From the Ludwig Institute for Cancer Research, Box
595, S-751 24 Uppsala, Sweden and § Department of
Molecular Biophysics, University of Lodz, 90--237
Lodz, Poland
Received for publication, December 6, 2000, and in revised form, January 19, 2001
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ABSTRACT |
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Smad proteins are major components in the
intracellular signaling pathway of transforming growth factor- Discovery of Smad proteins provided insights into the
intracellular signaling by
TGF- Since expression of Smad7 is up-regulated after TGF- Phosphorylation has been found to be a potent regulatory mechanism of
Smad functions (10-13). Sequential phosphorylation of R-Smads at two
C-terminal serine residues by activated receptors is the triggering
event in ligand-dependent intracellular signaling (13).
Phosphorylation in the linker region, presumably by mitogen-activated protein kinase, may affect nuclear translocation of R-Smads (12). Moreover, phosphorylation of R-Smads by other kinases has also been
shown to influence intracellular signaling (14, 15). Phosphorylation of
mammalian Smad4 has not been reported, but Xenopus Smad4 We show here that Smad7 is phosphorylated, and we identify Ser-249 as a
major phosphorylation site. Mutation of Ser-249 did not affect the
inhibitory effect of Smad7 on TGF- Constructs, Cells, and Reagents--
pcDNA3-based expression
vectors for full-length mouse Smad7, as well as for its N-terminally
truncated (Smad7C) and C-terminally truncated (Smad7N) mutants, were
described previously (5). Expression vectors for Smad7 point mutants
were generated by a polymerase chain reaction-based approach
(QuickChange, Stratagene, La Jolla, CA) and sequenced to confirm
absence of undesired mutations. The luciferase reporter constructs
CAGA(12)-luc and SBE(4)-luc were described
earlier (18, 19), GCCG(12)-luc was obtained from Kohei
Miyazono, p800-luc from Kunihiro Matsumoto, pH6-luc from Howard
Goldberg, E2F1-luc and CycE-luc from Kristian Helin, and p21p-luc and
p15p751-luc from Xiao-Fan Wang.
Fusion constructs of the DNA-binding domain of GAL4 with wild-type
Smad7 and Smad7 mutants were obtained by subcloning Smad7 into the pM
expression vector (CLONTECH, Palo Alto, CA). The
GAL4-binding site-containing luciferase reporter GAL4-TGTA-luc,
containing a mutated TATA box from retinoid acid receptor
COS-1, Mv1Lu, and NIH-3T3 cell lines were obtained from ATCC, Manassas,
VA. DR-26 clone of Mv1Lu cells, lacking TGF-
All data in figures are presented as average ± S.E. Standard
tests were used to evaluate statistical significance of the obtained results.
Identification of Phosphorylation Sites in
Smad7--
[32P]Orthophosphate labeling and mapping of
tryptic phosphopeptides, as well as phosphoamino acid analysis, were
performed as described (13). FLAG-Smad7 protein was immunoprecipitated
with M2 anti-FLAG antibody (Sigma). Visualization of phosphopeptides was carried out using the Fujix BAS2000 imaging system.
[35S]Methionine Metabolic Labeling--
Detection
and quantification of transiently expressed or stably transfected
wild-type and mutant FLAG-Smad7 was performed by
[35S]methionine metabolic labeling (10 µCi/ml, 4 h), followed by immunoprecipitation with M2 anti-FLAG antibody. To
determine protein stability, cells were pulse-labeled with
[35S]methionine (100 µCi/ml, 1 h), chased with
medium containing unlabeled methionine, and immunoprecipitated with M2
anti-FLAG antibody. The immunoprecipitated material was resolved by
SDS-PAGE and quantified using the Fujix BAS2000 imaging system.
Luciferase Reporter Assays--
Luciferase reporter constructs
were cotransfected into cells together with Smad7 expression plasmids
using various transfection techniques (DEAE-dextran, LipofectAMINE, or
Fugene6) according to the manufacturer's instructions. Cells were
incubated and treated as described in the figure legends. Luciferase
activity in stimulated and nonstimulated cells was determined using a
luciferase detection kit (Promega, Madison, WI). Luminescence was
measured in a Victor multilabel plate reader (EG & G Wallac, Turku,
Finland). Cells were cotransfected with a
For transcriptional activation studies, GAL4 DNA-binding domain fusion
constructs were cotransfected into cells together with GAL4-binding
site-containing reporter constructs, and the level of luciferase
expression was determined as described above.
