ski-interacting protein (skip) interacts with smad proteins to
TRANSCRIPT
SKIP interacts with Smads to activate TGF-β signalling
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Ski-interacting protein (SKIP) interacts with
Smad proteins to augment TGF-β-dependent
transcription**.
Gary M. Leong∞∗ , Nanthakumar Subramaniam∞, Jonine
Figueroa∆, Judith L. Flanagan∞, Michael J. Hayman∆, John A.
Eisman∞ and Alexander P. Kouzmenko∞.
∞Bone & Mineral Research Program, Garvan Institute of Medical Research,
Sydney, Australia and ∆Dept. of Molecular Genetics & Microbiology, State
University of New York, Stony Brook, New York, U.S.A.
∗To whom correspondence should be addressed: Bone & Mineral
Research Program, Garvan Institute of Medical Research, 384 Victoria
Street, Darlinghurst, 2010, Australia. Tel.:61-2-9295-8247; Fax:61-2-
9295-8241;E-mail:[email protected].
Running Title: SKIP interacts with Smads to activate TGF-β signalling
Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
JBC Papers in Press. Published on March 6, 2001 as Manuscript M010815200 by guest on M
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Abstract
Transforming growth factor-β (TGF-β) signalling requires the action
of Smad proteins in association with other DNA-binding factors and
coactivator and corepressor proteins to modulate target gene
transcription. Smad2 and Smad3 both associate with the c-Ski and
Sno oncoproteins to repress transcription of Smad target genes via
recruitment of an N-CoR-repressor complex. Ski-interacting protein
(SKIP), a nuclear hormone receptor coactivator, was examined as a
possible modulator of transcriptional regulation of the TGF-β
responsive promoter from the plasminogen activator inhibitor gene-1
(PAI-1). SKIP augmented TGF-β-dependent transactivation in
contrast to Ski/Sno-dependent repression of this reporter. SKIP
interacted with Smad 2 and 3 proteins in vivo in yeast and in
mammalian cells through a region of SKIP between aa201-333. In
vitro deletion of the MH2 domain of Smad3 abrogated SKIP binding,
like Ski/Sno, but the MH2 domain of Smad3 alone was not sufficient
for protein-protein interaction. Overexpression of SKIP partially
overcame Ski/Sno-dependent repression, while Ski/Sno
overexpression attenuated SKIP augmentation of TGF-β−dependent
transcription. Our results suggest a potential mechanism for
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transcriptional control of TGF-β signalling that involves the opposing
and competitive actions of SKIP and Smad MH2-interacting factors,
such as Ski and/or Sno. Thus, SKIP appears to modulate both TGF-β
and nuclear hormone receptor signalling pathways.
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INTRODUCTION
Transforming growth factor-β (TGF-β) superfamily members are
multifunctional cell-cell signalling proteins which include the TGF-
βs, bone-morphogenetic proteins (BMPs), activins and inhibins,
mullerian-inhibiting substance and growth differentiation factors (1).
Members of this superfamily mediate many key cellular events in
growth and development and are evolutionarily conserved from
Drosophila to mammals (2). TGF-β signalling requires the action of a
family of DNA-binding proteins called Smads, including TGF-β-
specific (Smad2 and Smad3), BMP-specific (Smad1, Smad5 and
Smad8), a common Smad4 and anti-Smads (Smad6 and Smad7).
TGF-β signals through sequential activation of two cell surface
receptor serine-threonine kinases which phosphorylate Smad2
and/or Smad3. Phosphorylated Smad2 or Smad3, together with
Smad4, translocates into the nucleus where the Smad heterodimer
binds Smad-binding elements (SBEs) in association with other
nuclear factors in promoters of target genes (1,3,4).
