Ski-interacting protein (SKIP) interacts with Smad proteins to ...

SKIP interacts with Smads to activate TGF-β signalling

1

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.

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