Immunofluorescence--
Intracellular localization of FLAG-Smad7
was determined by immunofluorescence as described earlier (5).
FLAG-Smad7 protein in fixed cells was detected using M5 anti-FLAG
antibody (Sigma) and visualized with tetramethylrhodamine
isothiocyanate-labeled anti-mouse antibody (DAKO, Gostrup, Denmark).
Nuclei were counterstained with 4',6-diamidino-2-phenylindole.
Smad7 Is a Phosphoprotein--
To investigate whether Smad7 is a
phosphoprotein, full-length and N-terminal or C-terminal deletion
mutants of FLAG-tagged Smad7 were transiently expressed in Mv1Lu and
COS-1 cells. The cells were incubated with
[32P]orthophosphate, treated or not with 10 ng/ml
TGF-
Unlike the case for R-Smads, phosphorylation of Smad7 was not affected
by TGF-
The fact that the phosphorylation of Smad7 was TGF- Smad7 Is Phosphorylated at Serine 249--
To identify
phosphorylation sites, Smad7 was immunoprecipitated from
[32P]orthophosphate-labeled cells and subjected to
tryptic digestion; the generated peptides were separated by
two-dimensional mapping. We identified three major phosphopeptides in
full-length FLAG-Smad7 and four in FLAG-Smad7C (Fig.
2, A and C). Spots
1 and 2 were reproducibly detected in more than 20 repeated
phosphopeptide maps of full-length Smad7, whereas spot 4 was not always
seen. Spot 4 was also weak in maps of FLAG-Smad7C (Fig. 2C).
The highly phosphorylated phosphopeptide giving rise to the diffuse
spot 3 of FLAG-Smad7C was not found in full-length protein in any of the experiments. The phosphopeptides from spots 1-3 contained only
phosphoserine, and neither phosphothreonine nor phosphotyrosine were
detected (Fig. 2F). Phosphopeptides were also subjected to radiochemical sequencing. However, the sequencing of spots 1 and 2 did
not provide reliable data, probably due to the fact that the
phosphoserine residue(s) was located too far from the N termini of the
phosphopeptides to be detected in radiochemical sequencing by Edman
degradation (see below). Therefore, an alternative strategy to localize
the phosphorylated residues was used. Since the phosphopeptides found
in full-length FLAG-Smad7 (spots 1 and 2) were found also in
FLAG-Smad7C (Fig. 2, A and C), we constructed a
series of expression vectors containing serine-to-alanine mutations of
each of the serine residues present in FLAG-Smad7C. All these mutants
were expressed in COS-1 cells that were subjected to
[32P]orthophosphate labeling; Smad7C and full-length
Smad7 mutants were then trypsinized and subjected to two-dimensional
phosphopeptide analysis. Unequivocally, mutation of Ser-249 to an
alanine residue resulted in disappearance of the phosphopeptide
corresponding to spot 1 (Fig. 2, B and D).
Interestingly, in FLAG-Smad7C, mutation of Ser-249 to an alanine
residue also led to disappearance of spot 2 (Fig. 2D),
suggesting that in this mutant protein, Ser-249 phosphorylation may
also affect phosphorylation at another site. Ser-249 is located at
position 36 in the corresponding tryptic peptide (Fig. 2G).
This explains the lack of detection by radiochemical sequencing, which
gives reliable results only for phosphorylated residues in the
N-terminal 20 amino acid residues of each peptide.
The composite spot 3, consisting of three differently migrating
phosphopeptides unique to FLAG-Smad7C and not seen in full-length FLAG-Smad7, was very prominent. The phosphorylated residue in this
group of phosphopeptides was found to be Ser-206, since upon mutation
of Ser-206 to an alanine residue, all three phosphopeptides of spot 3 disappeared (Fig. 2E). Interestingly, Ser-206 is neighbored by a PPPPY sequence, which may be involved in regulation of
ubiquitin-dependent degradation (20). Therefore,
phosphorylation of Ser-206 in the truncated Smad7 may interfere with
its degradation, which may explain the higher level of FLAG-Smad7C
protein compared with full-length FLAG-Smad7. However, because
phosphorylation at Ser-206 was found only in the truncated mutant and
not in the full-length protein, we considered it an artifact caused by
the truncation. Thus, we have identified Ser-249 as a major
phosphorylation site in Smad7.