Recently it has been shown that Smad proteins also interact with
other nuclear factors such as c-Ski and the Ski-related novel (Sno)
protein and nuclear hormone receptors (NHRs), including the
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vitamin D receptor (VDR) to modulate TGF-β signalling (5-10). Ski
and Sno are involved in oncogenic transformation and enhancement
of muscle differentiation by blocking TGF-β signalling (11-14). The
mechanism of Ski/Sno repression of TGF-β signalling appears to
involve an interaction with a complex consisting of the nuclear
corepressor (N-CoR) and a histone deacetylase enzyme (HDAC)
(15,16). N-CoR, and its related co-repressor silencing mediator for
retinoic acid and thyroid receptors (SMRT), interact with a wide
variety of other nuclear factors to mediate transcriptional repression
(17-19). Interestingly, the Ski-interacting protein (SKIP) was initially
identified in a two hybrid screen using v-Ski as a bait, and was later
independently identified as a VDR- and CBF1-interacting factor (20-
22). Thus, the recent observation that SKIP modulates CBF1 and
Notch-dependent signalling suggests that SKIP may play a role in
the regulation of a number of different and distinct cellular signalling
pathways (23).
As Ski and Sno can modulate TGF-β-dependent signalling, it was of
interest to determine whether SKIP could also modulate the TGF-β-
signalling pathway through interaction with the Smad proteins. In
this study, in contrast to Ski- and Sno-mediated repression, SKIP
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augmented TGF-β-dependent transcription. A region of SKIP,
aa201-333, appeared to be required for the Smad interaction. SKIP
interacted in vitro and in vivo with Smad3 and partially counteracted
Ski- and Sno-dependent repression, while Ski/Sno attenuated SKIP
transactivation of TGF-β signalling. These results suggest that SKIP
may play an opposite role to Ski and Sno in the control of TGF-β-
dependent transcription.
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MATERIALS and METHODS
Plasmid constructs
SKIP wildtype cDNA was PCR cloned with the forward primer 5’-
GGG AAT TCC CGG GGT CTA GAA CCA CCA TGG CGC TCA
CCA GCT TTT TA-3’ and reverse primer 5’- GCG GGA TCC CTA
TTC CTT CCT CCT CTT-3’. The PCR product was ligated into
pGEM-T Easy plasmid (Promega, Madison, WI) from which an
EcoR1/BamH1 insert was excised and sub-cloned into a modified
GAL4AD pACTII plasmid (pACTIIb) and the vector pSG5
(Stratagene, La Jolla, CA). The pACTIIb plasmid was created by
replacing the BglII polylinker fragment of pACTII (Clontech, Palo
Alto, CA) with the double-stranded oligonucleotide: 5’-GAT CTG
TGA ATT CCC GGG GAT CCG TCG ACC TA-3’. The GAL4 DBD-
wild-type Smad-pBridge yeast two hybrid constructs were made by
EcoR1/Xho1 digestion of Smad2-, Smad3- and Smad4-pcDNA3
plasmids (3) and MH2 (400-425aa of hSmad3)-pBridge by PCR and
sub-cloning of Smad cDNAs into the EcoR1/Sal1 sites of pBridge
(Clontech). The GST-MH2 construct was made by cloning cDNA of
PCR product into the EcoR1/Xho1 sites of modified pGEX-4T2
plasmid (Pharmacia). The SKIP deletion constructs (aa1-200, aa1-333,
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aa201-536 and aa334-536) were made by PCR and cloned into the
EcoR1/BglII site of pACTIIb and the Xba1/BamH1 site of pCGN. The
Sno cDNA was amplified by PCR using the forward primer 5’ GCA
ATC TAG AGA AAG CCC ACA AGC AAA TTT CCC-3’ and reverse
primer 5’-GCA AGG ATC CCT ATT TTC CAT TTC CAT TTT TG-3’
and the PCR product ligated into the Xba1/BamH1 site of pCGN.
The GST-SKIP construct was made by PCR and cloned into the EcoR1
and Sal1 sites of pGEX-KG (24). All PCR primer sequences not listed
are available on request. All constructs were sequenced by
automated fluorescent sequencing and confirmed to be in frame and
correct. The wildtype SKIP-pCGN, c-Ski-pMT2, GST-Smad3, Smad2-,
Smad3, Smad4-pcDNA3 constructs and the 3TP-Lux reporter have
been described previously (3,20,25-27).
