From the Laboratory of Molecular Genetics,
Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki
305-0074, Japan, the § Institute of Medical Sciences,
University of Tsukuba, 1-1-1 Tennoudai, Tsukuba, Ibaraki 305-0006, Japan, the ** National Institute of Bioscience and Human Technology,
Agency of Industrial Science and Technology, 1-1 Higashi, Tsukuba,
Ibaraki 305-0046, Japan, the
Chugai
Research Institute for Molecular Medicine, 153-2 Nagai, Niihari,
Ibaraki 300-4101, Japan, the §§ Iatron
Laboratories Inc., 1460-6 Mitodai, Tako, Katori, Chiba 289-2247, Japan,
and the ¶ CREST (Core Research for Evolutional Science and
Technology), JST
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ABSTRACT |
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The mechanism by which the viral
oncogene ski (v-ski) transforms chicken embryo
fibroblasts is currently unknown. Recently, the c-ski gene
product (c-Ski) was found to bind to N-CoR (nuclear hormone receptor
co-repressor), an element implicated in transcriptional repression
mediated by multiple transcriptional repressors including the nuclear
hormone receptors and Mad. c-Ski is required for transcriptional repression mediated by Mad involved in negative regulation of cellular
proliferation. v-Ski abrogates Mad-induced transcriptional repression
in a dominant negative fashion. Here we report that v-Ski also inhibits
transcriptional repression mediated by Rb, another tumor suppressor
gene product. Rb forms a complex with c-Ski, Sin3A, and
histone deacetylase (HDAC) via direct binding to c-Ski and
HDAC. c-Ski is required for the transcriptional repression mediated by
Rb. These results suggest that inhibition of Rb activity contributes,
at least partly, to transformation by v-Ski.
The oncogene v-ski was originally identified in avian
Sloan-Kettering viruses and found to transform chicken embryo
fibroblasts (1). The human
c-ski1
proto-oncogene product (c-Ski) is a 728-amino acid nuclear protein, and
the N- and C-terminal regions of c-Ski possess a cysteine-rich and a
coiled-coil region, respectively (2, 3). The v-Ski protein lacks a
292-amino acid region from the C terminus of c-Ski, but still contains
the N-proximal cysteine-rich region (4). This N-proximal region is
responsible for the cellular transformation capacity of ski
(5). The ski gene family comprises two members, ski and sno
(ski-related novel gene)
(2), and both have been shown to share clear homology in their N- and
C-terminal regions (2, 6). Recently we found that c-Ski directly binds to N-CoR (nuclear hormone receptor co-repressor) (28). N-CoR was
originally identified as a co-repressor that binds to and mediates
transcriptional repression by nuclear hormone receptors (8). Another
co-repressor, SMRT, shows striking homology to N-CoR (9). N-CoR also
forms a complex with mammalian Sin3 orthologues (mSin3A and mSin3B).
The binding of mSins to histone deacetylase (HDAC) suggested that
transcriptional repression through N-CoR involves deacetylation of
nucleosomal histones (10-14). The basic helix-loop-helix proteins of
the Mad family act as transcriptional repressors after
heterodimerization with Max (15). Mad interacts with the HDAC complex
through direct binding to mSin3, and N-CoR is required for Mad-induced
transcriptional repression (10-14). We demonstrated that N-CoR binds
to the N-terminal region of c-Ski and that this interaction is also
required for transcriptional repression mediated by Mad and the thyroid
hormone receptor In addition to Mad, the retinoblastoma protein (Rb) encoded by another
tumor suppressor gene also binds to HDAC (18-20). Rb regulates the
G1/S transition in the cell cycle by silencing a group of
target genes regulated by E2F transcription factors (21, 22). Rb binds
to the activation domain of E2F and then actively represses the
promoter by recruiting HDAC. The pocket region of Rb, which contains
two subdomains, termed A and B, are responsible for interaction with
HDAC (18-20). Although HDAC forms a complex with mSin3, N-CoR, and
Ski, it remains unknown whether Rb can form a complex with any of these
components of the N-CoR complex. To understand the molecular mechanism
of transformation by v-Ski, we examined whether c-Ski forms a complex
with Rb and whether v-Ski abrogates Rb-induced transcriptional
activation as in the case of Mad. Our results indicate that c-Ski is
needed for the transcriptional repression mediated by Rb and that v-Ski
abrogates Rb-induced transcriptional repression.