Smad7-dependent Inhibition of TGF-
Because CAGA(12)-luc and GCCG(12)-luc reporters
have artificially designed promoters, we also tested how reporters with
natural promoters, responsive to the ligand, were affected by
interference with Smad7 phosphorylation. When reporters containing 800 base pairs of the PAI-1 promoter (p800-luc), 296 base pairs of collagen Phosphorylation of Smad7 Does Not Affect Its Stability and Nuclear
Localization--
Since regulation of Smad biosynthesis and
degradation has been shown to be an important mechanism of modulation
of TGF-
In two recent reports, the nuclear localization of Smad7 has been
described (5, 6). These observations also suggested that the nuclear
localization is a regulated process. Therefore, we tested whether
phosphorylation at Ser-249 affects the intracellular distribution of
Smad7. Fig. 5 shows that mutation of
Ser-249 did not lead to significant differences in intracellular
localization, since S249A and S249D mutants and wild-type of FLAG-Smad7
all localized to the cell nucleus to a similar extent. Thus, the
intracellular distribution of Smad7 is not affected by its
phosphorylation at Ser-249.
Transcriptional Activity of Smad7 Is Dependent on Phosphorylation
at Ser-249--
The findings that Smad7 is a nuclear protein, together
with the observation that a GAL4-Smad7 fusion protein showed
transcriptional activity in PC3U
cells,2 prompted us to
investigate whether the phosphorylation of Smad7 affects its
transcription-regulating activity. We tested the effect of full-length
Smad7, fused to the DNA-binding domain of GAL4, on the response of
various reporters containing different minimal promoters downstream of
the GAL4-binding elements (GAL4-TGTA-luc, GAL4-SV40-luc, and
GAL4-TK-luc). We found that, in NIH-3T3 cells, wild-type Smad7
up-regulated the GAL4-TGTA-luc reporter containing a point-mutated TATA
box to decrease the background activity (Fig. 6A). Abrogation of Smad7
phosphorylation at Ser-249 by its substitution to an alanine residue
significantly inhibited this stimulation (p < 0.01).
Substitution of Ser-249 to aspartic acid residue resulted in an effect
similar to the wild-type Smad7, probably due to partial mimicking of
phosphorylation by introduction of a negative charge (Fig.
6A). Similar results were obtained for the wild-type and S249A mutant when this reporter was used in COS-1 (Fig. 6C)
and Mv1Lu cells (data not shown). For Mv1Lu cells, however, the effect was weaker than for COS-1 cells. Interestingly, using the reporter containing an SV40 minimal promoter instead of TGTA, wild-type Smad7
was found to repress luciferase expression; in this case the S249D
mutant of Smad7 was mimicking the wild-type Smad7 effect on
transcription, and the S249A mutant even induced it (Fig.
6D). Results obtained with the GAL4-TK-luc reporter were
similar to the data for the GAL4-SV40-luc reporter (data not shown).
Thus, Smad7 can act as a transcriptional activator or as a repressor depending on the type of promoter. Our data suggest that the
phosphorylation at Ser-249 regulates the transcriptional activity of
Smad7, probably through regulation of its interaction with other
components in transcriptional complexes.
Here we report the identification of Ser-249 as a major
phosphorylation site in Smad7. Unlike the case for R-Smads, the
phosphorylation of Smad7 was found not to be dependent on TGF- Ser-249 is one of the two major phosphorylation sites in Smad7 (Fig.
2); the second major site is currently under investigation. Ser-249 is
located in the C-terminal part of the region corresponding to the
linker in other Smads. Interestingly, a Smad activation domain (SAD)
has been identified in the corresponding part of Smad4; SAD interacts
with MSG-1, a transcriptional coactivator of Smad4, and has an
important role for the transcriptional activity of Smad4 (22, 23). We
have not observed any homology between SAD and the corresponding region
of Smad7; however, given the importance of SAD in Smad4 transcriptional
regulation, it is interesting that phosphorylation in the corresponding
region of Smad7 affects its transcriptional activity.
