From the Interdepartmental Program in Vascular
Biology and Transplantation, Boyer Center for Molecular Medicine, Yale
University School of Medicine, New Haven, Connecticut 06536-0812 and
from the
Department of Medicine, University of Cambridge,
Cambridge CB2 2QQ, United Kingdom
Received for publication, August 7, 2000, and in revised form, December 5, 2000
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ABSTRACT |
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Tumor necrosis factor (TNF) receptor-associated
factor (TRAF) 2 is an intracellular adapter protein, which, upon TNF
stimulation, is directly recruited to the intracellular region of TNF
receptor 2 (TNFR2) or indirectly, via TRADD, to the intracellular
region of TNF receptor 1 (TNFR1). In cultured human umbilical vein
endothelial cells, endogenous TRAF2 colocalizes with the
membrane-organizing protein caveolin-1 at regions of enrichment
subjacent to the plasma membrane as detected by confocal fluorescence
microscopy. Both endogenous and transfected TRAF2 protein
coimmunoprecipitate with caveolin-1 in the absence of ligand. Upon TNF
treatment, the TRAF2-caveolin-1 complex transiently associates with
TRADD, and upon overexpression of TNFR2, the TRAF2-caveolin-1 complex
stably associates with and causes redistribution of this receptor as
detected by confocal fluorescence microscopy. In human embryonic kidney
293 cells, which have minimal endogenous expression of caveolin-1,
cotransfection of TRAF2 and caveolin-1 results in spontaneous
association of these proteins which can further associate with and
redistribute transfected TNFR2 molecules. The association of caveolin-1
with TNFR2 depends upon TRAF2. Cotransfection of caveolin-1 protein increases TRAF2 protein expression levels in HEK 293 cells, which correlates with enhancement of TNF and TRAF2 signaling, measured as
transcription of a NF- TNF1 is a critical
mediator of innate and adaptive immunity (1, 2). It exerts its
biological effects through binding to either of two receptor molecules,
designated TNFR1 (CD120a) and TNFR2 (CD120b) (3). TNF exists as a
stable homotrimer, and ligand binding induces homotypic clustering of
the transmembrane receptor proteins (4-6). The cytoplasmic tails of
the clustered receptors initiate signaling by recruitment of adapter
proteins (7). Clustered TNFR1 binds TNF receptor-associated death
domain (TRADD) protein. TRADD, in turn, binds receptor-interacting
protein (RIP) and TNF receptor-associated factor (TRAF) 2 (8-12). The TRADD·RIP·TRAF2 complex initiates new gene expression
through protein kinase cascades that result in activation of the
transcription factors NF- Many receptor signaling pathways have been linked to cholesterol- and
sphingomyelin-enriched invaginations of the plasma membrane called
caveoli (17-19). The inner surface of these membrane regions is coated
with a protein scaffolding formed by members of the caveolin family
(caveolin-1, -2, and -3). Caveolins have also been associated with
other membrane organelles, such as the Golgi, and they may play a role
in localizing signaling molecules to these intracellular compartments
(19). Such an association has been observed for endothelial
nitric-oxide synthase (called eNOS; also called NOS-3), which can exist
association with either plasma membrane or Golgi (20). Since TNFR1 can
also reside either in the plasma membrane or in the Golgi of human
umbilical vein endothelial cells (HUVEC) (21), we wondered if it too
might associate with caveolin. Although we did not find a direct
association of TNFR1 with caveolin, we did find an interaction of
caveolin-1 with TRAF2. This association is independent of ligand and
results in the recruitment of TRAF2-caveolin-1 complexes to either
TRADD or TNFR2.
