Báo cáo Y học: Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4)

doi:10.1046/j.1432-1033.2002.03180.x

Eur. J. Biochem. 269, 4819–4829 (2002) (cid:1) FEBS 2002

Intracellular localization and transcriptional regulation of tumor necrosis factor (TNF) receptor-associated factor 4 (TRAF4)

Heike Glauner1, Daniela Siegmund1, Hassan Motejadded2, Peter Scheurich1, Frank Henkler2, Ottmar Janssen3 and Harald Wajant1 1Institute of Cell Biology and Immunology and 2Institute of Industrial Genetics, University of Stuttgart, Germany; 3Institute of Immunology, Christian-Albrechts-University of Kiel, Germany

tact via its C-TRAF domain. The expression of some TRAF proteins is regulated by the NF-jB pathway. Thus, we investigated whether this pathway is also involved in the regulation of the TRAF4 gene. Indeed, in primary T-cells and Jurkat cells stimulated with the NF-jB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidly up-regulated. In Jurkat T-cells deficient for I-jB kinase c (IKKc, also known as NEMO), an essential com- ponent of the NF-jB-inducing–IKK complex, induction of TRAF4 was completely inhibited. In cells deficient for RIP (receptor interactive protein), an essential signaling inter- mediate of TNF-dependent NF-jB activation, TNF-, but not PMA-induced up-regulation of TRAF4 was blocked. These data suggest that activation of the NF-jB pathway is involved in up-regulation of TRAF4 in T-cells.

Keywords: IKKc; NF-jB; T-cells; localization; TRAF4.

To gain insight in the subcellular localization of tumor necrosis factor receptor-associated factor (TRAF4) we analyzed GFP chimeras of full-length TRAF4 and various deletion mutants derived thereof. While TRAF4–GFP (T4– GFP) was clearly localized in the cytoplasm, the N-terminal deletion mutant, T4(259–470), comprising the TRAF domain of the molecule, and a C-terminal deletion mutant consisting mainly of the RING and zinc finger domains of TRAF4 were both localized predominantly to the nucleus. Passive nuclear localization of T4(259–470) can be ruled out as the TRAF domain of TRAF4 was sufficient to form high molecular weight complexes. T4(259–470) recruited full- length TRAF4 into the nucleus whereas TRAF4 was unable to change the nuclear localization of T4(259–470). Thus, it seems that individual T4(259–470) mutant molecules are sufficient to direct the respective TRAF4–T4(259–470) heteromeric complexes into the nucleus. In cells forming cell–cell contacts, TRAF4 was recruited to the sites of con-

Correspondence to H. Wajant, Institute of Cell Biology and Immunology, University of Stuttgart, Allmandring 31, 70569 Stuttgart, Germany. Fax: + 49 711 685 7484, Tel.: + 49 711 685 7446, E-mail: harald.wajant@po.uni-stuttgart.de Abbreviations: CHX, cycloheximide; FLIP, fluorescence loss in photobleaching; IKK, I-jB kinase; NF-jB, nuclear factor jB; PBMNC, mononuclear cells; PMA, phorbol 12-myristate 13-acetate; RPA, RNAse protection assay; TNF, tumor necrosis factor; TRAF, TNF receptor-associated factor. (Received 24 April 2002, revised 10 July 2002, accepted 13 August 2002)

The tumor necrosis factor (TNF) receptor-associated factor (TRAF) family comprises a group of adaptor proteins that are involved in signal transduction by members of the TNF receptor and IL1/Toll-receptor family [1,2]. The TRAF proteins are characterized by a C-terminal homology domain of about 200 amino acids, called the TRAF domain. The TRAF domain mediates homo- and hetero- merization of TRAF proteins and is also responsible for the majority of protein–protein interactions that have been described for these molecules [1,2]. The TRAF domain can be subdivided into the highly conserved carboxy-terminal TRAF-C subdomain, consisting of an eight-stranded anti- parallel b-sandwich structure and a less conserved amino- terminal part, the TRAF-N domain, which is organized as a

coiled-coil [1,2]. The TRAF domains form trimeric trefoil- like structures, with the three TRAF-C domains as leaves and the trimerized TRAF-N domains as the stalk [3–5]. In mammalians six different TRAF proteins, designated as TRAF1–TRAF6, have been described. With respect to the architecture of the N-terminal domain, TRAF1 is clearly distinct from all other TRAFs. While the N-terminus of TRAF2–TRAF6 contains a highly conserved RING domain followed by a regularly spaced cluster of five or seven zinc fingers, the TRAF1 N-terminus only contains a single zinc finger and no obvious additional structural elements [1,2]. While TRAF1–TRAF5 have been implicated mainly in signaling by members of the TNF receptor family, TRAF6 primarily transduces signals initiated by IL1/Toll receptors. In particular, TRAF4 has been shown to interact with the lymphotoxin-b receptor and the p75 nerve growth factor receptor in in vitro binding assays [6,7] but the physiological relevance of these interactions remains to be elucidated. While there is ample experimental evidence, including the analyses of knockout mice, for an important role of TRAF2, TRAF5 and TRAF6 in TNF receptor and IL1/Toll-receptor induced activation of NF-jB and JNK (c-Jun N-terminal kinase) [1,2], the role of TRAF1 and TRAF3 for signal transduction by TNF receptors is poorly understood. In fact, B-cells from mice deficient in TRAF3 have a defect in immunoglobulin isotype switching in response to thymus-dependent antigens [8] and TRAF1 knockout mice show an increased TNF-R2-dependent

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Plasmids proliferation of CD8+ T-cells [9], but the molecular basis of these defects has not been identified.