The nuclear localization (Fig. 5) is consistent with the possibility
that Smad7 is involved in regulation of gene expression. R-Smads and
Smad4 are known as potent transcriptional regulators, and they are
translocated to the nucleus upon ligand addition. Their function is
dependent on interactions with coactivators and corepressors and on the
ability of Smads to bind DNA directly (1-3, 24). Recently, Bai
et al. (25) reported that inhibitory Smad6 also can act as a
transcriptional regulator through interaction with the homeobox
(Hox)c-8 protein, extending the suggestion of transcriptional regulator
function to inhibitory Smads. In line with previous observations (26),
we could detect only a weak effect of GAL4-FLAG-Smad7 on the activity
of GAL4-binding sequence-containing reporters in Mv1Lu cells of
epithelial origin (data not shown). However, in cells of mesenchymal
origin, GAL4-FLAG-Smad7 significantly induced the GAL4-TGTA-luc
reporter. Interestingly, a GAL4-binding sequence-containing reporter
with an SV40 or TK minimal promoter was inhibited by the wild-type
GAL4-FLAG-Smad7 fusion. Similar opposite effects on gene expression
were also described for Smad3; Smad3 was found to potently induce
CAGA(12)-luc (18) but inhibited stimulation of the
goosecoid reporter (27). In similarity to Smad3, the stimulatory or
inhibitory effects of Smad7 on the regulation of transcription may
depend on the interaction between Smad7 and other proteins. Our data
suggest that such interactions may be regulated by Smad7
phosphorylation, as the phosphorylation-deficient S249A mutant of Smad7
shows impaired effects when compared with the wild-type protein (Fig.
6). The S249D mutant of Smad7, partially mimicking the presence of a
phosphoryl group by introducing a negative charge, behaved similar to
wild-type Smad7.
Our findings suggest that Smad7 is not only involved in control of
signaling pathways of TGF-
(TGF-
), and phosphorylation is an important mechanism in regulation
of their functions. Smad7 was identified as a potent inhibitor of
TGF-
-dependent signaling. We have identified serine 249 in Smad7 as a major phosphorylation site, the phosphorylation of which
was not affected by TGF-
1. Abrogation of the phosphorylation by
substitution of Ser-249 with alanine or aspartic acid residues did not
affect the ability of Smad7 to inhibit TGF-
1 and BMP7 signaling. No
differences were found in the stability or in the intracellular
distribution of Smad7 mutants compared with the wild-type molecule.
However, Smad7 fused to the DNA-binding domain of GAL4 induced
transcription from a reporter with mutated TATA minimal promoter in a
Ser-249-dependent manner. Moreover, a reporter with the
SV40 minimal promoter was inhibited by GAL4-Smad7, and this effect was
also dependent on Ser-249 phosphorylation. The amplitude of effects on
transcriptional regulation was dependent on cell type. Our results
suggest that phosphorylation of Smad7, unlike phosphorylation of the
receptor-regulated Smads, does not regulate TGF-
signaling but
rather affects TGF-
-independent effects of Smad7 on transcriptional regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 family members.
Three groups of Smads have been described. Receptor-regulated Smads
(R-Smads) are direct targets of activated receptors and provide the
signaling specificity. Common mediator (Co-Smad) Smad4 forms complexes
with R-Smads and is involved in signal propagation. Finally, Smad7 and
Smad6 were identified as inhibitors (I-Smads) of signaling by members
of the TGF-
family (1-3). Smad7 has been suggested to inhibit
TGF-
signaling by inhibiting the phosphorylation of R-Smads by type
I receptors (1-4). Interestingly, Smad7 was found to occur abundantly
in the nuclei of certain cells and to be exported from the nucleus upon
TGF-
stimulation or a change in cell substrate (5, 6). This suggests
that Smad7 may also have a function in the nucleus, which may be
independent of the inhibition of ligand-induced signaling at the
receptor level.
stimulation, it
has been suggested that Smad7 is involved in negative feedback of
TGF-
signaling. Induction of Smad7 has also been described as the
pivotal mechanism whereby tumor necrosis factor-
and interferon
inhibit TGF-
signaling (7, 8). Smad7 has been found to be
up-regulated in human tumors, but no mutation in human cancers has yet
been described (9).
has been identified as a phosphoprotein (16). Among inhibitory Smads,
Smad6 is a phosphoprotein (17), whereas phosphorylation of Smad7 has
not been characterized.
or BMP7 signaling and did not
interfere with nuclear localization of Smad7. However, the
TGF-
-independent transcriptional activity of Smad7 was affected by
mutation of Ser-249, suggesting that phosphorylation of Smad7 at
Ser-249 is important for its ligand-independent ability to regulate transcription.
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
2, and
GAL4-TK-luc, containing a herpes simplex virus-thymidine kinase
basal promoter, were obtained from Johan Ericsson, and GAL4-SV40-luc,
containing an SV40 early promoter, was from Cory Abate.
type II receptor, was
obtained from Joan Massagué. For stable transfection, wild-type
FLAG-Smad7 was subcloned in pMEP4 vector. The construct and empty
vector were transfected in DR-26 and NIH-3T3 cells, followed by
selection in presence of hygromycin B.
