The Oncoprotein Ski Acts as an Antagonist of Transforming Growth Factor-
Signaling by Suppressing Smad2 Phosphorylation*
Céline Prunier
,
Marcia Pessah
,
Nathalie Ferrand
,
Su Ryeon Seo
,
Philip Howe
and
Azeddine Atfi
¶
From the
Department of Cell Biology, Lerner
Research Institute, Cleveland Clinic Foundation Cleveland, Ohio 44195 and
INSERM U482, Hôpital Saint-Antoine, 184
Rue du Faubourg Saint-Antoine, 75571, Paris Cedex 12, France
Received for publication, April 29, 2003
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ABSTRACT
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The phosphorylation of Smad2 and Smad3 by the transforming growth factor
(TGF)-
-activated receptor kinases and their subsequent
heterodimerization with Smad4 and translocation to the nucleus form the basis
for a model how Smad proteins work to transmit TGF-
signals. The
transcriptional activity of Smad2-Smad4 or Smad3-Smad4 complexes can be
limited by the corepressor Ski, which is believed to interact with Smad
complexes on TGF-
-responsive promoters and represses their ability to
activate TGF-
target genes by assembling on DNA a repressor complex
containing histone deacetylase. Here we show that Ski can block TGF-
signaling by interfering with the phosphorylation of Smad2 and Smad3 by the
activated TGF-
type I receptor. Furthermore, we demonstrate that
overexpression of Ski induces the assembly of Smad2-Smad4 and Smad3-Smad4
complexes independent of TGF-
signaling. The ability of Ski to engage
Smad proteins in nonproductive complexes provides new insights into the
molecular mechanism used by Ski for disabling TGF-
signaling.
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INTRODUCTION
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Members of the transforming growth factor-
(TGF-
)1
superfamily play a critical role in regulating many diverse biological
processes, including cell growth regulation, specification of development
fate, differentiation, and apoptosis
(13).
TGF-
signaling is initiated when the ligand induces formation of a
heteromeric complex, composed of type I (T
RI) and type II Ser/Thr kinase
receptors
(46).
Activation of the receptor complex occurs when the type II receptor
transphosphorylates the type I receptor in the GS domain, thus activating the
type I kinase that targets downstream substrates, such as the
receptor-activated Smads (R-Smads), Smad2 and Smad3
(46).
Smad2 and Smad3 are specifically recruited to the receptor complex by an
adapter molecule called SARA (Smad anchor for receptor activation)
(7). Receptor-mediated
phosphorylation of Smad2 and Smad3, which occurs within a conserved
carboxyl-terminal SS(M/V)S motif, induces their association with the common
mediator Smad4, and these complexes enter the nucleus, where they activate
transcription of specific genes
(5).
The ability of Smads to modulate transcription in response to ligand
results from a functional cooperativity with the transcriptional coactivators
CBP and p300, which are thought to mediate activation of some TGF-
target genes by bringing Smad2 or Smad3 within the proximity of the general
transcription machinery and by modifying the chromatin structure through
histone acetylation (5,
8). In addition to interacting
with coactivators, Smads can bind with nuclear transcriptional repressors as
well, including the oncoprotein Evi-1, TGIF, and SNIP1
(911).
Evi-1 has been shown to interact with Smad3 but not Smad2 and is able to
inhibit the activation of TGF-
-responsive promoters by disrupting the
binding of the Smad3-Smad4 complex to DNA
(9). TGIF is a DNA-binding
homeodomain protein that can interact with TGF-
-activated Smads and
repress expression of TGF-
target genes
(10). Transcriptional
repression by TGIF is dependent on its ability to recruit other
transcriptional corepressors, including histone deacetylases (HDACs), carboxyl
terminus-binding protein, and mSin3A
(10,
12,
13). The interaction of TGIF
with TGF-
-activated Smads also displaces the coactivators CBP/p300, thus
reducing the ability of Smads to activate transcription
(10). A similar mechanism has
been proposed to explain the action of the corepressors SNIP1, which interacts
with Smads on TGF-
-responsive promoters and represses their ability to
activate TGF-
target genes by competing with CBP/p300 for Smad
interaction (11).
Additional inhibitors of the transcriptional functions of Smad proteins
include c-Ski, the cellular counterpart of the v-Ski oncoprotein, and the
related protein SnoN (Ski-related novel gene)
(1419).
