From the Department of Molecular Pharmacology and The
Albert Einstein Cancer Center and the ¶ Departments of Medicine
and Molecular Genetics, Albert Einstein College of Medicine,
Bronx, New York 10461
Received for publication, September 12, 2000, and in revised form, November 30, 2000
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ABSTRACT |
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Transforming growth factor- The transforming growth factor
(TGF)1 As in numerous other cellular processes, TGF- However, surprisingly little is known about the interactions of
the TGF- Caveolae are ~50-100 nm vesicular invaginations of the plasma
membrane and are thought to form as a result of a local accumulation of
cholesterol, glycosphingolipids, and caveolin-1 (24-26). Caveolin-1, a
21-24-kDa integral membrane protein, is a principal component of
caveolae membranes in vivo (27-29). Structurally, a
hallmark of caveolin-1 and by extension, caveolae, is a
characteristically punctate staining pattern at the plasma membrane
(30). Although caveolae function in vesicular and cholesterol
trafficking (31, 32), they have also been implicated in signal
transduction (33, 34). Biochemical and morphological experiments have
shown that a variety of signaling molecules are concentrated within
these plasma membrane microdomains, such as Src family tyrosine
kinases, Ha-Ras, endothelial nitric oxide synthase, and
heterotrimeric G proteins (35-40). Furthermore, several independent
lines of evidence suggest that caveolin-1 plays a regulatory role in
signaling, i.e. by functioning as a direct inhibitor of a
variety of plasma-membrane initiated signaling cascades (reviewed in
Ref. 41).
Given the punctate plasmalemmal distribution of Cav-1 and its emerging
role in various signaling cascades, we set out to investigate its
relationship with the TGF- Materials and Cell Culture--
The caveolin-1 mAb 2297 (used
for immunoblotting) and mAb 2234 (used for immunofluorescence and
immunoprecipitation) (30) were the gifts of Roberto Campos-Gonzalez
(Transduction Laboratories, Inc.). The phospho-specific anti-SMAD2
rabbit pAb was a gift of Peter ten Dijke (Ludwig Institute for Cancer
Research). The anti-FLAG tag mAb (Sigma), the anti-SMAD2 mAb
(Transduction Laboratories, Inc.), and the anti-HA as well as the
anti-TGF- cDNA Expression Vectors and Transfections--
The cDNAs
encoding caveolin-1 (full-length and deletion mutants) were
C-terminally Myc-tagged and subcloned into the pCB7 mammalian
expression vector, as described previously (42-45). The cDNAs
encoding the HA-tagged wild-type, constitutively active (T204D), and
kinase-dead (K232R) TGF- Immunoblotting--
48 h post-transfection, cells were washed
with PBS and incubated with lysis buffer (10 mM Tris, pH
7.5, 50 mM NaCl, 1% Triton X-100, 60 mM octyl
glucoside) containing protease inhibitors (Roche Molecular
Biochemicals). Where indicated, protein concentrations were quantified
using the BCA reagent (Pierce). Samples were separated by SDS-PAGE
(12.5% acrylamide) and transferred to nitrocellulose. The
nitrocellulose membranes were stained with Ponceau S (to visualize protein bands) followed by immunoblot analysis. All subsequent wash
buffers contained 10 mM Tris, pH 8.0, 150 mM
NaCl, 0.05% Tween-20, which was supplemented with 1% bovine serum
albumin and 2% nonfat dry milk (Carnation) for the blocking solution
and 1% bovine serum albumin for the antibody diluent. Primary
antibodies (either polyclonal or monoclonal) were used at a 1:500
dilution. Horseradish peroxidase-conjugated secondary antibodies
(1:5000 dilution, Transduction Laboratory) were used to visualize bound primary antibodies with a chemiluminescent substrate (Pierce).
Immunofluorescence--
The procedure was performed as we
previously described (35). For colocalization studies, NIH-3T3 cells
(transfected with caveolin-1 and either T Purification of Caveolae-enriched Membrane
Fractions--
Caveolae-enriched membrane fractions were purified
essentially as previously described (44, 50). 293T cells plated on a
150-mm diameter plate were transfected with the appropriate plasmid(s).
36 h post-transfection, the cells were washed twice in cold PBS,
scraped into 2 ml of MBS (25 mM Mes, pH 6.5, 150 mM NaCl) containing 1% Triton X-100, passed 10 times
through a loose fitting Dounce homogenizer, and mixed with an equal
volume of 80% sucrose (prepared in MBS lacking Triton X-100). The
sample was then transferred to a 12-ml ultracentrifuge tube and
overlaid with a discontinuous sucrose gradient (4 ml of 30% sucrose, 4 ml of 5% sucrose, both prepared in MBS, lacking detergent). The samples were subjected to centrifugation at 200,000 × g (39,000 rpm in a Sorval rotor TH-641) for 16 h. A
light scattering band was observed at the 5/30% sucrose interface.
Twelve 1-ml fractions were collected, and 50-µl aliquots of each
fraction were subjected to SDS-PAGE and immunoblotting.
Coimmunoprecipitation of Caveolin-1 with TGF- Coimmunoprecipitation of SMAD2/SMAD4 Complexes--
Sets of
100-mm NIH-3T3 plates were transfected with the appropriate plasmids.
36 h post-transfection, cells were lysed in RIPA/Nonidet P-40
lysis buffer (150 mM NaCl, 50 mM Tris, pH 8.0, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, 50 mM NaF,
30 mM sodium pyrophosphate, 100 µM sodium
orthovanadate, 0.1 µg/ml okadiac acid, and protease inhibitors),
sonicated briefly to disrupt nuclei, and subjected to
immunoprecipitation with anti-FLAG M2 mAb. Immunoblot analysis with
anti-HA pAb visualized bound SMAD4 in the SMAD2/SMAD4 complex.
In Vivo Phosphorylation Experiments--
NIH-3T3 cells plated on
100-mm dishes were transfected with the appropriate plasmids. 36 h
post-transfection, cells were washed twice in Dulbecco's modified
Eagle's medium lacking phosphate and incubated for 3 h in
Dulbecco's modified Eagle's medium lacking phosphate supplemented
with 1 mCi [32P]orthophosphate/100-mm dish. Indicated
plates were then additionally treated with TGF- Intra-caveolar SMAD2 Phosphorylation--
293T cells plated on a
150-mm diameter plates were transfected with T Cytoplasmic/Nuclear Fractionation--
NIH-3T3 cells plated on
100-mm dishes were transfected with the appropriate plasmids. 36 h
post-transfection, cells were washed once with PBS, scraped in 1 ml of
Hypotonic lysis buffer (10 mM Hepes, pH 7.9, 10 mM KCl, 0.1 mM EDTA, 0.1 mM EGTA,
0.5% Nonidet P-40, 1 mM dithiothreitol, and protease
inhibitors), passed 10 times through a loose fitting Dounce
homogenizer, and centrifuged at 3000 rpm for 3 min. The supernatant
(i.e. cytoplasmic fraction) was saved, whereas the pellet
(nuclear fraction) was washed twice with hypotonic lysis buffer before
resuspending in the same buffer and sonicating to disrupt nuclear
membranes. Protein concentrations from both fractions were quantified
using the BCA reagent, and equal amounts of protein were subjected to
immunoblot analysis with the anti-FLAG mAb as the probe.
