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Originally published In Press as doi:10.1074/jbc.M510891200 on February 1, 2006

J. Biol. Chem., Vol. 281, Issue 13, 8565-8572, March 31, 2006
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NF{kappa}B Signaling Is Induced by the Oncoprotein Tio through Direct Interaction with TRAF6*

Stefanie Heinemann1, Brigitte Biesinger, Bernhard Fleckenstein, and Jens-Christian Albrecht

From the Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany

Received for publication, October 5, 2005 , and in revised form, January 12, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The transcription factor NF{kappa}B is a major regulator of genes involved in inflammation and oncogenesis. NF{kappa}B 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 I{kappa}B, the administrator of NF{kappa}B activity. Here we report on a strong NF{kappa}B activation by Tio, a unique herpesviral oncoprotein promoting transformation of human T cells in a Src-kinase-dependent manner. NF{kappa}B 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 NF{kappa}B activation. Cotransfection of a dominant negative TRAF6 construct led to a decrease in NF{kappa}B 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 NF{kappa}B through an oncoprotein.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
NF{kappa}B 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, 1-6). Three classical signaling pathways leading to the activation of NF{kappa}B are initiated by the stimulation of different receptors. Antigen recognition by the T cell receptor induces NF{kappa}B by recruiting protein tyrosine kinases Lck and Zap70 to the receptor complex. The signal is processed by several intermediate proteins, including protein kinase C{theta} (PKC{theta})2 and a multimolecular module containing CARMA, Bcl-10, MALT1, and TRAF6. Subsequently, the I{kappa}B kinase (IKK) complex and NF{kappa}B are activated (5, 7-10). Upon engagement by their cognate ligands, members of the tumor necrosis factor receptor (TNFR) family activate NF{kappa}B by recruiting TNFR-associated death domain protein (TRADD) and TNFR-associated factor 2 (TRAF2) (11, 12). The induction of NF{kappa}B 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 Toll-like receptor-4 is dependent on a module containing myeloid differentiation primary response gene 88 (MYD88) and interleukin-1 receptor-associated kinase-1 (IRAK1). The ubiquitin ligase TRAF6 links this module to a trimolecular complex consisting of transforming growth factor-beta-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 NF{kappa}B-inducing pathways is the activation of the IKK complex. This complex consists of two catalytic subunits, IKK-{alpha} (IKK1) and IKK-beta (IKK2), and the regulatory subunit IKK-{gamma} (NEMO), which is devoid of kinase activity. In the absence of IKK activity, cytoplasmic I{kappa}B binds NF{kappa}B and thereby prevents nuclear translocation of this transcription factor. The activated IKK complex phosphorylates I{kappa}B, which in turn gets ubiquitinated, dissociates from the NF{kappa}B complex, and is degraded by the proteasome. Free NF{kappa}B then enters the nucleus and activates transcription (1, 4, 16).

Induction of the transcription factor NF{kappa}B 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, 17-19). In this context, activation of NF{kappa}B 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-{gamma} to the upstream kinases MEKK1 and NIK, which in turn phosphorylate IKK-{alpha} and IKK-beta. In addition, Tax supports the oligomerization of IKK-{gamma} to enhance the activity of the IKK complex (22). LMP-1 induces NF{kappa}B 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 (25-27). TRAF6 also affects NF{kappa}B 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 NF{kappa}B by direct modification of the IKK complex, whereas LMP-1 mimics a constitutively active receptor.

Herpesvirus saimiri and Herpesvirus ateles are members of the {gamma}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 (35-37). 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 (43-45). 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 NF{kappa}B 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 NF{kappa}B induction and transformation of primary human T lymphocytes (46).

In this report, we describe a novel mode of constitutive NF{kappa}B activation by demonstrating a direct interaction between the viral oncoprotein Tio and TRAF6, a key modification enzyme and adapter molecule of multiple NF{kappa}B-inducing signaling cascades.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
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 106 cells/ml.