Materials--
-Recombinant human TNF- Plasmid Constructs--
cDNAs encoding mouse TRAF1,
full-length human TRAF2, and a dominant-negative of human TRAF2
(TRAF2DN, which has 86 amino acids deleted from the N-terminal end)
were each amplified by polymerase chain reaction (PCR) from mammalian
expression constructs pRK-TRAF1-Flag and pRK-TRAF2-Flag (both gifts
from Dr. V. Dixit, Genentech Inc., South San Francisco, CA). The
amplified cDNA fragments were directly inserted between
BamHI and KpnI sites of the expression vector pBK-CMV (Stratagene, La Jolla, CA) to generate pBK-CMV-TRAF1, pBK-CMV-TRAF2, and pBK-CMV-TRAF2DN. To generate enhanced green fluorescent protein (EGFP) fusion construct, the EGFP coding sequence minus its stop codon was first amplified from the plasmid pEGFP-N1 (CLONTECH) using 5'-oligonucleotide primer
5'-GTGAACCGTCAGATCCGCTAG-3' (based on the sequence of pEGFP-N1 from 575 to 595) and 3' primer 5'-CCATCTTGCGCGCCTTGTACAGCTCGTTCCATGC-3' (with the native
sequence of pEGFP-N1 from 1376 to 1396 underlined). The PCR-amplified
cDNA fragment, containing the EGFP cassette, was gel-purified,
digested with BamHI and BssHII, and directly
inserted between the BamHI and BssHII sites of
plasmids pBK-CMV-TRAF1, pBK-CMV-TRAF2, or pBK-CMV-TRAF2DN (in which the
BssHII site is located three base pairs upstream of the ATG
start codon of TRAF1 or TRAF2). To make TNFR2( Cell Culture--
HUVEC were isolated and serially cultured as
described previously on gelatin (J. T. Baker Inc., Phillipsburg,
NJ)-coated tissue culture plastic (Falcon, Lincoln Park, NJ) in Medium
199 supplemented with 20% (v/v) fetal bovine serum, 200 µM L-glutamine (all from Life Technologies,
Inc.), 50 µg/ml endothelial cell growth factor (Collaborative
Biomedical Products, Bedford, MA), 100 µg/ml porcine heparin (Sigma),
and 100 units/ml penicillin and 100 µg/ml streptomycin (from Life
Technologies, Inc.) (23). Transient transfection of HUVEC was performed
using a DEAE-dextran method as described previously (24). HEK 293 cells
were obtained from the American Type Culture Collection (ATCC,
Manassas, VA) and were maintained in Eagle's minimum essential medium
(Life Technologies, Inc.) supplemented with 10% (v/v) fetal bovine
serum in a 5% CO2 incubator at 37 °C. Cells were seeded
at a density of 60-80% and transfected using a modified calcium
phosphate method with 1-12 µg of plasmid (25).
Immunoprecipitation--
Immunoprecipitation of control or
transfected cells was conducted as follows. Cell monolayers were washed
with cold PBS and solubilized in 0.5 ml of lysis buffer containing
protease inhibitors (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% Nonidet P-40, 10 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml
aprotinin, 10 µg/ml trypsin/chymotrypsin inhibitor, 5 µg/ml
pepstatin A, 1 mM phenylmethylsulfonyl fluoride) for 1 h. Lysates were cleared by centrifugation in Eppendorf tubes, then
precleared with Protein G Plus/Protein A-agarose beads with rocking for
1 h at 4 °C. 5 µg of the specific antibody was added to the
precleared lysates for 2-16 h at 4 °C, Protein G Plus/Protein
A-agarose beads were used to absorb immunoprecipitates with rocking for
1-8 h at 4 °C, and the beads were then washed four times with lysis buffer.
Immunoblotting--
Cell lysates or immunoprecipitates
were separated by SDS-polyacrylamide gel electrophoresis and
electrophoretically transferred onto nitrocellulose membranes
(Bio-Rad). The membranes were blocked in PBS with 0.05% Tween 20 and
5% dried milk (Nestle Food Co., Glendale, CA), probed with specific
antibodies and peroxidase-conjugated goat anti mouse or rabbit antibody
(1:10000 dilution) and exposed using the Supersignal® West
Femto Chemiluminescence Western blotting detection system (Pierce).
Indirect Immunofluorescence and Confocal
Microscopy--
Transfected cells were seeded on human plasma
fibronectin- or collagen type I (Sigma)-coated glass coverslips placed
in six-well culture dishes at a density of 5 × 105
cells/well for HEK 293 and 2.5 × 105 cells/well for
HUVEC. The cells were then rinsed briefly in PBS and fixed in 3.7%
paraformaldehyde for 10 min. The fixed cells were permeabilized in PBS
containing 0.2% Triton X-100 for 10 min and blocked in PBS containing
0.2% bovine serum albumin for 10 min. After 1 h of incubation
with specific antibody, the cells were washed and incubated with Texas
Red® dye-conjugated donkey or swine anti-rabbit lgG,
and/or FITC-conjugated donkey or goat anti-mouse lgG (1:100 dilution)
for 1 h. The coverslips were mounted onto glass slides, and
immunofluorescence was observed with a Zeiss LSM-410 laser scanning
microscope at 568 and 488 nm excitation, respectively.
NF- TRAF2 Associates with Caveolin-1 in HUVEC and HEK 293 Cells--
Our first approach to study the role of caveolin-1 in the
TNF signaling pathway was to examine HUVEC cell lysates for an
association of caveolin-1 with various signaling molecules by
immunoprecipitation followed by immunoblotting. We did not observe an
association of caveolin-1 with TNFR1, TRADD, or TNFR2 (data not shown).
However, as shown in Fig. 1A,
caveolin-1 protein was found in immunoprecipitates of TRAF2 by
immunoblotting, but not in mock immunoprecipitates using control serum.
