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Schwab, U., Stein, H., Gerdes, J., Lemke, H., Kirchner, H., Schaadt,. M. & Diehl, V. (1982) Nature (London) 299, 65–67. 5. Ellis, T. M., Simms, P. E., Slivnick, D. J., ...

Proc. Natl. Acad. Sci. USA Vol. 93, pp. 14053–14058, November 1996 Medical Sciences

Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation (Hodgkin diseaseyT-cell activationycytokine receptorysignal transductionyprotein motif)

´PHANE A NSIEAU*†, INKA SCHEFFRAHN†‡, GEORGE MOSIALOS†§, HEIKE BRAND‡, JUSTUS DUYSTER‡, STE ¨ BINGER‡, ELLIOTT K IEFF§, KENNETH KAYE§, JOSEPHINE HARADA§, BILL DOUGALL¶, GABI HU ‡ ¨ FRIEDHELM HERRMANN , ACHIM LEUTZ*, AND HANS-JURGEN GRUSS‡i ‡Department of Internal Medicine III, University of Ulm Medical Center, Robert-Koch-Strasse 8, D-89072 Ulm, Germany; *Department of Tumor Development and Differentiation, Max-Delbru ¨ck Center for Molecular Medicine, Robert-Ro ¨ssle-Strasse 10, D-13125 Berlin, Germany; §Department of Microbiology and Molecular Genetics, Brigham & Women’s Hospital, Harvard Medical School, 75 Francis Street, Boston, MA 02115; and ¶Department of Molecular Biology,

Immunex Research and Development Corporation, University Street 51, Seattle, WA 98112

Contributed by Elliott Kieff, September 24, 1996

Although the homology among TNFRs is largely restricted to the extracellular domain, TNFRs have similarities in their biological effects, and recent investigations have revealed some shared intracellular signaling pathways (11). TNFRI, FAS, and nerve growth factor receptor (NGFR) share a death domain (12) that can interact with other death domain proteins (e.g., RIP, FADDyMORT-1, and TRADD) in inducing cell death (13–16). CD27 and 4-1BB share a PCXXXCP (where X is any amino acid) motif that mediates cytoplasmic tyrosine kinase activation (17). TNFRI and TNFRII appear to share the phosphorylation of serine-threonine kinases, activation of lipid second messengers, and induction of similar transcription factors, including nuclear factor kappa B (NF-kB; reviewed in ref. 18). CD30 and CD40 signaling also activates kinases and induces NF-kB (9, 19–26). A family of TNF receptor-associated factors (TRAF-1, -2 and -3) have been identified that associate differentially with several TNFRs, including TNFRII, CD40, lymphotoxin b-receptor, and the Epstein–Barr virus (EBV) oncoprotein LMP1 (reviewed in refs. 27 and 28). TRAF-2 has been specifically implicated in NF-kB activation from these receptors (29). Having isolated a cDNA encoding the TRAF homology domain of TRAF2 in a yeast two-hybrid screen with the cytoplasmic domain of CD30, we set out to evaluate the interaction of TRAFs with CD30 and with CD30-mediated NF-kB activation.

ABSTRACT CD30 is a member of the tumor necrosis factor receptor superfamily, which can transduce signals for proliferation, death, or nuclear factor kappa B (NF-kB) activation. Investigation of CD30 signaling pathways using a yeast twohybrid interaction system trapped a cDNA encoding the tumor necrosis factor receptor-associated factor (TRAF)-2 TRAF homology domain. TRAF-1 and TRAF-3 also interacted with CD30, and >90% of in vitro-translated TRAF-1 or -2, or 50% of TRAF-3, bound to the CD30 cytoplasmic domain. TRAF-1, -2, and -3 bound mostly, but not exclusively, to the carboxyl-terminal 36 residues of CD30. The binding was strongly inhibited by a CD30 oligopeptide centered around a PXQXT (where X is any amino acid) motif shared with CD40 and the Epstein–Barr virus transforming protein LMP1, indicating that this motif in CD30 is an important determinant of TRAF-1, -2 or -3 interaction. At least 15% of TRAF-1, -2, or -3 associated with CD30 when coexpressed in 293 cells. The association was not affected by CD30 cross-linking. However, cross-linking of CD30 activated NF-kB. NF-kB activation was dependent on the carboxylterminal 36 amino acids of CD30 that mediate TRAF association. TRAF-2 has been previously shown to have a unique role in TRAF-mediated NF-kB activation, and NF-kB activation following CD30 cross-linking was blocked by a dominant negative TRAF-2 mutant. These data indicate that CD30 cross-linkinginduced NF-kB activation is predominantly TRAF-2-mediated.