The oncogenic v-Ski was originally identified in avian Sloan-Kettering viruses
and found to transform chicken embryo fibroblasts
(20). The v-Ski oncoprotein is
truncated at the carboxyl terminus by 312 amino acids relative to the c-Ski
protein, but this truncation does not play a role in the activation of
ski as an oncogene
(21). Overexpression of either
c-Ski or v-Ski induces morphological transformation and anchorage-independent
growth in chicken and quail embryo fibroblasts, indicating that the
transforming activity is attributable to overexpression, not truncation, of
the c-Ski protein (22,
23). Recombinant c-Ski protein
purified from bacteria cannot directly bind to DNA, but c-Ski in nuclear
extracts from mammalian cell cultures binds to DNA, suggesting that c-Ski
lacks a DNA ability on its own and instead regulates transcription via its
ability to associate with other proteins
(24,
25). Recently, diverse types
of studies converged on the conclusions that Ski binding to DNA is mediated in
part through its association with the R-Smad-Smad4 heteromeric complexes
(14,
15,
17). Because Ski was found to
be a component of the HDAC complex through binding to the nuclear hormone
receptor corepressors nuclear hormone receptor corepressor and mSin3A
(26,
27), it has been postulated
that one of the mechanisms used by Ski to repress Smad signaling involves the
formation of a transcriptional repressor complex
(14,
15,
17). However, alternative
mechanisms of repression of Smad signaling may also exist because Ski has been
shown to prevent the formation of a functional R-Smad-Smad4 heteromeric
complex, thereby inactivating its ability to activate transcription
(28). In this study, we report
an additional and heretofore unexpected role of Ski in the negative regulation
of Smad activity by showing that Ski can function to prevent the
phosphorylation of Smad2 and Smad3 by the activated type I receptor. Our
results suggest a new mechanism for the silencing of Smad signaling by the
oncoprotein Ski.
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EXPERIMENTAL PROCEDURES
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Plasmids and Cell CultureThe following plasmids have
previously been described: EF-FLAG-Smad4, EF-FLAG-Smad4.NES and
EF-FLAG-Smad.NLS (29),
pcDNA36xMyc-Ski and pcDNA36xMyc-Ski.delA
(14), pCMV5-FLAG-Ski
(15), pCMV5-HA-T
RI,
pCMV5-HA-T
RI act, pCMV5-FLAG-Smad2, pCMV5-FLAG-Smad2.3SA,
pcDNA36xMyc-Smad2, and pCMV5-HA-Smad4
(30), and
pSC26xMyc-Fast1
(31). COS-7, Mv1Lu, and
Mv1Lu-Ski13 cells were maintained in Dulbecco's modified Eagle's medium
supplemented with 10% heat-inactivated fetal calf serum, 5 mM
glutamine, and antibiotics.
Immunoprecipitation and ImmunoblottingCOS7 cells
transfected by LipofectAMINE Plus (Invitrogen) were lysed at 4 °C in TNMG
buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl, 5
mM MgCl2, 10% glycerol, 0.5% Nonidet P-40, 1
mM sodium vanadate, 1 mM phenylmethylsulfonyl fluoride,
20 µg/ml aprotinin, and 20 µg/ml leupeptin) and subjected to
immunoprecipitation with the appropriate antibody for 2 h, followed by
adsorption to Sepharose-coupled protein G for 1 h. The precipitates were
washed five times in TNMG, and the immunoprecipitates were separated by
SDS-PAGE, transferred to a nitrocellulose membrane, and probed with the
indicated primary antibody. The bands were visualized by an enhanced
chemiluminescent detection system according to the manufacturer's instructions
(ECL; Amersham Biosciences). For determination of total protein levels,
aliquots of cell lysates were subjected to direct immunoblotting. The
following antibodies were used for immunoprecipitation and Western blot
analysis: anti-FLAG mouse monoclonal antibody (Clone M2; Sigma), anti-Myc
mouse monoclonal antibody (Clone 9E10; Santa Cruz), anti-HA mouse monoclonal
antibody (Clone 12C5A; Roche Applied Science), anti-HA rabbit polyclonal
antibody (Santa Cruz), anti-phospho-Smad2 rabbit polyclonal antibody (Zymed
Laboratories Inc.), anti-Smad2 rabbit polyclonal antibody (Zymed Laboratories
Inc.), and anti-phospho-Smad3 rabbit polyclonal antibody (generous gift from
Dr. Ten Dijke).