All mutant Tio plasmids derived from the previously described FLAG-Tio (wild-type Tio, YYYY) construct (38). Deletion mutants were created by PCR amplification with oligonucleotide primers introducing an NheI recognition sequence, which translates into alanine and serine residues at the deletion site. Mutant D1 was created using primers SH9 (5'-GTTCGCTAGCCTTGTCATCGTCGTCCTTG-3') and SH10 (5'-GGTCGCTAGCCCGCTGGGCGACTCTGG-3'); mutant D2 with primers SH9 and SH11 (5'-AGCCGCTAGCAACATCCCTCAGGACCCGA-3'); mutant D4 with primers SH14 (5'-CGGTGCTAGCTTCTTCGTGTTCTTGTGGC-3') and SH15 (5'-CCACGCTA GCGGTGAAGAAGGGCCACC-3'); mutant D6 with primers SH9 and SH12 (5'-CCATGCTAGCAATTCTAAAAATGAAGATTACC-3'); mutant D7 with primers SH12 and SH16 (5'-CTTCGCTAGCGATGTTAGGTGGCCCTTC-3'); mutant D8 with primers SH12 and SH14 (see Fig. 2). Tio point mutants were created by PCR according to the QuikChange® protocol (Stratagene, Heidelberg, Germany). Mutant P1 was created using primers SH17 (5'-GACAAGATGGCTAACCAGCCACAACAACACCAACAAGGAAAACCCTTCTTC-3') and SH18 (5'-GAAGAAGGGTTTTCCTTGTTGGTGTTGTTGTGGCTGGTTAGCCATCTTGTC-3') to replace Glu-7, Glu-9, and Glu-10 by Gln; mutant P2 using SH19 (5'-GCTGGGCGACTCTGGTCAACAAGGGCCACCTAACATCCCTCAG-3') and SH20 (5'-CTGAGGGATGTTAGGTGGCCCTTGTTGACCAGAGTCGCCCAGC-3') to replace Glu-23 and Glu-24 by Gln; mutant P3 using SH21 (5'-GACAAGATGGCTAACGGGCCACAAGGACACGGAGGAGGAAAACCCTTCTTC-3') and SH22 (5'-GAAGAAGGGTTTTCCTCCTCCGTGTCCTTGTGGCCCGTTAGCCATCTTGTC-3') to replace Glu-7, Glu-9, and Glu-10 by Gly; mutant P4 using SH23 (5'-GCTGGGCGACTCTGGTGGAGGAGGGCCACCTAACATCCCTCAG-3') and SH24 (5'-CTGAGGGATGTTAGGTGGCCCTCCTCCACCAGAGTCGCCCAGC-3') to replace Glu-23 and Glu24 by Gly; mutant P5 using SH17, SH18, SH19, and SH20 to replace Glu-7, Glu-9, and Glu-10 by Gln plus Glu-23 and Glu-24 by Gln; mutant P7 using SH21, SH22, SH23, and SH24 to replace Glu-7, Glu-9, and Glu-10 by Gly plus Glu-23 and Glu-24 by Gly. The mutant PAPA was created using primers PAPA-1 (5'-GAAAACCCTTCTTCGCCGGCGTGGGCGACT-3') and PAPA-2 (5'-AGTCGCCCACGCCGGCGAAGAAGGGTTTTC-3') to change Pro-16 and Pro-17 to Ala, Tio mutant PAQK was created using PAQK-A (5'-GGCCACCTAACATCGCTAAGGACCCGACGCCAGG-3') and PAQK-B (5'-CCTGGCGTCGGGTCCTTAGCGATGTTAGGTGGCC-3') to change Pro-30 to Ala and Gln-31 to Lys; PAPALK was created using LK-A (5'-CCTTCTTCGCCGC GAAGGGCGACTCTGGTG-3') and LK-B (5'-CACCAGAGTCGCCCTTCGCGGCGAAGAAGG-3') to substitute Leu-18 for Lys within the PAPA mutant. The resulting nucleotide sequences were confirmed by automated sequence analysis on an ABI3100 sequencer (Applied Biosystems, Darmstadt, Germany). Tio mutants FYFF, YFYY, and PARG were described previously (42). Expression plasmids for dominant negative I{kappa}B{alpha} (I{kappa}B{alpha} DN) and constitutively active IKK2 (IKK2 EE) were kindly provided by R. Voll (University Erlangen-Nürnberg, Erlangen, Germany) (47, 48). The LMP-1 expression construct was a gift of W. Hammerschmidt (GSF, München, Germany). The TRAF6, the dominant negative TRAF6(300-524), and the TRAF6-HA expression plasmids (28) were kindly provided by A. Kieser (GSF, München, Germany). A plasmid expressing enhanced green fluorescent protein (EGFP), pEGFP-C2, (BD Biosciences) was used to verify transfection efficiency.

Cell Lysis, Immunoblotting, and Immunoprecipitation—Jurkat T cells were lysed for 30 min at 4 °C in TNE buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% Nonidet P-40) containing 5 mM NaF, 1 mM Na3VO4, and 10 µg/ml of each aprotinin and leupeptin (Sigma). Lysates were cleared at 13,000 revolutions/min for 10 min. Protein concentrations were determined by BCA assay (Pierce), and equal amounts of protein were separated by denaturating SDS-PAGE. Polyvinylidene difluoride membrane filters (Amersham Biosciences) were used for immunoblotting and incubated with antisera against Tio (38), TRAF6 (H-274; Santa Cruz Biotechnology; diluted 1/1000), TRAF2 (C-20; Santa Cruz, Biotechnology; diluted 1/1000), or with monoclonal antibodies directed against beta-actin (AC-15; Abcam, Cambridge, UK; diluted 1/20000), {alpha}-FLAG (anti-FLAG® M2-horseradish peroxidase; Sigma; diluted 1/2000), or {alpha}-HA (HA.11; Covance, Princeton, New Jersey; diluted 1/1000), diluted in phosphate-buffered saline, pH 7.4, containing 0,1% Tween 20 and 5% milk powder for 12 h at 4 °C. For detection of primary antibodies, horseradish peroxidase-conjugated antibodies, either against mouse (F(ab)2; Amersham Biosciences; diluted 1/5000) or rabbit (Dako, Hamburg, Germany; diluted 1/20000) immunoglobulins were used. Immunodetection was performed by enhanced chemiluminescence according to the manufacturer's protocol (Amersham Biosciences). For immunoprecipitation, 500 µl of cleared lysates (800-1300 µg of protein) were mixed with antisera against TRAF6, TRAF2 (2 µg of each), or Tio (2 µl) and rotated at 4 °C overnight. The next day, 20 µl of protein A-Sepharose (Sepharose CL-4B; Amersham Biosciences) were added for 1 h at 4°C. FLAG-tagged proteins were incubated with 20 µl of anti-FLAG-agarose (Sigma) at 4 °C overnight. The beads were washed five times with lysis buffer. Samples were boiled for 5 min in 20 µl of SDS-loading buffer and applied to SDS-PAGE.

