From the Laboratory of Cell Regulation and
Carcinogenesis and ¶ Laboratory of Receptor Biology and Gene
Expression, NCI, National Institutes of Health, Bethesda, Maryland
20892, the § Baylor College of Medicine, Houston, Texas
77030, and the
Department of Pharmacology, Uniformed Services
University of the Health Sciences, Bethesda, Maryland 20814
Received for publication, July 20, 2000, and in revised form, February 14, 2001
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ABSTRACT |
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Members of the transforming growth factor- Ligands of the TGF- In a recently described direct signaling pathway, the activated type-I
receptor associates with and phosphorylates a family of
receptor-activated Smad proteins, which then dissociate from the
receptor, hetero-oligomerize with a common partner Smad4, and
translocate to the nucleus, where they take part in transcriptional activation of target genes (4-6). In addition, extensive cross-talk between the Smad pathway and other signal transduction cascades has
been demonstrated, notably the JAK-STAT pathway (7), mitogen-activated protein kinase pathways (8-10) as well as the vitamin D
signaling pathway (11), the glucocorticoid receptor pathway (12), and the Wnt pathway (13).
Efforts to elucidate TGF- Recently, a novel protein called TRAP1 was identified in a yeast
two-hybrid screen for a protein interacting with a mutationally activated T To better understand the biological role of TRAP1, we have now
expressed the full-length molecule, and used biochemical and functional
assays to show that TRAP1 differs in its binding characteristics from
the previously published Cell Lines and Transfections--
COS-1, HepG2, and NMuMg cells
were maintained in high glucose-Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum, 100 units of penicillin/ml,
and 100 µg of streptomycin/ml. Cells were transfected with the
constructs indicated using LipofectAMINE (Life Technologies), Superfect
(Quiagen), or FUGENE (Roche Molecular Biochemicals) according to the
manufacturer's instructions. Transfection protocols were generated
using Cellputer
Software.2
Construction of Plasmids--
Epitope-tagged TRAP1 and TRAP1
deletion constructs were polymerase chain reaction-amplified from
pCDNA3-TRAP1 using the Roche Molecular Biochemical
ExpandTM High Fidelity PCR System and cloned into the
mammalian expression vector pEFcx (gift from Dr. C. Hill), a derivative
of pEF-BOS with a modified multiple cloning site, as well as into pEGFP
(CLONTECH). For bacterial expression of GST-tagged
fusion proteins, Immunoprecipitation and Immunoblotting--
COS-1 cells were
plated in 100-mm plates at 1.5 × 106 cells/plate and
grown overnight. Cells were then transiently transfected with
epitope-tagged receptors (HA), TRAP1 (Flag, Myc, or EGFP), and/or Smad4
(Myc) and/or Smad2 (Flag) as indicated and incubated for 24 h in
maintenance medium as described above, followed by another incubation
period of 12-16 h in low serum medium (0.2% fetal calf serum).
Subsequently, the cells were washed twice with ice-cold
phosphate-buffered saline and lysed in a buffer containing 25 mM HEPES (pH 7.5), 100 mM NaCl, 10% glycerol,
5 mM EDTA, and 1% Triton X-100 supplemented with the
protease inhibitors pepstatin (1 µg/ml),
4-(2-aminoethyl)benzenesulfonyl fluoride (500 µg/ml), phosphatase
inhibitors NaF (50 mM), and sodium orthovanadate (1 mM). Lysates were cleared by centrifugation and then
subjected to immunoprecipitation using monoclonal anti-HA antibody
(CA12-5), monoclonal anti-Myc (9E10) antibody, or polyclonal anti-Myc
(Santa-Cruz A14) antibody, followed by adsorption to protein
G-Sepharose. Protein-G beads were then washed in lysis buffer for 5 times, boiled with sample buffer (NOVEX, containing 5%
mercaptoethanol), and loaded onto precast SDS gels (NOVEX). Blotting
was done utilizing ImmobilonTM polyvinylidene difluoride
membranes, which were subsequently blocked overnight at 4 °C with
5% skim milk in TBS-T buffer. Proteins were visualized by Western
blotting with anti-HA, anti-Flag (M2), anti-Myc, and anti-GFP
(Zymed Laboratories Inc. mouse monoclonal) antibodies.
In Vitro Transcription/Translation--
In vitro
transcription/translation was carried out using commercially available
reticulocyte lysates (InVitrogen) according to the manufacturer's
instructions. In short, Smad4 constructs under the control of T7
promoters were incubated with the reticulocyte lysates in the presence
of 20 mCi of [35S]methionine (PerkinElmer Life Sciences)
for 90 min at 30 °C and subsequently incubated with GST-tagged
Subcellular Localization by Immunofluorescent Confocal
Microscopy--
COS-1 cells were plated onto sterilized glass
coverslips (Corning) on day 0, transiently transfected on day 1, serum-starved overnight on day 2, and processed on day 3. Following
treatment, cells were fixed in 3.5% paraformaldehyde, permeabilized
with 0.5% Triton X-100 in phosphate-buffered saline for 10 min, and incubated for 30 min at room temperature with either of the following primary antibodies: 12CA5 anti-HA mouse monoclonal antibody (1:1000, own production) or M2 anti-Flag mouse monoclonal antibody (1:1000, Sigma). Cells were washed 3 times with phosphate-buffered saline prior
to incubation with another primary antibody and then incubated for 30 min with the following secondary antibodies: fluorescein isothiocyanate-conjugated goat anti-mouse antibody (Kirkegaard & Perry,
1:1000) and rhodamine-conjugated goat anti-rabbit antibody (Jackson
ImmonoResearch, 1:1000). Cells were mounted with medium containing
4,6-diamidino-2-phenylindole and then visualized with a Zeiss confocal microscope.