Luciferase Reporter Assays for TGF- TGF- TGF-
Biochemically, we have previously shown that caveolar
microdomains can be separated from other cellular constituents using a
sucrose gradient ultracentrifugation procedure. Via this method, it is
possible to concentrate Cav-1, the caveolae marker protein, by over
2000-fold with respect to total cellular protein (45, 60). By
transfecting 293T cells with appropriate cDNAs, we applied this
separation scheme to various members of the TGF-
Because 293T cells do not endogenously express Cav-1, it should be
noted that the fractionation of T
As Cav-1 is a marker protein for caveolae and has been shown to
interact with certain signaling molecules in vivo (reviewed in Ref. 41), we next investigated its potential interaction with
T
Since these experiments were performed in a heterologous setting, we
immunoprecipitated T Caveolin-1 Inhibits TGF-
The receptor-activated SMADs (Smad-2 and -3) are the first step in the
propagation of TGF-
The role of Cav-1 in SMAD phosphorylation was also investigated in a
novel manner. Based on our sucrose density gradient experimentation above, we observed that T
We next investigated the effects of Cav-1 on Smad-2/-4
heteromerization. NIH-3T3 cells were transfected with FLAG-tagged
Smad-2, HA-tagged Smad-4, and a combination of T
Finally, we tested the ability of Cav-1 to suppress TGF- The Caveolin-1 Scaffolding Domain Mediates the Functional
Interaction with TGF-
We next investigated the relevance of the Cav-1 scaffolding domain to
TGF-
If the scaffolding domain is indeed the region of caveolin that binds
to T
To demonstrate that the scaffolding domain is also functionally
important in vivo, we utilized the previously tested
A3-lux/Fast-1 luciferase reporter system. NIH-3T3 cells were
cotransfected with the A3-lux/Fast-1 reporter and a combination of
T The Caveolin-1/TGF-
We also determined the phosphorylation state of Smad-2 and observed an
increase in phosphorylation with expected kinetics (i.e.
maximal at ~20-30 min and plateauing thereafter; Fig. 10, lower panel). Note that the peak of Cav-1/T
To test this hypothesis, we studied the kinetics of TGF- By using several independent and complementary approaches, we have
examined the role of Cav-1 in TGF- To date, a variety of physiological regulators of TGF- The regulation of signaling mediated by Cav-1 and its kinetics of
interaction with T At this time, we cannot rule out the presence of intervening proteins
which mediate the Cav-1/T What physiological roles might the inhibitory interaction of Cav-1 with
T Furthermore, TGF- (TGF-
)
signaling proceeds from the cell membrane to the nucleus through the
cooperation of the type I and II serine/threonine kinase receptors and
their downstream SMAD effectors. Although various regulatory proteins
affecting TGF-
-mediated events have been described, relatively
little is known about receptor interactions at the level of the plasma
membrane. Caveolae are cholesterol-rich membrane microdomains that,
along with their marker protein caveolin-1 (Cav-1), have been
implicated in the compartmentalization and regulation of certain
signaling events. Here, we demonstrate that specific components of the
TGF-
cascade are associated with caveolin-1 in caveolae and that
Cav-1 interacts with the Type I TGF-
receptor. Additionally,
Cav-1 is able to suppress TGF-
-mediated phosphorylation of Smad-2
and subsequent downstream events. We localize the Type I TGF-
receptor interaction to the scaffolding domain of Cav-1 and show that
it occurs in a physiologically relevant time frame, acting to rapidly dampen signaling initiated by the TGF-
receptor complex.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily
consists of a disparate group of polypeptide cytokines that regulate a
plethora of biological processes. Members of this superfamily include
the bone morphogentic proteins, the activins, and the TGF-
s,
each with distinct roles in cellular differentiation, proliferation, apoptosis, and migration/motility, among others (see Refs. 1 and 2 for
reviews). The prototype member of the group, TGF-
1, whose mechanism
of action has been a focus of research for the past decade, classically
initiates signaling at the plasma membrane by binding to a
heterotetrameric complex consisting of two transmembrane serine/threonine kinases, known as Type I and Type II TGF-
receptors (T
R-I and T
R-II) (3, 4). The activation of this membrane complex
occurs via the ligand-dependent phosphorylation of T
R-I by T
R-II (5, 6). In turn, T
R-I can act to phosphorylate its
immediate downstream effectors, Smad-2 and Smad-3, members of the SMAD
family of intracellular signaling molecules (7, 8). This
phosphorylation induces a conformational change in Smad-2 and -3, thereby facilitating their heteromerization with another member of the
family, Smad-4 (9). The SMAD complex then translocates to the nucleus,
where it acts to regulate the transcription of various target genes (7,
9).
-mediated
signaling is subject to regulation and inhibition by a variety
of mechanisms. The p42/44 MAP kinase pathway,
interferon-
/STAT cascade, NF-
B, SnoN/Ski oncoproteins,
among others have been shown to act as negative regulators of TGF-
signaling primarily by interacting with, modifying, or regulating the
SMAD proteins (10-15). Various groups have also observed regulation at
the receptor level by the showing the interaction of certain
intracellular proteins with the T
R-I. Of specific interest are the
inhibitory proteins, Smad-6 and Smad-7, a functionally divergent subset
of the SMAD protein family that can inhibit TGF-
signaling by
directly interacting with the T
R-I (16-18). Other proteins such as
FKBP12 have also been shown to interact with and negatively regulate
T
R-I (19).
receptor complex with other membrane proteins. Although the
compartmentalization of signaling molecules via scaffolding proteins is
an emerging theme in signal transduction biology (20), the behavior and
regulation of the TGF-
receptors at the membrane has thus far been a
matter of speculation. Previous reports addressing the
heterotetramerization of the T
R-I/II complex have shown a characteristically punctate membrane distribution (21, 22), and
recently, the cloning of a SMAD anchor protein, SARA, also revealed a
punctate distribution for both SARA and the TGF-
receptor complex
(23).
receptor complex and substrate SMADs.
Here, we demonstrate that Cav-1 and T
R-I are highly colocalized at
the membrane, that T
R-I, T
R-II, and Smad-2 (but not Smad-4) cofractionate with caveolin-1 in caveolae enriched microdomains, and
that caveolin-1 directly interacts with T
R-I in both heterologous and endogenous settings. We show that this interaction has functional consequences because Cav-1 is able to suppress TGF-
-mediated transcriptional activation. In addition, we demonstrate that Cav-1 diminishes the phosphorylation of Smad-2, disrupts its interaction with
Smad-4, and prevents Smad-2 translocation to the nucleus in the
ligand-activated state. This inhibition is mediated by an interaction
between T
R-I and the scaffolding domain of Cav-1 and occurs in a
physiologically relevant time frame. We show a rapid increase in the
T
R-I/Cav-1 interaction upon ligand binding, and, by using an
antisense strategy, we demonstrate that targeted down-regulation of
caveolin-1 is sufficient to hyperactivate TGF-
signaling.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
type I receptor rabbit pAb (Santa Cruz Biotechnology) were
obtained commercially. Cell culture reagents were from Life
Technologies, Inc. Recombinant human TGF-
1 was obtained from R&D
systems. NIH-3T3 cells were cultured in Dulbecco's modified Eagle's
medium supplemented with 10% donor calf serum, 2 mM
glutamine, 100 units/ml penicillin, and 100 µg/ml streptomycin at
37 °C and 5% CO2. 293T cells were similarly cultured,
with the exception of serum (10% fetal bovine serum in lieu of donor
calf serum).
type I receptors, the HA-tagged wild-type
TGF-
Type II receptor, FLAG-tagged SMAD2, and HA-tagged SMAD4 were
as previously described (5, 9, 18, 46, 47). All transient transfections
were performed using the calcium phosphate precipitation method, as
described previously (48, 49).