Co-immunoprecipitation--
HeLa cells were lysed in lysis
buffer consisting of PBS, 0.1% Nonidet P-40, 10% glycerol, and
protease inhibitor mixture (Boehringer Mannheim). CV-1 cells were lysed
in lysis buffer consisting of PBS, 1 mM NaF, 1 mM Na3VO4, 0.1% Nonidet P-40, 1 mM phenylmethylsulfonyl fluoride, 10% glycerol, and
protease inhibitor mixture (Boehringer Mannheim). Lysates were
immunoprecipitated using anti-c-Ski monoclonal antibodies (28),
anti-Sno monoclonal antibodies, anti-Rb antibody G3-245 or XZ91
(Pharmingen), anti-Gal4 antibody (Santa Cruz Biotechnology Inc.), or
control IgG, and the immune complex was analyzed by Western blotting
using the anti-Rb, anti-N-CoR (28), anti-c-Ski, anti-mSin3A (Santa Cruz
Biotechnology Inc.), or anti-HDAC1 (Santa Cruz Biotechnology Inc.)
antibodies and ECL detection reagents (Amersham Pharmacia Biotech).
Anti-c-Ski and anti-Sno monoclonal antibodies were prepared using
bacterially expressed full-length c-Ski and Sno proteins, respectively.
To examine the effect of v-Ski on complex formation among Rb, HDAC1,
and Ski, CV-1 cells were transfected with a mixture of 1 µg of the
c-Ski or v-Ski expression plasmid, 3 µg of the Gal4-Rb expression
plasmid, and 1 µg of the HDAC1 expression plasmid using LipofectAMINETM (Life Technologies Inc.). The plasmid to
express Gal4-Rb containing the DNA-binding domain of Gal4 (amino acids
1-174) and the repressor domain of Rb (amino acids 379-792) was
constructed using the cytomegalovirus promoter-containing vector
pcDNA3 (Invitrogen). The HDAC1 expression plasmid was constructed
using pcDNA3, and the c-Ski and v-Ski expression plasmids were
described (28). Lysates were prepared and immunoprecipitated using
anti-Gal4 antibody (UBI) or control IgG as described above, and the
immune complex was analyzed by Western blotting using the anti-HDAC1
antibody (Santa Cruz Biotechnology Inc.).
In Vitro Binding Assay--
To express the GST-Rb fusion protein
containing the pocket region of human Rb (amino acids 372-787) in
Escherichia coli, a plasmid was constructed by the
polymerase chain reaction-based method using the pGEX-2T vector
(Amersham Pharmacia Biotech). The modified pSP65 vector pSPUTK
(Stratagene) was used for in vitro transcription/translation
of the various forms of Rb and c-Ski. The various deletion mutants of
c-Ski have been described previously (28), and the Rb deletion mutants
were constructed using appropriate enzyme sites. The Rb mutants
contained deletions of one of the following regions; Single-cell Microinjection Assay--
The antibody injection
experiments using the Gal4-Rb expression plasmid and the anti-c-Ski and
anti-Sno polyclonal antibodies were performed as described (28).
Effect of Ski on Rb-induced Transcriptional Repression--
To
investigate the effect of v-Ski on Gal4-Rb-mediated transcriptional
repression, a mixture of 3 µg of the Gal site-containing luciferase
reporter, 0.02 µg of Gal4-Rb or the Gal4 expression plasmid, and 1, 2, or 3 µg of the plasmid to express c-Ski or v-Ski and 0.5 µg of
internal control plasmid pRL-TK was transfected into CV-1 cells using
the CaPO4 method, and luciferase assays were performed. The
total amount of plasmid DNA was adjusted to 15 µg by addition of the
control plasmid DNA lacking the cDNA. To examine the effect of Ski
on E2F-dependent transcriptional activation, a mixture of
0.1 µg of the E2F1 site-containing luciferase reporter, 0.05 µg of
the E2F1 expression plasmid or the control DNA, and 1.5, 2, or 2.5 µg
of the v-Ski expression plasmid and 0.1 µg of the internal control
plasmid pRL-TK was transfected into CV-1 cells using
LipofectAMINETM (Life Technologies, Inc.), and luciferase
assays were performed. The total amount of plasmid DNA was adjusted to
3 µg by addition of the control plasmid DNA lacking the cDNA. The
E2F site-containing reporter was described previously (23). The
pcDNA1 vector (Invitrogen) containing the cytomegalovirus promoter
was used to express E2F1.