ImmunofluorescenceCOS7 cells were plated to semiconfluency
and 24 h later transfected with the indicated expression vectors by the
LipofectAMINE method. 48 h after transfection, the slides were washed twice in
phosphate-buffered saline, fixed in 4% paraformaldehyde for 30 min at room
temperature, and permeabilized in 0.1% Triton X-100. The cells were incubated
overnight at 4 °C with a mixture of polyclonal anti-FLAG (Sigma) and
monoclonal anti-Myc 9E10 antibodies. The cells were washed with
phosphate-buffered saline, incubated with a mixture of Texas Red-conjugated
goat anti-rabbit and fluorescein isothiocyanate-conjugated goat anti-mouse
antibodies, and examined on a Leica fluorescent microscope.
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RESULTS
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Ski Interacts with Smad4 in the CytoplasmWe and others have
recently reported that Ski is uniformly distributed throughout cells both in
the absence and the presence of TGF-
signaling
(32,
33). To investigate the
potential of a mechanistic relationship between cytoplasmic localization of
Ski and Ski-dependent repression of Smad signaling, we further extended the
analysis of the interaction of Ski and Smad proteins. To do so, COS-7 cells
were transfected with Ski and Smad2 or Smad4, together with either wild-type
or constitutively activated T
RI, which contains a substitution of Thr
residue 204 to Asp and signals TGF-
responses in the absence of ligand
and the type II receptor (34).
As shown in Fig. 1A, a
weak interaction of Smad2 with Ski could be detected in unstimulated cells,
and this interaction was induced by expression of the activated type I
receptor, consistent with published results
(14,
15,
17,
19). In contrast, the
interaction of Ski and Smad4 occurred in both the absence and the presence of
TGF-
signaling (Fig.
1B).
Because the mechanism of repression of Smad signaling appears to involve
the formation of a transcriptional repressor complex on DNA
(14,
15,
17) and because Smad4 shuttles
continuously between the cytoplasm and the nucleus in the absence of
TGF-
signaling because of the combined activities of nuclear import and
export signals present in Smad4
(29), we wanted to investigate
whether Ski can form a complex with a Smad4 that is already nuclear prior to
TGF-
signaling. To test this possibility, we employed two Smad4 mutants,
one (Smad4.NLS) with the five conserved Lys residues in the NLS mutated to Ala
and another (Smad4.NES) with the two conserved Leu residues in the NES mutated
to Ala. The Smad4.NLS mutant was completely excluded from the nucleus, whereas
the Smad4.NES mutant was exclusively localized in the nucleus
(Fig. 2C and Ref.
29). To detect the
interaction, cell lysates from transiently transfected COS-7 cells were
subjected to immunoprecipitation with anti-FLAG antibody directed toward
tagged Smad4 mutants, followed by immunoblotting with anti-Myc antibody for
the presence of Ski. Unexpectedly, we found that mutation of NLS in Smad4
induced a strong increase in the association of Ski and Smad4
(Fig. 2A), suggesting
that Ski may associate with Smad4 in the cytoplasm. Consistent with this
hypothesis, we observed little or no interaction of Ski with Smad4.NES,
despite efficient expression of this Smad4 mutant
(Fig. 2A). As a
control, we investigated the ability of these Smad4 mutants to associate with
Smad2 in response to TGF-
signaling. Similar to wild-type Smad4, the
association of Smad4.NLS or Smad4.NES with Smad2 was strongly increased by
activation of TGF-
signaling (Fig.
2B), eliminating the possibility that mutations in NLS
and NES motifs may affect the folding of Smad4 protein.

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FIG. 2. Smad4 associates with Ski in the cytoplasm. A, COS-7 cells
were transfected with 6xMyc-Ski together with wild-type FLAG-Smad4,
FLAG-Smad4.NES, or FLAG-Smad4.NLS. The cell lysates were subjected to
anti-FLAG immunoprecipitation (IP) and then immunoblotted with
anti-Myc antibody ( -Myc). Expression of transfected DNA was
monitored by direct immunoblotting. B, FLAG-Smad4, FLAG-Smad4.NES, or
FLAG-Smad4.NLS were cotransfected in COS-7 cells with 6xMyc-Smad2 and
with HA-T RI or HA-T RI act. Association of Smad2 with various Smad4
mutants was analyzed by blotting the FLAG immunoprecipitates (IP)
with anti-Myc ( -Myc) antibody. Direct Western blotting
monitored expression of transfected proteins. C, COS-7 cells were
transfected with 6xMyc-Ski together with wild-type FLAG-Smad4,
FLAG-Smad4.NES, or FLAG-Smad4.NLS. 48 h after transfection, the cells were
fixed, and the localizations of 6xMyc-Ski (green) and/or Smad4
mutants (red) were visualized by a fluorescent microscope.