(A much smaller amount of caveolin-1 was seen at long exposures in the
absence of specific antibody, consistent with a weak nonspecific
association.) Furthermore, immunoprecipitates of caveolin-1 contained
TRAF2 as detected by immunoblotting (Fig. 1B). Treatment of
the cells with TNF prior to lysis did not affect the TRAF2-caveolin
association, but did cause the TRAF2-caveolin-1 complex to transiently
associate with TRADD (maximal 5 min). Interestingly, this time course
is consistent with previous studies of HUVEC which demonstrated
formation of a transient TNF-induced complex at the plasma membrane
containing TNFR1, TRADD, and TRAF2 (26, 27).
We next turned to confocal immunofluorescence microscopy to confirm
these biochemical findings. Both TRAF2 and caveolin-1 are diffusely
present throughout the cytoplasm of HUVEC (Fig. 2), limiting any conclusions that may be
drawn regarding association. However, in many (but not all) cells,
caveolin-1 is focally enriched in regions located subjacent to the
plasma membrane (Fig. 2B). Such regions are more common when
the cultures reach confluence. Interestingly, TRAF2 is also enriched
within these same regions (Fig. 2, A and C),
supporting our immunoprecipitation results of an associate between
TRAF2 and caveolin-1. We did not observe any redistribution of TRAF2 or
caveolin-1 induced by TNF treatment (data not shown).
To further explore the interaction of TRAF2 and caveolin-1, we turned
to HEK 293 cells, which contain a little endogenous caveolin-1 compared
with the level found in HUVEC (Fig. 1C). In these
experiments, HEK 293 cells were cotransfected with Flag-tagged TRAF2
and caveolin-1, and the cell lysates were immunoprecipitated with
antibody to Flag and then immunoblotted. As shown in Fig. 1D, exogenous caveolin-1 was readily detected in the
immunocomplex precipitated from the cell lysates expressing both
Flag-tagged TRAF2 and caveolin-1. Only a small amount of caveolin-1 was
found in the anti-Flag immunocomplex prepared from cells transfected with caveolin-1 only, again attributable to the intrinsic
stickiness of this protein. These results strongly support the findings
first made in HUVEC that TRAF2 associates with caveolin-1 prior to
ligand signaling.
We again employed fluorescence confocal microscopy to confirm the
results observed by immunoprecipitation. HEK 293 cells were transfected
with GFP-TRAF2 alone or, were cotransfected with caveolin-1 and
GFP-TRAF2. As shown in Fig. 3, GFP-TRAF2
alone exhibited diffuse fluorescence throughout the cytoplasm (Fig.
3A) (28). Caveolin-1 is not detectable in HEK 293 cells by
immunofluorescence in the absence of transfection with this protein.
Transfection of caveolin-1 alone showed diffuse cytosolic staining
(data not shown). Cotransfection of caveolin-1 and GFP-TRAF2 induced a
redistribution of TRAF2 into a punctuate pattern (Fig. 3B).
To explore the specificity of this interaction, we transfected HEK 293 cells with TRAF1 instead of TRAF2. Transfection with GFP-TRAF1 alone,
like GFP-TRAF2, resulted in diffused cytosolic fluorescence (Fig.
3C). However, in this case, caveolin-1 did not cause a
redistribution of GFP-TRAF1 (Fig. 3D).
To further explore the effects of TNF receptor signaling, we examined
HUVEC transfected with GFP-TRAF2 and TNF receptors. In single
transfected cells, GFP-TRAF2 was evenly distributed throughout
cytoplasm. Once again, endogenous caveolin-1 was also observed to be
diffusely expressed, except where it sometimes coalesced along regions
of the plasma membrane to form a characteristic "patch" pattern
(Fig. 3A). In contrast to our findings with endogenous TRAF2, the GFP-TRAF2 signal was too bright to observe enrichment of the
patch regions. We also treated the single transfected cells with TNF,
but this treatment did not cause any obvious redistribution of
GFP-TRAF2 or of caveolin-1 (data not shown), similar to our findings
with endogenous TRAF2. We then proceeded to overexpress TNF receptors
in the same cells. We have reported previously that transfected TNFR1
does not escape from the Golgi apparatus and desensitizes cell
signaling (22). In contrast, TNFR2 is readily expressed at the plasma
membrane and does not desensitize signaling. Therefore, we
overexpressed TNFR2 receptor in the GFP-TRAF2-expressing HUVEC. This
maneuver causes ligand-independent receptor clustering and stable
recruitment of TRAF2 to the clustered receptors (Fig. 4). Specifically, in cells transfected
only with TNFR2, the receptor localized to the perinuclear region
(probably Golgi and endoplasmic reticulum) as well as the plasma
membrane (Fig. 4B), and isolated receptor did not show
significant overlap with caveolin-1 expression. In HUVEC cells
transfected with GFP-TRAF2 and Flag-TNFR2, GFP-TRAF2 fluorescence was
both redistributed to plasma membrane and clustered into a punctate
pattern in the cytoplasm that was largely coincident with that of
transfected TNFR2 stained with antibody to Flag (Fig. 4C).