To investigate whether Rb forms a complex with c-Ski in
vivo, co-immunoprecipitation assays were performed (Fig.
1A). The cell lysates were
prepared from HeLa cells and immunoprecipitated with anti-c-Ski,
anti-Sno, or control anti-Gal4 antibody. Rb was co-precipitated with
anti-c-Ski or anti-Sno antibodies but not with the control anti-Gal4
antibody. To examine complex formation between the endogenous Rb
protein and mSin3A, co-immunoprecipitation was performed using CV-1
cells. The anti-Rb antibody XZ91 co-immunoprecipitated mSin3A, whereas
the anti-Rb antibody G3-245 or control IgG did not (Fig.
1B). These results indicate that Rb forms a complex in
vivo not only with HDAC but also with c-Ski and mSin3A. We could
not detect N-CoR or SMRT in the complex immunoprecipitated with anti-Rb
antibodies (data not shown). However, we cannot exclude the possibility
that this is due to the low level of expression of N-CoR and SMRT in
HeLa and CV-1 cells.
INTRODUCTION
Top
Abstract
Introduction
References
(28). The same target sequence of Mad/Max, the
so-called E-box, is also recognized by a heterodimer of Myc/Max that
activates transcription. It is believed that Myc/Max enhances cellular
proliferation or transformation, whereas Mad/Max leads to suppression
of proliferation or induction of terminal differentiation in a wide
range of cell types (16, 17). Our data indicated that v-Ski blocks
Mad-induced transcriptional repression in a dominant negative fashion
(28), suggesting that inhibition of Mad function contributes to
transformation by v-Ski.
MATERIALS AND METHODS
B (amino acids
600-928),
A (amino acids 302-600),
A + B (amino acids
414-928), or A + B (amino acids 1-371 and amino acids 789-928). The
preparation of GST fusion proteins, the in vitro translation
of various forms of Rb and c-Ski, and the binding assays were done
essentially as described (28) except for the use of a binding buffer
consisting of 10 mM Hepes (pH 7.6), 0.1 M KCl,
2.5 mM MgCl2, 0.5 mM
dithiothreitol, 0.05% Nonidet P-40, and 0.1 mg/ml bovine serum albumin
and the use of PBS for washing.
RESULTS
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Fig. 1.
Rb binds to the HDAC-mSin3A-c-Ski complex via
a direct interaction with c-Ski and HDAC. A,
co-immunoprecipitation of c-Ski and Rb. Lysates of HeLa cells were
immunoprecipitated with anti-c-Ski, anti-Sno antibodies, or control
anti-Gal4 antibody. The immunocomplex was analyzed on 9% SDS gels
followed by Western blotting using anti-Rb antibody. B,
complex formation between Rb and mSin3A. Lysates of CV-1 cells were
immunoprecipitated with anti-Rb antibodies (G3-245 or XZ91) or normal
IgG. The immunocomplexes were analyzed on 7.5% gel followed by Western
blotting using anti-mSin3A antibodies. C, v-Ski inhibits
association between Rb and HDAC1. CV-1 cells were transfected with a
mixture of the c-Ski or v-Ski expression plasmid together with the
plasmids to express HDAC1 and Gal4-Rb. Lysates prepared from
transfected cells were immunoprecipitated with anti-Gal4 or control IgG
antibody. The immunocomplex was analyzed on 9% SDS gels followed by
Western blotting using anti-HDAC1 antibody. In the two lanes on
the left, HDAC1 was detected by Western blotting using lysates to
confirm that almost the same amount of HDAC1 was expressed in the
presence of c-Ski or v-Ski.