Reporter Assays--
HepG2 cells or COS-1 cells (3.5 × 105 cells/well) were seeded into six-well tissue culture
plates. Cells were transfected using Superfect (Quiagen) or Fugene
(Roche Molecular Biochemicals), respectively, according to the
manufacturer's instructions with the indicated amounts of DNA. Cells
were co-transfected with either with a 3TP-Luciferase reporter (gift
from J. Massaguè) or a SBE driven Luciferase reporter (gift from
S. Kern). 24 h later, cells were shifted into low serum with or
without TGF- TRAP1 Interacts Predominantly with Inactive T
We also examined the intracellular localization of TRAP1 and TGF- TRAP1 Associates Only with Type II Receptors of the TGF-
Since the primary binding partner for TRAP1 is the type II receptor, we
tested the association of TRAP1 with various other type II receptors.
Interestingly, only T Full-length TRAP1 Contains Multiple Receptor-binding Domains, and
Single Domains Show Reduced Binding Specificity--
To establish the
domains of TRAP1 involved in its binding to T
Since the association data for the full-length TRAP1 described in the
legend to Fig. 1 differ from those previously described for the
C-terminal Full-length TRAP1 Slightly Stimulates TGF-
These results distinguish functionally the full-length TRAP1 molecule
and its N-terminal deletion mutant Activation of the TGF-
In order to determine whether the interaction of TRAP1 with Smad4 is
direct or dependent on bridging molecules, we performed in
vitro pull-down assays. Since full-length TRAP1 could not be expressed in our bacterial strains (DH5 TRAP1-Smad4 Association Is Transient and Is Disrupted by a
Receptor-activated Smad--
The association of TRAP1 with Smad4 in
the presence of an activated receptor led us to investigate whether
TRAP1, Smad4, and Smad2 are part of a common complex and whether the
observed association of Trap1 with Smad4 results from the artificial
state generated by overexpression of Smad4 in the absence of an
equivalent amount of a receptor-activated Smad. Based on the receptor
binding pattern of TRAP1 to TGF-
Importantly, we show in Fig. 7 that the
interaction of TRAP1 with Smad4 in the presence of an active receptor
(lane 3) is substantially diminished upon addition of
increasing amounts of Smad2 (lanes 4 and 5) to a
level comparable to that of the TRAP1-Smad4 association seen in the
absence of an activated receptor (lane 2). These data are
consistent with the expectation that, in the presence of high levels of
receptor-activated Smad2, the major binding partner of Smad4 is Smad2
rather than TRAP1 (lane 5). The data demonstrate that TRAP1,
Smad4, and Smad2 do not complex together, but rather suggest that the
binding of Smad4 to TRAP1 or the activated Smad2 is mutually exclusive.
We interpret these results to suggest that a putative endogenous
Smad4-TRAP1 interaction is likely only very transient in
vivo and is disrupted as soon as Smad4 binds to receptor-activated
Smad2/3. Lane 6 provides further evidence for this model by
showing that disruption of the TRAP1-Smad4 interaction is not merely a
consequence of the presence of Smad2, but requires that Smad2 be
activated by phosphorylation at its C terminus. This is demonstrated by
comparing the ability of Smad2 and the inactive Smad2(3SA) mutant,
which cannot be terminally phosphorylated, to disrupt the TRAP1-Smad4
interaction. In contrast to wild type Smad2, Smad2(3SA) slightly
increases the association of TRAP1 with Smad4 (lane 6),
possibly by blocking the activation of endogenous Smad2. Together,
these data suggest that receptor activation leads to association of
Smad4 and TRAP1, and that this likely transient state occurs prior to
the association of Smad4 with a receptor-activated Smad.
Mutated Forms of TRAP1 Inhibit TGF- The regulation of cellular signaling cascades involves a multitude
of processes on various levels, including transcriptional regulation of
signaling components, receptor trafficking, and phosphorylation events.
In the case of signaling from TGF- We now propose that, given its signal-dependent association
with both T
(TGF-
) superfamily signal through unique cell membrane receptor
serine-threonine kinases to activate downstream targets. TRAP1 is a
previously described 96-kDa cytoplasmic protein shown to bind to
TGF-
receptors and suggested to play a role in TGF-
signaling. We
now fully characterize the binding properties of TRAP1, and show that
it associates strongly with inactive heteromeric TGF-
and activin receptor complexes and is released upon activation of signaling. Moreover, we demonstrate that TRAP1 plays a role in the Smad-mediated signal transduction pathway, interacting with the common mediator, Smad4, in a ligand-dependent fashion. While TRAP1 has only
a small stimulatory effect on TGF-
signaling in functional assays,
deletion constructs of TRAP1 inhibit TGF-
signaling and diminish the
interaction of Smad4 with Smad2. These are the first data to identify a
specific molecular chaperone for Smad4, suggesting a model in which
TRAP1 brings Smad4 into the vicinity of the receptor complex and
facilitates its transfer to the receptor-activated Smad proteins.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1
superfamily regulate a variety of physiologic and pathologic processes
such as embryogenesis, wound healing, tissue homeostasis, fibrosis, and
immunomodulation (1). Signal transduction by these ligands is initiated
by their association with a type-II receptor serine-threonine kinase,
which then recruits and transphosphorylates a type-I receptor, also a
transmembrane serine-threonine kinase (2). Transphosphorylation of the
type I receptor by the type II receptor takes place on several residues in a glycine-serine-rich juxtamembrane domain and leads to activation of the receptor and downstream signaling events (3).
