Intracellular localization and transcriptional regulation of tumornecrosis factor TNF receptor-associated factor 4 TRAF4 Heike Glauner1, Daniela Siegmund1, Hassan Motejadded2, Peter Sche
Trang 1Intracellular 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 Janssen3and Harald Wajant1
1
Institute of Cell Biology and Immunology and2Institute of Industrial Genetics, University of Stuttgart, Germany;3Institute 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 clearlylocalized 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 mainlyof the RING and zinc finger domains of
TRAF4 were both localized predominantlyto 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 bythe NF-jB pathway Thus, we investigated whether this pathwayis also involved in the regulation of the TRAF4 gene Indeed, in primaryT-cells and Jurkat cells stimulated with the NF-jB inducers TNF or phorbol 12-myristate 13-acetate (PMA), TRAF4-mRNA was rapidlyup-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 completelyinhibited 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
The tumor necrosis factor (TNF) receptor-associated factor
(TRAF) familycomprises a group of adaptor proteins that
are involved in signal transduction bymembers of the TNF
receptor and IL1/Toll-receptor family[1,2] The TRAF
proteins are characterized bya 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
majorityof protein–protein interactions that have been
described for these molecules [1,2] The TRAF domain can
be subdivided into the highlyconserved 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 highlyconserved RING domain followed bya regularlyspaced cluster of five or seven zinc fingers, the TRAF1 N-terminus onlycontains a single zinc finger and no obvious additional structural elements [1,2] While TRAF1–TRAF5 have been implicated mainlyin signaling bymembers of the TNF receptor family, TRAF6 primarilytransduces signals initiated byIL1/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 byTNF 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 Biologyand
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 July2002,
accepted 13 August 2002)
Trang 2proliferation 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 identitybetween 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 familywith 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 respiratorytract,
the latter associated with an increase in pulmonary
inflammation [12,13] TRAF4 was cloned originallyin a
differential expression screen from a cDNA libraryof 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 studywe 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 possibilitythat 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
byTNF 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
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 byTing 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 secondaryantibodies 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 byproofreading 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 solelyof 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 byproofreading 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 healthydonors byFicoll densitycentrifuga-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 bytwo rounds of Ficoll densitycentrifugation 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
4C and then stimulated in 6 well plates for the indicated times at a densityof 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 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) stimulation of freshlyisolated PBMNCs for 3 days The cells were expanded with IL2-containing medium (10 UÆmL)1) for 3–5 days Dead cells were removed byFicoll densitygradient centrifugation and living cells were further expanded with IL2-supple-mented medium At the dayof restimulation (usuallyday 12–15) the population consisted of above 95% CD3+ T-cells
Trang 3RNAse 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 AssaySystem (PharMingen,
Hamburg, Germany) according to the manufacturer’s
recommendations After RNase treatment the protected
transcripts were resolved byelectrophoresis on a denaturing
polyacrylamide gel (5%) and analyzed on a
Phosphor-Imager with theIMAGEQUANTsoftware
Gelfiltration, subcellular, fractionation and Western
blotting
HEK293 cells (20· 106cells 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 mMHepes, 10 mMKCl, 0.1 mMEGTA, 0.1 mMEDTA,
pH 7.9 All the following procedures were performed on ice
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 photography16–36 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 bya pale graybox 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).
Trang 4or at 4C 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 bysize exclusion
chromato-graphyon a Superdex 200 HR10/30 column (Pharmacia,
Freiburg, Germany) in 10 mMHepes, 10 mMKCl, 0.1 mM
EGTA, 0.1 mMEDTA, 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 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 bySDS/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/Piand 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 mMMgCl2, 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 onlycentrifuged 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 4C and finallynuclear lysates were obtained byremoval of insoluble material bycentrifugation for
15 min at 14 000 g at 4C
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 (37C, 5% CO2) 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) were
mat-ched with the cDNA sequences encoding TRAF2 to TRAF6 [accession
numbers U12597 (TRAF2), U19260 (TRAF3), X80200 (TRAF4),
AB000509 (TRAF5) and U78798 (TRAF6)] Onlyexons encoding
parts of the cDNA translated into protein were considered in this
illustration Thus, exons numbered in this scheme with 1 do not
necessarilycorrespond to exon 1 of the respective gene in the database.
Exons encoding parts of the TRAF domain were summarized and
labeled TD The number of nucleotides encoded byeach exon was
indicated in the box representing the respective exon Exons containing
multiples of three nucleotides are indicated bygrayboxes (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 ) or remained untreated.
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 bysize exclusion chromatographyon 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.