-galactosidase expression
vector, and luciferase activity values were compensated for differences
in transfection efficiency by a colorimetric
-galactosidase assay.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1 for 2 h, and FLAG-Smad7 proteins were immunoprecipitated
with anti-FLAG antibodies; immunoprecipitates were analyzed by
SDS-PAGE. We found that full-length and an N-terminal deletion mutant
of Smad7 (FLAG-Smad7C) were phosphorylated (Fig.
1, A and C). No
phosphorylation of a mutant with deleted MH2 domain was found (Fig. 1,
E and F). The protein expression level was
monitored by immunoprecipitation of FLAG-Smad7 from
[35S]methionine-labeled cells (Fig. 1, B, D,
F, and G), which were transfected in parallel with the
cells used for [32P]orthophosphate labeling.
Significantly higher phosphorylation of FLAG-Smad7C correlated with the
higher expression of the protein; therefore, no difference in its
specific phosphorylation compared with full-length FLAG-Smad7 was
observed (Fig. 1).
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Fig. 1.
Smad7 is phosphorylated in vivo,
and its phosphorylation is not affected by
TGF- . Subconfluent COS-1 cells were
transiently transfected with full-length mouse FLAG-Smad7, FLAG-Smad7C,
or FLAG-Smad7N alone (A-D) or together with type I and type
II TGF-
receptors (E and F). Cells were
labeled with [32P]orthophosphate (A, C, E, and
G) or with [35S]methionine (B, D,
F, and G) and treated or not with 10 ng/ml of TGF-
1
for the last 2 h of incubation. Prior to labeling, cells were
cultured in medium containing 10% FBS (A-G) or were
starved for 24 h in medium without serum (A-D).
C and N show transfection of FLAG-Smad7 mutants
with deletion of MH1 and MH2 domains, respectively. G,
phosphorylation of stably transfected wild-type FLAG-Smad7 is not
affected by TGF-
1 in NIH-3T3 cells and in DR-26 clone of Mv1Lu
cells. FLAG-Smad7 expression was induced by 4 h of pretreatment
with 50 µM ZnCl2, and cells were
subjected to [32P]orthophosphate
(32P) or
[35S]methionine (35S)
labeling. As a control, cells transfected with empty vector were used.
FLAG-Smad7 proteins were immunoprecipitated with anti-FLAG antibodies,
resolved by SDS-PAGE, and visualized on a FujiX PhosphorImager.
Arrows show the position of full-length FLAG-Smad7,
FLAG-Smad7C, and FLAG-Smad7N. Double arrows show migration
position of molecular mass markers (30 and 46 kDa).
1 in COS-1 cells with cotransfected T
R-I and T
R-II
(Fig. 1E) or without cotransfection of the receptors (Fig. 1, A and C). We did not observe
TGF-
-dependent differences in Smad7 phosphorylation in
NIH-3T3 cells, which express endogenous receptors. Smad7
phosphorylation was not affected in DR-26 cells, which lack T
R-II,
and therefore are nonresponsive to TGF-
(Fig. 1G).
Moreover, the two-dimensional phosphopeptide maps of Smad7 from cells
treated or not with TGF-
1 showed a similar pattern (data not shown).
Thus, TGF-
has no effect either on the total phosphorylation level
or on the pattern of phosphorylated sites. Therefore, Smad7 is
phosphorylated by kinase(s) other than the TGF-
receptors.
-independent
prompted us to attempt to evaluate how this phosphorylation is
regulated. We observed that the level of Smad7 phosphorylation was
higher if cells were serum-starved and that the phosphorylation was
decreased if cells were cultured in the presence of serum. The effect
of serum starvation was found to be especially strong for FLAG-Smad7C.
No changes in the level of protein expression were detected, suggesting
that the differences in phosphorylation were not related to the
quantity of Smad7. The phosphopeptide patterns of Smad7 from
serum-starved and proliferating cells were also similar, as evaluated
by two-dimensional mapping (data not shown). Therefore, the activity of
kinase(s) and/or phosphatase(s) regulating Smad7 phosphorylation may be
dependent on the serum starvation of cells but is not directly
regulated by TGF-
1.
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Fig. 2.
Identification of phosphorylation sites in
Smad7. Two-dimensional phosphopeptide mapping of wild-type
(A) and S249A mutant (B) of the full-length
FLAG-Smad7, and of wild-type (C), S249A (D), and
S206A (E) mutants of the N-terminal deleted construct
FLAG-Smad7C. The proteins were expressed in
[32P]orthophosphate-labeled COS-1 cells. The
arrows show positions of major phosphopeptides numbered from
1-4, and arrowheads show application points.
