From the Department of Biochemistry, The Cancer
Institute of the Japanese Foundation for Cancer Research, 1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, the § Section of
Orthopedic Spinal Surgery, Department of Frontier Surgical
Therapeutics, Division of Advanced Therapeutical Sciences, Graduate
School, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku,
Tokyo 113-8549, the ¶ Department of Biological Sciences, Graduate
School of Bioscience and Biotechnology, Tokyo Institute of Technology,
4259 Nagatsuta-cho, Midori-ku, Yokohama 226-8501, the
Department
of Biotechnology, Graduate School of Agriculture and Life Sciences,
University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, the
** Department of Frontier Biosciences, Graduate School of
Frontier Biosciences, Osaka University, 2-2 Yamada-oka, Suita, Osaka
565, and the
Department of Molecular
Pathology, Graduate School of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan
Received for publication, December 12, 2002, and in revised form, January 7, 2003
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ABSTRACT |
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Smad ubiquitin regulatory factor 1 (Smurf1), a
HECT type E3 ubiquitin ligase, interacts with inhibitory Smad7
and induces translocation of Smad7 to the cytoplasm. Smurf1 then
associates with the transforming growth factor (TGF)- Members of the transforming growth factor- In contrast to R-Smads and Co-Smad, I-Smads, including Smad6 and Smad7,
bind to type I receptors and compete with R-Smads for activation by the
receptors, resulting in inhibition of TGF- The transport of many large proteins from the nucleus to the cytoplasm
is mediated by a short leucine-rich motif, known as the nuclear export
signal (NES) sequence. The consensus sequence is defined as a set of
critically spaced large hydrophobic residues, usually leucines
L(X/XX/XXX)L(XX/XXX)LX,
where X indicates any residue) (13), although some
variations exist. The NESs of human immunodeficiency virus-1 Rev,
cyclic AMP-dependent protein kinase inhibitor, and MAPK
kinase have been well characterized. Mutations of leucines in NES
disrupt the ability of the protein to locate in the cytoplasm. CRM1,
which belongs to the family of importin Smad1 and Smad3 contain a lysine-rich nuclear localization signal in
their MH1 domains that is required for ligand-induced nuclear
translocation. In Smad3, phosphorylation of the C-terminal SSXS motif results in conformational changes that expose the
nuclear localization signal, so that importin- In the present study, we have shown that Smurf1 has a functional NES in
the C-terminal region. The Smurf1 NES mutant did not transport Smad7
from the nucleus to the cytoplasm, and it reduced inhibition by Smad7
of TGF- DNA Construction and Transfection--
The original constructs
of the constitutively active form of T Immunofluorescence Labeling--
Immunohistochemical staining of
FLAG-tagged Smad7, 6Myc-tagged Smurf1(CA), or 6Myc-tagged
Smurf1(NES-mut) in transfected cells was performed using mouse
anti-FLAG antibody or mouse anti-Myc antibody followed by incubation
with fluorescein isothiocyanate-labeled goat anti-mouse IgG as
described (12). 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 fluorescein isothiocyanate-labeled goat anti-mouse IgG
or rhodamine isothiocyanate-labeled goat anti-rabbit IgG, respectively.
For double staining of Smurf1 and CRM1, immunohistochemical staining of
6Myc-Smurf1 and CRM1-HA was performed using mouse anti-Myc or rabbit
anti-HA antibody followed by incubation with fluorescein
isothiocyanate-labeled goat anti-mouse IgG or rhodamine isothiocyanate-labeled goat anti-rabbit IgG, respectively. Cell nuclei
were stained by 4,6-diamidino-2-phenylindole (PI). Intracellular localization was determined by confocal laser scanning microscopy. For
LMB treatment, cells were incubated with 20 ng/ml LMB for 1 h just
before fixation.
Immunoprecipitation and Immunoblotting--
Cells were lysed
with Nonidet P-40 lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1% Nonidet P-40). Immunoprecipitation and
immunoblotting were performed as described (24).