Colocalization of Ski and Smad4 mutants (merge) appears as
yellow.
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To provide further evidence that the association between Ski and Smad4 may
take place in the cytoplasm, we transfected COS-7 cells with Ski and wild-type
or Smad4 mutants and visualized the proteins by immunofluorescence. In cells
expressing c-Ski alone, the Ski immunoreactivity was diffuse and was found
throughout the transfected cells (Fig.
2C) as described previously
(32,
33). Analysis of cells
transfected with wild-type Smad4 and Ski revealed that Ski colocalized with
wild-type Smad4, and the distribution of Ski was indistinguishable from cells
transfected with Ski alone (Fig.
2C). The distribution of Ski both in the cytoplasm and
the nucleus was also not altered by the expression of Smad4.NES, which was
found exclusively in the nucleus (Fig.
2C), consistent with our biochemical analysis showing
that Smad4.NES is defective in its ability to interact with Ski
(Fig. 2A). In
contrast, coexpression of Smad4.NLS caused Ski to accumulate predominantly in
the cytoplasm, presumably because of the interaction of Ski and Smad4.NLS
(Fig. 2C). Similar
results were obtained in cells cotransfected with the constitutively activated
type I receptor (data not shown), suggesting that the constitutive interaction
of Ski and Smad4 may occur exclusively in the cytoplasm independent of
TGF-
signaling.
Ski Induces a Ligand-independent Association of Smad2 or Smad3 with
Smad4 The observations that Ski constitutively interacts with
Smad4 in the cytoplasm suggested the possibility that Ski might act to exert
its inhibitory function by interfering with the assembly of R-Smad-Smad4
heteromeric complexes in response to TGF-
signaling. To test this
hypothesis, COS-7 cells were transfected with Smad2 and Smad4 in the presence
or absence of Ski and wild-type or activated T
RI. As shown in
Fig. 3A, association
of Smad2 with Smad4 was strongly increased by the activated type I receptor,
similar to previous observation
(35,
36). Surprisingly,
cotransfection of Ski resulted in a ligand-independent increase in the amount
of Smad2 bound to Smad4 (Fig.
3A). A similar conclusion could be drawn when Smad3 was
used instead of Smad2, suggesting that Ski can support the formation of
R-Smad-Smad4 heteromeric complexes in a manner independent of TGF-
signaling.
The phosphorylation of Smad2 and Smad3 at the carboxyl-terminal SS(M/V)S
motif by the activated TGF-
receptor is required for subsequent
interaction with Smad4 and the nuclear translocation of the complexes
(5,
8). In principle, the formation
of the Smad2-Smad4 complex by coexpression of Ski could be achieved either by
the ability of Ski to induce phosphorylation of Smad2 at the carboxyl-terminal
SSMS motif through as yet to be identified mechanism or, alternatively, to
induce the formation of Smad2-Smad4 complexes without affecting the
phosphorylation of Smad2. To distinguish between these two possibilities, we
determined the effect of Ski on the association of Smad4 with either wild-type
Smad2 or Smad2(3SA) that contains Ala mutations in the three Ser of the
sequence SSMS. As anticipated, coexpression of the activated receptor resulted
in a strong increase in the amount of wild-type Smad2 that coprecipitated with
Smad4, whereas Smad2(3SA)-Smad4 complexes were not detected
(Fig. 3B). In
contrast, in cells expressing Ski, Smad2(3SA) strongly associates with Smad4
with an efficiency approaching that elicited by transfection of wild-type
Smad2 (Fig. 3B),
indicating that the ability of Ski to induce the association of Smad2 and
Smad4 is not due to activation of endogenous TGF-
receptors, which in
turn phosphorylates Smad2, leading to the formation of Smad2-Smad4
complexes.