R-I w.t. or T
R-I
(T204D)) were fixed for 30 min in PBS containing 2% paraformaldehyde,
rinsed with PBS, and quenched with 50 mM NH4Cl
for 10 min. The cells were then incubated in permeabilization buffer
(PBS, 0.2% bovine serum albumin, 0.1% Triton X-100) for 10 min,
washed with PBS, and double-labeled with a 1:400 dilution of
anti-caveolin-1 mAb 2234 and 1:200 dilution of anti-HA rabbit pAb for
60 min. After rinsing three times with PBS, secondary antibodies (7.5 µg/ml) [(lissamine-rhodamine-conjugated goat anti-rabbit and
fluorescein isothiocyanate-conjugated goat anti-mouse) antibodies]
were added for a period of 60 min. Cells were washed three times with
PBS, and slides were mounted with Slow-Fade anti-fade reagent
(Molecular Probes). A Bio-Rad MR600 confocal fluorescence microscope
was used for visualization of bound secondary antibodies. For
assessment of SMAD2 translocation to the nucleus, a similar procedure
was followed with the following differences: NIH-3T3 cells were
transfected with caveolin-1, serum-starved for 20 h, and treated
with TGF-
1 (4 ng/ml) for 45 min. A 1:400 dilution of anti-caveolin-1
mAb 2234 and a 1:200 dilution of anti-SMAD2 mAb were used for the
caveolin-1 and endogenous SMAD2 staining, respectively.
RI--
For
coimmunoprecipitation of heterologously expressed proteins, 293T cells
plated on 100-mm dishes were transfected with the appropriate plasmids.
36 h post-transfection, cells were lysed in lysis buffer (see
"Immunoblotting"), clarified by centrifugation at 15,000 × g for 15 min, and precleared by incubation with protein A-Sepharose (Amersham Pharmacia Biotech) for 1 h at 4 °C.
Supernatants were transferred to separate 1.5-ml microcentrifuge tubes
containing anti-HA pAb or appropriate control antibodies (beads alone,
preimmune serum) prebound to protein-A Sepharose. After incubation by
rotating overnight at 4 °C, immunoprecipitates were washed three
times with lysis buffer and subjected to immunoblot analysis with the anti-caveolin-1 2297 mAb probe. For coimmunoprecipitation of endogenous proteins, NIH-3T3 cells were plated on 100-mm dishes and lysed at
confluency. The procedure was as described above with the
anti-caveolin-1 2234 mAb, anti-T
R-I pAb, or preimmune serum pAbs
serving as the precipitating antibodies.
1 (4 ng/ml) for 45 min. Cells were washed in ice-cold PBS and subjected to lysis in
RIPA/Nonidet P-40 buffer and immunoprecipitation with anti-FLAG M2 mAb
as outlined above. SDS-PAGE and subsequent autoradiography visualized
the phosphorylated SMAD2 proteins.
R-I (T204D), SMAD2,
and either caveolin-1 or empty vector. Caveolae-enriched membrane
fractions were purified as outlined above. Dilution with 1× MBS and
subsequent centrifugation was used to concentrate the caveolar
membranes into a 50-µl volume. Approximately 5 µg of these
membranes was mixed 1:1 with 10 µl of 2× kinase reaction buffer (40 mM Hepes, pH 7.4, 10 mM MgCl2, and
2 mM MnCl2), and the reaction was initiated by
the addition of 4 mM ATP (Sigma) for 15 min. Extent of
phosphorylation was determined by immunoblot analysis with
anti-phospho-SMAD2 pAb.
Activity--
The A3-lux
reporter construct and the TGF-
transcriptional coactivator Fast-1
were the gifts of Malcolm Whitman (Harvard Medical School) (51) and the
3TP-lux reporter construct was the gift of Joan Massagué
(Memorial Sloan Kettering Cancer Center) (52). Briefly, 150,000 NIH-3T3
cells were seeded per well in 6-well plates 12-24 h prior to
transfection. For assays involving TGF-
1 stimulation, each plate was
transfected with the reporter of interest (A3-lux/Fast-1 or 3TP-lux),
caveolin-1 (pCB7-Cav-1), or empty vector (pCB7) and pRSV-Gal (Promega,
Madison, WI). 12 h post-transfection, the cells were rinsed with
PBS, incubated in 0.2% donor calf serum starvation medium for 20 h and then, where indicated, treated with TGF-
1 (4 ng/ml). 8 h
post-stimulation, the cells were lysed in 200 µl of extraction
buffer, 100 µl of which was used to measure luciferase activity as
described (53). Luciferase activities were normalized for galactosidase
activity assayed by a galactosidase assay system (Promega). For the
analysis of the effects of caveolin-1 and the deletion mutant Cav-1
61-100 on the constitutively active TGF-
type I receptor, all
points were additionally transfected with T
R-I (T204D), and the
cells were serum-starved but not stimulated with TGF-
1
post-transfection. Results were expressed as a ratio of luciferase
activity to galactosidase activity. Each experimental value represents
the average of three separate transfections performed in parallel;
error bars represent the observed S.D. All experiments were performed
at least three times independently and yielded virtually identical results.
RI in Vitro Kinase Assay--
The kinase substrate used
in this assay, the GST-SMAD2 fusion protein, was a gift of Mark de
Caestecker (National Cancer Institute) (54). The purification of
GST-SMAD2 was as we described previously (30, 40, 55). Briefly, after
expression in Escherichia coli (BL21 strain; Novagen, Inc.),
GST-SMAD2 was affinity purified on glutathione-agarose beads, using the
detergent sarcosyl for initial solubilization (56) and washed three
times with TNET buffer (50 mM Tris, pH 8.0, 150 mM NaCl, 5 mM EDTA, 1% Triton X-100). SDS-PAGE
followed by Coomassie staining was used to determine the approximate
molar quantities of the fusion protein/100 µl of packed bead volume.
GST-SMAD2 was then eluted with an appropriate volume of TNET buffer
containing 20 mM reduced glutathione (Sigma). An anti-HA
rabbit pAb was used to immunoprecipitate HA-tagged T
R-I (T204D) from
transiently transfected 293T cells. In vitro kinase assays
were performed as described previously (35), with minor modifications.
Briefly, immunoprecipitates and an appropriate fraction of the
GST-SMAD2 eluate were equilibrated with kinase reaction buffer (20 mM Hepes, pH 7.4, 5 mM MgCl2, and 1 mM MnCl2), and the reaction was initiated by
the addition of 4 mM ATP (Sigma). After 15 min of
incubation at 25 °C, the reaction was terminated by addition of 2×
SDS-PAGE sample buffer and boiling for 4 min. Phosphorylated GST-SMAD2
was detected by immunoblotting with the anti-phospho-SMAD2 rabbit pAb.