Interestingly, when the cells cotransfected both with GFP-TRAF2 and
TNFR2 were stained with antibody to caveolin-1, the subcellular localization of GFP-TRAF2 was now clearly colocalized with that of
caveolin-1 (Fig. 4D), indicating that both TRAF2 and
caveolin-1 were corecruited to the TNFR2 receptor. In other words, the
redistribution of TRAF2 induced by overexpressed TNFR2 allowed
a clear demonstration of corecruitment of caveolin-1 to the activated
TNF receptor in HUVEC.
The association of TNFR2, TRAF2, and caveolin-1 observed in HUVEC cells
was also detected in HEK 293 cells by immunofluorescence (data not
shown) and further analyzed by immunoprecipitation (Fig. 5). In these experiments, Flag-tagged
TNFR2 and caveolin-1 were coexpressed with or without GFP-TRAF2. In the
presence of TRAF2, TNFR2 was found to significantly associate with
caveolin-1 (Fig. 5, lane 4), whereas, in the
absence of TRAF2, much less interaction of TNFR2 with caveolin-1 was
detected (Fig. 5, lane 3). These findings
strongly suggest that the interaction of TNFR2 with caveolin-1 is
mediated via TRAF2. The role of TRAF2 in linking caveolin-1 to TNFR2
was further supported by a mutational analysis of the TRAF2 binding
region on the C-terminal region of TNFR2. To do this, we made a series
of Flag-tagged TNFR2 proteins in which deletions were constructed by
removing different numbers of amino acid residues from the C-terminal
end of the receptor (Fig. 6A). These C-terminal deletions had no significant effect on intracellular distribution of the receptors, all of which localized both to plasma
membrane and to cytoplasm concentrated in the perinuclear area (data
not shown). The capacity of TNFR2 mutants to interact with TRAF2 was
then examined by cotransfection of GFP-TRAF2 with individual wild type
or mutant TNFR2 molecules (Fig. 6B). Like wild type TNFR2,
coexpression of TNFR2( Effect of Caveolin-1 on TRAF2 Expression Levels and
Signaling--
Since caveolin-1 interacts with TRAF2 and TRAF2 plays
critical roles in TNF signaling via NF- In this report, we describe an association of the
membrane-organizing coat protein, caveolin-1, with the signal
transducing adapter protein TRAF2. We demonstrate the association
between endogenous TRAF2 and caveolin-1 in HUVEC and extend these
findings using transfected proteins in HUVEC and HEK 293 cells. These
proteins are associated in unstimulated HUVEC or in cotrasfected HEK
293. Upon receptor activation, either by TNF induction or by receptor overexpression, caveolin-1 is recruited to the receptor signaling complex through its interaction with TRAF2.
TRAF2 belongs to a family of adapter proteins including TRAF1, -2, -3, -4, -5, and -6 (29, 30). These proteins link clustering of receptors
belonging to the TNF receptor family and to the Toll/IL-1 receptor
family to downstream signaling events such as activation of the
transcription factors NF- Our findings, which establish a biochemical protein-protein
association, raise a number of questions about the functional implication of caveolin-1 binding to TRAF2. TRAF2 signaling may be
terminated, in some systems, by TRAF2 degradation (38, 39). In
transfected HEK 293 cells, the presence of caveolin-1 increases TRAF2
expression levels. This effect could be due to TRAF2 protein stabilization by prevention of degradation. If true, then caveolin-1 binding to TRAF2 could prolong TNF signaling. We did observe a significant enhancement of TNF signaling in the presence of caveolin-1, but the effect was decreased at the higher levels of caveolin-1 expression and was not seen at supra-optimal TNF concentrations. In any
event, caveolin-1 is not required for TRAF2 signaling since TRAF2
overexpression can activate a NF- A more likely role for the association of caveolin-1 and TRAF2 is the
targeting of activated TNF receptor complexes to caveolin-enriched membrane compartments such as caveoli. Many other receptor systems appear to localize to caveoli, which are defined by their cholesterol- and sphingomyelin- enriched lipid environments as well as by their morphological features (18). This microenvironment may alter the
efficiency or receptor signaling and may be uniquely suited to allow
cross-regulation by different types of hormone and cytokine receptors.
If caveolin functions to recruit activated TNF receptors to a specific
signaling environment, then excess free caveolin may inhibit TNF
receptor recruitment to caveoli or related structures. This may explain
why high levels of transfected caveolin-1 decreases ligand-induced
responses but not signals caused by overexpression of TRAF2. It may
also explain why TNF addition fails to cause a redistribution of TRAF2
molecules within the cell, i.e. it is the receptor rather
than the TRAF molecule that is redistributed by ligand binding. To
date, there are no reports that TNF-induced transcription requires the
microenvironment of the caveoli, but TNF induced apoptosis in U937
cells have been reported to be initiated in caveoli-like regions (40).
