From the Department of Biochemistry, The
Cancer Institute of the Japanese Foundation for Cancer Research, and
Research for the Future Program, the Japan Society for the Promotion of
Science, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, ¶ The
Tokyo Metropolitan Institute of Medical Science, 3-18-22 Honkomagome,
Bunkyo-ku, Tokyo 113-8613, and
Department of Molecular
Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, January 8, 2001, and in revised form, February 2, 2001
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ABSTRACT |
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Smad7 is an inhibitory Smad that acts as a
negative regulator of signaling by the transforming growth factor- Members of the transforming growth factor- R-Smads and Co-Smads positively regulate signaling by the
TGF- The third class of Smads are I-Smads, which include Smad6 and Smad7 in
mammals (6-8). I-Smads associate with activated TGF- Ubiquitin-dependent protein degradation plays a key role in
various biological processes, including signal transduction, cell cycle
progression, and transcriptional regulation (12). In the TGF- Here we demonstrate a novel function of Smurf1 in receptor degradation
in TGF- Transfection, Immunoprecipitation, and Immunoblotting--
COS7
cells or 293T cells were transiently transfected using FuGENE6 (Roche
Molecular Biochemicals). Immunoprecipitation and immunoblotting were
performed as described (15). For inhibition of proteasomal degradation,
cells were incubated with 50 µM MG132 (Peptide Institute)
or 10 µM lactacystin (Calbiochem) for 4 h. Each
experiment has been repeated at least three times with essentially similar results.
Affinity Cross-linking and Immunoprecipitation--
Recombinant
TGF- Immunofluorescence Labeling--
Immunohistochemical staining of
6Myc-Smad7 in transfected COS7 cells was performed using mouse anti-Myc
antibody followed by incubation with fluorescein isothiocyanate
(FITC)-labeled goat anti-mouse IgG as described (15). For double
staining of Smad7 and Smurf1, immunohistochemical staining of
FLAG-Smad7 and 6Myc-Smurf1 was performed using mouse anti-FLAG or
rabbit anti-Myc antibody followed by incubation with FITC-labeled goat
anti-mouse IgG or rhodamine isothiocyanate (RITC)-labeled goat
anti-rabbit IgG, respectively. Nuclei of the cells were stained by
4,6-diamidino-2-phenylindole. Intracellular localization was determined
by confocal laser scanning microscopy.
Pulse-Chase Analysis--
Cells were labeled for 10 min at
37 °C with 50 mCi/ml [35S]methionine and cysteine
(Amersham Pharmacia Biotech) in methionine- and cysteine-free
Dulbecco's modified Eagle's medium and chased as described (13).
Cells were then lysed and subjected to immunoprecipitation.
Luciferase Assay--
R mutant mink lung epithelial cells
were transiently transfected with an appropriate combination of a
p3TP-lux promoter-reporter construct, expression plasmids, and
pcDNA3. Total amounts of transfected DNAs were the same in each
experiment, and values were normalized using Renilla luciferase activity.
Smurf1 Interacts with Smad6 and Smad7--
Smurf1 has been
identified as an E3 ubiquitin ligase for BMP-specific Smads (14).
Smurf1 has two WW domains that facilitate protein-protein
interactions by binding to the PPXY sequence (PY motif) on
partner proteins. Of eight different Smads, not only R-Smads including
Smad1 and Smad5 but also I-Smads have a PY motif in their linker
regions (Fig. 1A). We
therefore examined whether Smurf1 binds to I-Smads. We first analyzed
the interaction of Smurf1 with different Smads in transfected COS7
cells. A Smurf1 mutant, Smurf1(C710A), which has a mutation in the HECT
domain and fails to recruit ligase activity, was used for this study. Of Smads 1 through 8, Smad6 and Smad7 strongly interacted with Smurf1(C710A) (Fig. 1B). Smurf1(C710A) interacted with Smad1
and Smad5 less efficiently than with Smad6 and Smad7. In contrast, it
bound to Smad3 only weakly and failed to bind to Smads 2 and 4. Because
Smad8 lacks the PY motif, Smurf1(C710A) did not bind to Smad8 either
(Fig. 1B).