To construct human TRAF4 and TRAF4 deletion mutants (75–470, 259–470, 1–268, 1–307, 259–307, 259– 387) with carboxyl-terminal GFP or YFP tags, TRAF4 cDNA fragments with 5¢-end BamH1 overhangs and 3¢- end Sac2 overhangs were generated by proofreading PCR and HeLa cDNA as template. The BamH1/Sac2 digested amplicons were ligated into the pEGFP-N1 and pEYFP- N1 vectors digested with Bgl2 and Sac2. To construct a deletion mutant consisting solely of the C-TRAF domain of TRAF4, an appropriate cDNA fragment of TRAF4 with a 5¢-end BamH1 overhang and 3¢-end Sac2 overhang was generated by proofreading PCR and inserted into Bgl2/Sac2 digested pEYFP-N1 vector (Clontech). To obtain non-GFP/YFP tagged TRAF4 expression con- structs, the GFP/YFP encoding cDNA stretch was removed from the corresponding GFP/YFP expression construct by Sac2/Not1 digest and subsequent religation of the blunt-ended vector–TRAF4 fragment. GFP/YFP chi- meras of TRAF1, TRAF2 and TRAF3 were prepared in a similar way. In case of TRAF3 a splice form was used lacking exons 7–10.

Purification and stimulation of primary human T-lymphocytes TRAF4 is the most conserved phylogenetically, but also the most distinct member of the TRAF family [1]. Indeed, the overall sequence identity between human TRAF4 and its counterpart in Drosophila melanogaster (DmTRAF1) is 45%, whereas the closest related human TRAF shares only 26% sequence identity [1]. In addition, expression of TRAF4 and DmTRAF1 can be detected throughout embryogenesis and is predominantly found in undifferen- tiated cells, e.g. neuronal precursors or epithelial progenitor cells [7,10,11]. Thus, it seems possible that DmTRAF1 and mammalian TRAF4 represent conserved members of the TRAF family with related functions in differentiation of vertebrate and invertebrate cells. According to the broad expression of TRAF4 in developing epithelial and neuronal tissue, the analysis of TRAF4-deficient mice revealed a neural tube closure defect as well as malformation of rib, sternum, the spinal column and the upper respiratory tract, the latter associated with an increase in pulmonary inflammation [12,13]. TRAF4 was cloned originally in a differential expression screen from a cDNA library of breast cancer-derived metastatic lymph nodes and was found to be located in the nucleus [14]. However, another study, using a different antibody, failed to detect TRAF4 in breast carcinomas and reported a cytosolic localization of the protein [7].

In this study we found that deletion of the zinc finger domain of TRAF4 results in nuclear localization without disturbing the oligomerization status of the molecule. This opens the possibility that a zinc finger-dependent mechan- ism retains TRAF4 in the cytoplasm and could provide an explanation for the conflicting reports on the subcellular localizations of TRAF4. TRAF4 is also recruited to sites of cell–cell contacts under critical involvement of its C-TRAF domain. Finally, we show that TRAF4 is induced in T-cells by TNF and treatment with phorbol ester under critical involvement of I-jB kinase c (IKKc, also known as NEMO), an essential component of the NF-jB signaling pathway.

