The N-Terminal Domains Target TNF
Receptor-Associated Factor-2 to the Nucleus and
Display Transcriptional Regulatory Activity
This information is current as
of June 17, 2017.
Wang Min, John R. Bradley, Jennifer J. Galbraith, Sally J. Jones,
Elizabeth C. Ledgerwood and Jordan S. Pober
J Immunol 1998; 161:319-324; ;
http://www.jimmunol.org/content/161/1/319
Subscription
Permissions
Email Alerts
This article cites 28 articles, 13 of which you can access for free at:
http://www.jimmunol.org/content/161/1/319.full#ref-list-1
Information about subscribing to The Journal of Immunology is online at:
http://jimmunol.org/subscription
Submit copyright permission requests at:
http://www.aai.org/About/Publications/JI/copyright.html
Receive free email-alerts when new articles cite this article. Sign up at:
http://jimmunol.org/alerts
The Journal of Immunology is published twice each month by
The American Association of Immunologists, Inc.,
1451 Rockville Pike, Suite 650, Rockville, MD 20852
Copyright © 1998 by The American Association of
Immunologists All rights reserved.
Print ISSN: 0022-1767 Online ISSN: 1550-6606.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
References
The N-Terminal Domains Target TNF Receptor-Associated
Factor-2 to the Nucleus and Display Transcriptional
Regulatory Activity1
Wang Min,2* John R. Bradley,† Jennifer J. Galbraith,† Sally J. Jones,†
Elizabeth C. Ledgerwood,†‡ and Jordan S. Pober3*
T
he TNF receptor-associated factors (TRAFs)4 are a family
of structurally related adaptor proteins that couple certain
receptors of the TNFR family to the activation of new
gene expression in a variety of cell types. The first members of the
TRAF family, TRAF1 and -2, were identified as proteins that interact with the intracellular portion of TNFR-II (also known as the
p75 TNFR) (1). To date, three of six TRAF proteins have been
implicated in intracellular signaling initiated by a number of other
TNFR family members, including CD40, CD30, and the lymphotoxin-b receptor, as well as from at least one receptor that is structurally unrelated to TNFR-II, namely the type I IL-1R (2–9). In
human endothelial cells, proinflammatory actions of TNF, such as
transcriptional induction of the leukocyte binding protein E-selectin, are mediated through TNFR-I (also known as the p55 TNFR)
but not TNFR-II (10, 11). We have previously shown that the
proinflammatory responses initiated through ligation of TNFR-I
are strikingly similar to those elicited through engagement of type
*Boyer Center for Molecular Medicine, Yale University School of Medicine, New
Haven, CT 06536; and Departments of †Medicine and ‡Clinical Biochemistry, University of Cambridge, Addenbrooke’s Hospital, Cambridge, United Kingdom
Received for publication October 29, 1997. Accepted for publication March 6, 1998.
The costs of publication of this article were defrayed in part by the payment of page
charges. This article must therefore be hereby marked advertisement in accordance
with 18 U.S.C. Section 1734 solely to indicate this fact.
1
This work was supported by grants from the National Institutes of Health (HL36003 to J.S.P.) and the National Kidney Research Fund (U.K.) (to J.R.B.). W.M. was
supported by National Institutes of Health Training Grant T32-AI07019. E.C.L. was
supported by the Wellcome Trust.
2
Present address: Wang Min, Genemedicine, 8301 New Trails Drive, The Woodlands, TX 77381-4248.
3
Address correspondence and reprint requests to Jordan S. Pober, Boyer Center for
Molecular Medicine, 295 Congress Avenue, New Haven, CT 06536-0812. E-mail
address: [email protected]
4
Abbreviations used in this paper: TRAF, TNF receptor-associated factor; TRADD,
TNF receptor-associated death domain protein; JNK, Jun N-terminal kinase.
Copyright © 1998 by The American Association of Immunologists
I IL-1R or of CD40, two receptors that utilize TRAF proteins (12,
13). Although TRAF proteins do not directly interact with
TNFR-I, TRAF proteins may still participate in TNFR-I-initiated
signaling via interactions with the TNFR-I-associated death domain (TRADD) adaptor protein (14). Consistent with this, we have
recently shown that overexpressed TRAF2 protein can mimic the
proinflammatory effects of TNF in endothelial cells, including activation of NF-kB, of Jun N-terminal kinase (JNK), and of Eselectin transcription (15). More convincingly, the effects of TNF
on these same three responses are inhibited in endothelial cells by
overexpression of a “dominant negative” TRAF2 mutant protein
that lacks the amino-terminal ring finger domain required for signal transduction (15). Recent studies of TRAF2 knockout mice or
transgenic mice expressing dominant negative TRAF2 mutant protein have established that TRAF2 is absolutely required for activation of JNK by TNF, although it may be redundant in some cell
types for TNF-mediated NF-kB activation (16, 17).