Expression of GFP-NESs in Cells--
Fusion of the NES-like
peptides to green fluorescence protein (GFP) was achieved by insertion
of a cassette of two annealed oligonucleotides into
EcoRI-XhoI-cut pEGFP-C1
(Clontech) in which the multicloning site was
changed. The six NES-like sequences with three upstream residues from
Smurf1, FFR-L33PDPFAKIVV42,
GWE-V288RSTVSGRIYF298,
VQK-L356KVLRHELSL365,
LSY-F463HFVGRIMGLAV474,
END-I521TPVLDHTFCV531, and
ELI-I606GGLDKIDL614, were fused to the C
terminus of GFP and transfected into cells using the FuGENE6
transfection kit. GFP images were recorded 24 h after
transfection. For LMB treatment, cells were incubated with 20 ng/ml LMB
for 1 h just before image recording.
In Vitro Protein Binding Assay--
GST fusion proteins were
prepared as described (25). COS7 cells were transfected with CRM1-HA
expression vector, and cell extracts were isolated in Nonidet P-40
lysis buffer 24 h after transfection. Binding was performed by
incubating GST fusion proteins immobilized on the beads with cell
lysates at the indicated temperatures for 1 h, and the protein
complexes were washed with the same buffer. CRM1 bound to GST fusion
proteins was detected by immunoblotting using anti-HA antibody.
Luciferase Assay--
Cells were transiently transfected with an
appropriate combination of a p3TP-lux promoter-reporter construct,
expression plasmids, and pcDNA3 as described previously (10). The
total amounts of transfected DNAs were the same in each experiment, and
values were normalized using Renilla luciferase activity.
Smurf1 Has a Functional NES--
To test whether Smad7 or Smurf1
contains an NES, we examined the subcellular localization of Smad7 and
Smurf1 using transfected HeLa cells (Fig.
1) treated with LMB. Smurf1 induces the
degradation of Smad7. Because the ligase activity of Smurf1 is not
required for cytoplasmic translocation of the Smurf1-Smad7 complex
(12), a Smurf1 mutant, Smurf1(CA), which has a mutation in the
HECT domain and lacks the ligase activity, was used in this
experiment. In the absence of Smurf1(CA), Smad7 was located
predominantly in the nucleus. In contrast, Smad7 was detected
predominantly in the cytoplasm in the presence of Smurf1(CA), although
it also weakly stained in the nucleus (Fig. 1A,
top and middle rows, and Fig. 1B; see
Ref. 12). Treatment with LMB dramatically blocked nuclear export of
Smad7 by Smurf1(CA) (Fig. 1A, bottom row,
and Fig. 1B). Double staining of Smad7 and Smurf1 revealed
co-localization of these proteins in both the absence and presence of
LMB (Fig. 1C). Smurf1(CA) alone localized in both the
nucleus and cytoplasm without LMB treatment (Fig. 1D,
top row, and Fig. 1E), and LMB induced nuclear
accumulation of Smurf1(CA) (Fig. 1D, bottom row, and Fig. 1E), suggesting that Smurf1 contains a functional
NES.
CRM1, the cellular export receptor for NESs, has been reported to
dramatically induce nuclear export of NES-containing proteins (14, 15).
We found that overexpression of CRM1 did not affect the subcellular
localization of nuclear Smad7 (data not shown). However, it induced an
almost exclusively cytoplasmic distribution of Smurf1(CA), and the
effect of CRM1 was prevented by LMB (Fig. 1F). Although the
experiments have been performed using HeLa cells in Fig. 1, similar
results were obtained using COS7 cells (data not shown).
Identification of a Functional NES in Smurf1--
To define the
NES in Smurf1 that is responsible for nuclear export of the
Smurf1-Smad7 complex, we scanned the amino acid sequence of Smurf1 and
identified six putative leucine-rich NES-like motifs: amino acids
33-42, 288-298, 356-365, 463-474, 521-531, and 606-614 (Fig.