M A T E R I A L S A N D M E T H O D S

Cells and reagents

stimulation of

The human cervical carcinoma cell line HeLa, the human embryonic kidney cell line 293, the human breast cancer cell line MCF-7 and the human epidermal carcinoma cell line A431 were obtained from the American Type Culture Collection (Rockville, MD, USA). The IKKc-deficient Jurkat cell line and respective control cells were a gift from S.-C. Sun (Pennsylvania State University, USA) and are described elsewhere [11]. The RIP-deficient Jurkat T-cell line and the corresponding parental Jurkat clone were a gift from B. Seed (Massachusetts General Hospital, USA) and were described by Ting et al. [12]. Cells were maintained in RPMI medium (Biochrom, Berlin, Germany) supplemented with 5% (HeLa and HEK293 cells) or 10% (Jurkat cells) fetal bovine serum. Chemicals and secondary antibodies were obtained from Sigma (Deisenhofen, Germany). The polyclonal TRAF4-specific IgG preparation (C-20) was from Santa Cruz (Heidelberg, Germany). Mononuclear cells (PBMNC) were isolated from periph- eral blood of healthy donors by Ficoll density centrifuga- tion. The resulting PBMNC were then incubated with neuraminidase-treated sheep red blood cells at a ratio of 30 · 106 PBMNC per ml sheep erythrocytes (10% sus- pension in RPMI). The mixture was separated by two rounds of Ficoll density centrifugation. After the first gradient, the interphase containing nonrosetting cells was aspirated and the pellet with rosetted T-cells was carefully resuspended and centrifuged on the second gradient. After aspirating the second interphase and ficoll, the sheep red blood cells were lyzed with ammonium chloride solution (Sigma, Deisenhofen, Germany) and the T-cells were washed twice with RPMI containing 10% fetal bovine serum. The resulting cell population consisted of highly purified CD3+ T-cells (with an average percentage of CD3+ cells of 94–97%). The cells were kept overnight at 4 (cid:4)C and then stimulated in 6 well plates for the indicated times at a density of 30–50 · 106 cells with or without PMA (5 ngÆmL)1, Sigma) and ionomycin (500 ngÆmL)1, Calbiochem) in 5 mL of RPMI/10% fetal bovine serum. Finally, cells were carefully resuspended, washed once with NaCl/Pi, pelleted and stored at )20 (cid:4)C until RNA preparation. As a control, untreated T-cells were collec- ted, washed and stored the same way. For stimulation periods of 2, 4, 6 and 8 days, additional culture medium (2.5 mL each) was added after the second and fourth day. T-cell blasts were generated by phytohaemag- glutinin (0.5 lgÆmL)1) freshly isolated PBMNCs for 3 days. The cells were expanded with IL2-containing medium (10 UÆmL)1) for 3–5 days. Dead cells were removed by Ficoll density gradient centrifugation and living cells were further expanded with IL2-supple- mented medium. At the day of restimulation (usually day 12–15) the population consisted of above 95% CD3+ T-cells.

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Fig. 1. Structure of fusion proteins used in this study. The C-TRAF domain (CTD) is shown in black and the N-TRAF domain (NTD) in gray. An open box denotes a zinc finger structure (Zn) and a RING domain is represented by a pale gray box. YFP or GFP are labeled. The amino acids are numbered according to the human TRAF sequences available on GenBank (accession numbers U19261 (TRAF1), U12597 (TRAF2), U19260 (TRAF3) and X80200 (TRAF4).

Fig. 2. Subcellular localization of TRAF4 deletion mutants in isolated single cells. HeLa cells were seeded on glass cover slides and transiently transfected with expression constructs for the indicated proteins. Iso- lated single growing cells were selected for photography 16–36 h after transfection.

RNAse protection assay (RPA) analysis

Cells were treated as indicated and total RNAs were isolated with peqGOLD RNAPure (PeqLab Biotechnologie GmbH, Erlangen, Germany) according to the manufac- turer’s recommendations. To detect transcripts for xIAP, TRAF1, TRAF2, TRAF4, NAIP, cIAP2, cIAP1, TRPM2, TRAF3, L32 and GAPDH total RNAs were analyzed using a customer Multi-Probe template set (PharMingen, Hamburg, Germany). Probe synthesis, hybridization and RNase treatment were performed with the RiboQuant Multi-Probe RNase Protection Assay System (PharMingen, Hamburg, Germany) according to the manufacturer’s recommendations. After RNase treatment the protected transcripts were resolved by electrophoresis on a denaturing polyacrylamide gel (5%) and analyzed on a Phosphor- Imager with the IMAGEQUANT software.

Gelfiltration, subcellular, fractionation and Western blotting HEK293 cells (20 · 106 cells per mL) were electroporated (4 mm cuvette, 250 V, 1800 lF, maximal resistance) in medium with 5% fetal bovine serum, seeded on to two 150 mm tissue culture plastic plates and expanded for two days. Cells were scraped with a rubber policeman into the medium, centrifuged (500 g, 5 min) and washed with medium. The pellet was resupended in 300 lL of ice-cold 10 mM Hepes, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, pH 7.9. All the following procedures were performed on ice

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cytochrome anhydrase and

(mouse IgG) (1 : 10000 dilution). For subcellular fraction- ation, cells were washed twice with NaCl/Pi and half of the cells were used for preparation of cytoplasmic and nuclear extracts, respectively. For preparation of cytoplasmic extracts, the cells were resuspended in 10 mM Tris/HCl, 2 mM MgCl2, pH 7.6 supplemented with protease inhibitors and incubated for 5 min on ice. Then Triton X-100 was added to a final concentration of 0.5%. After an additional 5 min cells were pressed 3–5 times through a 22-needle. After centrifugation for 10 min at 14 000 g the supernatant was used for analysis. For preparation of nuclear extracts, cells were treated as above, however, this time cells were only centrifuged for 10 min at 200 g. The pellet was washed twice in 10 mM Tris/HCl, 2 mM MgCl2, pH 7.6 and resuspended in 20 mM Hepes, 1.5 mM MgCl2, 420 mM KCl, 1 mM EDTA, 25% glycerol, pH 7.9, supplemented with protease inhibitors. The suspension was shaken gently for 30 min at 4 (cid:4)C and finally nuclear lysates were obtained by removal of insoluble material by centrifugation for 15 min at 14 000 g at 4 (cid:4)C.