MATERIALS AND METHODS Cells. 293 cells were obtained from the American Type Culture Collection and maintained in Dubecco’s modified Eagle’s medium (DMEM) containing 10% fetal calf serum, 100 mg of penicillinystreptomycin per ml, and 2 mmol of glutamine (GIBCO) per ml in an atmosphere containing 5% CO2 in humidified air at 378C. Antibodies and Peptides. The agonistic anti-human CD30 monoclonal antibody (mAb) M67 (murine IgG1) was kindly provided by Immunex Research and Development (30). Crosslinking of CD30 was achieved by rabbit anti-mouse IgG1-specific antibody (Dianova, Hamburg, Germany). Fluorescein isothiocyanate (FITC)-conjugated anti-CD30 mAb Ber-H2 and an irrelevant control mAb were purchased from Dako and used in flow cytometry analysis (30). A peptide of sequence HTPHY-

CD30 is a phosphorylated 120-kDa transmembrane glycoprotein that has cysteine-rich extracellular repeats characteristic of the tumor necrosis family receptor (TNFR) superfamily and a 188-aa cytoplasmic carboxyl-terminal domain (1, 2). CD30 was identified as a surface antigen characteristic of Hodgkin disease (HD) tumor cells, but it is also expressed on activated, CD45RO1, memory T lymphocytes and on some virustransformed T or B lymphocytes (3–5). The CD30 ligand (CD30L; ref. 6) costimulates peripheral blood T-cell proliferation or apoptosis, induces surface expression of activation and adhesion molecules (e.g., CD54, CD80, and CD86), and induces cytokine [e.g., interleukin 2, tumor necrosis factor (TNF), and interferon g] secretion (7). Recently, deletion of the murine CD30 gene revealed that CD30 may have a unique role in negative thymic selection following exposure to antigen (8). CD30 has also been implicated in HIV replication and associated T-cell depletion (9, 10).

Abbreviations: TNFR, tumor necrosis family receptor; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor; HD, Hodgkin disease; NF-kB, nuclear factor kappa B; b-gal, b-galactosidase; EBV, Epstein–Barr virus. †S.A., I.S., and G.M. contributed equally to this work. i To whom reprint requests should be addressed at: Department of Internal Medicine III, University of Ulm Medical Center, RobertKoch-Strasse 8, D-89081 Ulm, Germany.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ‘‘advertisement’’ in accordance with 18 U.S.C. §1734 solely to indicate this fact.



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PEQETEPPLG corresponding to amino acids 556–570 of the CD30 cytoplasmic domain or a random control peptide (PSTMVYDACRMIRERIPEA) were synthesized using the automatic multipeptide synthesizer (SYRO 150; MultiSynTec, Bochum, Germany) and purified by HPLC (purity .95%). Plasmid Constructions. A cDNA encoding the cytoplasmic tail of CD30 was generated by PCR from the full-length human CD30 plasmid (kindly provided by R. G. Goodwin, Immunex) using sense (59-CGCGGGGGATCCACCGGAGGGCCTGCA-39) and antisense (59-CGCGGGATCCTCACTTTCCAGAGGCAGCT-39) primers. The PCR products were cloned into the yeast two-hybrid vector pGBT9 (CLONTECH). The human TRAF-1, -2, and -3 full-length or deletion cDNAs were cloned in frame into the vector pACTII or pGAD10 (31, 32). A glutathione S-transferase (GST)-fusion construct for the cytoplasmic CD30 region (GST–CD30cyt) was generated by PCR with sense (59-CGGGATCCCACCGGAGGGCCTGCAG-39) and antisense (59-CGCGGGATCCTCACTTTCCAGAGGCAGCT-39) primers. Similarly, a cDNA for the CD30 cytoplasmic region without amino acids 560 –595 (GST– CD30cytD560-595) was generated using sense (59-CGGGATCCCACCGGAGGGCCTGCAG-39) and antisense (59-CGCGGATCCGTGGGGGGTATGGTCCGCCTC-39) primers. These PCR products were cloned into pGEX-2TK (33). Similarly, the full-length CD30 cDNA and the carboxyl-terminal CD30 deletion mutant (CD30D560-595) were subcloned into the expression plasmid pCI-neo (Promega). The dominantnegative TRAF-2 construct was prepared by deleting the RING finger motif (pTRAF-2D1-86) as described elsewhere (29) and was subcloned into the plasmid pcDNA1 (Invitrogen). Yeast Two-Hybrid System. The two-hybrid system, the yeast strains (SFY526, HF7c, or Y190) and the human EBVtransformed peripheral blood B-lymphocyte library were purchased from CLONTECH. Protein interaction was quantified by liquid b-galactosidase (b-gal) assays using ortho-nitro-b-Dgalactosidase as substrate (31). Plasmid DNA was sequenced and analyzed using the European Molecular Biology Laboratory (EMBL) data bank (February 1996). Production and Purification of GST-Fusion Proteins. GSTfusion proteins were expressed and purified from Escherichia coli and transformed with pGEX-2TK or pGEX-2TK with appropriate inserts (34). Fusion protein concentrations of 3–5 mg of glutathione-agarose beads (Pharmacia) per ml were routinely obtained. In Vitro Protein Binding Assay. The pSG5 vector (Stratagene) encoding the full-length TRAF-1, TRAF-2, and TRAF-3 clones were used for in vitro transcription and translation in the presence of [35S]methionine (specific activity, 5 mCiyml, 1 Ci 5 37 GBq; Amersham) using 12.5 ml of reticulocyte lysate (TNT-lysate, Promega). 35S-labeled translated proteins were diluted to 200 ml with binding buffer (50 mM Tris, pH 8.0y150 mM NaCly0.1% Nonidet P-40y5 mM DTTy2 mM EDTAy1 mM phenylmethylsulfonyl fluoridey1 mg of leupeptin per mly2 mg of aprotinin per mly5 mM benzamidine) and precleared with glutathione beads. For each in vitro binding assay, 10 ml of GST or the appropriate GST-fusion protein coupled to glutathione-Sepharose beads ('5 mg) were incubated with 10 ml of the diluted in vitro-translated proteins for 1 hr at 48C. In competition binding assays, peptides were used at 50-fold excess and preincubated with the in vitrotranslated proteins for 2 hr at 48C before binding assay. The beads were recovered by centrifugation, washed five times with 1 ml of binding buffer, and bound material was subjected to SDSyPAGE (35). Gels were autoradiographed or stained with amino black for equal protein loading. Transfection, Immunoblotting, and NF-kB Electrophoretic Mobility Shift Assay. 293 cells (2 3 106 cells per 10-cm dish) were seeded and transfected 18–24 hr later using lipofection. After 48 hr, cells were stimulated via CD30 cross-linking. Nuclear extracts were prepared for detection of NF-kB activation (36). Protein

