). NFB activation by Tio was blocked when dominant negative IB␣ (IB␣ DN) was cotransfected (Fig. 1). Suppression of NFB activation also reduced the expression of Tio, which is most likely explained by the positive regulatory effect of NFB on the cytomegalovirus promoter (55,56). Increased amounts of Tio plasmid augmented the expression level but did not reconstitute NFB activity in the presence of IB␣ DN (Fig. 1). This demonstrated that Tio acts upstream of IB␣ to induce NFB.
NFB Activation by Tio Is Independent of Src-Kinase Interaction-Src interaction and phosphorylation of Tyr-136 of Tio is essential for the transformation of human T lymphocytes in vitro (Fig. 2) (42). Tio mutants FYFF, YFYY, and PARG were tested for their ability to activate NFB. The mutants PARG and YFYY, which were deficient for phosphorylation of Tyr-136, and control mutant FYFF, which acts like wild-type Tio in Srckinase interaction and T cell transformation, did not differ in their ability to induce NFB activation (Fig. 3). Thus, NFB activation by Tio was inde-pendent of Src-kinase interaction and Tyr-136. These mutants were further used as positive controls in the reporter assays. LMP-1 of Epstein-Barr virus (20) and H. saimiri StpC/Tip (57) were included as additional positive controls. Transfections with empty vector DNA or an expression construct for EGFP served as negative and transfection efficiency controls, respectively.
Identification of Tio Elements Required for NFB Induction-NFB activation by other viral oncoproteins is based on specific protein-protein interactions with upstream regulators. The interaction of TRAF2 with a consensus binding motif in StpC was shown to be essential for the activation of NFB (46). For identification of the region in Tio substantial for NFB activation, sequence comparisons were performed between Tio, StpC, LMP-1, and other TRAF2-interacting proteins. The TRAF2 consensus binding sequence has been reported to be PXQX(T/S) (58), which is common among TRAF1, -2, -3, and -5 but differs from that of TRAF6 (59). StpC contains the motif PIEET, an incomplete consensus motif still binding to TRAF2. We identified two related motifs in Tio, PLGDS, and PQDPT, which were targeted for mutation of the important first proline residue (Fig. 2). The resulting Tio mutants PAPA, PAPALK, and PAQK induced NFB activity in a similar manner as the wild-type protein (Fig. 3 and data not shown). In addition, coimmunoprecipitation analysis failed to demonstrate an interaction of TRAF2 with Tio (data not shown). Thus, TRAF2 does not seem to be involved in Tio signaling to NFB.
To narrow down a suspected domain responsible for NFB activation by Tio, deletion mutants in the StpC homologous part were created (Fig.  2, D1, D2, D4, D6 -D8). All deletion mutants, except mutant D7, failed to induce NFB activity (Fig. 3A), indicating that the required region is located in the N-terminal segment of Tio. Within this segment, two glutamic acid-rich motifs were found. Acidic amino acids forming a region of net negative charge were shown to be important for proteinprotein interactions, like it was demonstrated for StpC (60). Mutation of glutamic acids Glu-7, Glu-9, and Glu-10 and/or Glu-23 and Glu-24 in Tio to glutamine (Gln) or glycine (Gly) resulted in Tio mutants P1-P5 and P7 (Fig. 2). NFB reporter assays indicated that mutation of Glu-23 and Glu-24 to Gln or Gly (P2, P4) had no impact on NFB induction. In contrast, mutation of Glu-7, Glu-9, and Glu-10 to Gln or Gly (P1, P3, P5, P7) abrogated NFB activation (Fig. 3, B and C). Western blot analysis revealed reduced expression levels of all NFB negative mutants, which were again attributed to the NFB sensitivity of the cytomegalovirus promoter. Expression of these Tio mutants was augmented by increasing the amount of plasmid transfected. However, this did not correlate with an increase of luciferase activity in the reporter assay (Fig. 3, A-C). Deletion mutants and point mutants with a conservative exchange of glutamic acid (Glu) against glutamine (Gln) were retested in triplicates, and the S.D. was calculated (Fig. 3D). These experiments confirmed that NFB activation depends on glutamic acid residues at the N terminus of Tio.
Tio Does Not Utilize TNFR or PKC-dependent Pathways for NFB Induction-NFB-activating signaling pathways in T cells may originate from the T cell receptor, the TNFR, or the toll-like receptors. To test for the influence of Tio on TNFR-mediated NFB activation, transfected cells were stimulated with different amounts of TNF␣. NFB activation by Tio appeared not to be affected by increasing amounts of TNF␣. However, TNF␣ induced a dose-dependent rise of NFB activity in cells transfected with the vector-DNA, which served as a negative control, as well as with P1 mutant (Fig. 4). To exclude TNFR activation by Tio-or transfection-induced TNF␣ expression, a potent TNF␣ inhibitor (Remicade) was added to transfected Jurkat T cells. There was no difference between treated and untreated cells. To control whether Remicade efficiently inhibits TNF␣ signaling, transfected cells were treated with TNF␣ and the inhibitor simultaneously. This treatment prevented the dose-dependent rise of NFB activity by TNF␣ (Fig. 4).