2A, left panel). To
identify a functional NES in Smurf1, we generated six GFP fusion
proteins using NES-like peptides with three upstream amino acid
residues flanking each NES: GFP-NES-(33-42)
(FFR-L33PDPFAKIVV42), GFP-NES-(288-298)
(GWE-V288RSTVSGRIYF298), GFP-NES-(356-365)
(VQK-L356KVLRHELSL365), GFP-NES-(463-474)
(LSY-F463HFVGRIMGLAV474), GFP-NES-(521-531)
(END-I521TPVLDHTFCV531), and GFP-NES-(606-614)
(ELII606GGLDKIDL614) (Fig. 2A,
right panel). In conformity with the classical Rev NES, each
motif contains four properly spaced large hydrophobic residues. In
transfected HeLa cells, although GFP alone and five other hybrids were
distributed diffusely in both the cytoplasm and nucleus, GFP fused with
the most C-terminal NES-containing amino acids 603 and 614 (GFP-NES-(606-614)) was located mainly in the cytoplasm of cells.
Furthermore, we confirmed that cytoplasmic localization of
GFP-NES-(606-614) could be inhibited by LMB (Fig. 2B).
Similar results were obtained using COS7 cells and 293T cells (data not
shown). Notably, the NES-like sequence corresponding to NES-(606-614)
is conserved in Smurf1 from Xenopus, mouse, and human and in
DSmurf in Drosophila (26).
Smurf1 Binds to CRM1 through its C-terminal NES--
To further
confirm the functional importance of the NES in Smurf1, we next
constructed an NES mutant of Smurf1 (Smurf1(NES-mut)) with a mutation
in the C-terminal NES (Fig.
3A). We first tested the
interaction of this mutant with Smad7 in transfected COS7 cells (Fig.
3B). Both Smurf1(CA) and Smurf1(NES-mut) bound to Smad7 in
both the absence and presence of c.a.T C-terminal NES of Smurf1 Is Responsible for Nuclear Export of the
Smad7-Smurf1 Complex--
We next examined the effect of the Smurf1
NES on subcellular localization of the Smad7-Smurf1 complex. Smurf1(CA)
alone localized in both the nucleus and cytoplasm (data not shown; see
Fig. 1, D and E),
whereas Smurf1(NES-mut) localized mainly
in the nucleus (Fig. 4, A and B, top
rows), confirming the findings shown in Figs. 2 and 3. We
next examined the effect of CRM1 on subcellular localization of
Smurf1(NES-mut). Overexpressed CRM1 accumulated Smurf1(CA) in the
cytoplasm (data not shown; see Fig. 1F) but did not affect
the localization of nuclear Smurf1(NES-mut) (Fig. 4, A and
B, bottom rows). Moreover, when Smad7 was
co-transfected, Smurf1(CA), but not Smurf1(NES-mut), induced nuclear
export of Smad7 (Fig. 4, C and D).
Smurf1 NES Mutant Prevents Inhibition by Smad7 of TGF- Smad7 inhibits TGF- To provide insights into the molecular mechanisms of nuclear export of
the Smurf1-Smad7 complex, we generated GFP fusion proteins containing
putative NES-like sequences of Smurf1. Interestingly, only the most
C-terminal NES-like sequence was found to function as NES in
Smurf1. The mutant Smurf1 containing alanine substitutions in the
C-terminal NES-like sequence, Smurf1(NES-mut), was able to interact
with Smad7 but failed to bind efficiently to CRM1. Also, nuclear export
of the Smurf1-Smad7 complex was not observed in Smurf1(NES-mut),
suggesting that the C-terminal NES of Smurf1 plays a critical role in
nuclear export of Smad7 by Smurf1. Furthermore, we demonstrated that
Smurf1(NES-mut) is more potent than Smurf1(CA) in reducing the
inhibitory activity of Smad7. These results suggest that
CRM1-dependent nuclear export of Smurf1 is required for the negative feedback regulation of TGF- Although we have demonstrated CRM1-dependent nuclear export
of Smurf1 in this study, the molecular mechanisms of nuclear import of
Smurf1 that are required for Smurf1 to bind to nuclear Smad7 are still
unclear. It is interesting to note that, although Smurf1 is too large
to pass through the nuclear pore by diffusion, Smurf1(NES-mut) accumulated in the nucleus following treatment with LMB. Moreover, in
our preliminary study, the C-terminal deletion mutant of Smurf1 localized predominantly in the cytoplasm and failed to accumulate in
the nucleus in the presence of
LMB.2 These results suggest
that not only NES but also the nuclear localization signal may exist in
the C-terminal region of Smurf1. The distribution of Smurf1 between the
nucleus and cytoplasm might therefore be driven by a dynamic balance
between nuclear import and nuclear export.