Proc. Natl. Acad. Sci. USA 93 (1996) concentrations were determined by the Bradford assay (37). Double-stranded oligodeoxynucleotides corresponding to the NF-kB binding site of the interleukin 6 promoter were annealed and end-labeled as previously described (38). The end-labeled oligonucleotides (1 ng; '10,000 cpm) were incubated with 10 mg of nuclear extracts in an incubation buffer containing 25 mM Hepes (pH 7.5), 5 mM MgCl2, 1 mM dithiothreitol, 1 mM EDTA, and 10% (volyvol) glycerol (Sigma) for 20 min at room temperature. For competition assays, a 50-fold molar excess of wild-type or mutant oligonucleotides were added. The reaction products were analyzed by electrophoresis on 5% polyacrylamide gels and autoradiography. For immunoprecipitation analysis, 293 cells were cotransfected with the full-length or deletion CD30 construct and with each of the FLAG-tagged TRAF-1, -2, and -3 vectors. Cells were lysed in 1 ml of lysis buffer [10 mM Tris (pH 7.4), 5 mM EDTA, 150 mM NaCl, 1% Triton X-100, and protease inhibitors, including phenylmethylsulfonyl fluoride, aprotinin, leupeptin, and pepstatin]. Cell debris were removed by centrifugation. The cell lysates were precleared with protein GSepharose beads (Pharmacia) for 1 hr at 48C. Aliquots of lysates were incubated with 10 mg of anti-CD30 antibody per ml and then mixed with slurry of protein G-Sepharose beads. After extensive washings in lysis buffer, the protein complexes were recovered by boiling in SDS sample buffer and analyzed by SDSyPAGE. After transfer to Immobilon P membrane (Millipore), the blots were preblocked and subjected to Western blot analysis with either anti-CD30 Ber-H2 mAb or FLAG M2 mAb (Kodak) followed by horseradish peroxidase-conjugated goat anti-mouse IgG and detected using enhanced chemiluminescence (Amersham).