A key effector of the T cell receptor pathway is PKC. To investigate whether Tio acts upstream or at the level of PKC to induce NFB, we applied PKC inhibitors to transiently transfected Jurkat T cells. The inhibitors Gö6983 and Ro-31-8220 are specific for different PKC isoenzymes, among them PKC. In the NFB reporter assay, no difference was observed between untreated T cells and samples exposed to Gö6983 or Ro-31-8220 (Fig. 4). To guarantee the functionality of the PKC inhibitors, cells were stimulated with PMA and ionomycin to mimic NFB activation via the T cell receptor pathway. The induction of NFB by these drugs in all transfectants was efficiently suppressed by the simultaneous addition of Gö6983 or Ro-31-8220 (Fig. 4). With these assays, we suggest that the signal for NFB activation by Tio is not mediated by components of the TNFR or the PKC-dependent pathway.
TRAF6(300 -524) Blocks NFB Activation by Tio-TRAF6 is an essential mediator in the NFB signaling cascades triggered by various receptors and oncoproteins. Mutational analysis of Tio revealed the functional relevance of glutamic acid residues at the N terminus, which are major constituents of a TRAF6 consensus binding motif (PXEXX(Ar/Ac)) (Ar, any aromatic residue; Ac, any acidic residue) (59)  ( Fig. 2). Therefore, we overexpressed TRAF6 or dominant negative TRAF6(300 -524) in Jurkat T cells and analyzed their effect on Tiomediated NFB activation. TRAF6 or TRAF6(300 -524) alone induced no NFB activity compared with vector-transfected cells. Furthermore, TRAF6 did not influence the Tio-induced activity. However, cotransfection of TRAF6(300 -524) caused a dramatic decrease of Tio-induced NFB activity (Fig. 5). This indicates that TRAF6 is a major factor transmitting the stimulatory signal from Tio to NFB.
Tio Binds to TRAF6-Because of the impact of dominant negative TRAF6(300 -524) on NFB activation by Tio and the essential role of a consensus TRAF6 binding motif in Tio, we supposed a direct interaction between TRAF6 and Tio. To examine this hypothesis, we performed coimmunoprecipitation analyses. Interaction of Tio with TRAF6 at the predicted TRAF6 binding motif PQEHEE was controlled by Tio mutant P1, where the essential glutamic acid residues were mutated to glutamine. Jurkat T cells were cotransfected with combinations of HA-tagged TRAF6 and FLAG-tagged Tio or P1, respectively. Because expression levels of the cytomegalovirus promoter-driven constructs appeared to be regulated by NFB, we complemented our experiment by cotransfection of constitutively active IKK2 (IKK2 EE) to ensure comparable expression levels of Tio and P1. Expression from the transfected plasmids was controlled by Western blot analysis of cell lysates using adequate antibodies (Fig. 6C).
Tio and P1 were immunoprecipitated using anti-FLAG-agarose (Fig.  6A). Presence of the precipitated proteins (Tio and P1) was verified with an anti-FLAG antibody. Coprecipitated TRAF6 was detected by HA monoclonal antibody only from samples cotransfected with Tio and TRAF6. The polyclonal TRAF6 antiserum detected both endogenous and exogenous TRAF6. Tio mutant P1 did not bind any TRAF6. Cotransfection of IKK2 EE provided higher expression levels of mutant P1 but gave the same results. In the reverse experiment, the TRAF6 polyclonal antibody was used for immunoadsorption leading to precipitation of exogenous, as well as endogenous, TRAF6 proteins (Fig. 6B). Western blot analysis of TRAF6 complexes with anti-FLAG antibody established the interaction of Tio with TRAF6. In contrast, mutant P1 was not able to bind to TRAF6. These experiments defined the motif PQEHEE of Tio, which corresponds to a TRAF6 consensus binding sequence, as a direct interaction site for TRAF6.
Interaction of TRAF6 with Tio in Virus-transformed Human T Cells-We next wanted to test for the association of TRAF6 and Tio in human T cells transformed by recombinant H. saimiri C488. Because of low expression levels, TRAF6 precipitation from post-nuclear lysates was not suitable to detect binding to Tio (data not shown). In an attempt to enrich TRAF6, we extracted cytosolic and membrane proteins from virus-transformed lymphocytes expressing either StpC/Tip or Tio and analyzed these fractions by Western blot. Enrichment of the protein tyrosine kinase Lck in the membrane preparations confirmed the integrity of fractionation. In the presence of Tio, the majority of TRAF6 was recruited to the membrane fraction, whereas TRAF6 was mainly cytoplasmic in T cells expressing StpC and Tip. (Fig. 7A). Immunoprecipitation analyses with these solubilized membrane preparations revealed binding of TRAF6 to Tio (Fig. 7B). These results demonstrated that Tio interacts with TRAF6 and thereby recruits this cellular regulator to the membrane fraction of T cells transformed by recombinant H. saimiri.