Transfection—Ten million Jurkat T cells per sample were electroporated with 25-100 µg 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 pNF{kappa}B-Luc contains five NF{kappa}B binding sites in front of a luciferase gene (Stratagene, Heidelberg, Germany). Experimental integrity was confirmed with alternative NF{kappa}B reporter constructs, pBIIxLuc with four NF{kappa}B 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 µg of reporter DNA. Cell pellets were resuspended in 300 µl of lysis buffer (25 mM Tris-H3PO4, pH 7.8, 2 mM dithiothreitol, 1% Triton X-100, 10% glycerol, 2 mM DCTA (trans-1,2-diaminocyclohexane-N,N,N',N'-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 µl; 100 mM K3PO4, 15 mM MgSO4, 5 mM ATP) was mixed with lysates (25 or 50 µl) in a 96-well plate. Assay buffer (100 µl) 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.


Figure 1
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  		FIGURE 1.
Tio induces NF{kappa}B activation. A, Jurkat T cells were transiently transfected with NF{kappa}B reporter and different amounts of a Tio expression construct. Cell lysates were subjected to an NF{kappa}B-dependent luciferase reporter assay 48 h post-transfection. Transfected vector DNA alone served as the negative control. I{kappa}B{alpha} was cotransfected to block the induction of NF{kappa}B by Tio. B, Western blot analysis was performed to confirm protein expression of Tio. Immunodetection of beta-actin served as a loading control.

 


Figure 2
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  		FIGURE 2.
Mutations within the N-terminal 47 amino acids of Tio. The top line of the figure shows the amino acid sequence of the wild-type Tio (wt), amino acids 1-47. Deletion (D) and point (P) mutants are listed in lines 2-13. Gray boxes indicate the glutamic acid-rich sequences of Tio. In the N-terminal negatively charged region of Tio, the TRAF6 binding motif and its consensus sequence (PXEX(E/D)(Ar/Ac)) is given, with X representing any residue, Ar any aromatic residue, and Ac any acidic residue. Open boxes depict two matched TRAF2 consensus motifs (PXQX(T/S)). Mutations within these motifs are listed in lines 14-16, named after the amino acids that were changed (PAPA, PAQK, PAPALK).

 
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{alpha} (ImmunoTools, Friesoythe, Germany), or the TNF{alpha} inhibitor Remicade® (Infliximab, Essex Pharma, München, Germany), as indicated in the legend to Fig. 4. The volume of lysis buffer was reduced to 100 µl/sample.

Subcellular Fractionation of Proteins—Transformed human cord blood lymphocytes were harvested by low speed centrifugation at 500 x 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 x g for 15 min. Cellular membranes were sedimented from the remaining supernatant at 20,000 x 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 x g for 10 min. Protein concentrations were determined and equal amounts were analyzed by Western blot or immunoprecipitation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NF{kappa}B Activity Is Induced by the Tio Oncoprotein in T Cells—NF{kappa}B 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 NF{kappa}B. After cotransfection of Tio expression constructs with NF{kappa}B-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 NF{kappa}B activity observed was directly proportional to the amount of Tio-DNA transfected (Fig. 1). Significant NF{kappa}B activation could be measured for amounts of Tio far below the detection limit of the immunoblot (Fig. 1). NF{kappa}B induction by Tio was confirmed with an alternative NF{kappa}B luciferase reporter construct. In contrast, Tio did not stimulate luciferase expression from reporter plasmids with either mutated NF{kappa}B binding sites or consensus AP-1 or NFAT response elements, respectively (data not shown). NF{kappa}B activation by Tio was blocked when dominant negative I{kappa}B{alpha} (I{kappa}B{alpha} DN) was cotransfected (Fig. 1). Suppression of NF{kappa}B activation also reduced the expression of Tio, which is most likely explained by the positive regulatory effect of NF{kappa}B on the cytomegalovirus promoter (55, 56). Increased amounts of Tio plasmid augmented the expression level but did not reconstitute NF{kappa}B activity in the presence of I{kappa}B{alpha} DN (Fig. 1). This demonstrated that Tio acts upstream of I{kappa}B{alpha} to induce NF{kappa}B.