F, two-dimensional phosphoamino acid analysis of major
phosphopeptides. The nomenclature corresponds to the designations of
spots in A-E. Migration positions of different phosphoamino
acids were determined using nonradioactive internal standards.
pY, pT, and pS correspond to phosphotyrosine,
phosphothreonine, and phosphoserine, respectively. G,
location of the identified phosphorylatable serine residues 249 and 206 in the sequence of mouse Smad7.
and BMP Signaling
Is Not Dependent on Smad7 Phosphorylation--
To investigate whether
phosphorylation of Smad7 affects its inhibitory action on TGF-
and
BMP signaling, we used luciferase reporter assays, which are dependent
on the respective intact R-Smad-dependent pathways.
CAGA(12)-luc is activated upon treatment of cells with
TGF-
but not BMP, and its activation requires a receptor-dependent phosphorylation of Smad3 (18). Wild-type Smad7 inhibits the receptor-dependent phosphorylation of
R-Smads and blocks TGF-
-induced activation of the
CAGA(12)-luc reporter (18). We found that S249A and S249D
mutants of Smad7 were as potent inhibitors as wild-type Smad7 (Fig.
3A). Neither was any significant effect of the mutation of Ser-249 found on inhibition of
BMP7-dependent activation of the GCCG(12)-luc
reporter (Fig. 3B). Stimulation of this reporter is
dependent on receptor-induced activation of Smad1 and Smad5, which are
specific for the BMP signaling pathway (21). In addition, the S249A and
S249D mutants of Smad7 were equally efficient as wild-type Smad7 in
inhibition of ligand-induced activation of another reporter responsive
to TGF-
and BMP, SBE(4)-luc (data not shown).
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Fig. 3.
TGF- and BMP
signaling is not affected by phosphorylation of Smad7.
TGF-
-responsive CAGA(12)-luc (A) or
BMP-responsive GCCG(12)-luc (B) reporter
constructs were transfected in Mv1Lu cells together with wild-type and
mutants of Smad7. Cells were stimulated with 10 ng/ml of TGF-
1
(A, C, and D) or 100 ng/ml of BMP7
(B); luciferase activity was measured after 20 h and
corrected for transfection efficiency. Natural promoter-derived
p800-luc (C) and p21p-luc (D) reporter
constructs, susceptible to TGF-
stimulation, were cotransfected with
Smad7 constructs and cells were treated with TGF-
1, as described for
A and B. In all panels, open bars
correspond to unstimulated cells and filled bars to cells
stimulated with ligand. Expression levels were normalized by measuring
-galactosidase activity. Data are presented as mean from
n >3 experiments, and error bars represent
S.D.
2(I) promoter (pH6-luc), promoters of the
cyclin-dependent kinase inhibitors p21 and p15 (p21-luc,
p15-luc), or an E2F1-luc reporter were analyzed, the inhibition of the
TGF-
-induced activation by Smad7 was not found to be affected by
mutations of Ser-249 (Fig. 3, C and D, data not
shown). The diversity of molecular mechanisms, by which Smads regulate
different promoters used in this study, supported the conclusion that
phosphorylation at Ser-249 does not affect the ability of Smad7 to
interfere with Smad-mediated TGF-
and BMP signaling
pathways.
signaling (20), we investigated whether phosphorylation of
Smad7 affects its stability. Transfection of similar quantities of
cDNAs of the wild-type and the S249A and S249D mutants of Smad7
resulted in comparable protein expression levels (data not shown).
Moreover, pulse-chase experiments showed that interference with
phosphorylation at Ser-249 did not affect the half-life of Smad7 (Fig.
4). In the presence of the protein
synthesis inhibitor cycloheximide, the half-life of FLAG-Smad7 was ~1
h, and no significant differences were found for the mutants. In
absence of cycloheximide the apparent half-life was estimated at ~4 h
both for wild-type Smad7 and for Smad7 mutants (data not shown). Thus,
the phosphorylation of Smad7 at Ser-249 appears not to affect its
stability and half-life.
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Fig. 4.
Phosphorylation status does not influence the
stability of Smad7 in vivo. COS-1 cells were
transiently transfected with FLAG-Smad7 (wild-type,
diamonds; S249A, squares; S249D,
triangles), pulse-labeled with [35S]methionine
for 1 h and chased with medium containing 30 µg/ml cycloheximide
to inhibit further synthesis of proteins. At specific time points,
remaining radiolabeled Smad7 protein was immunoprecipitated with an
anti-FLAG antibody, resolved by SDS-PAGE, and quantified by
phosphorimaging. Data are presented as mean from 3 experiments, and
error bars represent S.D.