Yeast two hybrid analysis
Yeast transformation was performed using a lithium acetate
transformation kit (BIO101, Vista, CA). Wildtype SKIP-pACTII
plasmid encoding the SKIP-GAL4-AD fusion protein was
transformed into the Y187 yeast strain and Smad2- , Smad3-, Smad4-
and MH2-pBridge encoding the Smad-GAL4 DBD fusion proteins
were transformed into the opposite yeast mating strain, CG1945. To
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co-express the two different fusion proteins yeast matings were
performed (Clontech Yeast Handbook, PT3024-1). Yeast ligand
experiments were performed as previously described (28). β-
galactosidase activity in protein lysates was measured with the
Tropix Galactolight chemiluminescence assay (Perkin-Elmer,
Brachenburg, NJ) using the Berthold LB953 luminometer (Berthold,
Bad Wildbad, Germany) and expressed as relative light units (RLUs).
All results are shown as mean ± SEM of at least 3 different yeast
colonies, from at least two experiments, and corrected for protein
concentration (Biorad protein assay, Hercules, CA).
Cell culture and reporter assays
COS-1 African Green Monkey kidney cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM) medium with 5% fetal calf serum
(FCS) at 37o in 5% CO2. Cells were plated the day before transfection
in 24 well plates at a cell density of 2x104 cells per well. Transfections
was performed with Fugene-6 transfection reagent (Boehringer-
Mannheim, Indianapolis, IN) as per the manufacturer’s instructions
using 1.5 µL Fugene with 0.75µg total DNA per well. Transfected
cells were left in Fugene transfection reagent for 16-20 hours, then
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treated with TGF-β (Sigma, St.Louis, MI) or vehicle (4mM HCl and
1mg/ml BSA) in 2% charcoal-stripped medium for 16-24 hours. Then
the medium was removed and cells lysed with 2x Promega lysis
buffer. Luciferase assays were performed in triplicate with the firefly
luciferase assay kit (Promega) and measured with a luminometer
(Berthold) .
Glutathione sepharose binding assays
Expression of appropriately sized GST-SKIP and GST-Smad3
wildtype or mutant fusion proteins was confirmed by SDS-PAGE.
GST-binding assays were performed in triplicate with equal amounts
of 35S-labelled SKIP, VDR or luciferase as a negative control (28).
Bound proteins were resolved on 10% SDS-PAGE gels and subjected
to autoradiography. In vitro translation and transcription was
performed according to the manufacturer’s instructions (Promega)
with 35S methionine (Amersham Pharmacia Biotech, Piscataway, NJ).
Far Western and immunoblot analysis
Far Western analysis and preparation of nuclear extracts were
essentially as previously described (5,28). COS1 nuclear extracts
overexpressing Smad3 were run on 10% SDS-PAGE and
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electroblotted onto a polyvinylidne fluoride membrane (PVDF)
(Millipore, Bedford, MA). Proteins were denatured with 6M
guanidine hydrochloride and renatured by the stepwise dilution of
guanidine hydrochloride. The Smad3 membrane was then blocked
and hybridized overnight at 4 degrees with 20 µg of COS1 nuclear
extracts containing HA-SKIP. The filter were rinsed three times in
HYB (20mM Hepes-KOH, pH 7.4, 75mM KCL, 0.1mM EDTA, 2.5 mM
Mg Cl2, 1% nonfat milk, 0.05% IPEGAL and 1mM DTT) and then
probed with an anti-HA antibody (Boeringher-Mannheim) which
detected HA-SKIP, followed by probing with a anti-mouse–HRP
secondary antibody (Santa Cruz) prior to ECL chemiluminescent
detection (Amersham) and autoradiography.
Electromobility Shift Assay (EMSA)
EMSA was performed with the PE2 radiolabelled probe from the
PAI-1 promoter (29), as previously described (10), using in vitro
translated cold Smad3 and Smad4 with COS1 nuclear extracts
overexpressing either SKIP, Sno or Ski or empty vector. A parallel
35S-labelled in vitro translated reaction was performed to ensure
correct translation of the Smad3/4 proteins.