Immunofluorescence and confocal microscopy

or at 4 (cid:4)C. For cell lysis 1/10 volumes of a protease inhibitor cocktail (Boehringer Mannheim, Germany) and Nonidet- P40 to a final concentration of 0.6% were added. After 30 min on ice, the lysates were centrifuged at 10 000 g for 10 min and the supernatants were further cleared by centrifugation at 50 000 r.p.m. for 1 h in a TL-100 rotor (Beckman, Munich, Germany). The S-100 supernatants (250 lL) were then separated by size exclusion chromato- graphy on a Superdex 200 HR10/30 column (Pharmacia, Freiburg, Germany) in 10 mM Hepes, 10 mM KCl, 0.1 mM EGTA, 0.1 mM EDTA, pH 7.9 with 0.5 mLÆmin)1. Sam- ples were collected in fractions of 0.5 mL and analyzed by immunoblotting. For calibration of the column thyroglo- bulin (669 kDa), apoferritin (443 kDa), alcohol dehydro- genase (150 kDa), bovine serum albumin (66 kDa), c (29 kDa) carbonic (12.4 kDa), all purchased from Sigma (Deisenhofen, Germany) were used. For Western blot analysis 250 lL of each fraction was precipitated with trichloroacetic acid and dissolved in 60 lL of sample buffer. Identical volumes (30 lL) of the precipitated gel filtration fractions were separated by SDS/PAGE and transferred to nitrocellulose. The GFP and the various TRAF4–GFP/YFP fusion proteins were detected with 1 lgÆmL)1 of a mixture of GFP-specific monoclonal antibodies (Roche, Mannheim, Germany) and alkaline phosphatase-labelled goat anti-

Fig. 4. Gel filtration analysis of TRAF4 variants. HEK293 cells (20 · 106) were electroporated with expression plasmids encoding the indicated proteins. Two days after transfection, cell lysates (200 lL) were prepared and separated by size exclusion chromatography on a HR10/30 Superdex 200 column. Fractions of 0.5 mL were collected and analyzed by immunoblotting with a mixture of two GFP/YFP- specifc mAbs. Elution volumes of molecular mass standards are indi- cated above.

Fig. 3. Exon–intron structure of human TRAF genes and Western blot analysis of endogenous TRAF4. (A) The exon–intron structures of the human genes encoding TRAF2 to TRAF6 according to the NCBI entries NT_025667 (TRAF2), NT_010019 (TRAF3), NT_030828 (TRAF4), NT_021877 (TRAF5) and NT_024229 (TRAF6) were mat- ched with the cDNA sequences encoding TRAF2 to TRAF6 [accession numbers U12597 (TRAF2), U19260 (TRAF3), X80200 (TRAF4), AB000509 (TRAF5) and U78798 (TRAF6)]. Only exons encoding parts of the cDNA translated into protein were considered in this illustration. Thus, exons numbered in this scheme with (cid:2)1(cid:3) do not necessarily correspond to exon 1 of the respective gene in the database. Exons encoding parts of the TRAF domain were summarized and labeled (cid:2)TD(cid:3). The number of nucleotides encoded by each exon was indicated in the box representing the respective exon. Exons containing multiples of three nucleotides are indicated by gray boxes. (B) The indicated cells were fractionated and lysates containing cytoplasmic (C) and nuclear proteins (N) were analyzed by Western blotting. TRAF4 was detected using an affinity-purified polyclonal anti- (TRAF4) goat Ig recognizing a carboxy-terminal peptide of TRAF4. Cem T-cells were activated for 6 h with a mixture of PMA (100 nM) and Ionomycin (1 lM) or remained untreated.

HeLa cells were seeded overnight on to 18 mm glass coverslips in square Petri dishes with 25 compartments. The following day, cells were transfected with the indicated expression plasmid using Superfect reagent (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. For microscopic analysis transfected cells were fixed in 35% paraformaldehyde. For bleaching experiments, cells were transfected in glass bottom dishes (MatTek Corporation, Ashland, MA, USA) and maintained during the experiment in a conditioned chamber (37 (cid:4)C, 5% CO2) for up to 2 h on

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the microscope stage. To prevent the synthesis of new protein during the bleaching experiments cycloheximide (25 lgÆmL)1) was added. Fluorescent specimens were analyzed with a Leica SP2 confocal microscope and imaged using the Leica TCS software.

R E S U L T S A N D D I S C U S S I O N

Fig. 5. Subcellular localization of TRAF4 deletion mutants in cells with cell–cell contacts. HeLa cells were seeded on glass cover slides and transiently transfected with expression constructs for the indicated proteins. Next day, representative transfected cells were selected for photography (A). For quantification cells showing increased localiza- tion of the proteins in cell–cell contacts were counted (B).