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Fig. 5.
Intracellular distribution of Smad7
phosphorylation site mutants. Mv1Lu cells were transiently
transfected with FLAG-Smad7 (wild-type and S249A and S249D mutants)
using Fugene6 reagent. After 48 h, cells were fixed and
immunostained with anti-FLAG antibodies. Intracellular localization of
Smad7 protein was visualized by immunofluorescence and is shown in
A (wild-type), C (S249A mutant), and E
(S249D mutant). B, D, and F show counterstaining
of nuclei with 4',6-diamidino-2-phenylindole in the same slide sections
shown in A, C, and E, respectively.
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Fig. 6.
Effects of phosphorylation site mutations on
transcriptional activity of Smad7. NIH-3T3 (A and
B) and COS-1 (C and D) cells were
transiently cotransfected with constructs expressing wild-type Smad7
and S249A and S249D mutants, C-terminally fused to GAL4 DNA-binding
domain, as well as with reporter constructs containing GAL4-binding
sequences and a mutated TATA box (GAL4-TGTA-luc, A and
C) or an SV40-derived promoter sequence (GAL4-SV40-luc,
B and D) as minimal promoters upstream of the
luciferase reporter gene. Plasmids expressing GAL4 DNA-binding domain
were used as control. Luciferase activity values were measured after
48 h and corrected for transfection efficiency. Data are presented
as mean from 3 experiments, and error bars represent S.D. *,
p < 0.001; #, p < 0.01; +,
p < 0.05 (compared with wild type (WT)).
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
treatment of cells. We show that phosphorylation at Ser-249 does not
affect the stability or subcellular localization of Smad7, nor does it
affect the ability of Smad7 to inhibit TGF-
signaling.
Interestingly, however, the presence of the phosphorylatable Ser-249 in
Smad7 was found to be important for a novel function ascribed to Smad7,
i.e. its ability to regulate transcription.
family members, but may also have direct
effects in the nucleus, possibly regulated by other regulatory
pathway(s). To explore this possibility, it will be important to
identify the kinase phosphorylating Smad7, components interacting with
Smad7 in the nucleus, as well as target genes for Smad7 as a
transcription factor. These studies are in progress.
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ACKNOWLEDGEMENTS |
---|
We are thank Johan Ericsson for valuable
advice and Kuber Sampath for BMP7 and Napoleon Ferrara for
TGF-1.
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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.
¶ Supported by a scholarship from Svenska Institutet.
Supported in part by grants from the Swedish Cancer Society
and the Swedish Medical Research Council.
** Supported in part by a grant from the Royal Swedish Academy of Sciences. To whom correspondence should be addressed: Ludwig Institute for Cancer Research, Box 595, S-751 24, Uppsala, Sweden. Tel.: 46-18-16 04 11; Fax: 46-18-16 04 20; E-mail: serhiy.souchelnytskyi@ licr.uu.se.
Published, JBC Papers in Press, January 19, 2001, DOI 10.1074/jbc.M011019200
2 M. Landström, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are:
TGF-, transforming growth factor-
;
BMP, bone morphogenetic protein;
MH
domain, mad homology domain;
SAD, Smad activation domain;
PAGE, polyacrylamide gel electrophoresis;
TK, thymidine kinase.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. | Miyazono, K., ten Dijke, P., and Heldin, C.-H. (2000) Adv. Immunol. 75, 115-157[Medline] [Order article via Infotrieve] |
2. | Massague, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve] |
3. |
Piek, E.,
Heldin, C. H.,
and Ten Dijke, P.
(1999)
FASEB J.
13,
2105-2124 |
4. | Nakao, A., Afrakhte, M., Moren, A., Nakayama, T., Christian, J. L., Heuchel, R., Itoh, S., Kawabata, M., Heldin, N. E., Heldin, C. H., and ten Dijke, P. (1997) Nature 389, 631-635[CrossRef][Medline] [Order article via Infotrieve] |
5. |
Itoh, S.,
Landstrom, M.,
Hermansson, A.,
Itoh, F.,
Heldin, C. H.,
Heldin, N. E.,
and ten Dijke, P.
(1998)
J. Biol. Chem.
273,
29195-29201 |
6. |
Zhu, H. J.,
Iaria, J.,
and Sizeland, A. M.