signaling pathways have resulted in the
identification of a number of other non-Smad proteins which interact
directly with T
RI and/or T
RII, including SARA (14), STRAP (15),
FKBP12 (16), the
subunit of farnesyl transferase (17), TRIP-1 (18),
and the B
subunit of phosphatase 2A (19). Functional roles in
TGF-
signal transduction have been proposed for them but remain
controversial (2, 19-22). Of these, SARA, which has been shown to
facilitate Smad2/3 recruitment to the activated T
RI (14), is the
only protein directly shown to modulate the Smad signaling pathway.
RI (23). Indeed, the C-terminal portion of TRAP1 (
TRAP1) was shown to interact specifically with mutationally or
ligand-activated T
RI but not with the quiescent receptor, suggesting
that it might participate in signal transduction from the T
RI. In
functional assays,
TRAP1 acted as an inhibitor of TGF-
-mediated
effects, suggesting that it might play a role in modulating this signal
transduction pathway.
TRAP1 in that it predominantly associates
with receptor complexes that are signaling deficient. Moreover, we show
that TRAP1 specifically interacts with Smad4 and provide data
suggesting that it functions as a chaperone for Smad4 in TGF-
signal transduction.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
TRAP1 was subcloned into the pGEX4T3 vector.
Smad4-MH2 was cloned into pCDNA4HisMax (InVitrogen) for in
vitro translation. All constructs were sequence confirmed.
TRAP1 bound to glutathione-agarose. Following washes in a buffer
described for the immunoprecipitation studies, beads were boiled with
sample buffer (NOVEX, containing 5% mercaptoethanol) and loaded onto
precast SDS gels (NOVEX). Visualization was done by autoradiography.
(5 ng/ml). Where indicated, luciferase activity was
measured following 12-18 h of incubation using a commercially
available kit (Pharmingen) and normalized to
-galactosidase expression.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
RI·T
RII
Complexes and Dissociates from Activated Receptor Complexes--
To
investigate interactions of TRAP1 with TGF-
receptors, we have
expressed the full-length molecule together with combinations of wild
type and kinase-deficient T
RI and T
RII in vivo. COS-1 cells were transiently transfected with epitope-tagged receptor (HA)
and TRAP1 (Flag) constructs, and cell lysates were analyzed by
immunoprecipitation and Western blotting. In cells that overexpress only T
RI, a weak interaction of full-length TRAP1 can be
demonstrated with the kinase-deficient mutant but not the wild type
receptor (Figs. 1A and
2A). In cells overexpressing only T
RII, TRAP1 interacts with either wild type or kinase-deficient receptors, showing a stronger
association with the kinase-deficient T
RII (Figs. 1A and
2B). When both T
RI and T
RII are overexpressed
simultaneously, in various combinations of wild type and
kinase-deficient forms, TRAP1 binds most strongly to those complexes in
which at least one of the receptors has been mutationally inactivated
(Fig. 1B), making the complex incapable of signaling. A
direct effect of TGF-
on association of TRAP1 with wild type
receptor combinations could not be demonstrated (Fig. 1B),
most likely due to a ligand-independent activation of the receptor
complex in the context of overexpression of wild type receptors (20,
24), a phenomenon which we have confirmed in functional assays (Fig.
3C, and data not shown).
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Fig. 1.
Interaction pattern of TRAP1 and various
TGF- superfamily
receptors. A and B, COS-1 cells were
transiently transfected with Flag-TRAP1 or Flag-
TRAP1 in combination
with HA-T
RI (wt), HA-T
RI (K232R, kinase deficient, KD)
or HA-T
RI (T204D, mutationally activated, a*), and/or
T
RII (wt) or T
RII (K277R, KD). 30 h after
transfection, cells were serum starved for 12 h in the absence or
presence of 10 ng/ml TGF-
1 as indicated (B only). Cell
lysates were immunoprecipitated with anti-HA antibody and blotted with
anti-Flag antibodies. C and D, TRAP1 co-localizes
with TGF-
receptors in the absence of signaling. COS-1 cells were
transiently transfected with both KD receptors (C) or with
T
RII and activated T
RI (a*) (D). After a
24-36-h incubation period, the cells were fixed, subjected to
immunochemistry with primary antibodies against the Flag and HA
epitopes, and secondary antibodies linked to fluorescein isothiocyanate
or rhodamine dyes, and mounted with medium containing
4,6-diamidino-2-phenylindole (DAPI). Magnification:
×63.
receptors using epitope-tagged proteins and indirect immunofluorescence to confirm the above observations. In order to avoid ligand-independent activation of the signaling cascade, we again utilized mutationally activated and kinase-deficient forms of T
RI to simulate the presence and absence of TGF-
signal, as described for the immunoprecipitation assays. As shown in Fig. 1C, TRAP1 co-localizes with TGF-
receptors when the receptors are mutationally inactivated, displaying
the same patchy distribution pattern previously reported for the
receptor complex (14, 25, 26). In the presence of constitutively activated receptors, however, the intracellular distribution of TRAP1
changes from the patchy pattern to a more diffuse pattern (Fig.