Figure 3
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  		FIGURE 3.
Mutations affecting the TRAF6 binding motif abrogate induction of NF{kappa}B by Tio. A-C, Jurkat T cells were electroporated with the NF{kappa}B reporter plasmid, and increasing amounts of Tio deletion (A) or point (B and C) mutant constructs (25,50, or 100 µg; indicated by wedges). The total amount of transfected DNA was adjusted by the addition of empty expression vector. Tio wild-type (YYYY), tyrosine phosphorylation-competent (FYFF), and -incompetent (YFYY) Tio mutants, non-Src kinase-binding Tio mutant (PARG), PAPA, D7, Epstein-Barr virus LMP-1, and H. saimiri C-488 StpC/Tip served as positive controls (25 µg each). Vector DNA provided the basic NF{kappa}B activity, and a transfection control with green fluorescent protein served as an additional negative control. The expression of the Tio proteins was confirmed by Western blot analysis. Experiments were repeated at least three times. D, equal amounts of Tio expression constructs (50 µg each) and the plasmid NF{kappa}B-Luc were electroporated into Jurkat T cells. Mean values and S.D. values were calculated from triplicate samples.

 
NF{kappa}B 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 NF{kappa}B. The mutants PARG and YFYY, which were deficient for phosphorylation of Tyr-136, and control mutant FYFF, which acts like wild-type Tio in Src-kinase interaction and T cell transformation, did not differ in their ability to induce NF{kappa}B activation (Fig. 3). Thus, NF{kappa}B activation by Tio was independent 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 NF{kappa}B Induction—NF{kappa}B 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 NF{kappa}B (46). For identification of the region in Tio substantial for NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B.


Figure 4
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  		FIGURE 4.
NF{kappa}B induction by Tio is not mediated by TNF{alpha} or PKC. Jurkat T cells were transiently transfected with NF{kappa}B reporter and expression plasmids coding for Tio (YYYY) or mutant P1 (P1). One day post-electroporation, the cells were treated with different inhibitors or activators for an additional 24 h, and a luciferase assay was performed. Treatments included the stimulation with TNF{alpha} at concentrations of 10 and 50 ng/ml (second and third bars from the left), inhibition with the TNF{alpha} antibody Remicade at 10 and 50 µg/ml (fourth and fifth bars from the left), the addition of 10 or 50 ng/ml TNF{alpha} and 10 or 20 µg/ml Remicade (sixth and seventh bars from the left), blocking of PKC activity with 2 µM Gö6983 or Ro-31-8220 (eighth and ninth bars from the left), stimulation with 250 or 500 ng/ml ionomycin and 10 or 20 ng/ml PMA (tenth bars form the left), and addition of 2 µM Gö6983 or Ro-31-8220 with 10 ng/ml PMA and 250 ng/ml ionomycin (eleventh and twelfth bars from the left). First bars from the left show untreated cells. Mean values and S.D. values were calculated from triplicate samples.

 
To narrow down a suspected domain responsible for NF{kappa}B 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 NF{kappa}B 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 protein-protein 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). NF{kappa}B reporter assays indicated that mutation of Glu-23 and Glu-24 to Gln or Gly (P2, P4) had no impact on NF{kappa}B induction. In contrast, mutation of Glu-7, Glu-9, and Glu-10 to Gln or Gly (P1, P3, P5, P7) abrogated NF{kappa}B activation (Fig. 3, B and C). Western blot analysis revealed reduced expression levels of all NF{kappa}B negative mutants, which were again attributed to the NF{kappa}B 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 NF{kappa}B activation depends on glutamic acid residues at the N terminus of Tio.


Figure 5
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  		FIGURE 5.
Dominant negative TRAF6 diminishes NF{kappa}B activation by Tio. Jurkat T cells were transiently transfected with NF{kappa}B-Luc and the expression vector or plasmids coding for Tio (YYYY), TRAF6, or TRAF6(300-524) in the combinations given at the bottom. Mean values and S.D. values of relative NF{kappa}B activity were calculated from triplicate samples.

 
Tio Does Not Utilize TNFR or PKC-dependent Pathways for NF{kappa}B Induction—NF{kappa}B-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 NF{kappa}B activation, transfected cells were stimulated with different amounts of TNF{alpha}. NF{kappa}B activation by Tio appeared not to be affected by increasing amounts of TNF{alpha}. However, TNF{alpha} induced a dose-dependent rise of NF{kappa}B 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{alpha} expression, a potent TNF{alpha} 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{alpha} signaling, transfected cells were treated with TNF{alpha} and the inhibitor simultaneously. This treatment prevented the dose-dependent rise of NF{kappa}B activity by TNF{alpha} (Fig. 4).

A key effector of the T cell receptor pathway is PKC{theta}. To investigate whether Tio acts upstream or at the level of PKC{theta} to induce NF{kappa}B, 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{theta}. In the NF{kappa}B 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 NF{kappa}B activation via the T cell receptor pathway. The induction of NF{kappa}B 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 NF{kappa}B activation by Tio is not mediated by components of the TNFR or the PKC-dependent pathway.