Although molecular biologic approaches have established that
TRAF2 can couple endothelial TNFR-I to intracellular signaling
pathways, the mechanisms of TRAF2 action are not completely
understood. It has been proposed that TRAF2 proteins reside in the
cytosol of unstimulated cells, and are recruited to the intracytoplasmic domains of transmembrane TNFR-I molecules as a consequence of ligand-induced receptor clustering and binding of
TRADD protein (14). The membrane-associated TRAF2 proteins
are then thought to recruit downstream effector molecules, such as
the protein kinase NIK (18), leading to activation of IkB kinase
(19), and, ultimately, IkB degradation and nuclear translocation of
NF-kB (20). Even less is known about how TRAF2 is coupled to
activation of JNK (15, 21). Moreover, the intracellular localization
of TRAF2 in resting cells and its physical translocation to the
membranes where TNFR-I resides, the key assumptions made by
this model, have not been directly examined. To investigate these
0022-1767/98/$02.00
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
The subcellular localization of the TNF receptor-associated factor-2 (TRAF2) adaptor protein in human endothelial cells, which
mediates proinflammatory responses of TNF, has been analyzed by confocal immunofluorescence microscopy and by Western
blotting of fractionated cell extracts. Rabbit antisera reactive with either amino- or carboxyl-terminal TRAF2 peptides frequently
but not uniformly stain nuclei of cultured HUVEC or the established human endothelial cell line, ECV304. However, Western
blotting reveals significant heterogeneity in the reactivities of these polyclonal Abs. Transiently transfected HUVEC expressing
FLAG epitope-tagged TRAF2 consistently show prominent nuclear localization, and deletion mutants of TRAF2 identify the
portion of the molecule responsible for nuclear localization as the amino-terminal ring finger domain. TNF treatment does not
appear to influence the localization of endogenous or transfected TRAF2 protein. Transfection of the amino-terminal half of the
TRAF2 molecule, containing the ring and zinc finger domains, which localizes to the nucleus, results in activation of E-selectin but
not of NF-kB promoter-reporter gene transcription or of c-Jun N-terminal kinase activation. These observations suggest that
TRAF2 may reside in the nucleus and directly regulate transcription, independent of its role in cytoplasmic signal transduction.
The Journal of Immunology, 1998, 161: 319 –324.
320
FIGURE 1. Schematic representation of the amino-terminal FLAG
epitope-tagged TRAF2 protein constructs used in this study. The numbers
below the full-length construct (TRAF (1-501)) refer to amino acid residues in the endogenous TRAF2 protein. The ring finger (RING), the five
zinc fingers (ZINC Fingers 1-5), and the two TRAF domains (TRAF-N and
TRAF-C) are also delineated in this figure.
Materials and Methods
Cells and reagents
HUVECs were isolated and serially passaged as described (15) in Medium
199 supplemented with FCS (20% v/v from Life Technologies, Grand Island, NY), endothelial cell growth factor/porcine intestinal heparin, glutamine, penicillin, and streptomycin (all other reagents from Collaborative
Research, Bedford, MA). ECV304, an immortalized cell line of human
umbilical vein origin (22), was obtained from I. Fritz (The Babraham Institute, Cambridge, U.K.) TNF was provided by Biogen (Cambridge, MA).
Polyclonal rabbit Ab to the amino-terminal 19-amino acid residue peptide
or to the carboxyl-terminal 20-amino acid residue peptide of TRAF2, along
with the relevant peptide immunogens, were purchased from Santa Cruz
Biotechnology (Santa Cruz, CA). Mouse mAb M5 to the free amino-terminal FLAG epitope was purchased from Kodak (New Haven, CT). FITCand phycoerythrin-conjugated secondary Abs to rabbit and mouse Ig for
indirect immunofluorescence microscopy were purchased from Jackson
Immunoresearch Laboratories (West Grove, PA). Horseradish peroxidaseconjugated secondary Abs to rabbit and mouse Ig for Western blotting
were purchased from Dako (Glostrup, Denmark).