To examine whether expression of v-Ski results in loss of HDAC in the Rb-HDAC complex, we performed the co-immunoprecipitation experiment (Fig. 1C). CV-1 cells were transfected with a mixture of the c-Ski or v-Ski expression plasmid together with the plasmids to express HDAC1 and the Gal4-Rb fusion protein made up of the Gal4 DNA-binding domain and the pocket region of Rb. The cell lysates were prepared and immunoprecipitated with anti-Gal4 or control IgG antibody. The amount of HDAC1 coprecipitated with Gal4-Rb in the presence of v-Ski was apparently less than that with c-Ski. These results suggest that v-Ski inhibits association between Rb and HDAC1 in a dominant negative fashion.
During the analysis of the interaction between Rb and the components of the N-CoR complex, we found that c-Ski directly binds to Rb in vitro. In the GST pull-down assays using in vitro translated Rb and the GST-c-Ski resin containing full-length c-Ski, a significant amount of in vitro translated Rb was found to bind to the GST-c-Ski resin (Fig. 2A). The results of binding assays using the different mutants of Rb indicated that the B subdomain in the pocket region of Rb is responsible for the interaction with c-Ski. To identify the region in c-Ski that interacts with Rb, the GST pull-down assay was performed using the GST-Rb fusion protein resin and various forms of in vitro translated c-Ski protein (Fig. 2B). The results indicated that two regions in c-Ski efficiently interact with Rb; one is the region between amino acids 197 and 330, which includes a part of the N-CoR-binding domain (amino acids 99-274) and the other is the C-terminal coiled-coil region (amino acids 556-728). Thus, Rb directly binds to c-Ski and HDAC.
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To further investigate whether c-Ski is required for transcriptional
repression by Rb, antibody injection experiments were done (Fig.
3). Injection into Rat-1 cells of a
lacZ reporter plasmid containing the lacZ gene
linked to the TK promoter and Gal4-binding sites gave rise to many
lacZ-positive cells. Co-injection of this lacZ
reporter with a plasmid encoding the Gal4-Rb fusion protein made up of
the Gal4 DNA-binding domain and the pocket region of Rb resulted in a
decrease in the number of lacZ-positive cells. This decrease
was relieved significantly by co-injection of anti-c-Ski antibody and
partially by anti-Sno antibodies. Co-injection of both antibodies also
significantly relieved the decrease in the number of
lacZ-positive cells, but not completely. The incomplete abrogation of Gal-Rb function by co-injection of both antibodies may be
due to the presence of other Ski-related protein(s) such as the third
member of the ski gene family which we identified recently.2 As a control
experiment, we used a Gal4 fusion protein containing the repressor
domain of EF1, which is thought not to utilize the N-CoR c-Ski
complex. Co-injection of anti-c-Ski or anti-Sno antibodies did not
alleviate the decrease in the number of lacZ-positive cells
induced by Gal4-
EF1 (28), indicating that the effect of anti-Ski/Sno
antibodies was specific for Rb.
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To investigate whether v-Ski mutants that lack the C-terminal region of
c-Ski could abrogate transcriptional repression by Rb in a dominant
negative fashion as in the case of Mad, we examined the effect of
overexpression of v-Ski on Gal4-Rb-induced transcriptional repression
(Fig. 4, A and B).
Gal4-Rb containing the pocket region of Rb strongly repressed
transcription from the Gal4 site-containing reporter. This
Gal4-Rb-induced repression was abrogated by v-Ski in a
dose-dependent manner. Furthermore, wild type c-Ski partly abrogated Gal4-Rb-induced transcriptional repression. We observed that
the microspeckle pattern of N-CoR was disrupted by coexpression of a
high amount of c-Ski but not by a low amount of
c-Ski.3 These two
observations are consistent with the idea that overexpression of wild
type c-Ski abrogates transcriptional repression by creating an
imbalance between the components of the co-repressor complex rather
than potentiating transcriptional repression. In control experiments,
repression by the Gal4-EF1 fusion protein was not abolished by
co-expression of either v-Ski or wild type c-Ski (28). Using the E2F1
site-containing luciferase reporter, we also examined the effect of
v-Ski on E2F1-mediated transcriptional activation (Fig. 4C).
v-Ski was also found to enhance E2F1-induced transcriptional activation
in a dose-dependent manner. These results indicate that
v-Ski inhibits Rb-dependent transcriptional repression.