TRAF6(300-524) Blocks NF{kappa}B Activation by Tio—TRAF6 is an essential mediator in the NF{kappa}B 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 Tio-mediated NF{kappa}B activation. TRAF6 or TRAF6(300-524) alone induced no NF{kappa}B 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 NF{kappa}B activity (Fig. 5). This indicates that TRAF6 is a major factor transmitting the stimulatory signal from Tio to NF{kappa}B.


Figure 6
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  		FIGURE 6.
TRAF6 directly interacts with Tio at the consensus TRAF6 binding motif. Jurkat T cells were transiently transfected with expression vector DNA or plasmids encoding HA-tagged TRAF6 and FLAG-tagged Tio or mutant P1 in the combinations given on top of the figure. In an additional set of transfections, constitutively active IKK2 (IKK2 EE) was cotransfected to override the influence of NF{kappa}B on transcription from expression vectors (column to the right). Proteins were immunoprecipitated with either anti-FLAG monoclonal antibody (IP FLAG) (A) or a polyclonal TRAF6 antibody (IP TRAF6) (B). Control precipitations without antibodies (Ø Ab) were included with each experiment. Immune complexes and cell lysates (C) were assayed for the presence of FLAG-Tio (FLAG), HA-TRAF6 (HA), or total TRAF6 (TRAF6) with the respective antibodies (shown on the left). hc, immunoglobulin heavy chain.

 
Tio Binds to TRAF6—Because of the impact of dominant negative TRAF6(300-524) on NF{kappa}B 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 NF{kappa}B, 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).


Figure 7
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  		FIGURE 7.
Membrane recruitment and Tio interaction of TRAF6 in transformed human T cells. A, cytoplasmic (cyt) and membrane (mem) proteins of transformed lymphocytes expressing either Tio (CBL-Tio) or StpC/Tip (CBL-M124) were separated and assayed for the presence of TRAF6 and Lck. Whole cell lysate (wcl) of Jurkat T cells served as a positive detection control. B and C, soluble proteins from membrane fractions of transformed T cells were subjected to immunoprecipitation with either anti-FLAG monoclonal antibody (IP FLAG) or a polyclonal TRAF6 antibody (IP TRAF6). Coprecipitated Tio or TRAF6 was detected with the corresponding antibodies (shown on the left).

 
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
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Several NF{kappa}B activation pathways converge at the level of TRAF6, a ubiquitin ligase regulating the IKK complex and thereby NF{kappa}B activity (1). We have now demonstrated that Tio, the oncoprotein of the T lymphotropic H. ateles, dramatically induced NF{kappa}B in T cells. NF{kappa}B 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 NF{kappa}B 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 NF{kappa}B (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-{gamma} (22) in that both viral oncoproteins directly address signaling intermediates common to different NF{kappa}B activation pathways.

NF{kappa}B 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 NF{kappa}B relied on a functional IKK complex and cytoplasmic NF{kappa}B, as dominant negative IKK-beta (IKK2 K44M) (65) and I{kappa}B{alpha} DN (47) blocked NF{kappa}B activation by Tio (Fig. 1 and data not shown). TRAF6 complexes activated by interleukin-1 receptor triggering not only induce NF{kappa}B 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 NF{kappa}B (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 NF{kappa}B 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 NF{kappa}B-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 Toll-like 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 NF{kappa}B activation.

Another open question addresses the biological relevance of TRAF6 interaction and NF{kappa}B activation in T cell transformation and oncogenesis by viruses expressing Tio. NF{kappa}B 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 NF{kappa}B activation and Tio-TRAF6 interaction in the promotion of T cell growth and tumor induction, progression, and maintenance.


   FOOTNOTES
 
* This work was supported by the Deutsche Forschungsgemeinschaft (SFB 466, TP C8), the BMBF (Interdisziplinäres Zentrum für klinische Forschung Erlangen, TP B5), and the German-Israeli Foundation (674/2000). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

1 To whom correspondence should be addressed: Institut für Klinische und Molekulare Virologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Schlossgarten 4, 91054 Erlangen, Germany. Tel.: 49-9131-8526495; Fax: 49-9131-8526493; E-mail: stefanie.heinemann{at}viro.med.uni-erlangen.de.