FIGURE 2. Confocal microscope images of indirect immunofluorescence staining for TRAF2 protein in HUVEC (A and B) and ECV304 cells
(C and D) using polyclonal sera reactive with TRAF2 amino-terminal (A
and C) and carboxyl-terminal (B and D) peptides. Note that many, but not
all cells display prominent nuclear staining. Some cells also display diffuse
or localized cytosolic staining as well as plasma membrane staining. All
reactivity of these sera could be competed by excess of the relevant immunizing peptide (not shown).
Transient transfection of HUVEC cultures was performed by the
DEAE-dextran method as described previously (15). Typically, 10 to 15%
of cells are transfected by this method. Growth hormone secretion from
promoter-reporter genes induced by transfection of TRAF2 expression
constructs or TNF treatment was measured by radioimmunoassay, according to the instructions of the manufacturer (Nichols, San Juan Capistrano,
CA), and corrected for transfection efficiency using a b-galactosidase expression construct, as described previously (15). Lysates of control vectortransfected, TNF-treated, and TRAF2 expression construct-transfected
HUVEC cultures were also tested for JNK activity by measuring their
capacities to phosphorylate an amino-terminal c-Jun-glutathione S-transferase fusion protein as previously described (13, 15).
Indirect immunofluorescence and confocal microscopy
Fixation, permeabilization, and staining of cultured HUVECs or ECV304
cells were performed as described elsewhere (24). Confocal immunofluorescence microscopy was performed using a Bio-Rad Model 600 (Hercules, CA) confocal microscope.
Cell fractionation and Western blotting
Nuclear and cytosolic fractions of HUVECs were prepared as described
elsewhere (25). Extracts were subjected to SDS-PAGE (all reagents from
Bio-Rad) and electrophoretically transferred to PVP membranes (Dassel,
Germany). Ab staining of membranes was performed as described (15),
and then developed using chemiluminescence with a kit from Kirkegaard
& Perry (Gaithersburg, MD), according to the instructions of the
manufacturer.
Expression constructs and transient transfection of HUVECs
Results
Amino-terminal FLAG epitope-tagged full-length TRAF2 (TRAF (1-501))
and amino-terminal-deleted TRAF2 (TRAF (87-501)) cDNA expression
plasmids (14) were obtained from Dr. D. Goeddel (Tularik, South San
Francisco, CA). Two other truncated versions of TRAF2, one missing carboxyl- terminal residues 98 to 501 (TRAF (i.e., 1-97)) and one missing
carboxyl-terminal residues 250 to 501 (i.e., TRAF (1-249) were generated
by PCR cloning from full-length TRAF2 and reinserted into amino-terminal FLAG epitope expression plasmids. All constructs were confirmed by
sequencing. The relationship of these various deletion constructs to the
domain structure of the TRAF2 protein (23) is shown in Figure 1. Promoter-reporter genes containing 578 base pairs of the E-selectin promoter
(ELAMp(2578)), the same promoter in which the c-Jun/ATF-2 binding
site has been deleted (ELAMp(2578)DCRE, both from Dr. T. Collins,
Brigham and Women’s Hospital, Boston, MA), and a third construct (kB)
containing two binding sites for NF-kB from the k-chain enhancer, each
coupled to a human growth hormone reporter, have been described
elsewhere (15).
Endogenous TRAF2 protein immunoreactivity is localized to the
nucleus as well as the cytoplasm of human endothelial cells
We used indirect immunofluorescence and confocal microscopy to
localize TRAF2 protein in cultured HUVEC and in the spontaneously transformed HUVEC-derived cell line, ECV304. Staining
with polyclonal Abs raised either to an amino-terminal or a carboxyl-terminal peptide of the TRAF2 protein, but not irrelevant
control Abs, showed frequent staining of nuclei as well as of cytoplasm and plasma membrane (Fig. 2). The pattern of staining
appeared independent of the degree of culture confluence, previously shown to affect nuclear localization of other endothelial proteins (25). The cytoplasmic and nuclear staining of TRAF2 observed with these sera could be effectively competed with an
excess of the relevant immunizing peptides and was not observed
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
questions, we applied both confocal immunofluorescence microscopy and cell fractionation approaches to localize TRAF2 within
human endothelial cells. To our surprise, native TRAF2 was frequently, and FLAG epitope-tagged transfected TRAF2 was consistently found in the cell nucleus, a distribution that is unaffected
by TNF treatment. This location raises the possibility of a previously unanticipated direct role of TRAF proteins in transcriptional
regulation, a hypothesis that is supported by the effects of transfected TRAF molecules on E-selectin promoter-reporter gene
activity.