1D). This correlates with a reduction in the degree of
TRAP1-receptor association consistent with that observed in
immunoprecipitation/Western blotting. Taken together, these data
suggest that TRAP1 associates with the TGF-
receptor complex and
that the type II receptor is the primary binding partner. The fact that
associations are strongest in the presence of kinase-deficient
receptors suggests that activation of signaling through the TGF-
receptor complex results in dissociation of TRAP1 from this complex, in
striking distinction to previously published data for the C-terminal
fragment of TRAP1,
TRAP1 (23).
and
Activin Pathways, Whereas Its Pattern of Binding to Type I Receptors Is
Less Restricted--
To determine whether the interactions of TRAP1
were specific to the TGF-
receptors T
RI and T
RII, we next
examined the ability of TRAP1 to bind to other members of the TGF-
superfamily of receptors. As shown in Fig.
2A, TRAP1 associates also with
several kinase-deficient type I receptors, specifically with ALK1,
BMPR-IA (ALK3), ActRI (ALK4), and the T
RI (ALK5), but not with the
ALK2 receptor and only minimally with BMPR-IB (ALK6). With the
exception of the binding of TRAP1 to ALK3, these data suggest that
TRAP1 is likely to be involved in both TGF-
and activin but less
likely in BMP signaling.
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Fig. 2.
TRAP1 associates with distinct type II and
type I receptors of the TGF- superfamily of
ligands. Using the same experimental conditions as in the legend
to Fig. 1, A and B, TRAP1 was co-transfected with
various KD type I (A) or wild type and KD type II
(B) receptors with HA or Flag tags and coimmunoprecipitated
with Flag- or Myc-tagged TRAP1.
RII and the activin type IIB receptor associate
with TRAP1, whereas the BMP type II receptor does not (Fig.
2B). To explore the binding of TRAP1 to the activin receptors more fully, we examined the pattern of binding of TRAP1 to
both wild type and kinase-deficient forms of ActRIIB. In contrast to
the pattern observed with T
RII, where the binding of TRAP1 was
clearly stronger to the kinase-deficient form of the receptor (Fig.
1A and Fig. 3A), we
observed equal binding of TRAP1 to either active or inactive forms of
ActRIIB (Fig. 3A). Similar to that observed with the TGF-
receptor complex, activation of the complex by overexpression of the
constitutively activated form of ALK4 led to nearly complete
dissociation of TRAP1 from the receptor complex. In an attempt to
understand the basis of the difference in the comparative binding of
TRAP1 to the wild type and kinase-deficient forms of T
RII and
ActRIIB, we examined the effect of overexpression of each of these
receptor constructs on the activity of the TGF-
/activin responsive
3TP-Lux reporter (28) (Fig. 3B). The ability of wild type
T
RII but not ActRIIB to activate signaling when overexpressed in
this cellular context mirrors the apparent differential binding of
TRAP1 to the wild type- and kinase-deficient forms of these two
receptors and suggests that partial dissociation of TRAP1 from wild
type T
RII underlies its apparently stronger binding to inactive
T
RII. Whether the selective ability of T
RII to activate signaling
in COS-1 cells results from an autocrine loop or from differential
expression of ALK5 compared with ALK4 is not known.
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Fig. 3.
The apparent binding of TRAP1 to
T RII and ActRIIB is
signal-dependent. A, GFP-tagged TRAP1 was
co-transfected either with the wild type (wt) or
kinase-deficient (KD) forms of T
RII (HA-tagged,
lanes 1 and 2) or ActRIIB (flag-tagged,
lanes 3-7) with or without either kinase-deficient (KD,
lane 6) or activated (a*, lane 7)
ALK4. Lysates were immunoprecipitated with either anti-HA (lanes
1 and 2) or anti-Flag antibody (lanes 3-7),
and co-precipitating TRAP1 was probed with anti-GFP. The expression
levels of TRAP1 and the receptor constructs are shown as loading
controls. Note that in contrast to the apparently stronger binding of
TRAP1 to the kinase-deficient form of T
RII, its binding to ActRIIB
is independent of this inactivating mutation. B, T
RII but
not ActRIIB activates the transcription of the
TGF-
/activin-responsive 3TP-Lux reporter. The ability of transfected
wild type and kinase-deficient forms of T
RII and ActRIIB to trigger
the corresponding signaling pathways was assessed by activation of the
3TP-Lux reporter, under the experimental conditions described in
panel A. Note that T
RII significantly activates
transcriptional responses with the same order of potency of activated
ALK4. In contrast ActRIIB elicits transcriptional responses only
modestly higher than that of negative control and kinase-deficient
ActRIIB and T
RII. Results are expressed as ratio of luciferase
activity (relative light units) to
-galactosidase
activity and are representative of three independent experiments.
Error bars represent standard deviation; where no error bars
are visible, the error is too small to be shown.
RII, C- and N-terminal
deletion constructs and a middle region construct (Fig.
4A) were used in
immunoprecipitation assays. As shown in Fig. 4B, full-length
TRAP1,
MC-TRAP1 (amino acids 1-215),
NM-TRAP1 (amino acids
651-860),
NC-TRAP1 (amino acids 238-536), and
TRAP1 (amino
acids 474-860) co-immunoprecipitate with T
RII. It is noteworthy
that binding of each of the deletion constructs is severalfold stronger
than that of the wild type molecule, suggesting a complex
conformational regulation of these binding domains. Even under
stringent washing conditions (500 mM NaCl buffer), strong
binding of all regions to T
RII(KD) was detectable (data not shown).