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RESULTS
SKIP augments TGF-β dependent-transcription
As Ski and Sno interact directly with Smad proteins (Smad2 and
Smad3) to repress TGF-β dependent transcription, the effects of SKIP
on the 3TP-lux TGF-β-responsive reporter construct (27) were tested
(Fig. 1). In COS1 cells this reporter responded to TGF-β with a 4-fold
increase in reporter activity, consistent with these cells expressing
endogenous Smad proteins (30). Smad3 alone, or Smad2 and Smad4
together (but neither alone) augmented both basal (2-fold) and TGF-β
responses (10-fold) of this reporter activity. Smad3 co-transfection
with Smad4 led to a 6-fold increase in basal and a 30-fold increase in
ligand-dependent reporter activity. This augmentation was similar to
that of SKIP alone on ligand-dependent reporter activity (Fig.1). An
interaction between SKIP and Smads was suggested in co-
transfection studies with the fold increase of basal activity
progressively increasing when SKIP was co-transfected with Smad2
(8-fold), Smad2 and 4 (20-fold), Smad3 (53-fold) and Smad3 and
Smad4 (164-fold). The comparable increases in TGF-β induced
activity were 39-fold, 116-fold, 96-fold and 323-fold, respectively.
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These data are consistent with a functional interaction primarily
occurring between SKIP and Smad3, with or without exogenous
Smad4.
Mapping of SKIP-Smad interaction domains in yeast and mammalian cells
SKIP interaction with Smad proteins was investigated by yeast two
hybrid interaction analysis. SKIP interacted with both Smad2 and
Smad3 (Fig. 1B). Smad4 induced a high level of reporter activity,
which was unaltered by co-expression of SKIP. However, Smad4, as
expected, interacted strongly with v-Ski-GAL4-AD in yeast (data not
shown). Domains of SKIP required for Smad interaction were
examined using deletion constructs of SKIP (Fig. 1B). The C-
terminally deleted 1-333aa and N-terminally deleted 201-536aa SKIP
mutants had comparable interaction with Smad2 to that of wildtype
SKIP. The interaction between these two mutants and Smad3 were
about 50% and 25% of wildtype SKIP, respectively. No interaction of
the SKIP N-terminal (1-200aa) or C-terminal (334-536aa) domain with
Smad2 or Smad3 was observed. Thus, these results suggest that the
region of SKIP between aa201-333 interacts with Smad2 and Smad3.
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The same SKIP deletion constructs were tested with the 3TP-lux
reporter in the COS1 mammalian cell line (Fig. 1C). Co-expression of
wildtype SKIP (1-536aa) with Smad3 caused a synergistic 3-fold
increase in reporter activity above SKIP or Smad3 alone. The N-
terminal domain of SKIP (1-200aa) had no effect on reporter activity,
while the other SKIP constructs had comparable transactivation to
that of wildtype SKIP. Western blot analysis of these deletion
constructs showed comparable expression with wildtype, except for
the 1-200aa construct, which despite its lack of transactivation, was 2-
3 times more highly expressed (data not shown). Thus, these
transfection data were consistent with the yeast interaction data and
suggest that expression of the 201-333aa region of SKIP with Smad3 is
sufficient for near maximal transactivation of the 3TP-lux reporter.
Surprisingly, the 334-536aa SKIP construct was able to activate the
3TP-lux reporter with Smad3, even though no interaction was
observed with Smad3 in yeast. This suggests that an additional C-
terminal domain may also be transcriptionally functional and
possibly recruits other Smad-interacting co-factors present in
mammalian cells, but not yeast.
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SKIP interaction with Smad2 and Smad3 in vitro
The potential direct physical interaction between the Smad proteins
and SKIP was explored using a GST “pulldown” assay. GST-SKIP
bound both Smad2 and Smad3 (Fig. 2A). In comparison there was
minimal, if at all any binding of Smad2 or Smad3 to GST-0 and no
binding of luciferase to GST-SKIP.
To determine which domains of Smad3 may be involved in SKIP
interaction a GST-Smad3 binding assay was performed with 35S
labelled in vitro translated SKIP (Fig. 2B). GST-wildtype Smad3
bound SKIP and the positive control VDR (9). Deletion of the MH1
domain of Smad3 (aa199-427aa) had no effect on SKIP binding, but as
expected, VDR binding was abolished. Both SKIP and VDR binding
was lost when both the MH1 and MH2 domains of Smad3 were
deleted (GST-Smad3 199-405aa). However, no binding of SKIP was
observed to a GST-MH2 construct which expressed only the last 26 aa
of hSmad3. This result was further supported by a lack of interaction
between SKIP-GAL4-AD and a MH2-GAL4-DBD construct
containing the same C-terminal 26aa of hSmad3 in vivo in yeast (data
not shown). These results indicate that while deletion of the C-
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terminal MH2 domain abrogates SKIP binding, the MH2 domain
alone is not sufficient for SKIP interaction.