RESULTS TRAF-2, TRAF-1, and TRAF-3 Interact with the CD30 Cytoplasmic Domain. To identify molecules that directly interact with the intracellular domain of human CD30, the carboxyl-terminal cytoplasmic domain was used as a bait in a yeast two-hybrid screen for interacting proteins encoded by cDNAs from a B-lymphoblast library. Approximately 1 3 106 transformants were analyzed and five clones were strongly positive for b-gal activity. A lamin C negative control construct failed to interact with the five clones. The five clones encoded the TRAF-N and -C homology domains of TRAF-2. Two clones differed from the other three at their 59 and 39 ends. The three independent TRAF-2 encoding clones bound strongly to the cytoplasmic domain of CD30 as determined by a quantitive b-gal assay (TRAF-2aa36/501 with 126 6 7, TRAF-2aa156/501 with 39 6 2, and TRAF-2aa250/501 with 56 6 2 b-gal units using the yeast strain SFY526; Table 1). Deletions of the TRAF homology domain abrogated b-gal activity (data not shown). In summary, the carboxyl-terminal TRAF homology domain of TRAF-2 is important in TRAF-2 binding to the cytoplasmic domain of CD30; the N-terminal ring and putative zinc finger domains of TRAF-2 are not important. Since TRAF-2 and -3 bind to CD40 and TRAF-1 and -2 bind to TNFRII, we also tested the interaction of TRAF-1 and TRAF-3 with the CD30 cytoplasmic domain in yeast. Surprisingly, the CD30 cytoplasmic domain interacted equally well with all three TRAF proteins in yeast two-hybrid assays (Table 1). TRAF-1 and -3 truncated at their amino termini showed comparable activities, while deletions of the TRAF homology domain failed to interact with CD30 (Table 1). In vitro-translated TRAF-1, -2, or -3 (Fig. 1A) also bound to GST–CD30 cytoplasmic domain fusion proteins (GST– CD30cyt), which had been expressed and purified from E. coli. All three TRAF proteins bound at a high level to GST–CD30cyt and did not bind to GST (Fig. 1B). More than 90% of the input TRAF-1 and TRAF-2 and 50–60% of TRAF-3 bound to GST– CD30cyt. TRAF-1, -2, and -3 Associate with CD30 in Vivo. TRAF-1, -2, or -3 associated, in vivo, with CD30 when each FLAG epitopetagged TRAF was coexpressed with CD30 in 293 cells. TRAF

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Proc. Natl. Acad. Sci. USA 93 (1996)


Table 1. The cytoplasmic domain of CD30 directly interacts with TRAF-1, -2, and -3 in yeast DNA binding hybrid (in pGBT9)

Transactivation hybrid (in pGAD10)

b-gal activity

His auxotrophy

CD30 — Lamin CD30 CD30 CD30 CD30 — Lamin CD30 CD30 CD30 CD30 CD30 CD30 — Lamin CD30 CD30 CD30 CD30 p53 (amino acids

— TRAF-1 TRAF-1 TRAF-1 TRAF-1D1-52 TRAF-1D1-184 TRAF-1D185-416 TRAF-2 TRAF-2 TRAF-2 TRAF-2D1-35* TRAF-2D1-155* TRAF-2D1-249* TRAF-2D1-264 TRAF-2D265-501 TRAF-3 TRAF-3 TRAF-3 TRAF-3D1-308 TRAF-3D1-345 TRAF-3D309-568 SV40 large T-antigen (amino acids 84y708)

2 2 2 1 1 1 2 2 2 1 1 1 1 1 2 2 2 1 1 1 2

2 2 2 1 1 1 2 2 2 1 1 1 1 1 2 2 2 1 1 1 2







Yeast was cotransformed with either pGBT9-CD30 or pGBT9lamin C and pGAD constructs with the activation domain of GAL4, as indicated. The b-gal activity was detected within 2 hr of incubation. *Isolated TRAF-2 clones from the yeast two-hybrid screen.

coexpression did not affect CD30 expression by Western blot (Fig. 1C) or by live cell staining and flow cytometry (data not shown). CD30 was immunoprecipitated from cell extracts using the mAb Ber-H2, which is directed against the extracellular domain of CD30. Coprecipitating FLAG epitope tagged TRAFs were detected by Western blotting with the anti-FLAG M2 mAb. TRAF-1, -2, and -3 specifically associated in vivo with CD30 (Fig. 1D). At least 15% of each of the FLAG-tagged TRAF proteins were detected in the CD30 Ber-H2 mAb immunoprecipitate. FLAG-tagged TRAF proteins were not detected in the Ber-H2 immunopreciptate from cells transfected with a FLAG-tagged TRAF-1, -2, or -3 expression vector without CD30 coexpression. In line with the in vitro binding, slightly less TRAF-3 was associated with CD30 than TRAF-1 or TRAF-2 (Fig. 1D and data not shown). Surprisingly, cross-linking of CD30 for 5–60 min before lysis of cotransfected 293 cells failed to either increase or decrease TRAF-1, -2, or -3 association with CD30 (Fig. 1D). Similarly cross-linking of transfected CD30 in 293 cells did not affect the extent of association of CD30 with endogenous TRAF-2 (data not shown). A CD30 Motif Shared with CD40 and LMP1 Is Important for TRAF Interaction. TRAF-1, TRAF-2, and TRAF-3 bind to a 12-aa sequence in the carboxyl-terminal cytoplasmic domain of the EBV oncoprotein LMP1 and alanine mutagenesis of each of the residues indicates that PQQATDD are critical for TRAF binding (39). A similar motif, PXQXT (where X is any amino acid), is also in the cytoplasmic domain of CD40 and mutation of the T to A in CD40 abrogates CD40 signaling and TRAF binding (40, 41). CD30 also has a PXQXT sequence at residues 561–565 (Fig. 2A). The PXQXT sequence motif is conserved between murine and human CD30 (Fig. 2 A). The role of this CD30 motif in TRAF binding was evaluated using GST-fusion proteins with the cytoplasmic domain of CD30 or with a 36-aa carboxyl-terminal truncation of the cytoplasmic domain that ends at residue 559 (GST–CD30cytD560-595) and therefore lacks the putative TRAF binding site. TRAF-1, -2,