Subcellular localization of GFP/YFP-tagged TRAF4 deletion mutants

Using an antiserum against a peptide derived from the C-TRAF domain of TRAF4, Regnier et al. [14] observed TRAF4 in the nucleus of malignant epithelial cells from invasive breast carcinomas. However, another group uti- lizing an antiserum generated against a peptide correspond- ing to the N-terminal 18 amino acids of TRAF4 localized TRAF4 in the cytoplasm of cells and even failed to detect it in most breast cancer samples [7]. It is possible that these conflicting results are caused by the existence of alternative forms of TRAF4 that could be generated by alternative splicing or by proteolytic events. An alternative possibility would be that a TRAF4-interacting protein secondarily regulates TRAF4 localization, eventually in a signal-regu- lated manner. To study the possibility that TRAF4 variants compartmentalize differently, we analyzed the subcellular localization of GFP or YFP chimeras of TRAF4 deletion mutants with changed domain architecture (Fig. 1). To determine the cellular localization of the GFP or YFP fusion proteins, HeLa cells were transiently transfected with the respective expression plasmids and analyzed by confocal microscopy the next day. Full-length GFP-tagged TRAF4 molecules were localized in the cytoplasm and were barely detectable in the nucleus (Fig. 2). In a portion of cells expressing rather high amounts of TRAF4–GFP, the molecules also accumulated in a few, but large round patches (data not shown). Such TRAF4–GFP patches are most likely to be artifacts caused by overexpression and are also found with other TRAF proteins containing a RING– zinc finger domain (data not shown and Fig. 6). Interest- ingly, both a C-terminal deletion mutant lacking the TRAF domain [T4(1–268)–YFP] as well as an N-terminal deletion mutant domain lacking the RING and zinc finger domains of the molecule [T4(259–470)–YFP] predominantly locali- zed to the nucleus (Fig. 2). By sequence analysis Regnier et al. [14] have identified two potential nuclear localization sequences in the N-terminal part of TRAF4 which are present in T4(1–268)–YFP. However, in T4(259–470)–YFP there is no obvious nuclear localization sequence. TRAF4 deletion mutants lacking the RING domain [T4(75–470)– YFP] or the C-TRAF domain [T4(1–307)–YFP] showed also predominant localization in the cytoplasm (Fig. 2) suggesting that the central parts of the molecule (zinc fingers plus N-TRAF domain) are sufficient to establish cytoplas- mic retention. The nuclear localization of the aforemen- tioned TRAF4 deletion mutants do not simply reflect the deletion of a putative nuclear export sequence, as treatment for up to 6 h with leptomycin B, a specific inhibitor of nuclear export, showed no effect on TRAF4 localization (data not shown). Thus, the RING and/or C-TRAF domain seem to be necessary to localize TRAF4 in the nucleus. Whether this relies on functional nuclear localiza- tion sequences within these parts of the molecule or on the interaction with associated proteins remains to be estab- the human TRAF2– lished. Remarkably, analyses of TRAF6 genes revealed that all these genes share a stretch of 3–6 consecutive exons with a multiple of three nucleotides encoding the zinc finger domains of these molecules (Fig. 3A). Thus, any combination of these exons results potentially in an in-frame splice form of the respective

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Fig. 6. Subcellular localization of TRAF1, TRAF2, TRAF3 and deletion mutants derived thereof. HeLa cells were seeded on glass cover slides and transiently transfected with expres- sion constructs for the indicated TRAF proteins. Representative cells were selected for photography 16–36 h after transfection.

form of TRAF4. Additional studies with independent TRAF4 sera should reveal in the future whether this is indeed the case.