The mode of interaction between Smad7 and Smurf1 was further studied.
In COS7 cells, weak interaction of wild-type Smad7 (Smad7(WT)) with
wild-type Smurf1 (Smurf1(WT)) was detected, and it was slightly facilitated in the presence of the constitutively active TGF- Smurf1 Interacts with T Smad7 Is Translocated to the Cytoplasm by Smurf1--
We next
examined the effect of Smurf1 on the subcellular localization of Smad7.
In the absence of Smurf1, both Smad7(WT) and Smad7
An E3 ubiquitin ligase, MDM2, has been reported to promote
ubiquitin-dependent degradation and nuclear export of p53
(16, 17). In this case, a mutation within the MDM2 RING-finger domain that cannot induce p53 ubiquitination also lacks the ability to promote
the p53 nuclear export. Thus, both Smurf1 and MDM2 promote not only
ubiquitin-dependent degradation but also nuclear export of
the substrates, although the mechanisms of nuclear export appear to
differ between them. Itoh et al. (18) reported that Smad7 is
predominantly located in the nucleus and that it is exported to the
cytoplasm after ligand stimulation. It is possible that Smurf1
functions as a carrier protein for Smad7 for nuclear export, although
it is currently not known whether ligand stimulation triggers the
nuclear export of Smad7 by Smurf1.
Smurf1 Induces Ubiquitination of Smad7 and T Smurf1 Induces Degradation of Smad7 and T Smurf1 Enhances the Inhibitory Activity of Smad7--
To examine
the effect of Smurf1 on the inhibitory activity of Smad7, we first
compared the effect of Smad7 Smurf-like Molecules Target TGF-
(TGF-
) superfamily proteins. Smad7 is induced by TGF-
, stably
interacts with activated TGF-
type I receptor (T
R-I), and
interferes with the phosphorylation of receptor-regulated Smads. Here
we show that Smurf1, an E3 ubiquitin ligase for bone morphogenetic
protein-specific Smads, also interacts with Smad7 and induces Smad7
ubiquitination and translocation into the cytoplasm. In addition,
Smurf1 associates with T
R-I via Smad7, with subsequent enhancement
of turnover of T
R-I and Smad7. These results thus reveal a
novel function of Smad7, i.e. induction of
degradation of T
R-I through recruitment of an E3 ligase to the receptor.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
(TGF-
)1 superfamily
initiate cellular responses (1) by binding to two different types of
serine/threonine kinase receptors, termed type I and type II. Type I
receptor is activated by type II receptor upon ligand binding and
mediates specific intracellular signals (2). Members of the TGF-
superfamily transduce intracellular signals by Smad proteins. Eight
different Smad proteins have been identified in mammals and are
classified into three subgroups, i.e.
receptor-regulated Smads (R-Smads), common-partner Smads (Co-Smads),
and inhibitory Smads (I-Smads) (3-5).
superfamily (3-5). R-Smads directly interact with type I receptors and become activated through phosphorylation of the C-terminal SSXS motif. R-Smads then form heteromeric
complexes with Co-Smads (Smad4) and translocate into the nucleus.
Nuclear Smad complexes bind to transcriptional coactivators or
corepressors and regulate transcription of target genes. Smad2 and
Smad3 act in the TGF-
/activin pathway, whereas Smad1, Smad5, and
Smad8 are thought to act as bone morphogenetic protein (BMP)-specific Smads.
superfamily
type I receptors, thereby preventing phosphorylation of R-Smads. In
addition, Smad6 has been demonstrated to interact with phosphorylated
Smad1 to prevent complex formation between Smad1 and Smad4 (9). Smad6
was also reported to interact with Hoxc-8 and function as a
transcriptional corepressor for inhibition of BMP signaling (10).
