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

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,

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

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

) 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

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.

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

) 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

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

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

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–

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

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

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-
regulation of TRAF4. Parental Jurkat cells (left panel) or clones derived