In gel filtration experiments full-length TRAF4 [T4(1– 470)–GFP], as well as deletion mutants of TRAF4 lacking the Ring domain [T4(75–470)–YFP] or the C-TRAF domain [T4(1–307)–YFP] eluted mainly in high molecular weight complexes of 443 kDa and more. YFP chimeras solely comprising the N-TRAF domain of TRAF4 [T4(259–307)–YFP] or the complete TRAF domain of the molecule [T4(259–470)–YFP] showed significant complex formation (Fig. 4). While the TRAF domain of TRAF4 was almost completely organized in complexes, the N-TRAF domain of TRAF4 eluted over the whole fractionation range of the Superdex 200 column. A deletion mutant only comprising the Ring and zinc finger domain of TRAF4 [T4(1–268)–YFP] eluted over the whole separation range of the gel filtration column, too (Fig. 4). The C-TRAF domain of TRAF4 [T4(304–470)–YFP] eluted as a monomer but has a stabilizing effect on the N-TRAF domain based aggregation of the TRAF domain (Fig. 4). A deletion mutant comprising the Ring and zinc finger domain and in addition the N-TRAF domain [T4(1–307)– YFP] eluted predominantly in high molecular weight fractions. Together, these gel filtration data suggest that both the N-TRAF domain and the zinc finger region of TRAF4 drive the formation of TRAF4-containing high molecular weight complexes. This is in good accordance with the crystal structures of the TRAF domains of TRAF2 and TRAF3 showing a trimeric trefoil-like structure of these is mainly based on the triple helical molecules that TRAF protein. In the case of TRAF3, the existence of such splice forms has indeed already been described [17,18]. It will be interesting to see in the future whether related splice forms also exist for TRAF4 and if so, whether these splice forms exert differential subcellular localization. To investi- gate the subcellular distribution of TRAF4 further we analyzed cytoplasmic and nuclear extracts with respect to the content of endogenous TRAF4. Remarkably, in vari- ance with the overexpression studies discussed above, we observed in all cell lines investigated (A431, MCF-7, Jurkat, Cem) that full-length TRAF4 is found mainly in nuclear lines Jurkat and Cem, lysates (Fig. 3B). In the T-cell TRAF4 was induced by PMA treatment (see below), but there were no qualitative differences in these cells in the distribution of TRAF4 between the cytoplasmic and the nuclear fraction (data not shown). This discrepancy cannot be attributed to the GFP part of the various TRAF4 fusion proteins, as several control experiments with nontagged proteins showed that the GFP part has no influence on the subcellular distribution of the respective fusion protein (data not shown). Thus, it is tempting to speculate that a limited endogenous factor, which is rapidly titrated by overex- pressed TRAF4 forms, is responsible for the putative nuclear localization of endogenous TRAF4. As shown in Fig. 2, TRAF4 can also be recruited to cell–cell contacts. Thus, it cannot be ruled out that TRAF4 detected in the nuclear lysates was released from insoluble TRAF4 con- taining cell–cell contact or cytoskeleton structures that copurify during the preparation of the nuclei. The Western blot analyses also regularly revealed a smaller than expected anti-TRAF4 reactive band, which could represent a splice

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organization of parallel N-TRAF domains [3–5]. However, in principle, it cannot be ruled out completely that the separation behavior of the various mutants was caused by interaction with endogenous TRAF4 or unknown endo- genous TRAF4-interacting proteins. Interaction with endogenous TRAF4 is most likely negligible because the expression level of endogenous TRAF4 in the HEK293 cells is significantly below the expression level of the transfected constructs (data not shown) but the possible impact of an endogenous TRAF4-binding protein remains to be established.

As discussed before in isolated cells transfected with T4– GFP a homogenous cytoplasmic staining was observed. In contrast, in transfected cells that have cell–cell contacts, a significant local increase of T4–GFP was observed in the contact sites. Analysis of the various TRAF4 deletion mutants showed that the C-TRAF domain part of the TRAF domain is sufficient to direct the molecule into the sites of cell–cell contacts (Fig. 5).

In contrast to T4(1–268)–YFP,

Fig. 7. Translocation of TRAF4 from the cytoplasm to the nucleus by T4(259–470). HeLa cells were transfected with the indicated combi- nation of vectors encoding TRAF4, T4–GFP, T4(259–470), T4(259– 470)–YFP, T1–GFP, and GFP–T1(185–416). Next day, representative transfected cells were selected for photography.

To verify whether other TRAF proteins have a latent to similar capability to translocate to the nucleus localization of TRAF4, we analyzed the subcellular GFP-tagged fusion proteins of full-length TRAF1– TRAF3, and C-terminal as well as N-terminal deletion mutants derived thereof. Similar to TRAF4–GFP, all other investigated TRAF proteins (T1–GFP, T2–GFP and T3–GFP) were localized mainly to the cytoplasm and were hardly detectable in the nucleus (Fig. 6, left panel). the deletion mutants of TRAF1–TRAF3 lacking the TRAF domain still localized in the cytoplasm (Fig. 6, middle panel). The GFP-tagged TRAF domains of TRAF2 and TRAF3 also localized to the cytoplasm whereas the TRAF domain of TRAF1 showed nuclear and cytoplasmic the other localization (Fig. 6, right panel). Like all TRAF domains the TRAF domain of TRAF1 is part of high molecular complex (data not shown). Therefore, the nuclear localization found for the respective TRAF1 deletion mutant should not be caused by a passive effect. As already discussed above, round patches with increased TRAF–GFP concentrations were observable in cells expressing high amounts of TRAF2– or TRAF3–GFP (Fig. 6). These structures were not found for deletion mutants solely comprising the TRAF domain of TRAF2 and TRAF3 but were detected regularly in cells trans- fected with TRAF2/3 deletion mutants consisting of the RING–zinc finger domain.