Our present study suggested that nucleocytoplasmic shuttling of Smurf1
is necessary for the function of Smad7. Recently, I-Smads have been
reported to have certain biological functions in the nucleus. For
example, Smads 6 and 7 have been demonstrated to act as transcriptional
co-repressors in the nucleus (27), and nuclear Smad7 has been shown to
induce apoptosis in prostatic cancer cells (28). Thus, Smurf1 may
regulate the nuclear and cytoplasmic functions of I-Smads by altering
their subcellular distribution.
In conclusion, we have demonstrated CRM1-dependent nuclear
export of the Smurf1-Smad7 complex and identified a functional NES in
Smurf1. Nuclear export of the Smurf1-Smad7 complex appears to be
crucial for Smad7 function in the negative feedback regulation of
TGF- type I
receptor, T
R-I, enhancing turnover. However, the mechanism of
nuclear export of Smad7 by Smurf1 has not been elucidated. Here we
identified a functional nuclear export signal (NES) in a C-terminal
region of Smurf1. In transfected cells, the Smurf1-Smad7 complex was accumulated in the cytoplasm by the nuclear export receptor, CRM1; this action was prevented by treatment with leptomycin B, a
specific inactivator of CRM1 function. A green fluorescence protein
fusion protein containing the C-terminal NES motif of Smurf1, located in the cytoplasm, accumulated in the nucleus following treatment with
leptomycin B. Moreover, Smurf1 was shown to bind physically to CRM1
through NES, and nuclear export of the Smurf1-Smad7 complex was
prevented by mutations of Smurf1 within the NES. Finally, the Smurf1
NES mutant reduced inhibition by Smad7 of the transcriptional activation induced by TGF-
. These results thus suggest that
CRM1-dependent nuclear export of Smurf1 is essential for
the negative regulation of TGF-
signaling by Smad7.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
(TGF-
)1
superfamily are multifunctional
cytokines that regulate growth, differentiation, apoptosis, and
morphogenesis (1). TGF-
superfamily proteins, including TGF-
s,
activins, and bone morphogenetic proteins, initiate cellular responses
by binding to type I and type II serine/threonine kinase receptors.
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 mainly through Smad
proteins. Eight different Smad proteins have been identified in mammals
and classified into three subgroups, i.e. receptor-regulated
Smads (R-Smads), common-partner Smads (Co-Smads), and inhibitory Smads
(I-Smads) (3-5). R-Smads are direct substrates of the type I
receptors. They are phosphorylated by type I receptors and form
heteromeric complexes with Co-Smad, Smad4. The Smad complexes then
translocate into the nucleus, where they regulate transcription of
various target genes together with transcriptional factors and
co-activators (3). Among R-Smads, Smad2 and Smad3 act in the TGF-
,
activin, and nodal pathways, whereas Smad1, Smad5, and Smad8 function
in the bone morphogenetic protein and anti-Müllerian hormone pathways.
superfamily signaling
(6-8). Interestingly, however, Smad7 has been reported to be located
in the nucleus in many types of cells (9), although it is observed in
the cytoplasm in certain cells (10). Recently, HECT type E3
ubiquitin ligases, Smad ubiquitin regulatory factor 1 (Smurf1) and
Smurf2, have been reported to interact with Smad7 in the
nucleus and to induce translocation of Smad7 to the cytoplasm. The
Smurf-Smad7 complexes then associate with TGF-
type I receptor
(T
R-I) enhancing its turnover (11, 12). However, the mechanism of
nuclear export of the Smurf-Smad7 complex has not been elucidated.