To further support the existence of a direct protein-protein
interaction in vitro, a Far western Assay was performed using
mammalian cell nuclear extracts overexpressing HA-SKIP (upper
panel) or Smad3 (middle panel) (FIG. 2C). In the Far Western
analysis (lower panel) Smad3 detected by western analysis co-
localised with HA-SKIP detected by using an anti-HA antibody, but
not with the negative empty vector control extracts. These results
together with the GST-binding studies thus strongly support the
existence of a protein-protein interaction in vitro between SKIP and
Smad3.
Ski and Sno competitively inhibit SKIP-dependent activation
The Smad3 transcriptional repressors, Ski and its related protein, Sno,
are known to bind to the MH2 domain of Smad3 (26). Since SKIP
also interacts with Ski and Sno we tested whether SKIP modulates
Ski/Sno repression of Smad3-dependnet transcription. As shown
above, SKIP increased basal and TGF-β-dependent transactivation,
particularly in the presence of Smad3 (Fig. 3). Both Ski and Sno
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attenuated this SKIP-dependent transactivation by about 80% and
40%, respectively (Fig. 3). SKIP transactivation, either alone or with
Smad3, was repressed in a dose-dependent manner by co-
transfection with Ski. Sno had a similar but weaker effect (Fig. 4).
These data suggest that SKIP and Ski/Sno may act as counteracting
regulators of the TGF-β transcriptional response.
As Ski/Sno interact with both Smad3 and SKIP one alternative
possibility other than a competitive interaction between these
proteins is that they form a ternary complex. To address this question
a gel shift analysis was performed (FIG. 5). Using the PE-2 probe
from the PAI-1 promoter which binds a Smad3/4 heterodimer (29)
(FIG.5 lane 2), we showed that with addition of increasing amounts
of SKIP nuclear extracts there was augmentation of binding of a
higher molecular weight complex which presumably contained SKIP
and Smad3/4 (lane 3 to 6). The addition of Sno nuclear extracts also
led to increased Smad3/4 binding with a similar mobility shift (lane
9-10 & 12 and 13). This complex was specific as it was abrogated by
addition of cold probe (lane 11). However, in the presence of both
SKIP and increasing Sno though we observed increased intensity of
the upper complex, no further supershift and hence no ternary
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complex was observed (lane 7 to 10). Similar results were obtained
using Ski-overexpressing nuclear extracts (data not shown).
DISCUSSION
T h e Ski and Sno oncoproteins have been shown to negatively
modulate TGF-β signalling through an interaction with a N-CoR
repressor complex (15). As SKIP, a nuclear-hormone receptor
interacting cofactor, also associates with both Ski and Sno, this study
was undertaken to determine the potential role of SKIP in TGF-β
signalling. In these studies SKIP augmented TGF-β −dependent
transcription and exhibited a direct interaction with Smad proteins.
This SKIP-Smad interaction was apparent both in vitro and in vivo, as
demonstrated by GST “pulldown” assays, Far Western analysis and
yeast two hybrid protein-protein studies. The region between aa201-
333 within SKIP appeared to act as the Smad-interacting domain,
while, SKIP, like Ski and Sno, interacted with the MH2 domain of
Smad3. Moreover, Ski and Sno attenuated SKIP transactivation,
while SKIP partially counteracted Ski- and Sno-mediated
transcriptional repression.