T4(259–470) (Fig. 7). As both TRAF mutants are able to form high molecular weight complexes, it seems that the N-TRAF domain together with the central part of the C-TRAF domain is sufficient to allow formation of TRAF4-containing complexes but is insufficient to enable nuclear retention by T4(259–470). However, it is unclear whether the deleted C-terminal part of the C-TRAF domain of T4(259–387)–YFP and T4(1–307)–YFP is necessary to interact with the TRAF domain of TRAF4 to allow formation of heteromeric complexes or whether the incom- plete C-TRAF domain(s) of these TRAF4 variants lead to a reduced affinity–avidity of respective heteromeric com- plexes [T4(259–387)–YFP–T4(259–470), T4(1–307) –YFP– Since TRAF proteins tend to form homo- and/or heteromers [1,2] we analyzed whether T4–GFP or T4(1– 268)–YFP change their localization upon coexpression with other nontagged TRAF4 proteins or heterologous TRAF proteins. As shown in Fig. 7, coexpression of the nontagged TRAF domain of TRAF4 [T4(259–470)] was sufficient to recruit full-length TRAF4 [T4(1–470)–GFP] or T4(75– 470)–YFP into the nucleus whereas cotransfected non- tagged TRAF4 showed no effect on T4(259–470)–YFP. These data suggest that one T4(259–470) molecule might be sufficient to direct a heteromeric complex of full-length TRAF4 and T4(259–470)–GFP into the nucleus. TRAF4 deletion mutants lacking the C-TRAF domain [T4(1–307)– YFP] or only consisting of the N-TRAF domain and the central part of the C-TRAF domain [T4(259–387)–YFP] were not recruited into the nucleus upon cotransfection with

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Fig. 8. Fluorescence loss in photobleaching (FLIP) analysis of T4(1–470)–GFP and T4(259–470)–YFP. HeLa cells were seeded on glass bottom dishes and were transiently transfected with expression constructs encoding T4(1–470)–GFP and T4(259–470)–YFP. Cells were maintained in a conditioned chamber (37 (cid:4)C; 5% CO2) on the microscope stage and areas with two representative cells were selected for FLIP analysis 16–36 h after transfection. In one cell GFP fluorescence in the indicated area (white box) of the cell was bleached repetitively for the indicated time (bleaching cell (cid:2)B(cid:3)) (A). Finally, the average fluorescence intensity (red surrounded areas) in the nucleus (circles) and cytoplasm (boxes) of bleached (filled symbols) and nonbleached (open symbols) (reference cell (cid:2)R(cid:3)) cell was determined using the Leica TCS software (B) and plot against the bleaching time. Both, GFP and YFP fusion proteins, were bleached and monitored using 488 nm.

cytoplasmic TRAF4 may represent functionally distinct populations of this molecule.

Analyses of the various deletion mutants of TRAF4 suggest that the zinc fingers of the molecule are responsible for the cytoplasmic retention of TRAF4. Interestingly, it has been recently shown that the oncogenic serine–threonine kinase Pim-1 induces translocation of the TRAF4-interact- ing protein-sorting Nexin 6–TRAF4–associated factor 2 from the cytoplasm to the nucleus [19]. Thus, it will be interesting to see in the future whether Pim-1 and sorting Nexin 6 regulate the subcellular distribution of TRAF4.

TRAF4 is up-regulated in activated T-cells

In former studies we and others have identified TRAF1 and TRAF2 as possible targets of the NF-jB pathway [20,21]. To find out whether TRAF4 can also be regulated by this pathway, we treated a variety of cell lines with the potent NF-jB inducers TNF and phorbol 12-myristate 13-acetate (PMA). In most of the investigated cell lines there was no induction, or only a modest, poorly reproducible induction, of TRAF4 mRNA by these stimuli. However, in the T-cell lines Jurkat (Fig. 9A) and D23II-7 (Fig. 9B) both TNF and PMA/Ionomycin, induced a significant and reproducible up-regulation of TRAF4 mRNA. TNF- and PMA-induced TRAF4 expression was already detectable 1 h after T4(259–470)] for a putative nuclear target structure. In addition, we found no evidence for a T4(259–470)-depend- ent recruitment of heterologous TRAFs to the nucleus (data not shown). In contrast to the TRAF domain of TRAF4, the TRAF domain of TRAF1 was not able to recruit its full- length counterpart to the nucleus (data not shown). Although there was a dominant localization of T4(259– 470)–YFP in the nucleus, a significant part remained in the cytoplasm (Fig. 8). T4(1–470)–GFP was predominantly found in the cytoplasm but a minor part was detectable in the nucleus (Fig. 8). To verify whether TRAF4 or the TRAF4-derived TRAF domain shuttles between nucleus and cytoplasm, we analyzed T4(1–470)–GFP and T4(259– 470)–YFP by fluorescence loss in photobleaching (FLIP). Repetitive bleaching for 5–10 times of a small area in the nucleus depleted the nuclear fluorescence of T4(1–470)– GFP and T4(259–470)–YFP but had only a minor effect on the respective cytoplasmic-located protein fraction (Fig. 8). Correspondingly, fluorescence of cytoplasmic T4(259–470)– YFP and full-length T4–GFP was already significantly reduced after 2 min of bleaching in a small area of the cytoplasm, whereas the fluorescence of nuclear localized TRAF4 proteins was almost not affected even after prolonged bleaching cycles (Fig. 8). Together, these data indicate that there is only a slow exchange of cytoplasmic and nucleus-localized TRAF4, indicating that nuclear and