Ski Blocks the Phosphorylation of Smad2 and Smad3The
finding that Ski induces the formation of Smad2-Smad4 complexes independent of
TGF-
signaling raised questions about the ability of the activated
TGF-
receptor to recognize and phosphorylate R-Smads that are already
engaged in complexes with Ski and Smad4. To investigate whether Ski could
interfere with the ligand-dependent phosphorylation of Smad2, we transfected
COS-7 cells with 6xMyc-Smad2 and FLAG-Ski together with either wild-type
or constitutively activated T
RI and assessed the phosphorylation of
6xMyc-Smad2 by an anti-phospho-Smad2 antibody that specifically
recognizes TGF-
receptor-phosphorylated Smad2
(30). As described previously
(30), coexpression of Smad2
with activated T
RI resulted in a strong increase in Smad2
phosphorylation (Fig.
4A). In contrast, overexpression of Ski completely
inhibited the phosphorylation of Smad2 in response to TGF-
signaling
(Fig. 4A). The
inhibitory effect of Ski on TGF-
-mediated phosphorylation of Smad2
depended on the amount of Ski expressed in the cells
(Fig. 4B). A similar
inhibitory effect of Ski was observed in cells cotransfected with
6xMyc-Smad3, suggesting that Ski may function to inhibit TGF-
signaling by preventing the ligand-dependent phosphorylation of Smad2 and
Smad3 (Fig. 4C).

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FIG. 4. Ski prevents receptor-dependent phosphorylation of Smad2 and Smad3.
A, COS-7 cells were transfected with the indicated combinations of
6xMyc-Smad2, HA-T RI, or HA-T RI act either in the absence or
the presence of FLAG-Ski. The cell lysates were subjected to anti-Myc
immunoprecipitation (IP) and then immunoblotted with
anti-phospho-Smad2 antibody ( -p-Smad2). The phosphorylation of
Smad2 was also assessed by immunoblotting total cell lysates with
anti-phospho-Smad2 antibody ( -p-Smad2). Expression of
transfected DNA was monitored by direct immunoblotting. B, COS-7
cells were transfected with 6xMyc-Smad2 and the indicated amounts of
FLAG-Ski together with HA-T RI or HA-T RI act, and total cell
lysates were subjected to immunoblotting with anti-phospho-Smad2 antibody
( -p-Smad2). The expression of transfected DNA was determined
by immunoblotting total cell lysates using anti-FLAG ( -FLAG),
anti-Myc ( -Myc), or anti-HA ( -HA) antibodies.
C, cell lysates from transiently transfected COS-7 cells were
subjected to immunoprecipitation with anti-Myc antibody (IP) directed
toward Smad3 and then immunoblotted using anti-phospho-Smad3
( -p-Smad3) antibody that specifically recognizes TGF-
receptor-phosphorylated Smad3. The relative levels of transfected proteins
were determined by direct Western blotting of total cell lysates. D,
Mv1Lu cells stably transfected with empty vector (Control) or Ski
(Ski13) were treated with TGF- for 15 min, and phosphorylation
of endogenous Smad2 was assessed by immunoblotting total cell lysates with
anti-phospho-Smad2 antibody ( -p-Smad2). For comparison, the
same membrane was reprobed with a polyclonal anti-Smad2 antibody
( -Smad2). E, COS-7 cells were transfected with
FLAG-Smad2 or FLAG-Smad2(3SA) in the presence or absence of 6xMyc-Ski
and HA-T RI or HA-T RI act. The cell lysates were subjected to
immunoprecipitation with anti-Myc antibody (IP) and then
immunoblotted with anti-FLAG antibody ( -FLAG). The expression
of transfected DNA was determined by immunoblotting total cell lysates using
anti-Myc ( -Myc), anti-FLAG ( -FLAG), or anti-HA
( -HA) antibodies.
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In the course of these experiments, we found that when the phosphorylation
of 6xMyc-Smad2 was monitored by immunoprecipitation followed by Western
blotting with anti-phospho-Smad2 antibody, a protein that was the size
expected for endogenous Smad2 associated with 6xMyc-Smad2 and became
phosphorylated by activated T
RI, and this TGF-
-dependent
phosphorylation is blocked by overexpression of Ski
(Fig. 4A). Because
Smad2 protein can form homodimer in response to TGF-
signaling, this
result suggested that Ski might also prevent the phosphorylation of endogenous
Smad2 in response to TGF-
signaling. To examine this directly, we
analyzed the phosphorylation of endogenous Smad2 in response to activation of
endogenous TGF-
receptors using the previously defined cell line
Mv1Lu-Ski13, in which the stable expression of Ski suppressed the ability of
Smads to mediate TGF-
-induced growth arrest and transcriptional
responses (17). Western
blotting analysis with anti-phospho-Smad2 antibody from the parental cell line
Mv1Lu that was untreated or treated with TGF-
for 15 min revealed a
ligand-dependent phosphorylation of endogenous Smad2
(Fig. 4D). In
contrast, in the Mv1Lu-Ski13 cell line, we could not observe any effect of
TGF-
on Smad2 phosphorylation (Fig.