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The C-terminal MH2 domain of Smad2 and Smad3 has been reported
to be a key region involved in multiple protein-protein interactions,
including those with the coregulators CBP/p300 and the Smad
repressors Ski and Sno (1). The N-terminal MH1 domain of the
Smads confers only low affinity DNA binding to a consensus Smad-
binding element (SBE) (1). Though natural TGF-β-responsive
promoters contain functional clusters of SBEs, other DNA-binding
factors, such as FAST-1, TFE3 and AP-1, as well as non-DNA binding
factors through protein-protein interaction with the C-terminal MH-2
domain are involved in determining the specificity and direction of
Smad target gene action (29,31,32). As such, SKIP appears to play a
role in augmentation of TGF-β-specific Smad transcriptional activity
via an interaction with the MH2 domain of Smad3. Our data also
suggest that as SKIP was unable to interact with an isolated MH2
domain (last 26aa) of Smad3 in GST binding assays and yeast two-
hybrid studies, that other regions within the Smad proteins possibly
within the context of the whole Smad protein may also modulate
Smad-SKIP interaction.
Though SKIP was able to interact with Smad2 and Smad3 in yeast,
SKIP co-transfection with Smad3 with or without Smad4, led to the
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greatest increases in reporter activity in mammalian cells,
presumably because the 3TP-lux reporter is Smad3-selective (27).
Thus, as SKIP interacted with Smad2 in vivo and in vitro, it is also
possible that SKIP in mammalian cells may be able to modulate TGF-
β signalling through Smad2 in certain situations (30). Furthermore,
in the transient transfections we observed that Smad3 augmented
basal reporter activity, as previously described with this promoter
(5), but this activity was further increased by SKIP. Further studies
will be required to address the specific reasons for this effect of SKIP.
The deletional analysis of SKIP in yeast and mammalian cells
suggests that the aa201-333 region of SKIP is required for Smad
interactions in vivo. However some functional differences were
observed between yeast and mammalian cells. Specifically, while the
C-terminal SKIP construct (aa334-536) did not interact with Smad2 or
Smad3 in yeast, its transactivation activity in mammalian cells was
comparable to wildtype SKIP. A C-terminal transactivation domain
of SKIP that functions in mammalian cells, distinct from the Smad
interaction domain, is consistent with the domain C-terminal to aa437
of murine SKIP (NcoA-62) being involved in vitamin-D-dependent
transactivation (21).
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As SKIP interacts with Ski/Sno and Smad3 and in turn Ski/Sno
interact with Smad3, to address the possibility that these proteins
form a ternary complex we performed a gel shift analysis using the
PE2 probe from the PAI-1 promoter, as used in the transient
transfections. The EMSA clearly showed that both SKIP and Ski/Sno
alone formed a slightly higher migrating complex with Smad3/4.
However we did not observe the formation of a ternary complex in
the presence of all three proteins. Thus these data are consistent with
the transient transfection results which suggest competition occuring
between SKIP and Ski/Sno for Smad3 transactivation, but do not
exclude the presence of a ternary complex forming between these
proteins.
In this study SKIP acted as a coactivator of TGF-β-dependent
transcription. SKIP similarly acts as a coactivator of nuclear hormone
receptor-dependent transcription, but also as a repressor of Notch
signalling through its interaction with SMRT and associated HDAC
proteins (21,23). These divergent effects of SKIP may depend on
interaction of SKIP with other, possibly cell-specific nuclear factors.
For example SKIP converts CBF1 from a transcriptional repressor to
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activator through switching its interaction between the corepressor
SMRT and Notch 1C (23). In our study SKIP and Ski/Sno modulated
each other’s opposing transcriptional activities, raising the intriguing
possibility that the relative cellular expression of SKIP versus Ski or
Sno may play a regulatory role on TGF-β−dependent transcription
and hence its effects on cell growth and differentiation. Interestingly,
the Smad-interacting domain of SKIP (201-333aa) appears to be also
involved in interaction with Ski and Sno (J.Figueroa and
M.J.Hayman, unpublished observations). These results and those
showing that SKIP, like Ski/Sno, interacted with the MH2 domain of
Smad3, suggests that the opposing transcriptional effects of SKIP and
Ski/Sno may involve competition for Smad3 binding between SKIP
and c-Ski/Sno, and/or other Smad3 MH2-interacting factors, such as
with CBP/p300 (7,33,34). Thus, the modulatory effects of SKIP
through the MH2 domain potentially increases the complexity and
diversity of Smad-dependent transcriptional effects. Furthermore, as
SKIP and Ski/Sno interact with each other and also with the related
corepressors N-CoR/SMRT, an additional mechanism could involve
SKIP-mediated derepression (1,15,20,23). This may possibly occur
via SKIP sequestration of corepressors such as SMRT or N-CoR from
the Ski/Sno repressor complex, a mechanism similar to that
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suggested for Hoxc-8 and Smad1 (35). Whatever the molecular
mechanism of SKIP action it is nevertheless clear that SKIP plays a
role in modulation of this important cellular and signalling pathway.