(1999)
J. Biol. Chem.
274,
32258-32264 |
7. |
Bitzer, M.,
von Gersdorff, G.,
Liang, D.,
Dominguez-Rosales, A.,
Beg, A. A.,
Rojkind, M.,
and Bottinger, E. P.
(2000)
Genes Dev.
14,
187-197 |
8. | Ulloa, L., Doody, J., and Massague, J. (1999) Nature 397, 710-713[CrossRef][Medline] [Order article via Infotrieve] |
9. | Kleeff, J., Ishiwata, T., Maruyama, H., Friess, H., Truong, P., Buchler, M. W., Falb, D., and Korc, M. (1999) Oncogene 18, 5363-5372[CrossRef][Medline] [Order article via Infotrieve] |
10. | Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224[Medline] [Order article via Infotrieve] |
11. |
Abdollah, S.,
Macias-Silva, M.,
Tsukazaki, T.,
Hayashi, H.,
Attisano, L.,
and Wrana, J. L.
(1997)
J. Biol. Chem.
272,
27678-27685 |
12. | Kretzschmar, M., Doody, J., and Massague, J. (1997) Nature 389, 618-622[CrossRef][Medline] [Order article via Infotrieve] |
13. |
Souchelnytskyi, S.,
Tamaki, K.,
Engstrom, U.,
Wernstedt, C.,
ten Dijke, P.,
and Heldin, C. H.
(1997)
J. Biol. Chem.
272,
28107-28115 |
14. |
Brown, J. D.,
DiChiara, M. R.,
Anderson, K. R.,
Gimbrone, M. A., Jr.,
and Topper, J. N.
(1999)
J. Biol. Chem.
274,
8797-8805 |
15. |
de Caestecker, M. P.,
Parks, W. T.,
Frank, C. J.,
Castagnino, P.,
Bottaro, D. P.,
Roberts, A. B.,
and Lechleider, R. J.
(1998)
Genes Dev.
12,
1587-1592 |
16. |
Masuyama, N.,
Hanafusa, H.,
Kusakabe, M.,
Shibuya, H.,
and Nishida, E.
(1999)
J. Biol. Chem.
274,
12163-12170 |
17. | Imamura, T., Takase, M., Nishihara, A., Oeda, E., Hanai, J., Kawabata, M., and Miyazono, K. (1997) Nature 389, 622-626[CrossRef][Medline] [Order article via Infotrieve] |
18. |
Dennler, S.,
Itoh, S.,
Vivien, D.,
ten Dijke, P.,
Huet, S.,
and Gauthier, J. M.
(1998)
EMBO J.
17,
3091-3100 |
19. |
Jonk, L. J.,
Itoh, S.,
Heldin, C. H.,
ten Dijke, P.,
and Kruijer, W.
(1998)
J. Biol. Chem.
273,
21145-21152 |
20. | Attisano, L., and Wrana, J. L. (1998) Curr. Opin. Cell Biol. 10, 188-194[CrossRef][Medline] [Order article via Infotrieve] |
21. |
Kusanagi, K.,
Inoue, H.,
Ishidou, Y.,
Mishima, H. K.,
Kawabata, M.,
and Miyazono, K.
(2000)
Mol. Biol. Cell
11,
555-565 |
22. |
de Caestecker, M. P.,
Hemmati, P.,
Larisch-Bloch, S.,
Ajmera, R.,
Roberts, A. B.,
and Lechleider, R. J.
(1997)
J. Biol. Chem.
272,
13690-13696 |
23. |
Shioda, T.,
Lechleider, R. J.,
Dunwoodie, S. L.,
Li, H.,
Yahata, T.,
de Caestecker, M. P.,
Fenner, M. H.,
Roberts, A. B.,
and Isselbacher, K. J.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9785-9790 |
24. | Derynck, R., Zhang, Y., and Feng, X. H. (1998) Cell 95, 737-740[Medline] [Order article via Infotrieve] |
25. |
Bai, S.,
Shi, X.,
Yang, X.,
and Cao, X.
(2000)
J. Biol. Chem.
275,
8267-8270 |
26. |
Topper, J. N.,
DiChiara, M. R.,
Brown, J. D.,
Williams, A. J.,
Falb, D.,
Collins, T.,
and Gimbrone, M. A., Jr.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
9506-9511 |
27. | Labbe, E., Silvestri, C., Hoodless, P. A., Wrana, J. L., and Attisano, L. (1998) Mol Cell 2, 109-120[Medline] [Order article via Infotrieve] |