Since both the C-terminal deleted construct
MC-TRAP1 and the
N-terminal deleted construct
NM-TRAP1 as well as the middle region
NC-TRAP1 all bind strongly to T
RII, there are at least three
binding sites for interaction of TRAP1 with the receptor.
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Fig. 4.
TRAP1 interacts with
T RII through multiple domains.
A, schematic representation of the truncation mutants in
relation to the full-length (wild type) TRAP1 molecule. The constructs
shown are used for experiments in B, and in Figs. 5 and 8.
B, HA-tagged, kinase-deficient T
RII was coexpressed with
Myc-tagged TRAP1 and Flag-tagged TRAP1 deletion constructs and
subjected to immunoprecipitation with anti-HA and probed with
peroxidase-linked anti-Myc (left IP panel) or anti-Flag
(right IP panel) antibody. Expression of all constructs is
shown by SDS-PAGE and Western blotting of equal amounts of total cell
lysates (left middle panel, anti-Myc; right middle
panel, anti-Flag; lower panel, anti-HA as indicated).
C, interaction pattern of
TRAP1 with various TGF-
superfamily receptors. In a similar experimental approach as outlined
for Fig. 1, cells were transfected with TGF-
superfamily receptors
as indicated and co-transfected with
TRAP1, followed by
immunoprecipitation and Western blotting. Cells in lane 5 were stimulated with TGF-
1 (10 ng/ml).
TRAP1, we also investigated the interaction pattern of
this truncated protein with T
RI and T
RII. In our hands,
TRAP1
neither showed any preferential binding to activated, wild type, or
inactivated TGF-
receptors nor did it discriminate between T
RI
and T
RII, as shown in Fig. 4C, in striking distinction from the pattern shown for the full-length molecule. Since
overexpression of either T
RII or the activated form of T
RI are
sufficient to induce signaling, these data demonstrate that
TRAP1,
unlike full-length TRAP1, does not dissociate from the active signaling
receptor complex.
Signaling--
Initial functional studies of
TRAP1 by Charng
et al. (23) showed an inhibitory effect on TGF-
induced
activity of the 3TP-Lux reporter, suggesting a functional role of the
molecule in TGF-
signaling. Based on the different interaction
patterns of full-length TRAP1 and
TRAP1 molecules with the TGF-
receptors, we investigated whether their functional roles might also
differ, and specifically whether
TRAP1 might function as a dominant
negative mutant of the full-length molecule. HepG2 cells were
transiently transfected with the indicated TRAP1 constructs and the SBE
luciferase reporter, consisting of 4 tandem repeats of the Smad-binding
element CAGA (29) or the 3TP-Lux reporter (data not shown). Stimulation with TGF-
resulted in activation of luciferase activity with either
promoter (Fig. 5 and data not shown).
Addition of TRAP1 slightly enhanced luciferase activity, whereas
TRAP1 consistently inhibited luciferase activity, as shown
previously (23). Similar results were observed in NMuMg cells with the
3TP-Lux reporter (data not shown).
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Fig. 5.
Expression of TRAP1 and
TRAP1, respectively, enhances or inhibits
TGF-
-dependent reporter
activity. HepG2 cells were transiently transfected with the
SBE4-lux reporter and increasing concentrations of
Flag-tagged TRAP1 and
TRAP1 constructs as indicated. pCDNA3 was
used to adjust the total amount of DNA to equal amounts in each well.
Cells were subsequently grown in Dulbecco's modified Eagle's medium
supplemented with 10% fetal calf serum for 24 h and then
incubated with and without TGF-
at 10 ng/ml for 16 h.
Luciferase activity was measured in arbitrary units (relative light
units) and the data expressed as relative light units ± S.E. For
a schematic of the deletion constructs, see Fig. 4A.
TRAP1. We propose that the
relatively minor effect of the full-length molecule might be due to its
abundance within the cell, making it a non-limiting component in the
signal transduction pathway. In this respect, TRAP1 behaves like SARA,
which was shown to have little effect on reporter gene activity
(3TP-Lux and ARE-Lux) as a full-length molecule, but which was
inhibitory in its truncated and functionally inactive forms (14).
Receptor Complex Leads to Dissociation of
TRAP1 from the Receptor and Association of TRAP1 with Smad4--
Based
on the fact that
TRAP1 has an inhibitory effect on TGF-
signaling
and fails to dissociate from active receptor complexes in contrast to
full-length TRAP1, we hypothesized that TRAP1 might play a fundamental
role in signaling from the TGF-
and activin receptors, and possibly
other receptors of the TGF-
superfamily. Smad4 was a potential
target for TRAP1 action, since it is an obligatory signaling
intermediate in Smad-mediated signaling from all TGF-
superfamily
receptors. We therefore investigated whether TRAP1 might interact with
Smad4 in vivo, and whether such an interaction might be
signal-dependent. COS-1 cells were transiently transfected with TRAP1 and Smad4 and with or without activated T
RI. As shown in
Fig. 6A, TRAP1 binds only
weakly to Smad4 in the absence of a TGF-
signal. However, upon
activation of the signaling cascade, a strong interaction can be
demonstrated between TRAP1 and Smad4. This interaction most likely
occurs off the receptor, as the activated receptor interacts only
weakly with TRAP1, as shown above. These data are also consistent with
the fact that a direct association of Smad4 with the activated receptor
complex has not thus far been demonstrated (30, 31). To address the
question whether TRAP1 might also interact with other Smad proteins, we
transfected Flag- and Myc-tagged Smad 1-8 constructs and utilized a
GFP-tagged TRAP1 construct as a potential binding partner (Fig.