In summary, our results support a model in which SKIP positively
modulates TGF-β-dependent-transcription and potentially competes
with other MH2-interacting factors, such as c-Ski and Sno, to
determine the transcriptional outcome of a TGF-β responsive target
gene. This suggests a potential role for SKIP in the regulation of
TGF-β effects on cell growth and differentiation.
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Acknowledgments
To Jean Massague, Takeshi Imamura, Kohei Miyazono and Robert
Weinberg for plasmids, Edith Gardiner, Michelle Henderson, Roger
Daly, and Nobuhide Nueki for critical reading of the manuscript, and
Colette Fong and other members of the Bone & Mineral Research
Program for general technical support, advice and camaraderie.
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Footnote
∗∗This work was supported by a National Health & Medical
Research Council (NHMRC) grant to the Bone & Mineral Research
Program at the Garvan Institute, in part through a NHMRC Medical
Post-graduate Scholarship (to G.M.L.) and by NIH Public service
Grants CA28146 and CA42573 (to M.J.H).
The abbreviations used are: SKIP, ski-interacting protein; TGF-
β, transforming growth factor-β; N-CoR, nuclear corepressor; PAI-1,
plasminogen activator inhibitor gene-1; MH1, mad-homology
domain 1; MH2, mad-homology domain 2; BMPs, bone-
morphogenetic proteins; SBEs, Smad-binding elements; NHR,
nuclear hormone receptor; VDR, vitamin D receptor; HDAC, histone
deacetylase enzyme; SMRT, silencing mediator for retinoic acid and
thyroid receptors; GAL4-AD & GAL4-DBD, GAL4 activation and
DNA-binding domain, respectively.
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Figure legends
FIG. 1. SKIP augments TGF-β dependent transactivation in
mammalian cells. A) Transient transfections of COS1 cells were
performed in 24 well plates with the 3TP-lux reporter (250ng per
well) and the following expression plasmids as indicated: SKIP-
pCGN and Smad2-, Smad3- and Smad4-pcDNA3. The 3TP-Lux
reporter contains 3 clusters of Smad-binding elements from the
plasminogen activator inhibitor-1 gene promoter (PAI-1) and is
predominantly a Smad3-responsive promoter reporter. Cells were
treated with vehicle or TGF-β ligand (1ng/mL). The total
amount of transfected DNA was kept constant by use of respective
empty vectors. The results are shown as the mean luciferase activity
SEM of three independent experiments performed in triplicate. B)
SKIP interacts in vivo with Smad2 and 3 in a yeast two hybrid system
through a domain between 201-333aa. Deletional analysis of SKIP-
GAL4-AD as indicated followed by co-expression with wildtype
Smad2-, Smad3- and Smad4-GAL4-DBD was performed as described
in Methods. Results of β-galactosidase activity are shown as mean ±
SEM from 3 independent colonies obtained in at least duplicate
experiments and are corrected for protein levels determined by the
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Biorad protein assay. C) SKIP domain between aa201-333 enhances
TGF-β transactivation in mammalian cells. Transient transfections of
COS1 cells were performed as described in FIG. 1A. with the 3TP-lux
reporter and the Smad3-pcDNA3 expression plasmid (100ng) and
wildtype SKIP-pCGN or deletion constructs as indicated (50ng).
Cells were treated with vehicle or TGF-β ligand (1ng/mL). The
results are shown as the mean luciferase activity ± SEM of two
independent experiments performed in triplicate.
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FIG. 2. SKIP interacts with Smad2 and Smad3 in vitro.