Because expression of Smad6 and Smad7 is induced by TGF-
and BMPs,
I-Smads inhibit TGF-
superfamily signaling by a negative feedback
system (11).
signaling pathways, R-Smads, e.g. Smad2 and
Smad1/5, have recently been shown to be degraded by the
ubiquitin-proteasome pathway. Smad2 activated by TGF-
is degraded by
the ubiquitin-proteasome pathway after translocation into the nucleus
(13). Smurf1, a member of the HECT family of E3 ubiquitin
ligases, ligand-independently induces the ubiquitination and
degradation of BMP-specific Smads 1 and 5 through binding to a
PY motif in the linker regions (14).
superfamily signaling. Inhibitory Smad7 associates with
Smurf1 in the nucleus and is exported to the cytoplasm. Smad7 thus
recruits Smurf1 to T
R-I, resulting in the degradation and rapid
turnover of the T
R-I protein.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
1 (R & D Systems) was iodinated using the chloramine T method.
Cross-linking was performed on ice to avoid degradation of the
receptors and other proteins. Subsequent immunoprecipitation and
analysis by SDS polyacrylamide gel electrophoresis (PAGE) were
performed as described (15).
RESULTS AND DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
REFERENCES
View larger version (48K):
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Fig. 1.
Smad6 and Smad7 interact with Smurf1 through
the PY motif. A, amino acid sequence alignment of PY
motif of Smads 1 through 8. B, interaction of I-Smads with
Smurf1. Binding of a Smurf1 mutant, Smurf1(C710A)
(Smurf1(CA)), to different Smads was analyzed in
vivo. Transfected COS7 cells were subjected to FLAG
immunoprecipitation (IP) followed by Myc immunoblotting
(Blot). The top panel shows the interaction, and
the lower two panels the expression of each
protein as indicated. C, binding of Smurf1(WT) to Smad7(WT)
but not to Smad7 PY. The binding between Smurf1(WT) and Smad7(WT) was
slightly facilitated by T
R-I(TD), which was further enhanced by the
presence of a proteasome inhibitor lactacystin. The top
panel shows the interaction of Smurf1 and Smad7. FLAG-Smad7 was
immunoprecipitated in lanes 2-7 from the left,
whereas FLAG-Smurf1 was precipitated in lanes 8-11. Note
that the expression levels of Smurf1 were lower than those of Smad7;
thus the degradation of Smad7 was not remarkable in this figure.
D, binding of Smurf1 to the T
R-II·T
R-I complex. COS7
cells were transfected or not with FLAG-tagged Smurf1(CA) and HA-tagged
T
R-I and T
R-II in the presence or absence of FLAG- or 6Myc-tagged
Smad7. Cells were affinity-labeled with 125I-TGF-
1, and
lysates were immunoprecipitated (IP) with anti-FLAG M2
antibody. Immune complexes were subjected to SDS-PAGE and analysis
using a Fuji BAS 2500 bio-imaging analyzer (Fuji Photo Film).
FLAG-Smurf1 was immunoprecipitated in lanes 4-6 from the
left. As controls, FLAG-Smad7 was precipitated in
lanes 1-3. Cross-linking analysis revealed that the
expression levels of T
R-II and T
R-I were similar in each lane
(data not shown).
type I
receptor, T
R-I(TD) (Fig. 1C). Moreover, the
interaction between Smurf1(WT) and Smad7 was enhanced by the proteasome
inhibitor lactacystin. In contrast, a Smad7 deletion mutant that lacks
the PY motif (amino acids 207-211) in the linker region (Smad7
PY) did not bind to Smurf1.
R-I via Smad7--
Smad7 interacts with
T
R-I activated by T
R-II, thereby competing with Smad2 and Smad3
for inhibition of TGF-
signaling. We therefore examined in an
affinity cross-linking assay whether Smad7 acts as an adapter molecule
that links T
R-I to the ubiquitin-proteasome pathway. Although Smurf1
alone did not efficiently bind to T
R-I in transfected COS7 cells,
Smad7 dramatically enhanced the interaction between Smurf1 and the
T
R-I·T
R-II complex (Fig. 1D, lanes 4 and
5). Moreover, Smurf1 failed to interact with the receptor complex in the presence of Smad7
PY (Fig. 1D, lane
6). These results indicate that Smurf1 is recruited to T
R-I
through Smad7.
PY were
predominantly located in the nucleus, although weak staining in the
cytoplasm was also detected (Fig.
2A). When transfected alone,
Smurf1 was detected in the cytoplasm (data not shown). In the presence
of Smurf1, Smad7(WT) was mainly observed in the cytoplasm. The
cytoplasmic staining of Smad7 was further enhanced by the presence of
proteasomal inhibitor MG132 or lactacystin (Fig. 2B).