As we have noted above, TRAF2 has not been implicated in TNF-induced
apoptosis (9) and may in fact inhibit through the induced expression of
antiapoptotic proteins and through the recruitment of inhibitor of
apoptosis proteins to the receptor complex (13, 15, 41). Activated TNF
receptors are rapidly removed from the membrane by endocytosis, but
this is thought to involve clathrin-coated pits and vesicles rather
than caveoli (21, 42). Further studies will be needed to determine
whether caveolin-1 alters this pattern of receptor transport.
In summary, we have made the unexpected finding that the TNF signaling
adapter protein TRAF2 exists in a complex with caveolin-1, allowing
caveolin-1 to be recruited into activated receptor signaling complexes.
The functional significance of this finding is unclear, and may relate
to TRAF2 stabilization and/or to targeting of activated receptors
within the cells. Indeed, our results may suggest that from a cell
biology perspective, it is more accurate to describe the recruitment of
ligand-receptor complexes to TRAF2-caveollin-1 binding sites than it is
to describe the recruitment of TRAF2 to the occupied receptor.
B promoter-reporter gene, although the caveolin-enhanced response to TNF is attenuated at higher caveolin levels. These findings suggest that intracellular distribution of
activated TNF receptors may be regulated by caveolin-1 via its
interaction with TRAF2.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and AP-1 (7, 9, 13, 14). Clustered TNFR2 directly binds TRAF2, initiating the same signaling pathways; TRAF2
recruits TRAF1 to the TNFR2 complex, and it is not known if RIP is
involved in signaling through this receptor (10, 15, 16).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
was purchased from R&D
Systems Inc. (Minneapolis, MN). Vent® DNA polymerase, T4
DNA ligase, and all restriction endonucleases were purchased from New
England Biolabs (Beverly, MA). Monoclonal antibody against green
fluorescence protein (GFP) was purchased from
CLONTECH (San Francisco, CA); monoclonal antibody
against human TRAF2 and polyclonal antibody against caveolin-1 were
purchased from BD Biosciences-PharMingen (San Diego, CA) and BD
Biosciences-Transduction Laboratories (Lexington, KY). Protein G
Plus/Protein A-agarose and Nonidet P-40 were purchased from Calbiochem
(La Jolla, CA). Mouse control serum IgG (K16/16) for
immunoprecipitation was a gift from Dr. D. Mendrick (Brigham and
Women's Hospital, Boston, MA), and rabbit control serum IgG was
purchased from Santa Cruz Biotechnology (Santa Cruz, CA).
Peroxidase-conjugated donkey anti-mouse lgG and anti-rabbit lgG, Texas
Red® dye-conjugated donkey anti-rabbit lgG, and
fluorescein isothiocyanate (FITC)-conjugated donkey anti-mouse lgG were
purchased from Jackson ImmunoResearch Laboratories (West Grove, PA).
Texas Red® dye-conjugated swine anti-rabbit IgG and
FITC-conjugated goat anti-mouse IgG were purchased from DAKO
(Carpinteria, CA). All other reagents are from Bio-Rad or Sigma.
16), TNFR2(
37), and
TNFR2(
59), which are C-terminal deletion constructs of wild type
TNFR2, nucleotides encoding 16, 37, and 59 amino acids, respectively,
of the C-terminal end of TNFR2 were removed from the cDNA encoding
the full-length receptor (in plasmid pRCX-3 (Ref. 22)) and a new stop
codon was introduced by PCR. Each of the TNFR2s with C-terminal
truncations was then directly inserted back into the same expression
vector as the full-length cDNA. A mammalian expression construct of
dog caveolin-1 (pCB7-caveolin-1) was a gift from Dr. W. C. Sessa
(Yale University, New Haven, CT). The pBIIXLuc plasmid which contains
two NF-
B sites from the Ig-
enhancer fused with a firefly
luciferase encoding cDNA, and the plasmid p
-actin-Rluc, which
contains the
-actin promoter fused to a Renilla
luciferase encoding cDNA, were both gifts from Dr. S. Ghosh (Yale
University). The sequences of all constructs were confirmed by DNA sequencing.
B Promoter Reporter Assay--
Cell lysates of reporter
gene-transfected cells were prepared and assayed using Promega
Dual-Luciferase® reporter assay system (Promega, Madison,
WI) according to manufacturer's instruction. Luciferase activity was
measured in triplicate using a Berthold (Schwarzwald, Germany) model
LB9501 luminometer according to the manufacturer's instructions.