FIG. 1. Association of human TRAF-1, TRAF-2, and TRAF-3 with the cytoplasmic domain of CD30. (A) TRAF-1, -2, and -3 proteins were [35S]methionine-labeled by in vitro translation (IVT). Ten microliters (20%) of each in vitro translation reaction was loaded. (B) The cytoplasmic domain of CD30 was fused to GST (GST–CD30cyt) and bound to glutathione beads. GST or GST–CD30cyt were incubated with 25% of the in vitro-translated TRAF proteins. The fraction bound to glutathione beads was analyzed by SDSyPAGE. Comparable GST loading was confirmed by staining with amino black and Western blotting against GST (data not shown). More than 90% of TRAF-1 and TRAF-2 and 50–60% of TRAF-3 respectively bound to GST– CD30cyt. (C) Western blot analysis of CD30 and FLAG expression in transfected 293 cells. 293 cells were transiently cotransfected with the full-length CD30 expression vector (5 mg) and the indicated FLAGtagged TRAF constructs (5 mg). After 48 hr, lysates were prepared from unstimulated and anti-CD30-stimulated (15 min) transfected 293 cells. Thirty microliters of lysates (1y30) were separated by SDSy PAGE, blotted, preblocked, and probed for CD30 andyor FLAGtagged TRAF proteins using anti-CD30 Ber-H2 or anti-FLAG M2 mAb followed by horseradish peroxidase-conjugated goat anti-mouse IgG and detection using enhanced chemiluminescence (FLAG indicates the transfected FLAG-tagged TRAF protein). The molecular weight (MW) standard is shown (in thousands). (D) In vivo interaction of TRAF-1, -2, and -3 with the cytoplasmic domain of CD30. One hundred microliters of the cell lysates analyzed in C were subjected to immunoprecipitation with the anti-CD30 Ber-H2 mAb. This mAb shows no cross-reactivity with the transfected TRAF proteins (data not shown). Coprecipitating FLAG-tagged TRAF proteins were detected by immunoblotting using the anti-FLAG M2 mAb (indicated by FLAG). KARPAS 299 is a large cell anaplastic lymphoma line.

and -3 bound much more efficiently to GST–CD30cyt than to GST–CD30cytD560-595 (Fig. 2B). Further CD30cytD560-595 did


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FIG. 2. A critical CD30 receptor sequence motif involved in TRAF binding. (A) A common sequence motif is involved in TRAF binding and is shared by CD30, CD40, and LMP1. Sequence aligment of the cytoplasmic domains of CD30, CD40, and LMP1 show the TRAF binding motif centered around the consensus sequence PXQXT (gray box). The mouse and human CD30 and CD40 sequences are highly conserved around the putative TRAF binding motif (1, 42– 44). (B) The distal region of the cytoplasmic domain of CD30 is involved in TRAF binding. GST or GST-fusion proteins containing the full-length cytoplasmic domain of CD30 (CD30cyt) or a carboxyl-terminal deletion (CD30 lacking the carboxyl-terminal 36 aa; CD30cytD560-595) bound to glutathione beads were incubated with in vitro-translated TRAF-1, -2, and -3 proteins (25%), respectively. Reaction mixtures were analyzed by SDSy PAGE and autoradiography. A proximal deletion mutant of the cytoplasmic domain of CD30 (CD30cytD408-430) showed similar interaction as the full-length GST–CD30cyt fusion protein with all three TRAF proteins (data not shown). Western blotting confirmed that equal amounts of GST were used (data not shown). I, input; B, bound; and F, free. (C) Interaction of TRAF proteins with the cytoplasmic domain of CD30 was blocked by a peptide overlapping the TRAF binding domain in CD30. [35S]Methionine-labeled TRAF-1, -2, and -3 proteins (25%) were preincubated with 50-fold excess of a random control peptide (PSTMVYDACRMIRERIPEA) or the ‘‘P-Q-T’’ peptide encompassing amino acids 556 –570 of the cytoplasmic domain of CD30 (HTPHYPEQETEPPLG) for 2 hr at 48C, followed by incubation with the full-length cytoplasmic domain of CD30 fused to GST or GST alone for 1 hr at 48C and analyzed by SDSyPAGE. Equal GST protein loading was confirmed by Western blotting (data not shown).