4D). Thus, in stably transfected cells and in transient
transfection assays, the presence of Ski caused a loss of Smad2
phosphorylation in response to TGF-
signaling.
Because Ski appears to prevent the ligand-dependent phosphorylation of
Smad2 on the SSMS motif (Fig. 4, A,
B, and D), we investigated how the formation of a
complex between Ski and Smad2 is increased in response to TGF-
signaling
(Fig. 1A). To do this,
we analyzed Ski interaction with wild-type Smad2 or the Smad2(3SA) mutant,
which is defective in its ability to be phosphorylated by the activated type I
receptor. As with wild-type Smad2, we observed a strong increase in the amount
of Smad2(3SA) that associates with Ski upon activation of TGF-
signaling
(Fig. 4E). These data
suggest that TGF-
signaling can induce the association of Ski and Smad2
independently of Smad2 phosphorylation by the activated type I receptor.
The Suppression of Smad2 Phosphorylation by Ski Is Linked to the
Association of Smad4 with SkiPrevious studies have shown that Ski
contains discrete binding sites for Smad2 and Smad4
(14,
15,
28). The availability of a
mutant of Ski (Ski.delA) that interacts with Smad2 but fails to bind to Smad4
(14) allowed us to address the
question of whether Ski repressing TGF-
-mediated activation of Smad2 is
dependent on the presence of Smad4. We first examined the ability of Ski.delA
to modulate the association of Smad2 and Smad4 in response to TGF-
signaling. Consistent with our previous analysis
(Fig. 3A),
overexpression of wild-type Ski resulted in a strong increase in the
association of Smad2 and Smad4 independent of TGF-
signaling
(Fig. 5A). In contrast
to wild-type Ski, Ski.delA, which was expressed efficiently, had little or no
ability to induce the association of Smad2 and Smad4 in the absence of
TGF-
signaling (Fig.
5A). More importantly, in the presence of TGF-
signaling, we found an
3-fold increase in the association of Smad2 and
Smad4 in cells transfected with Ski.delA relative to cells transfected with
control vector (Fig.
5A), raising the interesting possibility that deletion of
the Smad4 binding site might generate a specific dominant inhibitory form of
Ski. From these results, it can be suggested that Ski might mediate the
ligand-independent formation of Smad2-Smad4 heteromeric complex through its
association with Smad4.
We next evaluated the effect of Ski.delA on the ability of the activated
type I receptor to induce the phosphorylation of Smad2. As described above
(Fig. 4, A and
D), coexpression of wild-type Ski with Smad2 resulted in
a marked reduction in Smad2 phosphorylation induced by the constitutively
activated type I receptor (Fig.
5B). In contrast, expression of Ski.delA along with Smad2
increased the sensitivity of the cells, yielding a34-fold increase in
receptor-induced Smad2 phosphorylation
(Fig. 5B). Together,
these results suggest that the ability of Ski to prevent TGF-
-dependent
phosphorylation of Smad2 is linked to the association of Ski with Smad4 and
further suggest that Ski.delA might act as a dominant-negative inhibitor by
blocking the function of endogenous Ski protein.
Ski Induces the Formation of Inactive Smad2-Smad4
ComplexesFollowing TGF-
-mediated phosphorylation and
association with Smad4, Smad2 moves to the nucleus and activates expression of
specific genes through cooperative interactions with specific DNA-binding
proteins. For example, activation of the Mix2 gene by TGF-
or
activin-related ligands require the formation of a Smad2-Smad4-Fast1 complex
that binds to a specific promoter sequence known as the activin response
element (37,
38). To provide further
evidence that Ski can block the TGF-
-dependent phosphorylation of Smad2,
we tested whether it could interfere with the assembly of Smad2-Fast1 complex
in response to TGF-
signaling. For this, COS-7 cells were transfected
with Fast1 and Smad2 in the presence or absence of Ski and wild-type or
activated T
RI. In the absence of TGF-
signaling, weak interaction
between Fast1 and Smad2 could be detected
(Fig. 6). However, coexpression
with the activated T
RI enhanced the interaction of Fast1 with Smad2
(Fig. 6), supporting the notion
that receptor-dependent phosphorylation of Smad2 is required for subsequent
association with Fast1 (37,
38). Interestingly, in cells
expressing Ski, we observed almost a complete block in the formation of the
Smad2-Fast1 complex in response to TGF-
signaling
(Fig. 6). To determine whether
Ski inhibits the TGF-
-dependent association of Smad2 and Fast1 through
its constitutive association with Smad4, we investigated the function of the
mutant Ski.delA, which interacts with Smad2 but not with Smad4. In contrast to
wild-type Ski, expression of Ski.delA enhanced the ability of the activated
type I receptor to induce the association of Smad2 and Fast1
(Fig. 6), consistent with the
notion that Ski.delA might act as a dominant-negative inhibitor.