6B). The data show that Smad4 is the only Smad binding
partner of TRAP1.
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Fig. 6.
TRAP1 interacts selectively with Smad4.
A, TRAP1 associates with Smad4 in a
signal-dependent manner. COS-1 cells were transiently
transfected with Myc-tagged Smad4, HA-tagged TGF- receptor type I
(T204D) and type II (K277R), and Flag-tagged TRAP1 as indicated and
subjected to immunoprecipitation with anti-HA antibody (first
lane, positive control) and anti-Myc antibody (second
through fifth lanes), followed by SDS-PAGE and Western blotting
with anti-Flag antibody. Expression of all constructs is shown by
SDS-PAGE and Western blotting of equal amounts of total cell lysates.
Only two lanes were used for anti-HA controls, lanes not blotted with
anti-HA are crossed. B, TRAP1 interaction with
Smad4 is specific. COS-1 cells were transiently transfected with Myc-
and Flag-tagged Smad1-8 and GFP-tagged TRAP1 as indicated and
subjected to immunoprecipitation with anti-Myc or anti-Flag antibody
(lane 1, negative control), followed by SDS-PAGE and Western
blotting with anti-GFP antibody. C,
TRAP1 interacts
directly with Smad4 in vitro. Various Smad4 deletion
constructs were in vitro transcribed/translated using
[35S]methionine and subsequently incubated with
GST-tagged
TRAP1 bound to glutathione-agarose. Following SDS-PAGE,
proteins were visualized using autoradiography.
and BL21, data not shown), we utilized bacterially expressed GST-
TRAP1 bound to
glutathione-agarose and various in vitro
transcribed/translated Smad4 constructs. The Smad4 constructs were
labeled with [35S]methionine and incubated with
GST-agarose and GST-
TRAP1-agarose beads, followed by SDS-PAGE and
autoradiography. As shown in Fig. 6C,
TRAP1 pulls down
Smad4 full-length protein weakly and almost no Linker + MH2 and MH2
constructs. However, the MH1 domain, a MH1 + Linker construct, as well
as a SAD + MH2 construct bind strongly to
TRAP1. This suggests that
there are at least two binding sites in Smad4 that facilitate a direct
TRAP1-Smad4 interaction, and that these binding sites are
conformationally restricted and regulated in the full-length Smad4
in vivo (as shown above). Together, these results
demonstrate a signal dependent, direct TRAP1-Smad4 interaction.
and activin receptors, each of
which activate Smad2 and Smad3, we examined the association of Smad4
with TRAP1 in COS-1 cells in the presence of increasing concentrations
of Smad2 or the Smad2(3SA) mutant, which cannot be phosphorylated at
its C-terminal end and thus cannot bind Smad4 following receptor activation.
View larger version (42K):
[in a new window]
Fig. 7.
Activated Smad2 disrupts the TRAP1-Smad4
interaction. COS-1 cells were transiently transfected with
Myc-tagged Smad4, HA-tagged TGF- receptor type I (T204D,
a*), Flag-tagged TRAP1, and increasing concentrations of
Flag-tagged Smad2 or Smad2(3SA) as depicted. Cell lysates were
subjected to immunoprecipitation with anti-Myc antibody and blotting
with anti-Flag antibodies. Expression levels of TRAP1, Smad2,
Smad2(3SA), T
RI, and Smad4 are shown in the total lysates.
Signaling by Interfering
with Formation of the Smad2-Smad4 Complex--
Since
TRAP1 failed
to dissociate from activated receptors (Fig. 4C) and
inhibited TGF-
signaling (Fig. 5) (23), we hypothesized that the
dissociation of TRAP1 from active receptors and its association with
Smad4 might facilitate the interaction of Smad4 with Smad2/3. To test
this model, we determined whether
TRAP1 and other N- and C-terminal
deletion mutants of TRAP1 would decrease the association of Smad4 with
Smad2. As demonstrated in Fig. 8, the
interaction of Smad2 and Smad4 was analyzed in the absence (lane
2) or presence (lane 3) of activated receptor but in
the absence of overexpressed TRAP1, or in the presence of activated
receptor and full-length TRAP1 (lane 4) or various TRAP1
mutants (lanes 5-8). All mutant forms of TRAP1 inhibited
the Smad2-Smad4 interaction in the presence of activated receptor
(lanes 5-8) in comparison to the Smad2-Smad4 interaction in
the presence of activated receptor alone (lane 3).
NM-TRAP1, the shortest of all constructs (C-terminal 209 amino
acids), exhibited the strongest inhibition. The full-length TRAP1
molecule also showed a weak inhibitory effect (lane 4). We
have occasionally seen slight inhibition by overexpressed TRAP1 in
reporter gene assays as well, and suggest that its strong expression in
the experiment shown might result in sequestration of Smad4 by TRAP1.
In comparable immunoprecipitation experiments where TRAP1 expression
levels were lower, we did not see this effect of full-length TRAP1
(data not shown).
View larger version (31K):
[in a new window]
Fig. 8.
