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

Intracellular localization and transcriptional regulation of tumor
necrosis factor (TNF) receptor-associated factor 4 (TRAF4)
Heike Glauner
1
, Daniela Siegmund
1
, Hassan Motejadded
2
, Peter Scheurich
1
, Frank Henkler
2
,
Ottmar Janssen
3
and Harald Wajant
1
1
Institute of Cell Biology and Immunology and
2
Institute of Industrial Genetics, University of Stuttgart, Germany;
3
Institute of
Immunology, Christian-Albrechts-University of Kiel, Germany
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-
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-jBpathwayis
involved in up-regulation of TRAF4 in T-cells.
Keywords:IKKc;NF-jB; T-cells; localization; TRAF4.
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-jBandJNK
(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
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:
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)
Eur. J. Biochem. 269, 4819–4829 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03180.x
proliferation of CD8+ T-cells [9], but the molecular basis
of these defects has not been identified.
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-jBkinasec (IKKc, also known as

NEMO), an essential component of the NF-jB signaling
pathway.
MATERIALS AND METHODS
Cells and reagents
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).
Plasmids
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/Sac2digested
amplicons were ligated into the pEGFP-N1 and pEYFP-

N1 vectors digested with Bgl2andSac2. 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 Sac2overhang
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
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 · 10
6
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 °C and then stimulated in 6 well plates for the indicated
times at a density of 30–50 · 10
6
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/P
i
, pelleted and stored at )20 °CuntilRNA
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
) stimulation of 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.
4820 H. Glauner et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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 · 10
6
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 m
M
Hepes, 10 m
M
KCl, 0.1 m
M
EGTA, 0.1 m
M
EDTA,
pH 7.9. All the following procedures were performed on ice
Fig. 2. Subcellular localization of TRAF4 deletion mutants in isolated
single cells. HeLacellswereseededonglasscoverslidesandtransiently
transfected with expression constructs for the indicated proteins. Iso-
lated single growing cells were selected for photography 16–36 h after
transfection.
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).
Ó FEBS 2002 Localization and transcriptional regulation of TRAF4 (Eur. J. Biochem. 269) 4821
or at 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 m
M
Hepes, 10 m
M
KCl, 0.1 m
M
EGTA, 0.1 m
M
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),
carbonic anhydrase (29 kDa) and cytochrome c
(12.4 kDa), all purchased from Sigma (Deisenhofen,
Germany) were used. For Western blot analysis 250 lLof
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-
(mouse IgG) (1 : 10000 dilution). For subcellular fraction-
ation, cells were washed twice with NaCl/P
i
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 m
M
Tris/HCl,
2m
M
MgCl
2
, 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 m
M
Tris/HCl, 2 m

M
MgCl
2
,pH7.6and
resuspended in 20 m
M
Hepes, 1.5 m
M
MgCl
2
, 420 m
M
KCl, 1 m
M
EDTA, 25% glycerol, pH 7.9, supplemented
with protease inhibitors. The suspension was shaken gently
for 30 min at 4 °C and finally nuclear lysates were obtained
by removal of insoluble material by centrifugation for
15 min at 14 000 g at 4 °C.
Immunofluorescence and confocal microscopy
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 °C, 5% CO

2
) for up to 2 h on
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)weremat-
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 Ô1Õ 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 ÔTDÕ. 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 n
M
)
and Ionomycin (1 l
M
)orremaineduntreated.
Fig. 4. Gel filtration analysis of TRAF4 variants. HEK293 cells
(20 · 10
6

) 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.
4822 H. Glauner et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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.
RESULTS AND DISCUSSION
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
TRAF4inthecytoplasmofcellsandevenfailedtodetectit
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
forupto6hwithleptomycinB,aspecificinhibitorof
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-
lished. Remarkably, analyses of the human TRAF2–
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
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).
Ó FEBS 2002 Localization and transcriptional regulation of TRAF4 (Eur. J. Biochem. 269) 4823
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
lysates (Fig. 3B). In the T-cell lines Jurkat and Cem,
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
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
molecules that is mainly based on the triple helical
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.
4824 H. Glauner et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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).
To verify whether other TRAF proteins have a latent
capability to translocate to the nucleus similar to
TRAF4, we analyzed the subcellular localization of
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). In contrast to T4(1–268)–YFP, 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
localization (Fig. 6, right panel). Like all the other
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.
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
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–
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.
Ó FEBS 2002 Localization and transcriptional regulation of TRAF4 (Eur. J. Biochem. 269) 4825
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
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
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 °C; 5% CO
2
) 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
ÔBÕ) (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 ÔRÕ) 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.
4826 H. Glauner et al.(Eur. J. Biochem. 269) Ó FEBS 2002
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
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-jBtarget
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

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
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
)oramixtureofPMA(100 n
M
) and Ionomycin (1 l
M
). 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.
Ó FEBS 2002 Localization and transcriptional regulation of TRAF4 (Eur. J. Biochem. 269) 4827
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.
ACKNOWLEDGEMENTS
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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Fig. 10. The NF-jB pathway is involved in TNF- and PMA-induced up-
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