In reactions involving the use of caveolin-derived peptides, prior to
initiating the reaction, the immunoprecipitates were preincubated in
kinase reaction buffer with the indicated peptide for 30 min at
4 °C. The caveolin-based peptides were synthesized using standard
methodology and subjected to amino acid analysis and mass spectroscopy
(Massachusetts Institute of Technology Biopolymers Laboratory and
Research Genetics) to confirm their purity and composition, as we
described previously (35, 48, 49, 57, 58). Peptides were dissolved in
Me2SO, and 100× stock solutions were prepared for use in
experiments. In vitro kinase assays assessing the
autophosphorylation of T
R-I were exactly as described above except
for the addition of GST-SMAD2 and the use of 10 µCi of
[
-32P]ATP in lieu of ATP. Phosphorylated T
R-I was
visualized by autoradiography.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
Type I Receptor Colocalizes, Cofractionates, and Interacts
with Caveolin-1 in Caveolae-enriched Domains--
Given the punctate
membrane immunostaining previously reported for the T
R-I/II complex
(21-23), a distribution resembling that of caveolin-1 and caveolae
(30), we first investigated the possibility of colocalization between
the two proteins in vivo. We utilized two different T
R-I
cDNA constructs (the HA-tagged wild-type form or a constitutively
active T204D mutant). It is important to note that the T
R-I (T204D)
construct is a constitutively active mutant that can initiate TGF-
signaling independent of ligand binding or heteromerization with the
T
R-II (47). For most of the following studies we used NIH-3T3, cells
readily responsive to TGF-
/SMAD signaling, or 293T, a Cav-1-negative
cell line readily amenable to heterologous Cav-1 expression (50, 59).
NIH-3T3 cells cotransfected with Cav-1 and either T
R-I w.t. or
T
R-I (T204D) were immunostained and visualized by confocal
microscopy. Confocal slices of areas delineating the plasma membrane
revealed significant colocalization between the proteins (Fig.
1). T
R-I (T204D) was highly aligned
with Cav-1, indicating that the two proteins can colocalize independent
of ligand activation and T
R-II. The coalignment for cells
transfected with T
R-I w.t. was not complete, however, possibly
indicating that the base-line distribution of T
R-I is not solely
restricted to areas of Cav-1 expression (Fig. 1).
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Fig. 1.
Caveolin-1 colocalizes with
T R-I w.t. and T
R-I
(T204D). NIH-3T3 cells were cotransfected with the cDNAs
encoding caveolin-1 and either T
R-I w.t. or T
R-I (T204D). Cells
were then doubly immunostained with antibodies that specifically
recognize caveolin-1 and HA (the hemaglutanin tag fused to each
receptor). The bound primary antibodies were visualized with distinctly
tagged secondary antibody probes (see "Experimental Procedures").
Both cell populations revealed significant membrane colocalization
between caveolin-1 and T
R-I. Positive and negative controls omitting
a given primary antibody and with cells singly transfected with a given
cDNA were also performed and yielded the expected results (data not
shown).
/SMAD pathway. The
outputs of this centrifugation, which consists of 12 equal fractions
(of which fractions 4-6 and 9-12 are considered of caveolar and
noncaveolar origin, respectively), are shown in Fig.
2 (A and B).
Distinct pools of T
R-I (T204D), T
R-I w.t., and T
R-II w.t.
cofractionate with Cav-1 in caveolae enriched domains. Note that the
overwhelming majority of cellular proteins are noncaveolar, as observed
by Ponceau S staining (Fig. 2A, top panel).
Therefore, the enrichment of a signaling protein cofractionating with
Cav-1 is actually higher than indicated. A comparison of two of the above fractions (one of caveolar origin (fraction 5), and one of
noncaveolar origin ( fraction 11)) via equal protein (rather than equal
volume) serves to illustrate this point (Fig. 2C); note that
T
R-I is concentrated in the caveolar fractions. Similarly, our
fractionation scheme applied to the receptor-phosphorylated Smad-2 and
the common-mediator Smad-4 showed that although Smad-2 is enriched in
caveolae, Smad-4 is distinctly excluded from these fractions (Fig. 2,
B and C). The presence of Smad-2 in caveolae microdomains is intriguing in light of the cloning of SARA, an anchoring protein shown to recruit Smad-2 to subcellular locations enriched in the TGF-
receptors (23). It should be noted that as a
general rule, receptors and other transmembrane molecules are normally
excluded during the purification of caveolae, or they are washed out by
the harsh treatments employed (36, 57, 61-67). In this regard, it is
intriguing that several members of the TGF-
/SMAD cascade remain
associated with caveolae.
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Fig. 2.
TGF- Type I receptor
and SMAD2 cofractionate with caveolin-1 in caveolae- enriched
microdomains. A and B, 293T cells were
individually transfected with the indicated cDNAs and subjected to
sucrose gradient centrifugation after homogenization in buffer
containing 1% Triton X-100 (see "Experimental Procedures"), a
method which separates Triton-resistant caveolae-rich domains
(fractions 4-6) from other cellular components
(fractions 9-12). Immunoblot analysis with anti-caveolin
2297 mAb, anti-HA pAb, and anti-FLAG mAb was with anti-caveolin 2297 mAb, anti-HA pAb, and anti-FLAG mAb was
used to detect the Cav-1, T
R-I w.t./T
R-I (T204D)/T
R-II
w.t./SMAD4, and SMAD2 proteins, respectively. The Type I and Type II
receptors and SMAD2 cofractionate with Cav-1 (fractions
4-6), whereas SMAD4 is entirely excluded from these
caveolae-enriched fractions. The distribution of total cellular protein
(as analyzed by Ponceau S staining) is shown in the top
panel, indicating that only a minute portion of total cellular
protein actually exists in caveolae. C, analysis of two
fractions from selected gradients in A (fraction
5, caveolar origin; fraction 11, noncaveolar origin)
using equal protein quantities. Note that Cav-1 as well as T
R-I
w.t., T
R-I (T204D), and Smad-2 are concentrated in caveolar
fractions in contrast to Smad-4, which is entirely noncaveolar.
R-I, T
R-II, and Smad-2 to
caveolar microdomains occurs independently of Cav-1 expression. This
indicates that these signaling molecules are more generally localized
to caveolae and caveolae-related domains (a term used to denote
membrane regions rich in cholesterol and glycosphingolipids irrespective of the presence of Cav-1). This observation raises the
issue as to the function of Cav-1 in TGF-
signaling, a point we
address in the following experiments.
R-I. 293T cells were transfected with HA-tagged T
R-I (T204D) and
Cav-1 either separately or together and subjected to
immunoprecipitation using either anti-HA pAb or relevant negative
controls (preimmune serum pAb and beads alone). Cav-1 is
immunoprecipitated only in cells coexpressing T
R-I (T204D) and Cav-1
(Fig. 3A). It should be noted
that this interaction occurs to a similar extent with T
R-I w.t. (see
Fig. 9A), indicating that Cav-1 does not discriminate between the constitutively active and wild-type forms of the
receptor.
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Fig. 3.
Caveolin-1 interacts with
TGF- Type I receptor in both heterologous and
endogenous settings. A, 293T cells were transfected
with HA-tagged T
R-I (T204D) and Cav-1 either separately or together,
as indicated. Cell lysates were prepared and immunoprecipitated
(IP) with either anti-HA pAb or relevant negative controls
(preimmune serum pAb and beads alone). Immunoprecipitates were resolved
by SDS-PAGE and subjected to immunoblot analysis with anti-caveolin-1
2297 mAb. Note that Cav-1 is immunoprecipitated only in cells
coexpressing T
R-I (T204D) and Cav-1 (top panel).
B, NIH-3T3 cells grown to confluence were subjected to
immunoprecipitation with either anti-Cav-1 2234 mAb, anti-T
R-I pAb,
or appropriate controls (irrelevant mAb, preimmune serum pAb, and beads
alone). Immunoblotting with anti-Cav-1 2297 mAb reveals an endogenous
interaction between T
R-I and Cav-1. As compared with
immunoprecipitated Cav-1 (first lane), the amount of Cav-1
associated with T
R-I can be estimated to be on the order of
~5-10%.