TRAF2 IS A NUCLEAR PROTEIN
The Journal of Immunology
with irrelevant control rabbit sera (data not shown). At least three
separate batches of the anti-carboxyl-terminal peptide sera were
found to produce similar staining patterns. However, Western blots
using whole lysates of HUVEC or ECV304 cells or isolated nuclei
from these cell types revealed multiple immunoreactive species of
varying sizes, both larger and smaller than the expected size of
TRAF2. Moreover, the immunoreactive species observed with antiamino-terminal peptide sera did not overlap with the patterns observed with anti-carboxyl-terminal peptide sera, although the relevant immunizing peptides completely blocked all reactivity in
both cases. We conclude that the pattern of cell staining is consistent with nuclear as well as cytoplasmic localization of TRAF2
protein, but that the antipeptide sera are insufficiently specific to
draw firm conclusions.
Nuclear localization of overexpressed exogenous TRAF2 protein
in human endothelial cells
To address the ambiguous findings noted above from the studies
using antipeptide TRAF2 Abs, we introduced an epitope-tagged,
exogenous TRAF2 protein into HUVEC by means of transient
transfection. Four different constructs were made (Fig. 1), including full-length TRAF2 (TRAF (1-501)) and three different truncated proteins that lack the amino-terminal 86 amino acids (TRAF
(87-501), the carboxyl-terminal 252 amino acids (TRAF (1-249),
or the carboxyl-terminal 404 amino acids (TRAF (1-97)). Each
construct had the FLAG epitope attached to its respective amino
terminus, which has been previously shown not to interfere with
TRAF2 function (15). Using indirect immunofluorescence with antiFLAG mAb and confocal microscopy, a prominent and consistent
nuclear localization was observed for all three TRAF constructs
that contained the amino terminus, i.e., TRAF (1-97), TRAF (1249), and TRAF (1-501) (Fig. 3). These constructs additionally
showed a less intense, granular cytoplasmic staining pattern.
TRAF (1-501), the full-length protein, also gave a diffuse cytosolic
staining pattern, which was less conspicuous in the cells transfected with the other constructs (i.e., with TRAF (1-97) or TRAF
(1-249)). In contrast, cytosolic staining was intense and nuclear
staining was strikingly reduced in cells transfected with the construct that lacked the amino-terminal ring finger domain (i.e.,
TRAF (87-501)).
The localizations of transfected epitope-tagged TRAF proteins
observed by confocal immunofluorescence microscopy were confirmed by cell fractionation and Western blotting. When transfected HUVEC were fractionated into nuclear and cytosolic extracts, Western blotting with anti-FLAG Ab identified TRAF (1501) in both fractions, and identified TRAF (87-501) exclusively
in the cytosol and TRAF (1-249) exclusively in the nucleus (Fig.
4). The differences in localization detected by Western blotting
with anti-FLAG Ab appear more extreme than those observed by
microscopy, but are qualitatively consistent with the immunofluorescence images. Treatment of cells with TNF did not affect the
localization of transfected TRAF2 proteins as detected by confocal
immunofluorescence microscopy (not shown) or by cell fractionation and Western blotting (Fig. 4).
The generation of transfected HUVECs overexpressing TRAF2
proteins that contain or lack the amino-terminal of carboxyl-terminal peptide sequence allowed us to reexamine the specificity of
anti-TRAF2 peptide Abs purchased from the same vendor as those
used earlier in this study. In Western blots of whole cells, antiamino-terminal peptide Ab appropriately recognized transfected
TRAF (1-501) and TRAF (1-249) but not TRAF (87-501), whereas
anti-carboxyl-terminal peptide Ab appropriately recognized TRAF
(1-501) and TRAF (87-501) but not TRAF (1-249) (Fig. 5). These
Abs also recognized what appeared to be breakdown products of
FIGURE 4. Western blot analysis with anti-FLAG mAb of cytosolic (cyto) and nuclear (nucl) extracts prepared from HUVEC following transient
transfections with FLAG epitope-tagged TRAF2 constructs. (TRAF (1-97) was excluded from this analysis because it was too small for resolution by
SDS-PAGE.) Lanes designated “2” were mock treated and those designated “TNF” were pretreated with TNF (100 U/ml) for 15 min before harvest. Note
that TRAF (87-501) was excluded from the nucleus, that TRAF (1-249) was exclusively localized to the nucleus, and that TRAF (1-501) was found in the
nucleus and the cytosol. Note also that TNF treatment did not cause redistribution of any TRAF proteins.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 3. Confocal microscope images of indirect immunofluorescence staining of FLAG epitope with monoclonal anti-FLAG Ab following
transient transfection of HUVEC with FLAG epitope-tagged TRAF2 constructs: A, TRAF (1-97); B, TRAF (1-249); C, TRAF (89-501); and D,
TRAF (1-501). Note that all TRAF constructs containing the amino-terminal ring finger show prominent nuclear localization, whereas the one
construct lacking this domain, namely TRAF (89-501), fails to enter the
nucleus.