Truncated TRAP1 mutants interfere with
formation of the receptor activated Smad2-Smad4 complex. COS-1
cells were transiently transfected with Myc-tagged Smad4, Flag-tagged
Smad2, HA-tagged TGF- receptor type I (T204D, a*) and
identical TRAP1 and TRAP1 deletions as utilized in Fig. 4B
as indicated. Immunoprecipitations were carried out using anti-Myc
antibody and subsequent blotting was done against the Flag-epitope.
Expression levels of the transfected proteins are shown in total
lysates.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
superfamily receptors,
phosphorylation on serine and threonine residues of both the receptors
themselves and of Smad proteins activates signal transduction. Other
emerging mechanisms of signal regulation are the chaperone, anchoring,
and scaffolding proteins that regulate the folding, the subcellular
distribution, and the recruitment of signaling molecules (27). Such
mechanisms have been identified in a variety of signaling pathways such
as the steroid/glucocorticoid pathway, mitogen-activated protein kinase
signaling cascades, protein kinase A and C signaling pathway, and
others (32). Very recently, such mechanisms have been shown to be
important in the TGF-
signaling pathway as well, in that a FYVE
domain protein SARA has been shown to regulate the subcellular
localization of two of the R-Smad proteins, Smad2 and Smad3 (14).
However, despite evidence for such anchoring proteins and for
identification of certain amino acids within the Smad and receptor
proteins that confer specificity for the coordinated action of the
pleiotropic receptors of the TGF-
superfamily (33), it cannot be
ruled out that other, as yet unidentified mechanisms might not also contribute to the fine tuning of response in this cascade. It is
therefore reasonable to assume that other anchoring and scaffolding proteins will be found which regulate and promote assembly of signaling
intermediates in the TGF-
pathway.
RII and ActRIIB and its interaction with Smad4, the previously identified TRAP1 protein plays such a chaperone role in
signaling downstream of not only TGF-
, but likely also activin. Our
working hypothesis is that TRAP1 facilitates interaction of Smad4 with
Smad2/3 proteins by binding to Smad4 in the vicinity of the activated
receptor and mediating its transfer to the phosphorylated Smad2/3 (Fig.
9). In support of this model, we have
shown that TRAP1 associates most strongly with receptors that are not
actively signaling, as is the case either in the absence of ligand or, experimentally, by use of kinase-deficient receptors. Consistent with
our data, we propose that activation of the receptor might then lead to
a conformational change in TRAP1 such that it dissociates from the
receptor and forms a transient complex with Smad4. Most importantly,
the strong signal-dependent association of TRAP1 and Smad4
is seen only in the absence of co-expressed Smad2, suggesting that it
might be a transient complex which can be visualized only in the
experimental condition of overexpression of Smad4 in the absence of an
acceptor R-Smad. TRAP1 and the activated, phosphorylated Smad2 bind
Smad4 in a mutually exclusive fashion. The C-terminal phosphorylation
deficient 3SA mutant of Smad2 does not interfere with Smad4-TRAP1
association, presumably because it does not leave the receptor and
cannot function as an acceptor for TRAP1-activated Smad4. For the
R-Smads, phosphorylation of the C-terminal SSXS motif has been proposed
to relieve the autoinhibitory interaction of the MH1 and MH2 domains,
freeing the MH2 domain to interact with the MH2 domains of other
R-Smads or of Smad4 (30, 34). At present, there is no known
phosphorylation-dependent mechanism for activation of Smad4
similar to that of the R-Smads. While the simpler explanation of our
data is that Smad4 can bind either receptor-activated TRAP1 or Smad2/3,
we propose instead that the interaction of receptor-activated TRAP1
with Smad4 might function to reduce the autoinhibitory MH1/MH2 domain
interaction of Smad4 and thereby make it competent to interact with
activated R-Smads. Whether there is a complementary TRAP1-related
mechanism operative downstream of BMP receptors remains to be
demonstrated.
View larger version (24K):
[in a new window]
Fig. 9.
Proposed model of TRAP1 function in
TGF- signal transduction.
The only other molecule described so far that serves as a chaperone in
TGF- signaling is SARA (14). Although there are no sequence
homologies between TRAP1 and SARA, there are some strikingly similar
characteristics. Both proteins associate with the TGF-
receptor
complex, both are regulated in a ligand-dependent fashion,
and both display mutually exclusive binding to their Smad protein
partner with respect to the acceptor Smad. While TRAP1 binds Smad4 only
in the absence of bound Smad2, SARA binds Smad2 only when it is not
associated with Smad4. However, unlike domain binding data for SARA, we
are unable to delineate any specific domain within TRAP1 necessary for
the binding of the receptor and Smad4, but rather show that N- and
C-terminal and middle regions all have binding activity. However, this
is not without precedent in this field. Another chaperone/scaffolding
protein, RAP, involved in folding and escorting certain low density
lipoprotein receptor family proteins, has also been shown to
bind multiple sites in the target receptor through multiple sites in
the chaperone (35). Again with the TRIP-1 protein, shown previously to
bind strongly to the type II TGF-
receptor, deletion studies showed
that the receptor-binding domain could not be localized and that
multiple regions of the molecule participated in the interaction (22). Moreover, the binding of multiple Smad4 domains to TRAP1 and vice versa
supports our hypothesis that TRAP1 might serve as a scaffold that
separates the Smad4-MH1 from the Smad4-MH2 domain in order to present
Smad4-MH2 to the MH2 domain of Smad2. However, in the absence of
three-dimensional structural information about TRAP1, we cannot, for
the present, address the question of its binding properties in detail.