Proc. Natl. Acad. Sci. USA 93 (1996) not interact significantly in yeast two-hybrid assays with GADTRAF-1, -2, or -3 fusion proteins. Moreover, when 293 cells were cotransfected with a TRAF-1, -2, or -3 expression vector and a CD30D560-595 expression vector, ,10% of the TRAF-1, -2, and -3 that coimmunoprecipitated with wild-type CD30 was in the CD30D560-595 immunoprecipitate. These results are all consistent with the major TRAF binding activity being within CD30 amino acids 560–595. However, a small fraction of in vitro-translated TRAF-1, -2, or -3 still bound to the GST– CD30cytD560-595, and the binding was clearly above that obtained with the GST negative control (Fig. 2B). This is the most sensitive of the three assays used and the residual binding could be due to a weak TRAF binding site in the CD30 cytoplasmic domain, amino-terminal to residue 559. To evaluate more precisely the role of the CD30 PXQXT motif in binding each of the TRAFs, a pentadecapeptide (HTPHYPEQETEPPLG) that included this motif was examined for its ability to block TRAF-1, -2, or -3 binding to CD30. As shown in Fig. 2C, a 50-fold excess of this peptide blocked most of the binding of in vitro-translated TRAF-1, -2, or -3 to the CD30 cytoplasmic domain (Fig. 2C). In contrast, a control peptide had no effect on TRAF-1, -2, or -3 binding to CD30. These data are compatible with the CD30 PXQXT site being an important and potentially dominant CD30 domain in binding TRAF-1, -2, and -3. TRAF-2 Is a Mediator of NF-kB Activation by CD30. The role of the CD30 TRAF binding site in CD30-induced NF-kB activation was evaluated in CD30-transfected 293 cells in which NF-kB induction is dependent on CD30 ligand or agonistic antibody (Fig. 3 A and B). Equal amounts of CD30 or CD30D560595 were expressed in 293 cells (Fig. 3C). Agonistic M67 antiCD30 mAb and rabbit anti-mouse IgG1 antibody treatment increased NF-kB levels 8- to 10-fold in 293 cells expressing full-length CD30 but had no significant effect on 293 cells expressing CD30D560-595, which lacks the TRAF binding motif or on 293 cells that had been transfected with the empty expression vector (Fig. 3 A and B). These data implicate the TRAF binding domain in CD30-mediated NF-kB activation. TRAF-2 has been specifically implicated in NF-kB activation by TNFRII and CD40 (29). Overexpression of TRAF-2 but not of TRAF-1 or TRAF-3 induced NF-kB activation in 293 cells, and a deletion mutant of TRAF-2 lacking the RING finger had a dominant negative effect on TNFRII or CD40 induced NF-kB activation (29). Coexpression of the dominant negative mutant of TRAF-2 (pTRAF-2D1-86) with CD30 into 293 cells did not affect CD30 expression but prevented CD30 cross-linking from activating NF-kB (Fig. 3 B and C). Since TRAF-2 can specifically mediate NF-kB activation in 293 cells (29), the blocking effect of the dominant negative TRAF-2 specifically implicates TRAF-2 binding in CD30-mediated NF-kB activation.

DISCUSSION In demonstrating CD30 interaction and association in vivo with the TNFR-associated factors TRAF-1, -2, and -3, these data extend the similarity of CD30 to other members of the TNFR superfamily and to the EBV transforming protein, LMP1. CD30 appears to be unique among TNFRs and similar to LMP1 in interacting directly with TRAF-1, -2, and -3; while CD40 and TNFR II interact with TRAF-2 and TRAF-3 but not directly with TRAF-1 (29, 31, 32, 41, 45–47). CD30 cross-linking has costimulatory and survival effects in T lymphocytes, which are similar to the effects of CD40 cross-linking or of LMP1 expression in B lymphocytes. CD30 or CD40 cross-linking or LMP1 expression can induce expression of activation and adhesion molecules, stimulate cytokine secretion, and activate NF-kB (7, 29, 48). Given the coincidence of similarity in intracellular interaction with TRAF-2 and -3 and in effects on cell growth and survival, TRAF-2 and -3 are likely mediators of these effects. This is particularly likely, since LMP1 interacts with TRAFs and medi-

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Proc. Natl. Acad. Sci. USA 93 (1996)