321
322
TRAF2 IS A NUCLEAR PROTEIN
FIGURE 6. JNK activity in HUVEC lysates prepared from control,
TNF-treated, or TRAF2 expression construct transfected cells. Note that
full-length TRAF2 appears as effective as TNF at activation of JNK,
whereas the TRAF2 fragments, including TRAF (1-249), appear
ineffective.
Function of transfected TRAF2 fragments in HUVECs
transfected TRAF2 proteins derived from the amino terminus (labeled N*) and from the carboxyl terminus (labeled C*). Interestingly, these Abs also reacted with several proteins in empty vector
as well as TRAF-transfected HUVECs that migrated at an appropriate size for intact TRAF2 (56 kDa, labeled a) as well as for
large carboxyl-terminal (40 kDa, labeled b) and amino-terminal
(18 kDa, labeled c) fragments. However, the precise identity of
these bands as TRAF2 breakdown products has not been confirmed by other means.
Discussion
The present study was designed to test the hypothesis that TNF
causes a translocation of cytosolic TRAF2 protein to the plasma
membrane of human vascular endothelial cells, i.e., the organelle where TNFR-I molecules become clustered as a consequence of ligand binding. This may, in fact, occur, but such an
effect was not observed by confocal immunofluorescence microscopy, perhaps because there are too few TNFR-I molecules
in the plasma membrane (11) to cause significant redistribution
of TRAF2 molecules, which appear to be expressed at significantly higher levels.
The most surprising finding of the present study is that TRAF2
prominently localizes to the nucleus as well as the cytoplasm. This
was first suggested in studies using polyclonal Abs to TRAF2 peptides (of uncertain specificity) and confirmed using transfected
epitope-tagged TRAF2 molecules. The transfection studies were
critical because of the unexpectedly broad reactivates displayed by
both antipeptide sera, and this broad reactivity was noted in multiple batches of these Abs. Our experience points to the need for
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
FIGURE 5. Western blot analysis using antisera reactive with aminoterminal (aN19) or carboxyl-terminal (aC20) TRAF2 peptide antisera, or
both, of extracts prepared from HUVEC that were transiently transfected
with empty vector (mock) or TRAF2 constructs. Note that these sera detect
both full-length and discrete breakdown products of the various transfected
TRAF2 proteins. The breakdown products are derived from both the amino
terminus (N*) or the carboxyl terminus (C*) of transfected TRAF proteins
that contain the relevant peptides. These sera also appear to react with
full-length endogenous TRAF2 (labeled a) as well as with bands that may
represent an amino-terminal (labeled c) and a carboxyl-terminal (labeled b)
breakdown product of the endogenous TRAF2 protein.
Previous studies from our laboratory had demonstrated that fulllength TRAF2 (TRAF (1-501)) acted as a dominant activator of
NF-kB or E-selectin promoter-reporter genes, and that the magnitude of the effect on the E-selectin promoter was larger if the
ATF-2/c-Jun binding element was present in the promoter (15).
These observations are confirmed in the experiments reported in
Table I. Our previous report also showed that TRAF (87-501) did
not trans-activate, and was actually a dominant negative regulator
of NF-kB and E-selectin promoter reporter gene transcription in
response to TNF. In the present study, we find that TRAF (1-97),
which largely localizes to the nucleus, is not a transactivator of
these promoter-reporter genes. Remarkably, TRAF (1-249), which
also localizes to the nucleus, is an even more potent activator of
wild-type E-selectin than is full-length TRAF2, yet it has no effect
on the E-selectin promoter lacking the ATF-2/c-Jun binding site or
on the NF-kB promoter. Two additional observations suggest that
TRAF (1-249) may act by directly regulating gene transcription
rather than influencing cytoplasmic signal transduction. First,
TRAF (1-249), unlike TRAF (1-501), does not activate JNK (Fig.
6). Second, as shown in Table I (Expt. 2), coexpression of TRAF
(87-501) does not inhibit transcription of E-selectin caused by
TRAF (1-249), whereas it does inhibit transcription caused by
TRAF (1-501). These observations suggest that nuclear TRAF2
may have specific functions as a transcriptional activator or, more
likely, coactivator (e.g., in conjunction with c-Jun/ATF-2) distinct
from its previously described role as a receptor-associated adaptor
molecule for coupling to cytosolic signaling pathways.