-related nuclear transport
receptors, directly and specifically associates with NES and mediates
nuclear export of the protein containing NES (14, 15). Leptomycin B
(LMB) directly binds to CRM1 and disrupts the interaction between CRM1
and NES, resulting in the inhibition of the effects of CRM1 (16).
1 can bind Smad3 and
mediate its nuclear import (17-19). Smad1 contains a C-terminal
leucine-rich NES that mediates its constant nuclear export by
interacting with CRM1 (20). Smad1 is therefore continuously shuttling
between the nucleus and the cytoplasm. Smad4 has an NES at the linker region, which is not conserved in R-Smads. Smad4 does not accumulate in
the nucleus by itself, because of the presence of the NES. Only upon
complex formation with R-Smads is the NES masked, enabling Smad4 to
accumulate inside the nucleus (21, 22). In I-Smads, neither the NES in
Smad4 nor that in Smad1 is conserved, and the putative NES of I-Smads
has not been identified.
transcriptional activity, suggesting that
CRM1-dependent nuclear export of Smurf1 is essential for negative regulation of the TGF-
pathway by Smad7.
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
R-I (c.a.T
R-I),
Smurf1(WT), Smurf1(CA), and Smad7 were generated as described
previously (6, 12, 23). Construction of the Smurf1 NES mutant
(Smurf1(NES-mut)) and CRM1 were performed by a polymerase chain
reaction (PCR)-based approach. Smurf1(NES-mut) does not contain the CA
inactivation mutation. COS7 cells, 293T cells, HeLa cells, and R mutant
mink lung epithelial (Mv1Lu) cells were transiently transfected using
FuGENE6 (Roche Molecular Biochemicals) as described (23).
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Smurf1 contains a functional NES.
A-C, treatment of LMB blocks nuclear export of
Smad7 in the presence of Smurf1. HeLa cells were transiently
transfected with FLAG-Smad7 alone (A and C,
top row) or together with 6Myc-Smurf1(CA) (A and
C, middle and bottom rows). In the
bottom row, cells were treated with 20 ng/ml LMB for 1 h. Cells were fixed and stained as described under "Materials and
Methods." Anti-FLAG staining for Smad7 (green) and nuclear
staining by PI (red) were performed in A, whereas
anti-FLAG staining for Smad7 (green) and anti-Myc staining
for Smurf1(CA) (red) were done in C. Similar
results were obtained in COS7 cells (data not shown). In B,
the distribution of Smad7 in transfected cells was scored as nuclear
(N), nuclear and cytoplasmic (NC), or cytoplasmic
(C) and graphed (mean ± S.D. from three experiments).
More than 150 cells were counted in each experiment. D and
E, LMB induces nuclear accumulation of Smurf1. HeLa cells
transiently transfected with 6Myc-Smurf1(CA) were treated with LMB
followed by anti-Myc staining for Smurf1(CA) (green) and
nuclear staining by PI (red). In E, the graph
shows the percentage of cells with the indicated Smurf1(CA) staining
pattern in the presence and absence of LMB. The distribution of
Smurf1(CA) was scored as in B. F, cytoplasmic
accumulation of Smurf1 is enhanced by CRM1, and blocked by LMB. HeLa
cells were transiently transfected with 6Myc-Smurf1(CA) alone
(top row) or together with CRM1-HA (middle and
bottom rows). In the bottom panels, cells were
treated with LMB. Anti-Myc staining for Smurf1(CA) (green)
and anti-HA staining for CRM1 (red) were performed.