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DISCUSSION |
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The oncogene v-ski can transform chicken embryo fibroblasts. Our results indicate that v-Ski abrogates transcriptional repression mediated not only by Mad but also by Rb. c-Ski has two regions that are conserved in related proteins, the N-terminal cysteine-rich region and the C-terminal coiled-coil region. N-CoR binds to the N-terminal cysteine-rich region, while the C-terminal coiled-coil region binds to mSin3 (28). The C-truncated c-Ski protein lacking the coiled-coil region cannot bind to mSin3 and disrupts the dot-like structure of N-CoR (28), suggesting that this form of c-Ski acts as in a dominant negative fashion. Because v-Ski also lacks the C-terminal coiled-coil region, v-Ski probably inhibits Mad- and Rb-mediated transcriptional repression in a dominant negative fashion. In our co-transfection assay, overexpression of normal c-Ski also partly abrogated the transcriptional repression mediated by Rb (Fig. 4). This is consistent with the fact that overexpression of wild type c-Ski also leads to transformation (24). Mutation of the human Rb gene occurs in a wide variety of tumors (25). In addition, one of the mad-related genes, mxi1, was recently demonstrated to act as a tumor suppressor using mutant mice (26). Therefore, abrogation of Rb and Mad activity by v-Ski may contribute, at least partly, to transformation by v-ski.
Rb was recently reported to directly bind to HDAC (18-20). Our results
indicate that Rb also directly interacts with c-Ski. Furthermore, Rb
forms a complex with mSin3, although it is not clear whether the
Rb-HDAC-mSin3A-Ski complex contains N-CoR. The antibody injection
experiments showed that c-Ski is required for Rb-mediated
transcriptional repression (Fig. 3). At present, it remains unknown
whether N-CoR and mSin3 are needed for the transcriptional repression
mediated by Rb. Thus, c-Ski is required for the transcriptional repression mediated by at least Mad, thyroid receptor, and Rb. It is
possible that other transcriptional repressors that utilize the
N-CoR-mSin3-HDAC complex also require c-Ski. The complex containing mSin3 consists of multiple proteins such as SAP30 and the
histone-binding proteins RbAp46 and RbAp48 (27). Interestingly, SAP30
is required for the transcriptional repression mediated by the estrogen
receptor but not by thyroid receptor or the retinoic acid receptor (7). To understand the molecular mechanism of v-Ski-induced transformation, it will be important to determine whether c-Ski acts in specific transcriptional repression mediated by a limited number of repressors or in transcriptional repression in general.
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ACKNOWLEDGEMENTS |
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We thank Dr. T. Dryja for the human Rb cDNA, Drs. K. Ohtani and M. Ikeda for the E2F site-containing luciferase reporter and the E2F1 expression plasmid, and Dr. S. L. Schreiber for the HDAC1 cDNA.
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FOOTNOTES |
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* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
On leave from the Dept. of Oncology, Institute of
Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku,
Tokyo 108-0071, Japan.
¶¶ To whom correspondence should be addressed: Laboratory of Molecular Genetics, Tsukuba Life Science Center, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305, Japan. Tel.: 81-298-36-9031; Fax: 81-298-36-9030; E-mail: sishii{at}rtc.riken.go.jp.
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ABBREVIATIONS |
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The abbreviations used are: c-ski, cellular ski gene; c-Ski, c-ski gene product; HDAC, histone deacetylase; N-CoR, nuclear hormone receptor co-repressor; Rb, retinoblastoma gene product; v-Ski, viral ski gene product; sno, ski-related novel gene; PBS, phosphate-buffered saline; GST, glutathione S-transferase.
2 M. M. Khan, T. Nomura, and S. Ishii, unpublished data.
3 T. Nomura and S. Ishii, unpublished data.
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REFERENCES |
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