The Journal of Immunology
323
Table I. Transactivation in mammalian reporter gene assaya
Reporter Gene
Expt.
Transfection/Treatment
ELAM p(-578)
ELAM p(-578)DCRE
kB
1
Vector
TNF (100 U/ml)
TRAF (1-501)
TRAF (1-249)
TRAF (1-97)
1.0
24.0
2.6
5.0
0.8
1.0
4.0
1.9
1.1
0.9
1.0
16.0
2.1
1.2
0.8
2
Vector
TNF (100 U/ml)
TRAF (1-501)
TRAF (1-249)
TRAF (87-501)
TRAF (1-501) 1 TRAF (87-501)
TRAF (1-249) 1 TRAF (87-501)
1.0
25.0
5.8
7.9
1.1
2.0
7.3
1.0
5.0
2.0
1.3
0.9
1.1
1.5
ND
ND
ND
ND
ND
ND
ND
better, possibly monoclonal anti-TRAF Abs before investigations
of endogenous TRAF2 protein localization are extended to other
systems.
The distribution of the partially deleted TRAF2 molecules identifies the nuclear targeting region within the first 97-amino acid
residues of the amino terminus, which encodes the ring finger domain, and establishes that the two TRAF domains, contained
within residues 247-501, are devoid of nuclear targeting propensity. There are no identifiable candidate sequences for nuclear localization within the first 97 amino acids that conform to the expected basic residue-rich motif (26). However, such motifs show
wide variability, and we cannot completely exclude the possibility
of a nuclear localization sequence in TRAF2. Alternatively, it is
possible that TRAF2 reaches the nucleus by associating with some
other protein that contains a conventional nuclear localization
sequence.
The propensity of TRAF2 to localize in the nucleus was partly
diminished by the presence of TRAF domains, i.e., more of fulllength TRAF2 was found outside the nucleus than of TRAF (1249), which was essentially confined to the nucleus. The extranuclear location of full-length TRAF2 was equivalent in cells that
were not exposed to TNF as in those that were, suggesting that
interactions of the TRAF domains with cytosolic or membrane
molecules other than clustered TNFRs probably occurs constitutively. The identity of these other putative TRAF-interacting proteins is also unknown.
The second surprising finding of this study is the activating
effect that the TRAF (1-249) protein displays on transcription of
a cotransfected E-selectin promoter-reporter gene, which is
greater than that produced by full-length TRAF2. This activity
of the TRAF2 amino-terminal regions is specific in that there
was no effect on transcription of an NF-kB promoter-reporter
gene or even on the E-selectin construct in which the c-Jun/
ATF-2 element was deleted. In contrast, both of these latter
constructs are activated by full-length TRAF2. Since TRAF (1249) does not appear to activate JNK, our data are most consistent with the possibility that the amino-terminal half of the
TRAF2 protein, including the ring and zinc finger domains (but
not the ring domain alone contained in TRAF (1-97)) can act as
a transcriptional activator or co-activator. This activity is consistent with nuclear localization. This idea was further sup-
ported by the observation that cytosolic TRAF (87-501) can act
as a dominant negative inhibitor of TRAF (1-501) but not of
TRAF (1-249) function. In other words, the TRAF domains,
which are essential for TNF signaling, are irrelevant for the
observed effects on E-selectin transcription. Since less transcriptional activity is found in cells transfected with the fulllength TRAF2 protein, these data further suggest that it may be
necessary to cleave the TRAF2 molecule for such transcriptional regulation to operate in normal cells. This putative proteolytic event could occur in the nucleus or in the cytoplasm,
since once formed, an amino-terminal TRAF2 fragment would
rapidly enter the nucleus. Interestingly, HUVECs do appear to
have a TRAF2-derived amino-terminal fragment, labeled as “c”
in Figure 5, which may have arisen from such a cleavage event.
However, neither the function nor the identity of this fragment
has been rigorously established.
The possibility that a cytosolic adaptor molecule could regulate
gene expression is not without precedent. The most obvious examples are the signal transducer and activator of transcription proteins, which mediate cytokine receptor signals (27). Another recently described example of such regulation is b-catenin, an
adaptor protein that associates with the cytoplasmic tail of cadherins or the APC protein and may enter the nucleus to act as a
co-activator of transcription mediated by the DNA binding protein
LEF (28, 29). It is premature, on the basis of the available evidence, to categorize TRAF2 as a transcriptional activator or coactivator, but the parallels are intriguing.