DISCUSSION
Several NFB activation pathways converge at the level of TRAF6, a ubiquitin ligase regulating the IKK complex and thereby NFB activity (1). We have now demonstrated that Tio, the oncoprotein of the T lymphotropic H. ateles, dramatically induced NFB in T cells. NFB activation correlated with binding of Tio to TRAF6. This interaction relied on the PQEHEE motif in Tio (Fig. 2) that conforms to the TRAF6 consensus binding motif (PXEXX(Ar/Ac)) (59). Direct targeting of TRAF6 distinguishes Tio from LMP-1, an oncoprotein of Epstein-Barr virus that is involved in several forms of human malignancies (61). LMP-1 also engages TRAF6 for NFB activation but depends on direct interactions with upstream regulators TRADD and TRAF2 (25,26,28,29). Stable multimers of LMP-1 are localized in the plasma membrane and are considered to form constitutively active signaling complexes related to those induced by activated members of the TNFR family, which also utilize TRADD and TRAF2 to induce growth-promoting signals via NFB (11,12,62,63). A similar model of receptor mimicry has been established to explain the oncogenic properties of H. saimiri StpC (46,64). Tio also forms oligo-or multimers, and its primary amino acid sequence suggests membrane localization (38). Therefore, Tio likely induces TRAF6-containing signaling complexes that are without precedent among known membrane receptors or viral oncoproteins. However, Tio-TRAF6 binding is reminiscent of the interaction between human T cell leukemia virus type-1 Tax and IKK-␥ (22) in that both viral oncoproteins directly address signaling intermediates common to different NFB activation pathways.
NFB induction by the Tio-TRAF6 complex may be explained by Tio-mediated TRAF6 multimerization and ubiquitin ligase activation. In accordance with this, the activated complex was still sensitive to, but not dependent on, upstream signals from PKC or the TNFR (Fig. 4). Downstream signaling to NFB relied on a functional IKK complex and cytoplasmic NFB, as dominant negative IKK-␤ (IKK2 K44M) (65) and IB␣ DN (47) blocked NFB activation by Tio ( Fig. 1 and data not shown). TRAF6 complexes activated by interleukin-1 receptor triggering not only induce NFB but also JNK, a kinase of the MAPK family known to activate the transcription factor AP-1 (11, 62, 66 -69). However, we did not detect the activation of AP-1 response elements by luciferase reporter assays in Tio-transfected Jurkat T cells (data not shown). Thus, Tio-TRAF6 complexes appear to be functionally distinct from TRAF6 complexes induced by interleukin-1 receptor ligation.
We previously reported that Tio directly interacts with Src family kinases via an SH3 binding motif in the C-terminal region of the protein (38). This interaction is necessary for the phosphorylation of Tio on tyrosine residue 136. Both the SH3 binding motif and the phosphorylation site are essential for Tio to support growth transformation of primary human T cells (42). Here we document that both sites were dispensable for Tio signaling to NFB (Fig. 3, mutants YFYY and PARG). These results indicate that two independent signals are originating from the Tio molecule. Furthermore, our findings suggest that Tio does not induce Src-kinase signaling pathways leading to the activation of PKC, AP-1, or NFAT (Figs. 3 and 4 and data not shown).
A major question arising from these observations concerns the composition and localization of Tio-induced protein complexes. Tio-TRAF6 and Tio-Src complexes may represent specific signaling entities segregated into different cellular compartments. However, the organization of the binding sites on Tio would also allow for the simultaneous binding of both Src kinases and TRAF6. Due to the modular structure of the proteins involved, additional binding partners are likely to be recruited into the complex(es). With respect to NFB induction, inclusion or exclusion of known regulators and effectors of TRAF6 will be of special interest. So far, TRAF2 may be excluded, as mutation of suspected TRAF2 binding sites did not alter the NFB-inducing ability of Tio (Figs. 2 and 3, mutants PAPA, PAQK, and PAPALK and data not shown) and no binding to Tio was detected (data not shown). In Tolllike receptor-4 signaling, TRAF6 directly binds to interleukin-1 receptor-associated kinase-1 (70), and further downstream a complex is formed by TAB1, TAB2, TAK1, and TRAF6 (14). In addition to TAK1, the MAPKKKs, NIK, and MEKK1 were reported to activate IKK (71,72). In this context, Tio might act as a scaffold and induce or stabilize protein interactions that facilitate IKK phosphorylation and thus NFB activation.
Another open question addresses the biological relevance of TRAF6 interaction and NFB activation in T cell transformation and oncogenesis by viruses expressing Tio. NFB is a ubiquitous transcription factor involved in numerous cellular events leading to cell proliferation and/or apoptosis inhibition (2,3). Thus, it is conceivable that Tio, similar to Tax, LMP-1, and StpC, exploits this pathway to exert its oncogenic potential. Further studies will be required to analyze the role of NFB activation and Tio-TRAF6 interaction in the promotion of T cell growth and tumor induction, progression, and maintenance.