Smad7
PY failed to accumulate in the cytoplasm even in the presence
of Smurf1, although there is a little leakage of Smad7
PY out of the
nucleus (Fig. 2A); these results strongly suggest that
interaction of Smurf1 with Smad7 is required for the cytoplasmic
localization of Smad7. Consistent with this, Smurf1 and Smad7
colocalized in the cytoplasm (Fig. 2B). Interestingly, similar findings were obtained using Smurf1(C710A), suggesting that
recruitment of ligase activity is not required for cytoplasmic translocation of the Smad7·Smurf1 complex (Fig. 2B).
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Fig. 2.
Cytoplasmic translocation of Smad7 by
Smurf1. A, translocation of Smad7 from the
nucleus to the cytoplasm by Smurf1. Subcellular localization of Smad7
in the presence or absence of Smurf1 was analyzed. Anti-Myc staining
for Smad7(WT) or Smad7 PY (green) and nuclear staining by
4,6-diamidino-2-phenylindole (PI; red) were
performed in transfected COS7 cells. B, proteasomal
inhibitors MG132 and lactacystin facilitated the cytoplasmic staining
of Smad7. Similar findings were obtained using Smurf1(C710A) in the
absence of proteasomal inhibitors.
R-I--
To
determine whether Smurf1 acts as an E3 ubiquitin ligase for Smad7,
ubiquitination of Smad7 by Smurf1 was investigated in vivo.
Smad7 was transfected into COS7 cells, together with Smurf1 and
HA-tagged ubiquitin. Smurf1 efficiently induced the ubiquitination of
Smad7 (Fig. 3A). Notably,
Smad7 ubiquitination occurred more efficiently than that of Smad1 or
Smad4. Polyubiquitination of Smad7 was not observed when Smad7
PY or
Smurf1(C710A) was used (Fig. 3B). We also tested the effect
of Smad7 on T
R-I ubiquitination by Smurf1 in 293T cells. Although
Smurf1 alone ubiquitinated T
R-I weakly, Smad7 enhanced the receptor
ubiquitination by Smurf1 (Fig. 3C).
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Fig. 3.
Ubiquitination of Smad7 and
T R-I by Smurf1 in vivo.
COS7 cells (A and B) and 293T cells
(C) were transfected with the indicated plasmids and treated
with 50 µM MG132 for 4 h before cell lysis. Lysates
from cells were subjected to anti-FLAG immunoprecipitation followed by
anti-HA immunoblotting. Polyubiquitination species of Smad7
(A and B;
[HA-Ub]n-FLAG-Smad7) and those of T
R-I
(C;
[HA-Ub]n-FLAG-T
R-I(TD)) are
indicated in the top panel.
R-I--
To
investigate whether Smurf1 regulates degradation of Smad7 and T
R-I,
we analyzed turnover of these proteins by pulse-chase experiments.
Smurf1(WT), but not Smurf1(C710A), enhanced the degradation of Smad7
(Fig. 4, A and B),
suggesting that Smurf1-induced Smad7 degradation is dependent on the
HECT catalytic activity and through the proteasome. Smurf1(WT), but not
Smurf1(C710A), was also rapidly degraded (Fig. 4B).
Moreover, Smad7 and Smurf1 induced the degradation of T
R-I (Fig.
4C). Our results thus demonstrate that Smad7 accelerates turnover of the T
R-I protein by recruitment of an E3 ubiquitin ligase, Smurf1.
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Fig. 4.
Smurf1 induces rapid turnover of Smad7 and
T R-I and inhibits transcriptional activity
induced by T
R-I. A and
B, degradation of Smad7 is enhanced by Smurf1(WT)
(A) but not by Smurf1(C710A) (B). In panel
B, turnover of Smurf1(WT) or Smurf1(CA) is also shown. COS7
cells were transfected with FLAG-tagged Smad7 with or without
FLAG-tagged Smurf1(WT) or Smurf1(CA). Cell lysates were
immunoprecipitated by FLAG antibody and analyzed by SDS-PAGE.