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Fig. 9. TNF and PMA up-regulate TRAF4 mRNA in T-cells. Jurkat (A) and D23II-7 T-cells (B) as well as primary human T-cells and human T-cell blasts (C) were stimulated for the indicated times with TNF (20 ngÆmL)1) or a mixture of PMA (100 nM) and Ionomycin (1 lM). Jurkat and D23II- 7 cells were analyzed in addition in the presence of CHX (50 lgÆmL)1) that was added 1 h prior to PMA/TNF stimulation. Total RNAs were isolated for RPA analysis and 10 lg of each RNA sample were analyzed with a Multi-Probe template set to detect the indicated mRNAs in particular TRAF4. L32 and GAPDH were included as internal controls. Protected transcripts were resolved by electrophoresis on a denaturing polyacrylamide gel (5%) and quantified on a PhosphorImager with the IMAGEQUANT software. For quantification each TRAF4 or L32 band was individually corrected for background intensities by an area of corresponding size in close neighborhood of the respective mRNA signal. To obtain relative TRAF4 and L32 expression values the ratio between the signal intensities of bands of treated cells and the corresponding band of the untreated group were calculated. Finally, relative TRAF4 expression values were normalized according to the respective values of relative L32 expresssion. The position of protected TRAF4-specific mRNA bands are indicated with an arrow.

TRAF4 mRNA in Jurkat cells after 6 h compared to a 2.7- fold induction in the absence of CHX (Fig. 9A). Thus, TNF- and PMA-initiated signaling events directly lead to the induction of TRAF4. This is also in good agreement with the rapid kinetics of TRAF4 induction (Fig. 9). The increased induction of NF-jB regulated genes in the presence of CHX might reflect that some NF-jB target genes (e.g. A20, I-jBa) are involved in the termination of the NF-jB response itself, but this possibility was not investi- gated further here.

TNF and PMA up-regulate TRAF4 under essential involvement of signaling components of the NF-jB pathway

stimulation in both cell lines, reached its maximum after 3– 6 h and dropped down near to basal levels after 24 h. PMA- induced up-regulation of TRAF4 was also found to a comparable extent in primary T-cells and in T-cell blasts (Fig. 9C). Six hours after stimulation, primary T-cells showed a 7.8-fold, and day 13 T-cell blasts a 6.1-fold, induction of TRAF4 mRNA. Basal TRAF4 mRNA expression was roughly comparable in primary T-cells and T-cell blasts. To verify whether TRAF4 mRNA is directly up-regulated by TNF- and PMA-induced signaling path- ways, we analyzed the effect of the protein synthesis inhibitor cycloheximide (CHX) on TRAF4 induction. We found no evidence for an inhibitory effect of CHX on TRAF4 up-regulation. Moreover, in the presence of CHX the induction of TRAF4, and also the induction of the known NF-jB targets TRAF1 and cIAP2, was enhanced significantly (Fig. 9A,B) whereas CHX alone did not change basal mRNA levels (data not shown). For example, in the presence of CHX, PMA induced a 15-fold increase of TNF and PMA/I were chosen for the studies described above, as both are potent inducers of NF-jB. To finally verify whether this pathway is involved in TRAF4 induction in T-cells, we analyzed a mutant Jurkat cell

4828 H. Glauner et al. (Eur. J. Biochem. 269)

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Fig. 10. The NF-jB pathway is involved in TNF- and PMA-induced up- regulation of TRAF4. Parental Jurkat cells (left panel) or clones derived thereof deficient in IKKc (middle panel) or RIP (right panel) expres- sion were stimulated with TNF (20 ngÆmL)1) or PMA (100 nM) for 6 h and analyzed by RPA analysis as described in Fig. 9.

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line deficient in expression of IKKc/NEMO [15], an essential component of the NF-jB-inducing IKK complex [22]. Both TNF and PMA/I-induced up-regulation of TRAF4 was completely inhibited in this mutant Jurkat cell line (Fig. 10). Moreover, in a Jurkat clone deficient for RIP, a molecule involved in TNF but not in PMA- induced NF-jB activation [16], TNF-induced but not PMA-induced TRAF4 expression was blocked (Fig. 10). These data clearly argue for an essential role of the NF- jB pathway in TNF- and PMA-induced up-regulation of TRAF4.

A C K N O W L E D G E M E N T S

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16. Ting, A.T., Pimentel-Muinos, F.X. & Seed, B. (1996) RIP medi- ates tumor necrosis factor receptor 1 activation of NF-jB but not Fas/APO-1-initiated apoptosis. EMBO J. 15, 6189–6196.

We thank Brian Seed (Massachusetts General Hospital, USA) for the RIP-deficient Jurkat clone and S.-C. Sun (Pennsylvania State Univer- sity, USA) for the IKKc-deficient Jurkat cell line. This work was supported by Deutsche Forschungsgemeinschaft Grant Wa 1025/3–1 and Sonderforschungsbereich 495 project A5.

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