2 The abbreviations used are: PKC{theta}, protein kinase C{theta}; IKK, I{kappa}B kinase; TNFR, tumor necrosis factor receptor; TRADD, TNFR-associated death domain; TRAF, TNFR-associated factor; MYD88, myeloid differentiation primary response gene 88 protein; LMP-1, latent membrane protein-1; TAB, TAK1-binding protein; TAK1, transforming growth factor-beta-activated kinase-1; IRAK1, interleukin-1 receptor-associated kinase-1; MAPK, mitogen-activated protein kinase; MEKK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase kinase; StpC, saimiri transformation-associated protein of subgroup C; Tip, tyrosine kinase-interacting protein; Tio, two-in-one; SH3, Src homology 3; CBL, cord blood lymphocyte; DN, dominant negative; IKK2 EE, constitutively active IKK2; HA, hemagglutinin; EGFP, enhanced green fluorescent protein; Luc, luciferase; PMA, phorbol 12-myristate 13-acetate; NIK, NF{kappa}B-inducing kinase; NFAT, nuclear factor of activated T cells. Back


   ACKNOWLEDGMENTS
 
We thank Arnd Kieser, Wolfgang Hammerschmidt, and Reinhard Voll for providing expression constructs.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Ghosh, S., and Karin, M. (2002) Cell 109, (suppl.) S81-S96[CrossRef][Medline] [Order article via Infotrieve]
  2. Greten, F. R., and Karin, M. (2004) Cancer Lett. 206, 193-199[CrossRef][Medline] [Order article via Infotrieve]
  3. Lin, A., and Karin, M. (2003) Semin. Cancer Biol. 13, 107-114[CrossRef][Medline] [Order article via Infotrieve]
  4. Li, Q., and Verma, I. M. (2002) Nat. Rev. Immunol. 2, 725-734[CrossRef][Medline] [Order article via Infotrieve]
  5. Thome, M., and Tschopp, J. (2003) Trends Immunol. 24, 419-424[CrossRef][Medline] [Order article via Infotrieve]
  6. Baldwin, A. S. (2001) J. Clin. Investig. 107, 241-246[CrossRef][Medline] [Order article via Infotrieve]
  7. Li, Y., Sedwick, C. E., Hu, J., and Altman, A. (2005) J. Biol. Chem. 280, 1217-1223[Abstract/Free Full Text]
  8. Li, Z. W., Rickert, R. C., and Karin, M. (2004) Mol. Immunol. 41, 701-714[CrossRef][Medline] [Order article via Infotrieve]
  9. Lin, X., and Wang, D. (2004) Semin. Immunol. 16, 429-435[CrossRef][Medline] [Order article via Infotrieve]
  10. Sun, L., Deng, L., Ea, C. K., Xia, Z. P., and Chen, Z. J. (2004) Mol. Cell 14, 289-301[CrossRef][Medline] [Order article via Infotrieve]
  11. Dempsey, P. W., Doyle, S. E., He, J. Q., and Cheng, G. (2003) Cytokine Growth Factor Rev. 14, 193-209[CrossRef][Medline] [Order article via Infotrieve]
  12. Wajant, H., Pfizenmaier, K., and Scheurich, P. (2003) Cell Death Differ. 10, 45-65[CrossRef][Medline] [Order article via Infotrieve]
  13. Tsan, M. F. (2005) Semin. Cancer Biol.
  14. Akira, S. (2003) J. Biol. Chem. 278, 38105-38108[Free Full Text]
  15. Martin, M. U., and Wesche, H. (2002) Biochim. Biophys. Acta 1592, 265-280[Medline] [Order article via Infotrieve]
  16. Bonizzi, G., and Karin, M. (2004) Trends Immunol. 25, 280-288[CrossRef][Medline] [Order article via Infotrieve]
  17. Akagi, T., and Shimotohno, K. (1993) J. Virol. 67, 1211-1217[Abstract/Free Full Text]
  18. Grassmann, R., Berchtold, S., Radant, I., Alt, M., Fleckenstein, B., Sodroski, J. G., Haseltine, W. A., and Ramstedt, U. (1992) J. Virol. 66, 4570-4575[Abstract/Free Full Text]
  19. Kaye, K. M., Izumi, K. M., and Kieff, E. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 9150-9154[Abstract/Free Full Text]
  20. Laherty, C. D., Hu, H. M., Opipari, A. W., Wang, F., and Dixit, V. M. (1992) J. Biol. Chem. 267, 24157-24160[Abstract/Free Full Text]
  21. Leung, K., and Nabel, G. J. (1988) Nature 333, 776-778[CrossRef][Medline] [Order article via Infotrieve]
  22. Jeang, K. T. (2001) Cytokine Growth Factor Rev. 12, 207-217[CrossRef][Medline] [Order article via Infotrieve]
  23. Gires, O., Zimber-Strobl, U., Gonnella, R., Ueffing, M., Marschall, G., Zeidler, R., Pich, D., and Hammerschmidt, W. (1997) EMBO J. 16, 6131-6140[CrossRef][Medline] [Order article via Infotrieve]