Activity of the NF-
B promoter-firefly luciferase reporter was
normalized to the activity of the cytokine-unresponsive
-actin
promoter Renilla luciferase reporter.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Identification of an association of TRAF2
with caveolin-1 by immunoprecipitation. A, immunoblot
of TRAF2 immunoprecipitates from 200 µg of proteins from HUVEC cell
lysates using 5 µg of anti-TRAF2 antibody (right) or
control mouse IgG (left) probed with antibody to caveolin-1.
B, immunoblots of caveolin-1 immunoprecipitates using 1 mg
of proteins prepared from TNF-induced HUVEC cells were probed with
anti-TRAF2 (top panel) and anti-TRADD (bottom
panel). HUVEC cells were pretreated with 10 ng/ml TNF for 0, 5, 10, and 30 min. C, immunoblot from HEK 293 cell lysates
(left lane) and HUVEC lysates (right lane) probed
with antibody to caveolin-1. D, immunoblots of
immunoprecipitates with 1 µg of antibody to Flag (M2) from HEK 293 lysates transfected with 5 µg of pCB7-caveolin-1 with (right
lane) or without (left lane) 5 µg of pRK-TRAF2-Flag
probed with antibody to Flag for TRAF2 (top panel) and
caveolin-1 (bottom panel). All experiments were performed at
least three times with similar results. IP,
immunoprecipitation; WB, western blot.
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Fig. 2.
Identification of an association of TRAF2
with caveolin-1 by fluorescence confocal microscopy in untreated
HUVEC. Representative thin section confocal fluorescence
micrographs showing localization of TRAF2 in HUVEC by indirect
immunofluorescence in fixed and permeable HUVEC using monoclonal
antibody to human TRAF2 and FITC-conjugated goat anti-mouse IgG
(left panel), localization of caveolin-1 using rabbit
polyclonal anti-caveolin-1 and Texas Red® dye-conjugated
swine anti-rabbit IgG (middle panel) and the merged image
(right panel). The micrographs shown are representative of
over 75% of the cells in those fields where coalescence of caveolin-1
staining was observed in four separate experiments.
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Fig. 3.
Subcellular localization of GFP-tagged TRAF1
and TRAF2 in HEK 293 cells in the presence or absence of overexpressed
caveolin-1. Representative thin section confocal fluorescence
micrographs of HEK 293 cells transiently transfected with 5 µg of
pBK-CMV-GFP-TRAF2 (A and B) or pBK-CMV-GFP-TRAF1
(C and D), alone (A and C)
or together with 5 µg of pBC-caveolin-1 (B and
D). GFP fluorescent (green) signal was visualized
directly, and caveolin-1 was detected by indirect immunofluorescence
(red) under a confocal microscope. Observations were made on
5-10 cells in each of three different experiments. The micrographs are
representative of more than 75% of the cells observed.
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Fig. 4.
Subcellular distribution of GFP-tagged TRAF2
and caveolin-1 proteins in HUVEC cells in the presence and absence of
overexpressed TNFR2. Representative thin section confocal
fluorescence micrographs of HUVEC cells transiently transfected with 5 µg of pBK-CMV-GFP-TRAF2 (A), pRCX-Flag-TNFR2
(B) or both (C and D). GFP fluorescent
signal (green) was visualized directly and TNFR2 and
endogenous caveolin-1 were visualized by indirect immunofluorescence
(red) staining using antibody to Flag tag for TNFR2 or
antibody to caveolin-1. Experiments were performed on 5-10 cells in
three independent experiments. The micrographs are representative of
more than 75% of the cells observed.
16) with TRAF2 caused a redistribution of
TRAF2, consistent with the fact that TNFR2(
16) retains the TRAF2
binding region (Fig. 6, B and C). In contrast, TRAF2 remained evenly distributed in the cells in which TNFR2(
37) or
TNFR2(
59) was expressed, indicating that loss of the ability of
TNFR2(
37) and TNFR2(
59) to associate with TRAF2 (Fig. 6, D and E). Analysis of the same transfectants by
indirect immunofluorescence showed that caveolin-1 was only
redistributed by TNFR2 molecules (wild type and TNFR2(
16)), which are
capable of interacting with TRAF2 (Fig.
7, A and B). In
contrast, the distribution of caveolin-1 in cells transfected with
TNFR2(
37) or TNFR2(
59) did not show any receptor association (Fig.
7, C and D). Similar results were also found in
HEK 293 cells transiently transfected with GFP-TRAF2, caveolin-1, and
different Flag-tagged TNFR2 and its deletion mutants (data not shown).
Collectively, these data demonstrate that TRAF2 binding to activated
TNFRs results in corecruitment of caveolin-1 into the receptor
complex.
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Fig. 5.