R-I from untransfected NIH-3T3 cells in an
attempt to assess the endogenous Cav-1/T
R-I interaction. Fig.
3B shows that a significant amount of Cav-1 is associated with T
R-I at base-line. As compared with the immunoprecipitation of
Cav-1 using a high affinity mAb-clone 2234, an antibody capable of
immunodepleting Cav-1 from cell
lysates,2 we estimate the
association of endogenous Cav-1 with T
R-I to be on the order of
~5-10% in vivo (Fig. 3B).
/SMAD Signaling by Blocking SMAD
Activation--
Because T
R-I plays a pivotal role in the
propagation of TGF-
signaling from the membrane to the nucleus, we
were interested in the functional consequences of its interaction with
Cav-1. First, we investigated the response of two commonly used TGF-
transcriptional reporter assays to heterologous Cav-1 expression. The
A3-lux/Fast-1 and 3TP-lux systems utilize TGF-
-responsive promoter
elements to drive the expression of a luciferase reporter gene (51,
52). NIH-3T3 cells were cotransfected with the appropriate luciferase
reporters and a combination of T
R-I (T204D), Cav-1, or empty vector
controls. Both reporters displayed robust activation in the
presence of the constitutively active T
R-I, an effect that was
dramatically reverted in cells coexpressing Cav-1 (Fig. 4A). In lieu of T
R-I
(T204D), we used the same reporters to look at the activation of the
endogenous TGF-
receptor complex by treating NIH-3T3 cells with
TGF-
1 (4 ng/ml) for 8 h. Cav-1 again displayed inhibitory
capacity in this respect, diminishing the ligand-activated state
3-4-fold (Fig. 4B). A K232R (kinase dead) mutant of the
T
R-I had previously been shown to lack kinase activity and to act in
dominant negative fashion by dimerizing with the wild-type T
R-I
(46). A comparison of the effects displayed by Cav-1 with those of
T
R-I (K232R) reveals a similar inhibitory capacity for both proteins
(Fig. 4B). We have independently performed these assays
using COS-7 cells yielding similar results,2 indicating
that the Cav-1-mediated inhibition is not necessarily cell
type-specific.
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Fig. 4.
Caveolin-1 functionally regulates
TGF- /SMAD signaling at the transcriptional
level. A, NIH-3T3 cells were transfected with either
A3-lux/Fast-1 (left panel) or 3TP-lux (right
panel) (the TGF-
-responsive luciferase reporters) and a
combination of T
R-I (T204D), Cav-1, or empty vector controls. Note
that Cav-1 inhibits signaling mediated by the constitutively activate
T
R-I in both reporter systems. B, as in A, the
A3-lux/Fast-1 and 3TP-lux reporter systems were utilized. However,
instead of the constitutively active receptor, TGF-
1 ligand (4 ng/ml) was used to stimulate endogenous T
R-I in NIH-3T3 cells for
8 h, as indicated. In addition, the ability of Cav-1 to diminish
signaling was compared with that of T
R-I (K232R), a kinase-dead
receptor mutant. Note that Cav-1 significantly diminishes the
transcriptional response, an effect on the same order of potency as the
dominant negative K232R mutant receptor. In both panels, luciferase
activities are expressed as ratios normalized to
-galactosidase
activity, and each experimental value represented graphically is the
average of three separate transfections performed in parallel.
Error bars represent the observed S.D.
signals from the plasma membrane to the nucleus.
The ligand-activated T
R-I directly phosphorylates Smad-2 and -3 at a
C-terminal SSXS motif (7, 68), a modification that
facilitates Smad-2/-3 release and subsequent heteromerization with
Smad-4 (9, 69). This interaction is followed by nuclear translocation
of the complex with pleiotropic effects on the transcription of target
genes (reviewed in Ref. 2). Because T
R-I activation is the
initiating event in this cascade, the interaction of Cav-1 with the
receptor should by extension disrupt Smad-2/-3 phosphorylation, heteromerization with Smad-4, and translocation to the nucleus. We
first investigated ligand-activated phosphorylation of SMADs by
transfecting NIH-3T3 cells with FLAG-tagged SMAD2 and either Cav-1 or
empty vector, labeling with 32PO4, and
selectively treating with TGF-
1. As previously reported, the
phosphorylation of Smad-2 was readily apparent in the
TGF-
1-stimulated cells (54) (Fig.
5A). However, in cells
coexpressing Cav-1, a dramatic reduction of this phosphorylation was
observed. Note that although base-line levels are reduced, Cav-1 more
dramatically attenuates Smad-2 phosphorylation in the
TGF-
1-stimulated state.
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Fig. 5.
Caveolin-1 inhibits base-line and
TGF- 1-stimulated phosphorylation of the
receptor-linked SMAD (SMAD2). A, NIH-3T3 cells were
cotransfected with FLAG-tagged SMAD2 and either Cav-1 or empty vector,
incubated in phosphate/serum-free medium supplemented with
32PO4, and selectively treated with TGF-
1 (4 ng/ml) for 45 min. Immunoprecipitation of SMAD2 via the anti-FLAG mAb
revealed the phosphorylated protein. Note that although Cav-1 reduces
base-line levels, it dramatically affects SMAD2 phosphorylation in the
TGF-
1-stimulated state (top panel). Total SMAD2 levels
are indicated in the bottom panel. B, 293T cells
were transfected with T
R-I (T204D), SMAD2, and either caveolin-1 or
empty vector. Caveolae-enriched membrane fractions were purified,
concentrated, and subjected to in vitro kinase assays by the
addition of 4 mM ATP (see "Experimental Procedures").
The presence of Cav-1 in these microdomains is sufficient to inhibit
SMAD2 phosphorylation by the constitutively active T
R-I.
R-I and Smad-2 are localized to
caveolae-enriched microdomains, independent of Cav-1 coexpression. To
test the functional significance of Cav-1, we cotransfected 293T cells
with T
R-I (T204D), Smad-2, and either Cav-1 or empty vector and
subjected the purified and concentrated caveolar fractions to an
in vitro kinase reaction by adding 4 mM ATP.
Fig. 5B shows that the constitutively active receptor can
effectively phosphorylate cofractionated Smad-2 in these microdomains
in the absence of Cav-1. In contrast, the fractions also containing
Cav-1 significantly attenuate this process.
R-I (T204D), Cav-1,
or empty vector controls. Immunoprecipitation of Smad-2 via anti-FLAG mAb revealed coprecipitated Smad-4. As predicted, cells expressing constitutively active T
R-I induced the formation of a Smad-2/Smad-4 complex, whereas cotransfection with Cav-1 completely abrogated this
association (Fig. 6).
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Fig. 6.
Caveolin-1 abrogates
TGF- -mediated heteromerization of SMAD2 with
SMAD4. NIH-3T3 cells were cotransfected with FLAG-tagged SMAD2,
HA-tagged SMAD4, and a combination of T
R-I (T204D), Cav-1, or empty
vector controls. Immuprecipitation of SMAD2 via anti-FLAG mAb and
blotting with anti-HA pAb revealed coprecipitated SMAD4. Note that the
T
R-I-induced complex formation between SMAD2/SMAD4 is disrupted in
the presence of Cav-1 (top panel). Total SMAD2 and SMAD4
levels are indicated in the middle and lower
panels.
-mediated
nuclear translocation of Smad-2 both biochemically and via microscopy.