In summary, we have demonstrated that TRAF2, an adaptor
protein that links occupancy by ligand at the plasma membrane to
cytoplasmic signaling pathways that initiate new gene expression
in vascular endothelial cells, is unexpectedly present in the cell
nucleus. We have also presented evidence that nuclear TRAF2
protein has the potential to function as a transcriptional activator or
co-activator of at least one relevant TNF target gene. It remains to
be seen whether other TRAF proteins have similar functions and
which other target genes are similarly regulated.
Acknowledgments
We thank Louise Benson and Gwendolyn Davis for assistance in endothelial cell culture. We also thank Dr. David Goeddel of Tularik, Inc. for
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
a
The capacity of varying TRAF2 expression constructs, used singly or in combination, are tested as activators of a wild-type
E-selectin promoter/reporter gene (ELAMp(-578)), which responds to TNF through a combination of three kB binding sites and
an ATF2/c-Jun binding site; of the same construct in which the JNK-dependent ATF-2/c-Jun binding site has been deleted
(ELAMp(-578)DCRE) from the promoter; or a promoter/reporter gene constructed with two tandem kB sites (kB). The numbers
represent the fold increase of secreted growth hormone at 24 h, corrected for transfection efficiency, using a b-galactosidase
vector, and normalized to vector control cells. TNF treatment is included as a positive control. The values in experiment 1 are
means of quadruplicate determinations from one of two similar experiments, whereas those in experiment 2 are means of
quadruplicate determination pooled from two separate assays. ND, not done.
324
provision of the full-length and amino-terminal-deleted TRAF2 expression
constructs.
References
14. Hsu, H., H. B. Shu, M. G. Pan, and D. V. Goeddel. 1996. TRADD-TRAF-2 and
TRADD-FADD interactions define two distinct TNF receptor 1 signal transduction pathways. Cell 84:299.
15. Wang Min, and J. S. Pober. 1997. TNF initiates E-selectin transcription in human
endothelial cells through parallel TRAF-NF-kB and TRAF-RAC/CDC42-JNKcJun/ATF-2 pathways. J. Immunol. 159:3508.
16. Lee, S. Y., A. Reichlin, A. Santana, K. A. Sokol, M. C. Nussenzweig, and Y.
Choi. 1997. TRAF2 is essential for JNK but not NF-kB activation and regulates
lymphocyte proliferation and survival. Immunity 7:703.
17. Yeh, W.-C., A. Shahinian, D. Speiser, J. Kraunus, F. Billia, A. Wakeham,
J. L. de la Pompa, D. Ferrick, B. Hum, N. Iscove, P. Ohashi, M. Rothe,
D. V. Goeddel, and W. W. Mak. 1997. Early lethality, functional NF-kB activation, and increased sensitivity to TNF-induced cell death in TRAF2-deficient
mice. Immunity 7:715.
18. Malinin, N. L., M. P. Boldin, A. V. Kovalenko, and D. Wallach. 1997. MAP3Krelated kinase involved in NF-kB induction by TNF, CD95 and IL-1. Nature
395:54.
19. Régnier, C. H., H. Y. Song, X. Gao, D. V. Goeddel, Z. Cao, and M. Rothe. 1997.
Identification and characterization of a IkB kinase. Cell 90:373.
20. Beg, A. A., T. S. Finco, P. V. Nantermet, and A. S. Baldwin. 1993. Tumor
necrosis factor and interleukin-1 lead to phosphorylation and loss of IkB: a mechanism for NF-kB activation. Mol. Cell. Biol. 13:3301.
21. Liu, Z. G., H. Hsu, D. V. Goeddel, and M. Karin. 1996. Dissection of TNF
receptor 1 effector functions: JNK activation is not linked to apoptosis while
NF-[kappa]B activation prevents cell death. Cell 87:565.
22. Takahashi, K., Y. Sawasaki, J. I. Hata, K. Mukai, and T. Goto. 1990. Spontaneous transformation and immortalization of human endothelial cells. In Vitro Cell.
Dev. Biol. 25:265.
23. Takeuchi, M., M. Rothe, and D. V. Goeddel. 1996. Anatomy of TRAF2. J. Biol.
Chem. 271:19935.
24. Bradley, J. R., S. Thiru, and J. S. Pober. 1994. Disparate localization of 55kd and
75kd TNF receptors in human endothelial cells. Am. J. Pathol. 146:27.
25. Sierra-Honigmann, M. R., J. R. Bradley, and J. S. Pober. 1996. “Cytosolic”
phospholipase A2 is in the nucleus of subconfluent endothelial cells but confined
to the cytoplasm of confluent endothelial cells and redistributes to the nuclear
envelope and cell junctions upon histamine stimulation. Lab. Invest. 74:684.