FITC, fluorescein isothiocyanate; RITC, rhodamine
isothiocyanate.
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Fig. 2.
Identification of a C-terminal functional NES
in Smurf1. A, schematic representation of six putative
NES-like sequences in Smurf1 (left panel) and their GFP
fusion protein constructs (right panel). B, The
subcellular localization of the GFP fusion proteins with NES-like
peptides was examined in transfected HeLa cells in the presence and
absence of LMB. Similar results were obtained using COS7 cells and 293T
cells (data not shown).
R-I. Next, we examined the
association of CRM1 with Smurf1(CA) or Smurf1(NES-mut) in transfected
cells. Smurf1(CA), but not Smurf1(NES-mut), bound to CRM1 in
co-immunoprecipitation assays (Fig. 3C). Moreover, when
physical interaction between Smurf1 and CRM1 in vitro was examined at different temperatures, Smurf1(CA), but not
Smurf1(NES-mut), bound to CRM1 at 37 °C but not at 4 °C (Fig.
3D). These findings suggest that Smurf1 binds to CRM1
through the NES and that the binding of the two proteins is
cold-sensitive.
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Fig. 3.
Smurf1 binds to CRM1 through C-terminal
NES. A, schematic representation of the functional NES
motif in Smurf1. Isoleucine-612 and leucine-614 were each mutated to
alanine to generate the Smurf1 NES mutant
(Smurf1(Mut)). B, the
interaction between Smad7 and Smurf1(CA) or Smurf1(NES-mut) was
examined. Transfected COS7 cells were subjected to
FLAG-immunoprecipitation (IP) followed by Myc-immunoblotting
(Blot). The top panel shows the interaction, and
the lower three panels show the expression of each protein
as indicated. C, mutation in NES abolishes interaction of
Smurf1 with CRM1 in transfected cells. 293T cells were transfected with
CRM1-HA together with FLAG-Smurf1(CA) or FLAG-Smurf1(NES-mut). Cell
lysates were subjected to FLAG immunoprecipitation followed by HA
immunoblotting. The top panel shows the interaction, and the
lower two panels show the expression of each protein as
indicated. D, there is a cold-sensitive interaction
between Smurf1 and CRM1 in vitro. GST-Smurf1(CA) or
GST-Smurf1(NES-mut) was mixed with cell lysates from COS7 cells in
which CRM1-HA had been transfected. The samples were then incubated at
the indicated temperatures, and the precipitates were subjected to
SDS-PAGE followed by immunoblotting using anti-HA antibody or anti-GST
antibody to detect GST-Smurf1(CA). As a control, cell lysate was
directly subjected to SDS-PAGE (Input).
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Fig. 4.
C-terminal NES in Smurf1 induces nuclear
export of the Smad7-Smurf1 complex. A and B,
subcellular localization of Smurf1(NES-mut) in the absence (top
rows) or presence of CRM1 (bottom rows) was examined in
transfected HeLa cells. Anti-Myc staining for Smurf1 (green
in A and B) and nuclear staining by PI
(red in A) or HA staining for CRM1
(red in B) were performed. FITC,
fluorescein isothiocyanate; RITC, rhodamine isothiocyanate.
C and D, mutation in NES abolishes cytoplasmic
localization of the Smad7-Smurf1 complex. HeLa cells were transfected
with FLAG-Smad7 together with 6Myc-Smurf1(CA) (top rows) or
6Myc-Smurf1(NES-mut) (bottom rows). Anti-FLAG staining for
Smad7 (green in C and D) and nuclear
staining by PI (red in C) or anti-Myc staining
for Smurf1(CA) or Smurf1(NES-mut) (red in D) were
performed. Similar results were obtained in COS7 cells (data not
shown).
Transcriptional Activity--
To examine the effect of Smurf1(NES-mut)
on the inhibitory activity of Smad7, we performed a reporter assay
using a TGF-
-responsive promoter-reporter construct, p3TP-lux.