NIH-3T3 or 293T cells were cotransfected with T
R-I (T204D),
FLAG-tagged Smad-2, and either Cav-1 or empty vector. Via hypotonic
lysis, cells were fractionated into distinct cytoplasmic and nuclear
fractions and the translocation of Smad-2 was analyzed. In both cell
lines, Cav-1 reduced Smad-2 levels in the nuclear fraction (Fig.
7A). We used
immunofluorescence confocal microscopy to corroborate these
observations by transfecting NIH-3T3 cells with Cav-1 and comparing the
TGF-
1-induced translocation of endogenous Smad-2 in transfected and
nontransfected cells. Fig. 7B shows a mid-line confocal
slice delineating the cytoplasms/nuclei of two closely juxtaposed
cells. Although there is near complete nuclear translocation of Smad-2
in the untransfected cell, the neighboring Cav-1-expressing cell has a
significant cytoplasmic pool of Smad-2.
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Fig. 7.
Caveolin-1 disrupts
TGF- -mediated translocation of SMAD2 to the
nucleus. A, NIH-3T3 cells were cotransfected with
T
R-I (T204D), FLAG-tagged SMAD2 and either Cav-1 or empty vector.
Cells were fractionated into cytoplasmic and nuclear components via
hypotonic lysis (see "Experimental Procedures") and immunoblotted
for the relative translocation of SMAD2 with anti-FLAG mAb. Note that
in the presence of Cav-1 a significant portion of SMAD2 remains in the
non-nuclear (cytoplasmic) fraction. B, NIH-3T3 cells were
transfected with Cav-1 and 36 h post-transfection were treated
with TGF-
1 (4 ng/ml) for 45 min to allow for the translocation of
endogenous SMAD2. Cells were then doubly immunostained with antibodies
that specifically recognize caveolin-1 (anti-Cav-1 N20 pAb) and SMAD2
(anti-SMAD2 mAb). The bound primary antibodies were visualized with
distinctly tagged secondary antibody probes (see "Experimental
Procedures"). Note that only the cell overexpressing Cav-1
(left panel) shows significant cytoplasmic SMAD2 staining,
whereas the untransfected neighboring cell shows nuclear SMAD2 staining
that is characteristic of TGF-
1-treated cells.
Type I Receptor--
The demonstration of an
association between caveolin-1 and T
R-I and its inhibitory effects
on downstream SMAD signaling led us to determine the Cav-1 domains
possibly mediating this functional interaction via in vitro
kinase assays. We affinity purified a GST-Smad-2 fusion protein and
used it as a physiologically relevant substrate for immunoprecipitated
T
R-I (T204D) in vitro. We have previously described a
series of caveolin-derived peptides spanning various regions of the
caveolin molecule (Fig. 8A)
(35, 70). Using these peptides in combination with T
R-I (T204D) and
GST-Smad-2, we were able to localize an inhibitory region in the Cav-1
molecule. Of the several peptides derived from the N- or C-terminal
regions, only two displayed a potent suppression of GST-Smad-2
phosphorylation, namely residues 61-101 (the Cav-1 oligomerization
domain) and its 82-101 truncation (the Cav-1 scaffolding domain).
Importantly, when the Cav-1 scaffolding domain is divided into two
halves (residues 84-92 and 93-101), this inhibition is completely
abrogated (Fig. 8B). The T
R-I (T204D) has been shown to
retain autophosphorylation activity in vitro (46, 47).
Therefore, we conducted kinase assays as above using only the
immunoprecipitated T
R-I (T204D). The same peptides that displayed
inhibition of Smad-2 phosphorylation also abrogated the
autophosphorylation of T
R-I (Fig. 8C), indicating that
the Cav-1 scaffolding domain acts to block T
R-I kinase activity.
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Fig. 8.
Peptides derived from the caveolin-1
scaffolding domain are selectively capable of inhibiting the
phosphorylation of GST-SMAD2 by T R-I (T204D)
and the autophosphorylation of T
R-I (T204D)
in vitro. A, the sequences of the
caveolin-derived peptides used in these in vitro kinase
experiment are shown. The Cav-1-(61-101) peptide contains the
caveolin-1 scaffolding domain (residues 82-101), whereas the
Cav-1-(53-81) peptide specifically lacks this region.
B, immunoprecipitated T
R-I (T204D) was used to
phosphorylate affinity purified full length SMAD2 fused to GST
(GST-SMAD2). The GST-SMAD2 and a panel of the caveolin-1 peptides (10 µM) were incubated with T
R-I (T204D) in the presence
of ATP, subjected to SDS-PAGE, and immunoblotted with the
phospho-specific anti-Smad2 pAb. Note that only the caveolin-1 peptides
that include the scaffolding domain (residues 82-101) inhibit Smad2
phosphorylation and that this effect is reversed if the scaffolding
domain is divided into two peptides. C,
[
-32P]ATP was added to immunoprecipitated T
R-I
(T204D) to observe receptor autophosphorylation. As in B,
only caveolin-1 peptides derived from the scaffolding domain inhibit
T
R-I kinase activity. DMSO, dimethyl sulfoxide;
TM, putative membrane-spanning domain.
signaling in vivo. We have previously described the
construction of a Cav-1 mutant containing a deletion of residues 61-100 (Cav-1
61-100), schematically shown in Fig.
9A (71). 293T cells
transfected with HA-tagged T
R-I w.t. and either c-Myc-tagged Cav-1
FL or Cav-1
61-100, were subjected to immunoprecipitation using either anti-HA pAb or control preimmune serum pAb. Note that
Cav-1 FL specifically interacts with immunoprecipitated T
R-I w.t.,
whereas the Cav-1
61-100 mutant does not (Fig. 9B).
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Fig. 9.
The region of caveolin-1 containing the
scaffolding domain (residues 61-100) mediates the ability of Cav-1 to
interact with T R-I w.t. and functionally
inhibit TGF-
signaling. A,
schematic diagram summarizing the domain organization of wild-type
full-length caveolin-1 (
-isoform residues 1-178) (Cav-1 FL) and the
deletion mutant (lacking residues 61-100) (Cav-1
61-100).
B, 293T cells were cotransfected with HA-tagged T
R-I w.t.
and either c-Myc-tagged Cav-1 FL or c-Myc-tagged Cav-1
61-100, as
indicated. Cell lysates were prepared and immunoprecipitated
(IP) with either anti-HA pAb or the negative control
(preimmune serum pAb). Immunoprecipitates were resolved by SDS-PAGE and
subjected to immunoblot analysis with anti-Myc mAb. Note that Cav-1 FL
specifically interacts with immunoprecipitated T
R-I w.t., whereas
the Cav-1
61-100 mutant does not. C, NIH-3T3 cells grown
to confluence were subjected to immunoprecipitation with anti-T
R-I
pAb. Where indicated, immunoprecipitates were coincubated with the
caveolin-derived peptides (20 µM), the sequences of which
are displayed in Fig. 8A. Immunoblotting with anti-Cav-1
2297 mAb reveals the endogenous interaction between T
R-I and Cav-1.
The only peptides that disrupt this interaction are ones containing the
Cav-1 scaffolding domain (i.e. peptides 61-101 and
82-101). DMSO, dimethyl sulfoxide. D, NIH-3T3
cells were transfected with the A3-lux/Fast-1 TGF-
-responsive
luciferase reporter system and a combination of T
R-I (T204D), Cav-1
FL, Cav-1
61-100, or empty vector controls. Note that as in Fig.