FIG. 3. NF-kB activation of CD30-stimulated cells is mediated through TRAF proteins. (A) 293 cells were transfected with either the full-length CD30 cDNA or a deletion mutant lacking the distal 35 aa (CD30D560-595). Forty-eight hours after transfection, 293 cells were cross-linked using the anti-CD30 mAb M67 in the presence of a rabbit anti-mouse IgG1-specific antibody for 2 hr. Nuclear proteins were prepared and gel mobility shift assays were performed using a double-stranded oligodeoxynucleotide containing a NF-kB binding site as a probe. (B) 293 cells were cotransfected with the full-length CD30 expression vector (CD30) and either a control plasmid (Vector) or the negative dominant TRAF-2 plasmid (pTRAF-2D1-86). Forty-eight hours after transfection, 293 cells were cross-linked using the anti-CD30 mAb M67 plus rabbit anti-mouse IgG1-specific antibody (anti-CD30) for 2 hr. Nuclear proteins were prepared and analyzed for NF-kB activation by electrophoretic mobility-shift assay. Specificity of the complex was confirmed by competition assays using 50-fold molar excess of unlabeled NF-kB binding oligodesoxynucleotide (WT Comp.) or a mutated NF-kB site (MT Comp.). (C) CD30 surface expression of transfected 293 cells was determined by flow cytometry using the fluorescein isothiocyanate (FITC)-conjugated anti-CD30 mAb Ber-H2.

ates transforming effects through a relatively short 45-aa sequence (ref. 39; for review see ref. 48). The CD30 cytoplasmic domain interacted strongly with TRAF-1, -2, and -3 in yeast two-hybrid assays and in binding assays using GST–CD30 cytoplasmic domain fusions with in vitro-translated TRAFs. After these binding studies were done, TRAF-1 and -2 were reported to interact with the CD30 cytoplasmic domain in yeast two-hybrid assays and with a GST–CD30 cytoplasmic domain fusion protein in 293 cells (49) and TRAF-1, -2, and -3 were reported to interact with the CD30 cytoplasmic domain in yeast and with a GST–CD30 cytoplasmic domain fusion protein in vitro (50). The data presented here however are the only demonstration of wild-type CD30 association with TRAF-1, TRAF-2, and TRAF-3 in vivo. Current and previous studies partially define the TRAF-1, TRAF-2, or TRAF-3 and CD30 domains which interact. As has been previously found for TRAF-2 interaction with TNFRII and TRADD, for TRAF-2 and TRAF-3 interaction with CD40, and for TRAF-1, TRAF-2, and TRAF-3 interaction with LMP1, the TRAF domains appear to mediate the interaction TRAF-1, TRAF-2, or TRAF-3 with CD30 (data herein and refs. 15, 29, 31, 39, 41, 45–47, and 49–51). The CD30 domains that interact with TRAFs are more precisely defined and are more complex. The data presented here and in recent publications (49, 50) indicate that the last 36 aa of CD30 are required for high-level interaction with TRAF-1, TRAF-2, or TR AF-3. Within these last 36 aa, P 561 EQET 565 . . . E581EGKE585 are similar to CD40 residues P250VQETLHGCQPVTQEDGKE268; CD40 T254 has been implicated in TRAF binding and in signaling (40, 41); and P206QQET210 is a LMP1 cytoplasmic motif important in TRAF-1, TRAF-2, and TRAF3 interaction and in NF-kB activation (39, 51, 52). Consistent with the importance of the CD30 P561EQET565 site in TRAF-1, TRAF-2, and TRAF-3 binding, a peptide consisting of CD30 amino acids 554–570 is sufficient for binding TRAF-3 (52). Our finding that CD30 amino acids 556–570 can block most of the TRAF-1, TRAF-2, and TRAF-3 binding to CD30 indicates the importance of this site in TRAF binding and the potential utility of this motif in blocking the interac-