  24. Mitchell, T., and Sugden, B. (1995) J. Virol. 69, 2968-2976[Abstract]
  25. Kaye, K. M., Devergne, O., Harada, J. N., Izumi, K. M., Yalamanchili, R., Kieff, E., and Mosialos, G. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 11085-11090[Abstract/Free Full Text]
  26. Kieser, A., Kaiser, C., and Hammerschmidt, W. (1999) EMBO J. 18, 2511-2521[CrossRef][Medline] [Order article via Infotrieve]
  27. Devergne, O., Hatzivassiliou, E., Izumi, K. M., Kaye, K. M., Kleijnen, M. F., Kieff, E., and Mosialos, G. (1996) Mol. Cell. Biol. 16, 7098-7108[Abstract]
  28. Schultheiss, U., Puschner, S., Kremmer, E., Mak, T. W., Engelmann, H., Hammerschmidt, W., and Kieser, A. (2001) EMBO J. 20, 5678-5691[CrossRef][Medline] [Order article via Infotrieve]
  29. Luftig, M., Prinarakis, E., Yasui, T., Tsichritzis, T., Cahir-McFarland, E., Inoue, J., Nakano, H., Mak, T. W., Yeh, W. C., Li, X., Akira, S., Suzuki, N., Suzuki, S., Mosialos, G., and Kieff, E. (2003) Proc. Natl. Acad. Sci. U. S. A. 100, 15595-15600[Abstract/Free Full Text]
  30. Falk, L., Wright, J., Wolfe, L., and Deinhardt, F. (1974) Int. J. Cancer 14, 244-251[Medline] [Order article via Infotrieve]
  31. Falk, L., Johnson, D., and Deinhardt, F. (1978) Int. J. Cancer 21, 652-657[Medline] [Order article via Infotrieve]
  32. Falk, L. A., Nigida, S. M., Deinhardt, F., Wolfe, L. G., Cooper, R. W., and Hernandez-Camacho, J. I. (1974) Int. J. Cancer 14, 473-482[Medline] [Order article via Infotrieve]
  33. Jung, J. U., Choi, J. K., Ensser, A., and Biesinger, B. (1999) Semin. Cancer Biol. 9, 231-239[CrossRef][Medline] [Order article via Infotrieve]
  34. Biesinger, B., Muller-Fleckenstein, I., Simmer, B., Lang, G., Wittmann, S., Platzer, E., Desrosiers, R. C., and Fleckenstein, B. (1992) Proc. Natl. Acad. Sci. U. S. A. 89, 3116-3119[Abstract/Free Full Text]
  35. Duboise, S. M., Guo, J., Czajak, S., Desrosiers, R. C., and Jung, J. U. (1998) J. Virol. 72, 1308-1313[Abstract/Free Full Text]
  36. Wehner, L. E., Schroder, N., Kamino, K., Friedrich, U., Biesinger, B., and Ruther, U. (2001) DNA Cell Biol. 20, 81-88[CrossRef][Medline] [Order article via Infotrieve]
  37. Medveczky, M. M., Geck, P., Sullivan, J. L., Serbousek, D., Djeu, J. Y., and Medveczky, P. G. (1993) Virology 196, 402-412[CrossRef][Medline] [Order article via Infotrieve]
  38. Albrecht, J. C., Friedrich, U., Kardinal, C., Koehn, J., Fleckenstein, B., Feller, S. M., and Biesinger, B. (1999) J. Virol. 73, 4631-4639[Abstract/Free Full Text]
  39. Albrecht, J. C., Biesinger, B., Muller-Fleckenstein, I., Lengenfelder, D., Schmidt, M., Fleckenstein, B., and Ensser, A. (2004) J. Virol. 78, 9814-9819[Abstract/Free Full Text]
  40. Biesinger, B., Tsygankov, A. Y., Fickenscher, H., Emmrich, F., Fleckenstein, B., Bolen, J. B., and Broker, B. M. (1995) J. Biol. Chem. 270, 4729-4734[Abstract/Free Full Text]
  41. Jung, J. U., Lang, S. M., Friedrich, U., Jun, T., Roberts, T. M., Desrosiers, R. C., and Biesinger, B. (1995) J. Biol. Chem. 270, 20660-20667[Abstract/Free Full Text]
  42. Albrecht, J. C., Muller-Fleckenstein, I., Schmidt, M., Fleckenstein, B., and Biesinger, B. (2005) J. Virol. 79, 10507-10513[Abstract/Free Full Text]
  43. Hartley, D. A., and Cooper, G. M. (2000) J. Biol. Chem. 275, 16925-16932[Abstract/Free Full Text]
  44. Heck, E., Lengenfelder, D., Schmidt, M., Muller-Fleckenstein, I., Fleckenstein, B., Biesinger, B., and Ensser, A. (2005) J. Virol. 79, 5713-5720[Abstract/Free Full Text]
  45. Bauer, F., Hofinger, E., Hoffmann, S., Rosch, P., Schweimer, K., and Sticht, H. (2004) Biochemistry 43, 14932-14939[CrossRef][Medline] [Order article via Infotrieve]
  46. Lee, H., Choi, J. K., Li, M., Kaye, K., Kieff, E., and Jung, J. U. (1999) J. Virol. 73, 3913-3919[Abstract/Free Full Text]