4B, Cav-1 FL inhibits signaling mediated by the
constitutively activated T
R-I, whereas the Cav-1
61-100 mutant
has no effect. Luciferase activities are expressed as ratios normalized
to
-galactosidase activity, and each experimental value represented
graphically is the average of three separate transfections performed in
parallel. Error bars represent the observed S.D.
R-I, it would be predicted that coincubation of NIH-3T3 cell
lysates with Cav-1 peptides containing this domain (as also utilized in
Fig. 8) would competitively disrupt the Cav-1/T
R-I complex. Fig.
9C shows that indeed only peptides containing the scaffolding domain (i.e. 61-101 and 82-101) are capable of
abrogating the interaction of Cav-1 with immunoprecipitated
T
R-I.
R-I (T204D), Cav-1 FL, Cav-1
61-100, or empty vector controls.
Cav-1 FL inhibits the signaling mediated by the constitutively activate
T
R-I (also see Fig. 4B), whereas the Cav-1
61-100
mutant has no effect (Fig. 9D).
Type I Receptor Interaction Occurs in a
Physiologically Relevant Time Frame and Is Important for Dampening
TGF-
Signaling--
Various investigators have reported the
phosphorylation kinetics of Smad-2 to occur gradually with a
t1/2 of ~5-10 min, peaking at 20-30 min (2, 54).
The demonstration of an endogenous interaction between Cav-1 and
T
R-I (Fig. 3B) led us to investigate whether this
association occurs with altered kinetics in the ligand-stimulated state
and whether it occurs within the time frame of SMAD phosphorylation.
Serum-starved NIH-3T3 cells (grown to confluence) were treated with
TGF-
1 (4 ng/ml) over an 80-min period and subjected to
immunoprecipitation with anti-T
R-I. The Cav-1/T
R-I interaction
gradually increases from base line, peaking at 40 min post-stimulation
(Fig. 10, top panel). The
total expression of Cav-1 is unaffected upon TGF-
1 treatment (Fig.
10, middle panel), indicating that the observed interaction is independent of transcriptional regulation. Furthermore, note that
the base-line Cav-1/T
R-I interaction in this serum-starved and
ligand-unstimulated setting is minimal in contrast to the ~5-10%
serum-stimulated interaction observed in Fig. 3B.
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Fig. 10.
The interaction of endogenous caveolin-1
with T R-I is enhanced upon
TGF-
1 stimulation and occurs in a
physiologically relevant time frame. Serum-starved NIH-3T3 cells
grown to confluence were treated with TGF-
1 (4 ng/ml) for the
indicated times and subjected to immunoprecipitation (IP)
with anti-T
R-I. Immunoblotting with anti-Cav-1 2297 mAb reveals the
kinetics of the interaction between endogenous T
R-I and Cav-1. The
Cav-1/T
R-I interaction is minimal at base line but gradually
increases, reaching a maximum at 40 min (top panel). Total
Cav-1 levels and the phosphorylation state of SMAD2 (as determined by
anti-phospho-SMAD2 pAb) are indicated in the middle and
bottom panels, respectively. Given the time frame of SMAD2
phosphorylation (t1/2 = ~5-10 min) (54), the
interaction of Cav-1 receptor could serve as a dampening mechanism for
TGF-
signaling.
R-I
interaction (~30-40 min) occurs slightly after the peak of
phosphorylation level of Smad-2 (~20-30 min). Given this time frame,
it is plausible that Cav-1 can act to dampen TGF-
signaling by
gradually sequestering more of the available T
R-I pool.
signaling
in cells with perturbed caveolin levels. We have previously described
the use of an antisense construct in down-modulating caveolin-1 levels
in NIH-3T3 cells (72). Cells harboring antisense caveolin-1 display
significantly reduced Cav-1 protein levels and a concomitant loss of
morphological caveolae (72). Confluent plates of serum-starved parental
NIH-3T3 cells and their antisense-Cav-1 counterparts were treated with
TGF-
1 (4 ng/ml) over a 60-min period and subjected to immunoblot
analysis. As expected, base-line phosphorylation of Smad-2 was
negligible in both cell types and gradually increased in the
TGF-
1-stimulated state (Fig.
11A). However, starting at
the 15-min time point, cells harboring antisense Cav-1 displayed
significantly higher Smad2 phosphorylation than the parental cells.
Note that the total Smad2 levels remain equal and unaltered in both
cell types. In addition, the antisense-expressing cells still produce
caveolin-1 but at dramatically reduced levels (Fig. 11A).
Quantitation of the Smad-2 phosphorylation levels in both cell types
was also conducted by densitometry of the above results (Fig.
11B). Given the time frame in which Smad2 is activated in
the antisense cells (i.e. unaltered at base-line but
hyperactivated thereafter), caveolin-1 can act to physiologically
dampen TGF-
signaling.
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Fig. 11.
Antisense-mediated down-regulation of
caveolin-1 protein levels is sufficient to hyperactivate
TGF- 1-induced SMAD phosphorylation.
A, serum-starved NIH-3T3 cells (either parental or ones
harboring the Cav-1 antisense construct) (72) were grown to confluence
and treated with TGF-
1 (4 ng/ml) for the indicated times. Cell
lysates containing phosphatase inhibitors were prepared and subjected
to immunoblot analysis with phospho-specific anti-Smad2 pAb. In the
nonstimulated state (time 0), both cell types have an identically low
Smad2 phosphorylation. Upon addition of TGF-
1, the Cav-1 antisense
cells show a distinct 2-2.5-fold hyperactivation of Smad2 activation.
Total Smad2 and Cav-1 levels in both cell types are also shown.
B, quantitation of the results shown in A were
performed by densitometric analysis.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
/SMAD signaling. We demonstrated
significant colocalization between the punctate distributions previously reported for both Cav-1 and T
R-I and observed that T
R-I, T
R-II, and Smad-2 (but not Smad-4) cofractionate with Cav-1
in caveolae-enriched domains. Support for a direct interaction between
Cav-1 and T
R-I was provided by coimmunoprecipitation studies in both
heterologous and endogenous settings. This interaction has functional
consequences because Cav-1 was able to suppress TGF-
-mediated
transcriptional activation. In addition, we showed that Cav-1
diminishes the phosphorylation of Smad-2, disrupts its interaction with
Smad-4 and prevents the nuclear translocation of Smad-2 in the
ligand-activated state. This inhibition was mediated via an interaction
between T
R-I and the scaffolding domain of Cav-1, because only
peptides derived from this region displayed potent inhibition of
T
R-I kinase activity in vitro and were able to disrupt
the Cav-1/T
R-I interaction in vivo. Furthermore, a Cav-1
mutant harboring a deletion of this domain was unable to either
interact with the receptor or functionally suppress TGF-
signaling.
We also demonstrated that the endogenous Cav-1/T
R-I association
occurs rapidly after ligand-activation and showed that
antisense-mediated down-regulation of caveolin-1 in NIH-3T3 cells was
sufficient to hyperactivate TGF-
-stimulated Smad-2 phosphorylation.
Taken together, our results support a novel role for caveolin-1 as an
important negative regulator of TGF-
signaling.
have been
identified. Many of these molecules (e.g. the p42/44 MAP kinases, SnoN/Ski oncoproteins, STAT proteins, etc) act to alter the function of the SMAD proteins by either modifying their
phosphorylation state, disrupting their interaction with downstream
partners, or preventing their capacity to affect transcription (11).