We observed both mild stimulatory and mild inhibitory effects of
exogenous full-length TRAP1 on TGF- signaling. Since TRAP1 is
ubiquitously expressed (23), we propose that these variable effects
might be dependent on the relative levels of endogenous TRAP1 expressed
by a particular cell. In cells expressing low levels of TRAP1,
exogenous TRAP1 might enhance TGF-
signaling, whereas in cells with
higher TRAP1 levels, additional exogenous TRAP1 might sequester Smad4
from endogenous R-Smads and inhibit signaling. This behavior is similar
to published data on the SARA protein and ARIP1, a molecule proposed to
act as a scaffold in the activin pathway (36). Both proteins are
thought to facilitate signaling, yet full-length SARA stimulated only
slightly or had no effect in functional assays (14), and full-length
ARIP1 inhibited signaling (36). In this context, it is noteworthy that
mutated forms of SARA or TRAP1 each inhibit signal transduction,
suggesting that these molecules suppress the activity of the endogenous
protein in a dominant-negative fashion. In the case of TRAP1, we have not yet determined whether this inhibitory activity might be due to the
inability of these constructs to dissociate from the activated receptor
complex, as shown for
TRAP1, their inability to associate with
Smad4, or possibly also their inability to release and/or activate
Smad4 to associate with Smad2. Our data showing that expression of
mutant forms of TRAP1 diminishes Smad2-Smad4 interaction do not
distinguish between these possibilities.
TRAP1 is a large protein (860 amino acids), and our data suggest that multiple subdomains contribute to its functional activity. Interestingly, we often observed two distinct TRAP1 bands. As the double band is observed only when utilizing a 3'-tagged full-length TRAP1 construct (such as Flag- and Myc-tagged TRAP1, e.g. Figs. 1, A and B, 2A, and 6A) but not a 5'-tagged construct (such as EGFP-TRAP1, Fig. 6B), the smaller molecule likely represents an N-terminally truncated TRAP1. Whether this is due to an alternative translation start site or proteolytic cleavage has yet to be determined.
During the course of this study, a report was published describing the
interaction of TRAP1 with 5-lipoxgenase in a yeast two-hybrid system
(37). However, no functional role was attributed to this binding
activity, nor was the finding confirmed in mammalian cells. We have no
data linking the effect of TRAP1 in TGF- signaling with the function
of 5-lipoxgenase; however, at this point, it should not be ruled out
that TRAP1 could serve other, as yet unknown, functions in the cells.
In this regard, the human ortholog of the yeast vacuolar sorting
protein, Vps39/Vam6p (38), previously published as a 3' cDNA named
KIAA0770 (39) shows a 25% identity and 40% similarity to TRAP1. This
new protein has now been shown to localize to the cytoplasmic face of
lysosomes, suggesting that it, and by inference possibly also TRAP-1,
may play a role in lysosome
biogenesis.3 In this regard,
another vesicular trafficking protein, caveolin-1, has been shown to
play a role in TGF-
signaling mediated by its direct interaction
with T
RI (40).
A recent data base search revealed that TRAP1 is localized on
chromosome 2 and encoded by 11 exons (the terminal 34 base pairs were
not part of the published contig). Given the functional importance of
TRAP1 in TGF- signaling, knowledge of its chromosomal localization will now enable investigation of whether this locus can be linked to
any diseases in which TGF-
superfamily members are known to play a
role. Studies addressing this question are under way in this laboratory.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Masa Kawabata for the HA-tagged
ALK 1, 2, 3, 4, and 6 constructs, Jeff Wrana and Liliana Attisano for
TGF- receptor constructs, Caroline Hill for pEF-CX, Rik Derynck for wild type Smad4, Mark deCaestecker and Seong-Jin Kim for Smad4 deletion
constructs, and Richard S. Kim, Pilar Frontelo, and Tony Parks for
helpful discussions. We also thank Drs. Steve Caplan and Juan
Bonifacino for sharing unpublished data.
![]() |
FOOTNOTES |
---|
* This work was supported by a stipend of the Deutsche Forschungsgemeinschaft (to J. U. W.).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.
** To whom correspondence should be addressed: Laboratory of Cell Regulation and Carcinogenesis, NCI, National Institutes of Health, Bldg. 41, Rm. C629, 41 Library Dr., MSC 5055, Bethesda, MD 20892-5055. Tel.: 301-496-5391; Fax: 301-496-8395; E-mail: Robertsa@dce41.nci.nih.gov.
Published, JBC Papers in Press, February 20, 2001, DOI 10.1074/jbc.M006473200
2 J. U. Wurthner, manuscript in preparation.
3 S. Caplan and J. S. Bonifacino, personal communication.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
TGF-, transforming growth factor-
;
TRAP1, TGF-
receptor-associated
protein 1;
T
RI, type I TGF-
receptor;
T
RII, type II TGF-
receptor;
R-Smad, receptor-activated Smad;
SARA, Smad anchor for
receptor activation;
FKBP12, FK506-binding protein 12;
TRIP1, TGF-
receptor interacting protein 1;
STRAP, serine-threonine kinase
receptor-associated protein;
Smad, MAD-related protein;
SBE, Smad
binding element;
GST, glutathione S-transferase;
HA, hemagglutinin;
EGFP, epidermal growth factor protein;
PAGE, polyacrylamide gel electrophoresis.
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