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DISCUSSION
|
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TGF-
is a potent natural antiproliferative agent that plays an
important role in suppressing tumorigenicity
(39). Tumor cells can acquire
resistance to the antiproliferative effect of TGF-
by a number of
different mechanisms including defects in TGF-
cell surface receptors
and mutational inactivation of downstream effector components of the signaling
pathways, such as Smad2 and Smad4
(2,
39). Elevated expression of
the proto-oncogene c-Ski may also provide an alternative mechanism whereby
tumor cells down-regulate TGF-
responsiveness and escape its tumor
suppressor function. Consistent with this notion, overexpression of Ski
resulted in suppression of TGF-
-induced transcriptional activation and
caused cells to resist the growth inhibition by TGF-
(14,
15,
17,
19). The proposed mechanisms
of Ski repression of TGF-
signaling appears to depend on its ability to
recruit a nuclear repressor complex consisting of nuclear hormone receptor
corepressor, mSin3A, and HDAC1 to TGF-
-responsive promoter elements
through its interaction with Smad proteins
(15,
17). Interaction of Ski with
TGF-
-activated Smads also prevents interaction with the coactivator
CBP/p300, thus reducing the ability of Smads to activate transcription
(14,
28). In addition to the
recruitment of a transcriptional complex and dissociation of CBP/p300, binding
of Ski to the Smads converts the Smad heterocomplex into an inactive
conformation (28). Recently,
we and others have reported that Ski can also reside in the cytoplasm
(32,
33,
40), suggesting that the
mechanism for repression of Smad functions by Ski may not be restricted to its
ability to repress transcription or to disrupt a functional Smad
heterocomplex. Indeed, we show here that Ski acts at a very early step to
suppress TGF-
-mediated activation of the Smad signaling pathway by
preventing the ligand-dependent phosphorylation of Smad2 and Smad3. This
inhibitory function of Ski is linked to its ability to constitutively
interacts with Smad4 in the cytoplasm, Furthermore, we demonstrate that
overexpression of Ski induces the assembly of nonproductive R-Smad-Smad4
heteromeric complexes independent of TGF-
signaling. These studies thus
provide a novel mechanism for the suppression of TGF-
signaling by the
Ski oncoprotein.
In the current model for TGF-
signaling, Smad4 is assumed to be
retained in the cytoplasm in the absence of a signal, but it accumulates in
the nucleus following stimulation of cells with TGF-
, presumably because
of formation of complexes with the R-Smads Smad2 and Smad3
(5,
6,
8). However, Ski was shown to
constitutively associate with Smad4 in both the presence and the absence of
TGF-
signaling. Our study indicates that this constitutive interaction
of Ski with Smad4 occurs in the cytoplasm in a TGF-
-independent manner.
Consistent with this, mutations in Smad4 NLS motif that completely excluded
Smad4 from the nucleus strongly enhance the association of Smad4 and Ski.
Further evidence for the association of Ski and Smad4 in the cytoplasm is
provided by the inability of Ski to interact with the Smad4.NES mutant, which
is exclusively localized in the nucleus.