The known repertoire of molecules affecting TGF-
signaling at the level of receptor is also expanding. Smad-6 and Smad-7, a functionally divergent subset of the SMAD protein family, can inhibit TGF-
signaling by directly interacting with T
R-I (16-18). Although Smad-6 is a more potent inhibitor of bone morphogentic protein signaling, Smad-7 seems to exclusively act on TGF-
pathways and in
fact participates in an inhibitory feedback loop with T
R-I, because
its transcription is rapidly induced upon TGF-
1 stimulation (17).
Recently, a T
R-I-related protein called BAMBI was cloned and shown
to inhibit activin, bone morphogentic protein, and TGF-
signaling by
acting as a pseudoreceptor (73). In addition, FKBP12 has been shown to
negatively regulate T
R-I by interacting with the receptor in the
inactive state. This inhibition is releaved upon ligand binding,
however, whereupon FKBP12 is released from the T
R-I/-II complex (19,
74).
R-I imply a distinct mechanism of TGF-
inhibition. In contrast to Smad-7, which is a TGF-
-inducible gene
(17), Cav-1 expression remains unaffected in the first 80 min of
TGF-
1 treatment. Because the transcriptional response of Smad-7
occurs maximally at 60 min (14, 17) and presumably longer for robust
protein expression, the negative regulation mediated by Smad-7 is
clearly different than that of Cav-1. The inhibitory effect of FKBP12
on T
R-I is releaved upon ligand binding, indicating that its
cellular function might be to control aberrant TGF-
signaling in the
ligand-independent state (19). This is again in contrast to Cav-1,
where its interaction with T
R-I actually increases upon ligand
activation and plateaus at 40 min. Various investigators have reported
the phosphorylation kinetics of Smad-2 to occur gradually with a
t1/2 of ~5-10 min, peaking at 20-30 min (2, 54).
This response rate is inversely correlated with the observed gradual
increase in Cav-1/T
R-I interaction, leading credence to the
possibility of a Cav-1-mediated dampening mechanism. In support of
these observations, we showed that NIH-3T3 cells harboring an
antisense Cav-1 construct behave similar to parental cells under
serum-starved conditions, but display a 2-2.5-fold hyperactivation of
Smad2 phosphorylation upon TGF-
-stimulation.
R-I interaction. Given the ability of
peptides derived from the Cav-1 scaffolding domain to potently inhibit
T
R-I enzymatic activity in vitro and disrupt the
Cav-1/T
R-I complex in vivo, a direct interaction between the two proteins is likely. By using phage display libraries, we have
previously identified ligands for the caveolin scaffolding domain.
These peptide ligands or "caveolin-binding motifs" are as follows:
X
XXXX
,
XXXX
XX
, and
X
XXXX
XX
, where
indicates an aromatic residue, Trp, Phe, or Tyr (75). More recent
analysis indicates that motifs with a mixture of appropriately spaced
aromatic and hydrophobic residues (i.e. Leu, Ile, and Val)
could also serve to bind caveolin (76). Because functional
caveolin-binding motifs have been deduced in tyrosine kinases,
serine/threonine kinases, and endothelial nitric oxide synthase
(reviewed in Ref. 41), the Cav-1/T
R-I interaction could presumably
occur in this manner. Indeed, there are several candidate
caveolin-binding motifs in T
R-I
(424YQLPYYDLV,
388INMKHFESF, and
393FESFKRADIY) (3).
Most of these motifs are present in the intracellular kinase domain of
the receptor (more specifically, subdomains IX and X as delineated in
Ref. 77). Therefore, the inhibitory effects of Cav-1 on T
R-I kinase
activity and downstream signaling could be mediated by a direct
interaction of the Cav-1 scaffolding domain with the T
R-I kinase domain.
R-I serve? Although we addressed only a subset of TGF-
growth
factors (i.e. TGF-
1), their pleiotropic effects on
cellular physiology includes some intriguing highlights. In contrast to
its role as an anti-mitogen in the early stages of tumor growth,
TGF-
1 appears to act as a promoter of metastasis and tumor cell
migration in the later stages (78). By cooperating with matrix
metalloproteinases on the deposition and remodeling of the
extracellular matrix, TGF-
signals appear to promote tumor invasion
and angiogenesis (79). These responses are in direct contrast to ones
elicited by Cav-1. We have recently shown that Cav-1 inhibits
lamellipod extension and cellular migration in a metastatic mammary
adenocarcinoma cell line (80). In addition, VEGF, a potent
angiogenesis factor, is capable of down-regulating Cav-1 expression in
an endothelial-derived cell line (81). Consequently, a loss of Cav-1
regulation on TGF-
signaling might be an important step in the
progression to cellular migration and metastasis.
signaling plays an extremely important role in
cellular differentiation. The progression of Schwann cell, myocyte,
adipocyte, endothelial cell, and other lineages are regulated by
TGF-
, and in many cases the attainment of a terminal phenotype depends on a cessation of TGF-
signaling (reviewed in Ref. 82). Cav-1 and other members of the caveolin family are up-regulated during
such processes and expressed at high levels in terminally differentiated cells, including many of the TGF-
-responsive lineages (83). For example, in the adipogenesis model system 3T3-L1, Cav-1
expression is up-regulated 25-fold in the transition from 3T3-L1
fibroblasts to adipocytes (84). In contrast, TGF-
signaling has been
shown to potently inhibit this adipocyte conversion (85). Therefore,
the attenuation of TGF-
signals by Cav-1 could be an important
mechanism for the controlled progression of developmental events.
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ACKNOWLEDGEMENTS |
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We thank Drs. Joan Massagué for the 3TP-lux plasmid, Malcolm Whitman for the A3-lux and Fast-1 plasmids, Mark de Caestecker for the GST-Smad-2 fusion construct, Peter ten Dijke for the phospho-specific Smad-2 pAb, and Roberto Campos-Gonzalez for the caveolin-1 mAb clones 2234 and 2297.
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FOOTNOTES |
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* This work was supported by grants from the the National Institutes of Health, the Muscular Dystrophy Association, American Heart Association, and the Komen Breast Cancer Foundation (to M. P. L.).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.
§ Supported by National Institutes of Health Medical Scientist Training Grant T32-GM07288.
Recipient of a research fellowship from the Deutsche Forschungsgemeinschaft.
** Recipient of the National Kidney Foundation/Kevin and Gloria Keily Fellow Research Fellowship Award.
Supported in part by National Institutes of Health Grant
DK-56077-01 and by a Young Investigator Award from National Kidney Foundation of New York/New Jersey.
§§ To whom correspondence should be addressed: Dept. of Molecular Pharmacology and the Albert Einstein Cancer Center, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-8828; Fax: 718-430-8830; E-mail: lisanti@aecom.yu.edu.
Published, JBC Papers in Press, December 1, 2000, DOI 10.1074/jbc.M008340200
2 B. Razani, X. L. Zhang, M. Bitzer, G. von Gersdorff, E. P. Bottinger, and M. P. Lisanti, unpublished observations.
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ABBREVIATIONS |
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The abbreviations used are:
TGF, transforming
growth factor;
TR-I, Type I TGF-
receptor;
T
R-II, Type II
TGF-
receptor;
Cav-1, caveolin-1;
mAb, monoclonal antibody;
pAb, polyclonal antibody;
HA, hemagglutinin;
PBS, phosphate-buffered saline;
PAGE, polyacrylamide gel electrophoresis;
w.t., wild type;
Mes, 4-morpholineethanesulfonic acid;
GST, glutathione
S-transferase;
FL, full length.
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