A) Glutathione sepharose beads were coated with bacterially
expressed GST-SKIP wildtype fusion proteins or GST alone and were
incubated with equal amounts of 35S-labelled wildtype Smad3 and
luciferase. Bound proteins were resolved by SDS-PAGE. Input
proteins show one-fifth of loaded lysate. B) Sepharose beads were
coated with bacterially expressed fusion proteins consisting of GST-
Smad3, or deletion mutants of Smad3, including an MH1 and MH2
domain double mutant (aa199-405) and a phosphorylated MH1
domain deletion mutant which retains the MH2 (aa199-425), a mutant
containing the MH2 domain consisting of the last 26aa of the C-
terminus of Smad3 (aa400-425), or GST alone. Beads were incubated
with equal amounts of 35S-labelled wildtype SKIP, and hVDR (9) and
luciferase as positive and negative controls, respectively. Bound
proteins were resolved on SDS-PAGE. Input proteins show one-fifth
of loaded lysate. C) Far western analysis. Twenty µg of nuclear
extracts containing HA-SKIP (upper panel) or Smad3 (middle panel)
was detected by western analysis. COS1 nuclear extracts were
prepared from cells transfected with the empty vector pcDNA3 or
pCGN (indicated by -) or Smad3-pcDNA3 or HA-SKIP-pCGN
expression plasmid (indicated by +). Proteins in the Smad3
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containing membrane were resolved on SDS-PAGE, electroblotted to
a PVDF membrane, denatured, and renatured with serial dilutions of
6M guanidine hydrochloride and was probed with an anti-HA
antibody (lower panel). On Far western analysis anti-HA antibody
detected a band co-migrating with Smad3, but not the negative
control extract.
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FIG. 3. SKIP augments TGF-β−dependent transcription and
partially counteracts Ski- and Sno-dependent repression. Transient
transfections of COS1 cells were performed with the 3TP-lux reporter
(250ng), Smad3-pcDNA3 (100ng), and increasing amounts of
wildtype SKIP-pCGN as indicated, with either A) c-Ski-pMT2 (100ng)
or B) Sno-pCGN (100ng) expression plasmids. Cells were treated
with vehicle or TGF-β ligand (1ng/ml). The results are shown
as the mean luciferase activity ± SEM of triplicate wells relative to
ligand-dependent reporter activity of Smad3 transfection alone set at
1, and are representative of three independent experiments.
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FIG. 4. Ski and Sno attenuates SKIP augmentation of TGF-
β−dependent transcription. Transient transfections of COS1 cells
were performed with the 3TP-lux reporter (250ng) and Smad3-
pcDNA3 (100ng), wildtype SKIP-pCGN (50ng) and increasing
amounts of either A) c-Ski-pMT2 or B) Sno-pCGN expression
plasmids as indicated. Cells were treated with vehicle or TGF-β
ligand (1ng/ml). The results are shown as the mean luciferase
activity ± SEM of triplicate wells relative to maximal ligand-
dependent reporter activity with SKIP and Smad3 co-transfection set
at 10, and are representative of three independent experiments.
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FIG. 5. SKIP and Sno independently bind to Smad3/4 DNA
complexes. In vitro translated cold Smad3 and Smad4 (2µL each) with
COS1 nuclear extracts of SKIP and Sno were added, as indicated (µL),
with radiolabelled PE2 probe, a fragment of the PAI-1 promoter and
analysed by gel shift assay. Lane 1 contains untranslated lysate and
lane 2 Smad3/4 lysate alone (lower arrow). Empty vector COS1
nuclear extracts were added to reactions equalise the total amount of
COS1 nuclear extracts used in each reaction. The SKIP/Smad3/4
(lanes 3-6) are indicated by the upper arrow and the Sno/Smad3/4
higher molecular weight complexes (lanes 7-10) are indicated by *.
CP= cold probe (lane 11).
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J. Hayman, John A. Eisman and Alexander P. KouzmenkoGary M. Leong, Nanthakumar Subramaniam, Jonine Figueroa, Judith L. Flanagan, Michael
TGF-{b}-dependent transcriptionSki-interacting protein (SKIP) interacts with Smad proteins to augment
published online March 6, 2001J. Biol. Chem.
10.1074/jbc.M010815200Access the most updated version of this article at doi:
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