tions of TRAFs with CD30 and possibly other TNFRs. A second TRAF-1 and TRAF-2 binding site has also been defined within CD30 amino acids 566–586 (50), which includes the CD40 homologous sequence E581EGKE585. CD30 lacking both amino acids 561–565 and amino acids 587–595 engaged TRAF-1 and TRAF-2 but not TRAF-3 in yeast two-hybrid assays, while CD30 lacking amino acids 560–595 did not interact (50). The ability of the CD30 amino acids 556–570 to block high-level TRAF-1, -2, and -3 interaction despite the absence of the E581EGKE585 domain is consistent with the notion that P561EQET565 and E581EGKE585 interact with adjacent regions of the same domain of TRAF-1 or TRAF-2. Also, we note that the CD30 cytoplasmic domain deleted for the last 36 amino acids has residual, albeit weak, TRAF-1, TRAF-2, and TRAF-3 binding activity in vitro, indicative of a third TRAF binding site in the CD30 cytoplasmic domain. Our data indicate that high-level TRAF-1, -2, and -3 association and NF-kB activation are dependent on the last 36 aa of the CD30 cytoplasmic domain and that TRAF-2 mediates CD30-induced NF-kB activation. TRAF-2 had previously been specifically implicated in NF-kB activation from TNFRII and CD40 in 293 cells (29). Interestingly, LMP1 has two NF-kB-activating domains, and only the weaker domain is strongly inhibited by the dominant negative TRAF-2 mutant (51). The dominant negative TRAF-2 mutant blockade of CD30 cross-linking-induced NF-kB activation, the strong binding of TRAF-2 to the last 36 aa of CD30, which are necessary for NF-kB activation, and the previous demonstration of the unique role of TRAF-2 in TRAF-mediated NF-kB activation (29) indicate that TRAF-2 is the principal mediator of CD30 cross-linking-induced NF-kB activation. The ability of TRAF-2 overexpression to induce ligandindependent NF-kB activation (29), the ability of TRAF-1 and TRAF-2 to heteroaggregate (45), the ability of LMP1 to constitutively associate with TRAF-1 and TRAF-2, probably through TRAF-1yTRAF-2 heteroaggregates (39), and the effect of dominant negative TRAF-2 on LMP1-mediated NF-kB activation (51) are compatible with the notion that the aggregation of TRAF-2 or of a TRAF-2-associated protein is


Medical Sciences: Ansieau et al.

the critical event in TNFR- or LMP1-mediated signal transduction. We were therefore surprised to find no increase in TRAF association with CD30 following treatment with agonistic antibody despite NF-kB activation. Perhaps liganddependent cross-linking of CD30 alters the conformation or posttranslational modification of TRAF-2, thus allowing recruitment and activation of other signaling components. Alternatively, ligand-dependent activation of CD30 may result in increased receptor-associated TR AF-1, TR AF-2, and TRAF-3 that we were unable to demonstrate because CD30 overexpression results in substantial ‘‘basal’’ association of non-cross-linked CD30 with TRAFs. The last 66 aa of the CD30 cytoplasmic domain are essential for T-cell death following simultaneous CD30 and T-cell receptor cross-linking (49). Thus, TRAF-1, TRAF-2, and TRAF-3 may also be mediators of CD30-induced cell death or NF-kB activation, growth, or survival functions. These various effects may be specifically mediated by TRAF-1, TRAF-1y TRAF-2 heteroaggregates, TRAF-2, or TRAF-3. Further delineation of the role of specific TRAF complexes in each of these effects is critical for understanding the role of CD30 in T-cell negative selection (8). The genesis of HD remains poorly understood, but EBV has been implicated by an apparent greater risk for HD after EBVrelated infectious mononucleosis and by higher EBV antibody titers and altered antibody patterns among patients with HD (for review, see ref. 53). EBV DNA and RNA and LMP1 are frequently detected in HD tumor cells. Cell activation is a characteristic feature of HD, and CD30 cross-linking or LMP1 expression can promote that process. CD30 is consistently expressed in HD tumor cells and is also believed to be important in tumor cell growth. The interaction of TRAF proteins with both CD30 and LMP1 is consistent with an important role for those adaptor proteins in HD tumor cell growth. We thank Harald Pankow for performing the sequence analysis. This work was supported in part by the Deutsche Krebshilfe (J.D. and F.H.), the Bundesministerium fu ¨r Bildung, Wissenschaft, Forschung und Technologie (H.-J.G.), the Deutsche Forschungsgemeinschaft (S.A.), the Leukemia Society of America (G.M.) and the U.S. Public Health Service Grant CA47006 (E.K. and G.M.). 1. 2. 3. 4. 5. 6.

7. 8.

9. 10. 11. 12.

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Medical Sciences. An author (Hans-Ju ¨rgen Gruss) of the article ‘‘Tumor necrosis factor receptor-associated factor (TRAF)-1, TRAF-2, and TRAF-3 interact in vivo with the CD30 cytoplasmic domain; TRAF-2 mediates CD30-induced nuclear factor kappa B activation’’ by Ste´phane Ansieau, Inka Scheffrahn, George Mosialos, Heike Brand, Justus Duyster, Kenneth Kaye, Josephine Harada, Bill Dougall, Gabi Hu ¨binger, Elliott Kieff, Friedhelm Herrmann, Achim Leutz, and Hans-Ju ¨rgen Gruss, which appeared in number 24, November 26, 1996, of Proc. Natl. Acad. Sci. USA (93, 14053–14058), has admitted scientific misconduct in misrepresenting data including Figs. 2C and 3. Because the experiments of Professor Gruss are a major part of this publication, I request that the paper be withdrawn. Elliott Kieff

Proc. Natl. Acad. Sci. USA (1997)

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