C, degradation of T
R-I is enhanced in the presence of
both Smad7 and Smurf1. COS7 cells were transfected with HA-tagged
T
R-I with or without Smad7 and Smurf1. Cell lysates were
immunoprecipitated by HA antibody and analyzed by SDS-PAGE.
D, Smad7
PY is less potent than Smad7(WT) in inhibiting
TGF-
signaling. Effects of Smad7(WT) and Smad7
PY on the
transcriptional activity of constitutively active T
R-I
(T
R-I(TD)) were examined. R mutant Mv1Lu cells
that lack functional T
R-I were co-transfected with p3TP-lux,
together with various combinations of T
R-I(TD) and Smad7 cDNAs.
E, Smurf1 enhances inhibitory activity of Smad7. R mutant
Mv1Lu cells were co-transfected with p3TP-lux, together with various
combinations of T
R-I(TD), Smad7, Smurf1(WT), and Smurf1(CA)
cDNAs.
PY with that of Smad7(WT) using a
TGF-
-responsible promoter-reporter construct, p3TP-lux (Fig.
4D). Smad7
PY suppressed activation of the reporter gene
in a dose-dependent manner, but its inhibitory effect was less potent than that of Smad7(WT), suggesting that the interaction of
Smad7 with Smurf-like molecules is important for efficient inhibition
of TGF-
signaling by Smad7. Next, we tested the effect of Smurf1 on
the inhibitory activity of Smad7 using p3TP-lux (Fig. 4E).
Smurf1(WT), but not Smurf1(C710A), enhanced the inhibitory activity of
Smad7. These data indicate that E3 ligase activity of Smurf1 is crucial
for its effect on the inhibitory activity of Smad7.
Receptors for Degradation via
I-Smads--
I-Smads have been shown to regulate TGF-
superfamily
signaling through multiple mechanisms, e.g.
competition with R-Smads for type I receptor interaction, inhibition of
complex formation between R-Smads and Co-Smads, and transcriptional
repression by interaction with transcription factors, such as Hoxc-8
(6-10). Our present findings revealed a novel mechanism for the
inhibitory activity of Smad7. Although degradation of receptor
complexes by Smurf1 may not be absolutely required for the action of
I-Smads, it may play an important role in the negative regulation of
TGF-
superfamily signaling by I-Smads. The present findings also
suggest that E3 ligases of the Smurf family regulate TGF-
superfamily signaling through dual mechanisms. (i) By interaction with
and degradation of R-Smads, Smurf1 negatively regulates BMP signaling. (ii) Smurf1 also interacts with Smad7 and inhibits TGF-
signaling by
receptor degradation. Recently, another Smurf, Smurf2, has been
suggested to exhibit similar dual specificities. Lin et al. (19) reported that Smurf2 interacts with Smad2, as well as other R-Smads, and induces the degradation of Smad2. Moreover, Kavsak et al. (20) reported that Smurf2 binds to TGF-
receptor complex via Smad7 and causes degradation of receptors and
Smad7. It will be important to determine in the future whether there
are some functional differences between Smurf1 and Smurf2
in vivo, especially in the interaction with I-Smads or receptors.
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ACKNOWLEDGEMENT |
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We thank G. H. Thomsen for Smurf1 cDNA.
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FOOTNOTES |
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* This research was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture of Japan and by special coordination funds for promoting science and technology from the Science and Technology Agency of Japan.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.
§ Contributed equally to this work.
** To whom correspondence should be addressed: Dept. of Biochemistry, The Cancer Inst. of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Tel.: 81-3-5394-3866; Fax: 81-3-3918-0342; E-mail: miyazono-ind@ umin.ac.jp.
Published, JBC Papers in Press, February 13, 2001, DOI 10.1074/jbc.C100008200
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
R-Smad(s), receptor-regulated Smad(s);
Co-Smad(s), common-partner Smad(s);
I-Smad(s), inhibitory Smad(s);
BMP(s), bone morphogenetic protein(s);
PAGE, polyacrylamide gel
electrophoresis;
FITC, fluorescein isothiocyanate;
RITC, rhodamine
isothiocyanate;
HA, hemagglutinin;
WT, wild type.
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