Identification of an association of TNFR2
with caveolin-1 in the presence of TRAF2 in HEK 293. Immunoblots
of immunoprecipitates of TNFR2 using anti-Flag antibody from HEK 293 cells transfected with 4 µg of pCB7-caveolin-1 alone (lane
1), or with 3 µg of pBK-CMV-GFP-TRAF2 (lane 2), or
with 3 µg of pRCX-Flag-TNFR2(lane 3) or with 3 µg of
pBK-CMV-GFP-TRAF2 and pRCX-Flag-TNFR2 together (lane 4). The
blots were sequentially probed with antibody to Flag (for TNFR2),
caveolin-1, and GFP (for TRAF2). Three individual experiments
were performed with similar results. IP,
immunoprecipitation; WB, western blot.
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Fig. 6.
Subcellular localization of GFP-tagged TRAF2
in HUVEC transfected with wild type TNFR2 and its deletion
mutants. A, schematic of a set of deletions of TNFR2
generated by removing various lengths of C-terminal amino acid residues
as indicated by the negative number. Also indicated is the
filled rectangular area responsible
for TRAF binding. B-E, representative thin section confocal
fluorescence micrographs of HUVEC cells transiently transfected with 6 µg of pBK-CMV-GFP-TRAF2 together with 6 µg of pRCX-Flag-TNFR2
(A), pRCX-Flag-TNFR2( 16) (B),
pRCX-Flag-TNFR2(
37) (C), and pRCX-Flag-TNFR2(
59)
(D) respectively. GFP fluorescent signal was visualized
directly, and TNFR2 was detected by indirect immunofluorescence
(red) staining using anti-Flag primary antibody under a
confocal microscope. The experiments were performed on 5-10 cells in
three independent experiments. The micrographs are representative of
more than 75% of the cells observed.
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Fig. 7.
Subcellular localization of GFP-TRAF2 and
caveolin-1 in HUVEC transfected with wild type TNFR2 or its deletion
mutants. Representative thin section confocal micrographs of HUVEC
cells transiently transfected with 6 µg of pBK-CMV-GFP-TRAF2 together
with 6 µg of pRCX-Flag-TNFR2 (A), pRCX-Flag-TNFR2( 16)
(B), pRCX-Flag-TNFR2(
37) (C), and
pRCX-Flag-TNFR2(
59) (D), respectively. GFP fluorescent
signal (green) was visualized directly, and caveolin-1 was
detected by indirect immunofluorescence (red) staining using
anti-caveolin-1 primary antibody under a confocal microscope. The
experiments were performed on 5-10 cells in three independent
experiments. The micrographs are representative of more than 75% of
the cells observed.
B pathway (7, 15), a possible functional linkage between interaction of TRAF2 and caveolin-1 and
TRAF2-mediated NF-
B activation was investigated. First, protein levels of GFP-TRAF2 in HEK 293 cells cotransfected with a constant amount of GFP-TRAF2 cDNA and varying amounts of caveolin-1 cDNA were examined by immunoblotting (Fig. 8).
Under the condition of equal protein loading, the amount of TRAF2
protein was significantly increased by coexpression of caveolin-1 in a
dose-dependent manner, even though the amount of cDNA
encoding TRAF2 was held constant. In a final series of experiments, we
examined the influence of caveolin-1 expression on TNF induced NF-
B
activation in HEK 293 using a NF-
B promoter luciferase-based
reporter system. At a suboptimal concentration of TNF (1 ng/ml), the
effect of caveolin-1 on activation of NF-
B was biphasic, being
initially augmented by increasing expression of caveolin-1 and then
decreased at the highest level of transfected caveolin cDNA. The
peak of this enhancement was about 2-3-fold over the cells not
expressing exogenous caveolin-1 (Fig.
9A). At supra-optimal TNF
concentrations (100 ng/ml), the effect of caveolin-1 was no longer
observed (data not shown). However, the enhancement of TNF-induced
NF-
B activity was mimicked by ligand-independent increases of
NF-
B activity triggered by overexpression of TRAF2, but not
with an inactive form of TRAF2 (TRAF2DN) (Fig. 9B). In this
case, increasing caveolin-1 simply increased TRAF2 signaling without
showing a diminution at higher levels. Thus the interaction of
caveolin-1 with TRAF2 can potentiate TNF signaling mediated by TRAF2.
It seems likely that this effect is explained by the increased level of
TRAF2 protein observed in the presence of caveolin-1.
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Fig. 8.
Effect of caveolin-1 on the expression of
TRAF2 protein in HEK 293. Shown are immunoblots of HEK 293 lysates
transfected with 1 µg of pBK-CMV-GFP-TRAF2 together with varying
amounts (0, 1, 2, and 3 µg, respectively) of pBC-caveolin-1. pBK-CMV
was added to individual transfections to make up to 4 µg of total
transfected DNA in each case. The cells were harvested after 24-h
transfection. 20 µg of cell lysates were loaded on each lane, and the
blots were sequentially probed with antibody to GFP for TRAF2,
caveolin-1. Similar results were obtained in two independent
experiments. WB, western blot.
View larger version (22K):
[in a new window]
Fig. 9.