  47. Schwarz, E. M., Van Antwerp, D., and Verma, I. M. (1996) Mol. Cell. Biol. 16, 3554-3559[Abstract]
  48. Voll, R. E., Jimi, E., Phillips, R. J., Barber, D. F., Rincon, M., Hayday, A. C., Flavell, R. A., and Ghosh, S. (2000) Immunity 13, 677-689[CrossRef][Medline] [Order article via Infotrieve]
  49. Saksela, K., and Baltimore, D. (1993) Mol. Cell. Biol. 13, 3698-3705[Abstract/Free Full Text]
  50. Rincon, M., and Flavell, R. A. (1996) Mol. Cell. Biol. 16, 1074-1084[Abstract]
  51. Gschwendt, M., Dieterich, S., Rennecke, J., Kittstein, W., Mueller, H. J., and Johannes, F. J. (1996) FEBS Lett. 392, 77-80[CrossRef][Medline] [Order article via Infotrieve]
  52. Catley, M. C., Cambridge, L. M., Nasuhara, Y., Ito, K., Chivers, J. E., Beaton, A., Holden, N. S., Bergmann, M. W., Barnes, P. J., and Newton, R. (2004) J. Biol. Chem. 279, 18457-18466[Abstract/Free Full Text]
  53. Ballard, D. W., Bohnlein, E., Lowenthal, J. W., Wano, Y., Franza, B. R., and Greene, W. C. (1988) Science 241, 1652-1655[Abstract/Free Full Text]
  54. Ruben, S., Poteat, H., Tan, T. H., Kawakami, K., Roeder, R., Haseltine, W., and Rosen, C. A. (1988) Science 241, 89-92[Abstract/Free Full Text]
  55. Prosch, S., Staak, K., Stein, J., Liebenthal, C., Stamminger, T., Volk, H. D., and Kruger, D. H. (1995) Virology 208, 197-206[CrossRef][Medline] [Order article via Infotrieve]
  56. Sambucetti, L. C., Cherrington, J. M., Wilkinson, G. W., and Mocarski, E. S. (1989) EMBO J. 8, 4251-4258[Medline] [Order article via Infotrieve]
  57. Merlo, J. J., and Tsygankov, A. Y. (2001) Virology 279, 325-338[CrossRef][Medline] [Order article via Infotrieve]
  58. Pullen, S. S., Dang, T. T., Crute, J. J., and Kehry, M. R. (1999) J. Biol. Chem. 274, 14246-14254[Abstract/Free Full Text]
  59. Ye, H., Arron, J. R., Lamothe, B., Cirilli, M., Kobayashi, T., Shevde, N. K., Segal, D., Dzivenu, O. K., Vologodskaia, M., Yim, M., Du, K., Singh, S., Pike, J. W., Darnay, B. G., Choi, Y., and Wu, H. (2002) Nature 418, 443-447[CrossRef][Medline] [Order article via Infotrieve]
  60. Jung, J. U., and Desrosiers, R. C. (1995) Mol. Cell. Biol. 15, 6506-6512[Abstract]
  61. Izumi, K. M. (2004) Prog. Mol. Subcell. Biol. 36, 269-288[Medline] [Order article via Infotrieve]
  62. Inoue, J., Ishida, T., Tsukamoto, N., Kobayashi, N., Naito, A., Azuma, S., and Yamamoto, T. (2000) Exp. Cell Res. 254, 14-24[CrossRef][Medline] [Order article via Infotrieve]
  63. Li, H. P., and Chang, Y. S. (2003) J. Biomed. Sci. 10, 490-504[Medline] [Order article via Infotrieve]
  64. Choi, J. K., Ishido, S., and Jung, J. U. (2000) J. Virol. 74, 8102-8110[Abstract/Free Full Text]
  65. Mercurio, F., Zhu, H., Murray, B. W., Shevchenko, A., Bennett, B. L., Li, J., Young, D. B., Barbosa, M., Mann, M., Manning, A., and Rao, A. (1997) Science 278, 860-866[Abstract/Free Full Text]
  66. Kundu, J. K., and Surh, Y. J. (2004) Mutat. Res. 555, 65-80[Medline] [Order article via Infotrieve]
  67. Baud, V., Liu, Z. G., Bennett, B., Suzuki, N., Xia, Y., and Karin, M. (1999) Genes Dev. 13, 1297-1308[Abstract/Free Full Text]
  68. Bradley, J. R., and Pober, J. S. (2001) Oncogene 20, 6482-6491[CrossRef][Medline] [Order article via Infotrieve]
  69. Song, H. Y., Regnier, C. H., Kirschning, C. J., Goeddel, D. V., and Rothe, M. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 9792-9796[Abstract/Free Full Text]
  70. Takeda, K., and Akira, S. (2004) Semin. Immunol. 16, 3-9[CrossRef][Medline] [Order article via Infotrieve]
  71. Malinin, N. L., Boldin, M. P., Kovalenko, A. V., and Wallach, D. (1997) Nature 385, 540-544[CrossRef][Medline] [Order article via Infotrieve]
  72. Yin, M. J., Christerson, L. B., Yamamoto, Y., Kwak, Y. T., Xu, S., Mercurio, F., Barbosa, M., Cobb, M. H., and Gaynor, R. B. (1998) Cell 93, 875-884[CrossRef][Medline] [Order article via Infotrieve]

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