26. Hicks, G. R., and N. Raukhel. 1995. Protein imports into the nucleus: an integrated view. Annu. Rev. Cell. Dev. Biol. 11:155.
27. Ihle, J. N., B. A. Witthuhn, F. W. Quelle, K. Yamamoto, and O. Silvennoinen.
1995. Signaling through the hematopoietic cytokine receptors. Annu. Rev. Immunol. 13:369.
28. Gumbiner, B. M. 1995. Signal transduction of beta-catenin. Curr. Opin. Cell Biol.
7:534.
29. Behrens, J., J. P. von Kries, M. Kuhl, L. Bruhn, D. Wedlich, R. Grosschedl, and
W. Birchmaier. 1996. Functional interaction of beta-catenin with the transcription
factor LEF-1. Nature 382:638.
Downloaded from http://www.jimmunol.org/ by guest on June 17, 2017
1. Rothe, M., S. C. Wong, W. J. Henzel, and D. V. Goeddel. 1994. A novel family
of putative signal transducers associated with the cytoplasmic domain of the
75kDa tumor necrosis factor receptor. Cell 78:681.
2. Hu, H. M., K. O’Rourke, M. S. Boguski, and V. M. Dixit. 1994. A novel RING
finger protein interacts with the cytoplasmic domain of CD40. J. Biol. Chem.
269:30069.
3. Rothe, M., V. Sarma, V. M. Dixit, and D. V. Goeddel. 1995. TRAF-2-mediated activation of NF-kB by TNF receptor 2 and CD40. Science 269:1424.
4. Cheng, G., A. M. Cleary, Z. S. Ye, D. Hong, S. Lederman, and D. Baltimore.
1995. Involvement of CRAF-1, a relative of TRAF, in CD40 signaling. Science
267:1494.
5. Gedrich, R. W., M. C. Gilfillan, C. S. Duchett, J. L. Van Dongen, and C. B.
Thompson. 1996. CD30 contains two binding sites with different receptor specificities for members of the tumor necrosis factor receptor-associated factor family of signal transducing proteins. J. Biol. Chem. 271:12852.
6. Lee, S. Y., S. L. Lee, G. Kandala, M. L. Liou, H. C. Liou, and Y. Choi. 1996.
CD3D-TRAF interaction: NF-kB activation and binding specificity. Proc. Natl.
Acad. Sci. USA 93:9699.
7. Ishida, T., T. Tojo, T. Aoki, N. Kobayashi, T. Ohishi, T. Watanabe,
T. Yamamoto, and J. I. Imue. 1996. TRAF5, a novel tumor necrosis factor receptor-associated family protein, mediates CD40 signaling. Proc. Natl. Acad. Sci.
USA 93:9437.
8. Nakano, H., H. Oshima, W. Chung, L. C. F. Ware, H. Yagita, and K. Okumura.
1996. TRAF5, an activator of NF-kB and putative signal transducer for the lymphotoxin-b receptor. J. Biol. Chem. 271:14661.
9. Cao, Z., J. Xiong, M. Taheuchi, T. Kurama, and D. V. Goeddel. 1996. TRAF6 is
a signal transducer for interleukin-1. Nature 383:443.
10. Mackay, F., H. Loetscher, D. Stueber, G. Gehr, and W. Lesslauer. 1993.
Tumor necrosis factor a (TNF-a)-induced cell adhesion to human endothelial
cells is under dominant control of one TNF receptor type, TNF-R55. J. Exp.
Med. 177:1277.
11. Slowik, M. R., L. G. DeLuca, W. Fiers, and J. S. Pober. 1993. Tumor necrosis
factor (TNF) activates human endothelial cells through the p55 TNF receptor but
the p75 receptor contributes to activation at low TNF concentration.
Am. J. Pathol. 134:1724.
12. Karmann, K., C. C. W. Hughes, J. Schechner, W. C. Fanslow, and J. S. Pober.
1995. CD40 on human endothelial cells: inducibility by cytokines and functional regulation of adhesion molecule expression. Proc. Natl. Acad. Sci. USA
92:4342.
13. Karmann, K., W. Min, W. C. Fanslow, and J. S. Pober. 1996. Activation and
homologous desensitization of human endothelial cells by CD40 ligand, tumor
necrosis factor, and interleukin 1. J. Exp. Med. 184:173.
TRAF2 IS A NUCLEAR PROTEIN