Trang 5the 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 LeicaTCSsoftware
R E S U L T S A N D D I S C U S S I O N
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 cy toplasm of cells and even failed to detect it
in most breast cancer samples [7] It is possible that these
conflicting results are caused bythe existence of alternative
forms of TRAF4 that could be generated byalternative
splicing or byproteolytic events An alternative possibility
would be that a TRAF4-interacting protein secondarily
regulates TRAF4 localization, eventuallyin a
signal-regu-lated manner To studythe possibilitythat 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 transientlytransfected with
the respective expression plasmids and analyzed by confocal
microscopythe 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 likelyto be artifacts caused byoverexpression 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]
predominantlylocali-zed to the nucleus (Fig 2) Bysequence 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 simplyreflect the
deletion of a putative nuclear export sequence, as treatment
for up to 6 h with leptomy cin 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 necessaryto 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, anycombination of these exons results potentiallyin 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 transientlytransfected 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).
Trang 6TRAF protein In the case of TRAF3, the existence of such
splice forms has indeed alreadybeen 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 mainlyin nuclear
lysates (Fig 3B) In the T-cell lines Jurkat and Cem,
TRAF4 was induced byPMA 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 discrepancycannot
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 rapidlytitrated
byoverex-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
copurifyduring 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 mainlyin high molecular weight complexes of 443 kDa and more YFP chimeras solelycomprising 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 completelyorganized in complexes, the N-TRAF domain of TRAF4 eluted over the whole fractionation range of the Superdex 200 column A deletion mutant onlycomprising 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 predominantlyin 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 mainlybased on the triple helical
Fig 6 Subcellular localization of TRAF1, TRAF2, TRAF3and deletion mutants derived thereof HeLa cells were seeded on glass cover slides and transientlytransfected with expres-sion constructs for the indicated TRAF proteins Representative cells were selected for photography16–36 h after transfection.
Trang 7organization of parallel N-TRAF domains [3–5] However,
in principle, it cannot be ruled out completelythat 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 likelynegligible because the
expression level of endogenous TRAF4 in the HEK293 cells
is significantlybelow 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 verifywhether other TRAF proteins have a latent
capabilityto 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 mainlyto the cytoplasm
and were hardlydetectable 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 bya passive effect
As alreadydiscussed 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 solelycomprising the TRAF domain of TRAF2
and TRAF3 but were detected regularlyin 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 onlyconsisting 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 byT4(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 necessaryto 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.
Trang 8T4(259–470)] for a putative nuclear target structure In
addition, we found no evidence for a
T4(259–470)-depend-ent recruitmT4(259–470)-depend-ent 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 verifywhether TRAF4 or the
TRAF4-derived TRAF domain shuttles between nucleus
and cytoplasm, we analyzed T4(1–470)–GFP and T4(259–
470)–YFP byfluorescence 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 onlya 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 alreadysignificantly
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 onlya slow exchange of cytoplasmic
and nucleus-localized TRAF4, indicating that nuclear and
cytoplasmic TRAF4 mayrepresent functionallydistinct 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 recentlyshown that the oncogenic serine–threonine kinase Pim-1 induces translocation of the TRAF4-interact-ing protein-sortTRAF4-interact-ing 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 bythis 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 onlya modest, poorlyreproducible induction,
of TRAF4 mRNA bythese 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 alreadydetectable 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 transientlytransfected 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 repetitivelyfor the indicated time (bleaching cell
B) (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 R) 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.
Trang 9stimulation 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 primaryT-cells and in T-cell blasts
(Fig 9C) Six hours after stimulation, primaryT-cells
showed a 7.8-fold, and day13 T-cell blasts a 6.1-fold,
induction of TRAF4 mRNA Basal TRAF4 mRNA
expression was roughlycomparable in primaryT-cells and
T-cell blasts To verifywhether TRAF4 mRNA is directly
up-regulated byTNF- 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 inhibitoryeffect 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 directlylead 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 possibilitywas 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 verifywhether this pathwayis 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 primaryhuman 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 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 byelectrophoresis on a denaturing polyacrylamide gel (5%) and quantified on a PhosphorImager with the IMAGEQUANT software For quantification each TRAF4 or L32 band was individuallycorrected for background intensities byan 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.
Trang 10line 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 completelyinhibited 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-PMA-induced but not
PMA-induced TRAF4 expression was blocked (Fig 10)
These data clearlyargue for an essential role of the
NF-jB pathwayin TNF- and PMA-induced up-regulation of
TRAF4
A C K N O W L E D G E M E N T S
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 byDeutsche 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
thereof deficient in IKKc (middle panel) or RIP (right panel)
expres-sion were stimulated with TNF (20 ngÆmL)1) or PMA (100 n M ) for 6 h
and analyzed by RPA analysis as described in Fig 9.