Effect of expression of caveolin-1 in HEK 293 on NF- B reporter activity. A,
TNF-induced NF-
B reporter activity was measured in HEK 293 cells
transfected transiently with 0.5 µg of pBIIXLuc and 0.1 µg of
p
-actin-Rluc together with increased amount of pBC-caveolin-1 (0, 1, 2, and 3 µg, respectively). Then the transfected cells were divided
into two replicate cultures. One culture was stimulated with 1 ng/ml
TNF, whereas the other culture remained unstimulated. The firefly and
Renilla luciferase activity from the transfected cells were
measured as described under "Experimental Procedures." Shown are
ratios of normalized NF-
B reporter activity with treatment by TNF
versus without treatment by TNF. Similar results were
obtained in two independent experiments. B, TRAF2-mediated
receptor-independent NF-
B activity was measured in HEK 293 cells
were transiently transfected with 0.5 µg of pBIIXLuc, 0.1 µg of
p
-actin-Rluc, and 1 µg of pBK-CMV, or pBK-CMV-GFP-TRAF2 or
pBK-CMV-GFP-TRAFDN together with increased amount of pBC-caveolin-1 (0, 1.5, 3.0, and 4.5 µg, respectively). The cells were harvested after
24-h transfection, and firefly and Renilla luciferase
activity were measured as described under "Experimental
Procedures." Shown are the representative results obtained in three
independent experiments.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
B and AP-1. The sequences and in some
cases, the partial three-dimensional structure of TRAF proteins have
been solved (10, 31-36). Caveolin-1 has been observed to bind to
various target proteins through consensus binding sequences ØXØXXXXØ and
ØXXXXØXXØ, where Ø is aromatic amino acid
Trp, Phe, or Tyr (37). The primary sequence of TRAF2 obtains a possible caveolin binding motif within its conserved TRAF domain
(Phe354-Ile-Trp-Lys-Ile-Ser-Asp-Phe361).
A similar sequence is present in TRAF6, but not in TRAF1. This may
explain why TRAF1, which we used as a specificity control for
caveolin-1 association, did not interact with caveolin-1 in cotransfected HEK 293 cells. Substitution of this putative caveolin binding site of TRAF2 with the nonfunctional sequence from TRAF1 appears to reduce but not eliminate the association with
caveolin-1.2 Therefore,
although this sequence may contribute to caveolin-1 association, it
does not appear to fully account for it. Furthermore, the binding site
is located within a region that is near the contact region of TRAF2
with the receptor (35), and it is not clear whether caveolin-1 binding
to this site and receptor binding are simultaneously possible.
B reporter gene in the absence of
significant caveolin expression (e.g. in transfected HEK 293 cells). We do not know if caveolin-1 plays a role in TNF-mediated apoptosis since HEK 293 cells cannot be killed by TNF whether or not
caveolin-1 is present.3 An
effect on apoptosis seems unlikely in any event since TRAF2 is not
involved in the TNF-activated death pathway (9). Overall, our data
suggest that caveolin-1 may modulate TRAF2-mediated signaling, but it
is not essential for TNF responses.
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ACKNOWLEDGEMENTS |
---|
We thank Drs. V. Dixit, W. C. Sessa, and S. Ghosh for provision of cDNA constructs. We also thank Louise Camera Benson and Gwendolyn Davis for excellent assistance in cell culture.
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FOOTNOTES |
---|
* This work was supported in part by grants from the National Institutes of Health (to J. S. P.) and from the National Kidney Research Fund (to J. R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Present address: Molecular Staging Inc., New Haven, CT 06511.
¶ Supported by a fellowship from the Pediatric Scientist Development Program.
** Supported by the National Kidney Research Fund.
To whom correspondence should be addressed. Tel.:
203-737-2292; Fax: 203-737-2293; E-mail: jordan.pober@yale.edu.
Published, JBC Papers in Press, December 8, 2000, DOI 10.1074/jbc.M007116200
2 X. Feng and J. S. Pober, unpublished observations.
3 L. A. Madge and J. S. Pober, unpublished observations.
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ABBREVIATIONS |
---|
The abbreviations used are:
TNF, tumor necrosis
factor;
TNFR1 and TNFR2, tumor necrosis factor receptors 1 and 2, respectively;
TRADD, tumor necrosis factor receptor 1-associated death
domain protein;
TRAF, tumor necrosis factor receptor-associated factor;
RIP, receptor-interacting protein;
PCR, polymerase chain reaction;
GFP, green fluorescent protein;
EGFP, enhanced green fluorescent protein;
NF-B, nuclear factor
B;
AP-1, activator protein-1;
HUVEC, human
umbilical vein endothelial cell;
HEK 293, human embryonic kidney cell;
CMV, cytomegalovirus;
FITC, fluorescein isothiocyanate;
PBS, phosphate-buffered saline.
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