Smurf1(WT), but not Smurf1(CA), reduced transcriptional activity
induced by c.a.T
R-I in the presence of Smad7. Interestingly,
Smurf1(NES-mut) prevented the inhibitory activity of Smad7 in a
dose-dependent manner, and it was more potent than
Smurf1(CA) in preventing the inhibitory activity of Smad7 (Fig.
5). These findings thus confirm the
involvement of Smurf1 in the negative regulation of TGF-
signaling
by Smad7.
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Fig. 5.
Smurf1 NES mutant reduces inhibition by Smad7
of TGF- transcriptional activity. Effects
of Smurf1(WT), Smurf1(CA), and Smurf1(NES-mut) on the transcriptional
activity of c.a.T
R-I in the presence of Smad7 were examined
using a p3TP-lux assay. R mutant Mv1Lu cells were co-transfected
with the p3TP-lux luciferase construct and various combinations of
c.a.T
R-I, Smad7, Smurf1(WT), Smurf1(CA), and Smurf1(NES-mut)
cDNAs. + and ++ are 0.2 and 0.4 µg of Smurf1(WT) DNA, 0.4 and 0.8 µg of Smurf1(CA) DNA, and 0.7 and 1.4 µg of Smurf1(NES-mut) DNA,
respectively, transfected into R mutant Mv1Lu cells. This
experiment was performed five times with essentially the same results.
Similar results were obtained using COS7 cells and HeLa cells (data not
shown).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
signaling by binding to T
R-I and
preventing activation of R-Smads. Because Smad7 is located
predominantly in the nucleus in many transfected mammalian cells (9,
12), nuclear export of Smad7 may play an important role in the negative feedback regulation of TGF-
signaling. We recently reported that Smurf1 binds to Smad7 and induces its nuclear export (12). Because endogenous expression of Smurf1 was negligible in the transfection experiments (data not shown), Smad7 was exported to the cytoplasm only
in cells co-transfected with Smurf1 in the present study. Generally,
the nuclear export of many large proteins is mediated by NESs, through
which CRM1 binds to such proteins in an LMB-sensitive manner. In this
study, we showed that nuclear export of Smad7 by Smurf1 is enhanced by
co-expression of CRM1 in an LMB-sensitive fashion, suggesting that
Smurf1 and/or Smad7 have functional NESs. Furthermore, we found that
CRM1 does not affect localization of nuclear Smad7, whereas Smurf1
accumulates in the cytoplasm in the presence of CRM1, and the effect of
CRM1 on Smurf1 is prevented by treatment with LMB. These findings
strongly suggest that Smurf1, but not Smad7, has a functional NES.
signaling.
signaling. It will be important to determine how the nuclear
import and export of Smurf1 are regulated under physiological and
pathological conditions.
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ACKNOWLEDGEMENTS |
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We are grateful to Yuri Inada and Aki Hanyu for technical help.
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FOOTNOTES |
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* This study was supported by grants-in-aid for scientific research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan and also by a grant from Boehringer Ingelheim.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.
§§ To whom correspondence should be addressed: Dept. of Biochemistry, Japanese Foundation for Cancer Research,1-37-1 Kami-ikebukuro, Toshima-ku, Tokyo 170-8455, Japan. Tel.: 81-3-3918-0342; Fax: 81-3-3918-0342; E-mail: miyazono-ind@umin.ac.jp.
Published, JBC Papers in Press, January 7, 2003, DOI 10.1074/jbc.M212663200
2 Y. Tajima, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
R-Smad, receptor-regulated Smad;
Co-Smad, common-partner Smad;
I-Smad, inhibitory Smad;
Smurf, Smad
ubiquitin regulatory factor;
T
R-I, TGF-
type I receptor;
c.a.T
R-I, constitutively active form of T
R-I;
NES, nuclear export
signal;
LMB, leptomycin B;
HA, hemagglutinin;
PI, 4,6-diamidino-2-phenylindole;
GFP, green fluorescence protein;
CRM1, chromosomal region maintenance 1.
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