The present work also demonstrates that Ski can induce the formation of
inactive R-Smad-Smad4 heteromeric complexes independent of TGF-
signaling, thereby interfering with the phosphorylation of Smad2 and Smad3 by
the activated TGF-
type I receptor. The Smad proteins contain two well
conserved domains, the amino-terminal MH1 domain and the carboxyl-terminal MH2
domain, which are separated by a proline-rich linker that differs
substantially between the different classes of Smad
(5). Receptor-mediated
phosphorylation appears to relieve these two domains from a mutually
inhibitory interactions, and this conformation is required for the formation
of R-Smad-Smad4 heteromeric complexes
(41). One possibility is that
conformational changes in unphosphorylated R-Smad, as a consequence of their
constitutive interaction with Smad4 caused by overexpression of Ski, may in
turn interfere with the access of R-Smad to the activated receptor and lead to
the suppression of R-Smad phosphorylation. Another not mutually exclusive
possibility is that Ski-mediated association of unphosphorylated R-Smad with
Smad4 may alter the affinity of R-Smad to the adaptor molecule SARA, which
recruits unphosphorylated R-Smad to the receptors for phosphorylation
(7). Indeed, recent evidence
has suggested that the binding of Smad4 to R-Smads serves as a dual signal for
dissociation of R-Smad from SARA and subsequent accumulation of the Smad
heterocomplexes in the nucleus
(7).
Because the interaction of Ski with R-Smad was increased in response to
TGF-
signaling, it is tempting to speculate that the ligand-dependent
phosphorylation of Smad2 at the carboxyl-terminal serines that serve as
TGF-
receptor phosphorylation sites is required for their association
with Ski (14,
15,
17,
19). Paradoxically, we found
that expression of Ski completely blocks the T
RI-dependent
phosphorylation of Smad2. This discrepancy is resolved by the evidence
presented here that activation of TGF-
can also lead to the association
of Ski and Smad2(3SA), which is defective in its ability to be phosphorylated
by the activated type I receptor. These observations not only suggest that Ski
could interact with the unphosphorylated form of R-Smad but also raised the
interesting possibility that TGF-
signaling might enhance the
association of Ski and Smad2 independent of phosphorylation of Smad2 by the
activated type I receptor. At present we do not understand the mechanism
whereby activation of TGF-
signaling induces the association of Ski with
the unphosphorylated form of Smad2.
It was recently proposed that Ski interacts with Smads on
TGF-
-responsive promoters and represses their ability to activate
TGF-
target genes, based on the reported ability of Ski to associate
with nuclear hormone receptor corepressor, mSin3A, and HDAC1
(14,
15,
17,
26,
27). In contrast, our
molecular model and the supporting data strongly suggest that Ski suppresses
TGF-
-Smad signaling through its ability to interfere with the
phosphorylation of R-Smad by the activated type I receptor. Because
overexpression of Ski suppressed the majority of TGF-
-mediated
phosphorylation of R-Smad, we suggest that perhaps only a small proportion of
R-Smad proteins is incorporated into a transcriptional corepressor complex
containing Ski and its associated HDAC. Consistent with this interpretation,
suppressing the activity of HDAC by the inhibitor trichostatin A did not
interfere with the ability of Ski to repress a TGF-
-inducible promoters
containing the Smad3/4-binding element SBE
(19). Furthermore,
overexpression of Ski in melanomas cells has been shown to prevent Smad3
nuclear translocation in response to TGF-
signaling
(32), reinforcing the notion
that the inhibitory effect of Ski on TGF-
signaling is not attributable
to transcriptional repression in the nucleus. From these molecular studies, we
concluded that the inhibitory effects of the oncogenic Ski on TGF-
signaling derive in part through its ability to prevent the ligand-dependent
phosphorylation of R-Smads.
 |
FOOTNOTES
|
---|
* This work was supported by INSERM, CNRS, la Ligue contre le Cancer
Comité de Paris, and l' Association pour la Recherche sur le Cancer.
The costs of publication of this article were defrayed in part by the payment
of page charges. This article must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section 1734
solely to indicate this fact. 
¶
To whom correspondence should be addressed. Tel.: 33-1-49-28-46-11; Fax:
33-1-40-19-90-62; E-mail:
atfi{at}pop.st-antoine.inserm.fr.
1 The abbreviations used are: TGF-
, transforming growth factor
;
HDAC, histone deacetylases; T
RI, TGF-
type I receptor; R-Smad,
receptor-activated Smad; CBP, cAMP-responsive element-binding protein-binding
protein; HA, hemagglutinin; NLS, nuclear localization signal; NES, nuclear
export signal; HA-T
RI act, constitutively active TGF-
type I
receptor. 
 |
ACKNOWLEDGMENTS
|
---|
We thank Drs. C. Hill, K. Miyazono, K. Luo, P. tenDijkle, and R. Weinberg
for providing reagents.
 |
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