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INTRODUCTION |
Activin and transforming growth factor-
(TGF-)1 are structurally
related, multipotent growth and differentiation factors. These peptides
are not only among the most potent known cellular growth inhibitors but
also regulate other diverse biological processes including early
embryonic patterning and cell fate determination (1). Signaling by
these proteins is initiated by ligand-induced hetero-oligomerization of
type I and type II receptor serine kinases. Transphosphorylation of a
type I receptor serine kinase, ALK4 for activin and ALK5 for TGF-,
by the type II receptor serine kinase activates the type I receptor,
which then phosphorylates the C-terminal Ser residues of Smad2 and -3. At present, no difference has been established between the
intracellular signaling pathways of activin and TGF-. Once
phosphorylated, Smad2 and -3 form heteromeric complexes with Smad4,
followed by translocation of the complexes into the nucleus. There they
modulate target gene transcription either by associating with various
DNA binding partners or by inducing ubiquitin-mediated protein
degradation (2-5).
In addition to Smad activation by activin/TGF--dependent
phosphorylation (6), other interactions are also likely to regulate Smad signaling. We have revealed previously that calmodulin binds with
Smad2 and acts as a Smad modulator (7). Scherer and Graff (8) have also
suggested that calmodulin and extracellular signal-regulated kinase
(ERK) may interact in their effects on modulating Smad signaling during
Xenopus embryogenesis. Furthermore, cross-talk between the
activin/TGF- pathway and the MAP kinase pathway is well known in
various biological processes; a dominant negative ras mutant
blocked mesoderm induction by activin, whereas a constitutively active
ras mutant mimicked the inducing activity (9). In addition, TGF- accelerated epithelial-fibroblastoid conversion of mammary epithelial cells in a Ras-dependent manner (10), and
signaling by c-Jun N-terminal kinase (JNK) was necessary for
TGF--induced fibronectin synthesis in fibrosarcoma cells (11).
Oncogenic Ras has also been shown to block the growth inhibitory effect of TGF- (12). Furthermore, Dok-1, a rasGAP-binding protein that
inhibits the Ras pathway (13), was required for activin-induced apoptosis in B-lineage cells (14).
The present study was performed to establish a mechanistic basis for
the regulation of Smad signaling by these other major intracellular
signaling pathways. Here we report that activated ERK phosphorylates
Smad2 and that this in turn leads to enhanced transcription of a
promoter containing an activin/TGF--responsive element. In addition,
we have located one of the ERK phosphorylation sites on Smad2. That
site lies within the primary calmodulin binding sequence, and we have
therefore been led to examine the role of calmodulin in ERK-induced
modulation of Smad2 function.
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EXPERIMENTAL PROCEDURES |
cDNA Constructs--
Constitutively active MEK1, (MEK1(ED)),
with Ser218 and Ser222 replaced by Glu and Asp,
respectively, and (MEK1*), with amino acid residues 32-51 deleted and
Ser218 and Ser222 replaced by Ala, as well as
GST-MEK1(ED), GST-ERK1, GST-Elk1 (305-425) (15), HVH2 (16), and
GST-Smad4 (6) cDNAs were kindly provided by Dr. K.-L. Guan. Dr. M. Whitman provided AR3-lux (17) and FoxH3 (18) cDNAs, TRII
cDNA (19) was given by Dr. H. F. Lodish, ALK5 cDNA (20)
was given by Dr. K. Miyazono, constitutively active ALK5 (ALK5(TD))
cDNA (21) was given by Dr. X.-F. Wang, C-terminal FLAG-tagged human
Smad4 cDNA (22) was given by Dr. R. Derynck, HA-pcDNA3 and
FLAG-pcDNA3 expression vectors (23) were given by Drs. N. Inohara
and T. Koseki, and calmodulin cDNA (7) was given by Dr. A. R. Means. The human Smad2 mutants used as expression vectors were
constructed by one step or two step PCR. The human Smad2 cDNAs were
subcloned into the EcoRI and XbaI sites of
HA-pcDNA3 to produce N-terminal HA-tagged proteins. For GST-Smad2
protein expression, Smad2 cDNA was subcloned into the vector
pGEX-2T using the SmaI and EcoRI sites.
Cell Culture and DNA Transfection--
Cell line L17, a
derivative of the mink lung epithelial cell line (Mv1Lu) (24), obtained
from by Dr. J. Massagué, and COS7 cells from ATCC were cultured
as described previously (6, 25). For transient transfection, cells in
24- or 6-well plates, or in 6-cm dishes, were transfected by the
DEAE-dextran method.
Metabolic Labeling--
For metabolic 32P labeling
of COS7 cells, cultures were transfected with HA-tagged Smad2. At
36 h after transfection the cells were transferred to
phosphate-free medium supplemented with 1% dialyzed fetal bovine serum
for 30 min, followed by labeling with 1.0 mCi/ml of
[32P]orthophosphate (ICN, Costa Mesa, CA) for 4 h.
The MEK1 inhibitor PD98059 (50 µM) was added to the
indicated lanes 30 min prior to EGF addition. After EGF stimulation (50 ng/ml) for 15 min, they were rinsed three times with HEPES dissociation
buffer (25 mM HEPES, pH 7.4, 150 mM NaCl, 5 mM KCl, 0.7 mM
Na2HPO4). They were then lysed in TNE buffer
(50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 0.5%
Nonidet P-40) containing protease inhibitors (1 mM phenylmethylsulfonyl fluoride, 1% aprotinin) for 30 min at 4 °C, in
the presence of heat-killed Staphylococcus aureus
(Pansorbin; Calbiochem, La Jolla, CA) preadsorbed with normal rabbit
serum. The lysates were centrifuged at 10,000 × g for
20 min at 4 °C. The supernatants were immunoprecipitated overnight
at 4 °C with anti-HA antibody (12CA5; Roche Molecular Biochemicals)
and incubated with protein A-agarose beads (Invitrogen) for
1 h at 4 °C. Samples were then washed two times in TNE buffer,
two times in 0.5 M LiCl, and two times in deionized water,
followed by elution in SDS-PAGE sample buffer.
For pulse-chase experiment, COS7 cells were transfected with HA-tagged
Smad2 with or without MEK1*. 36 h after transfection, they were
incubated for 30 min in Cys- and Met-free medium and then incubated in
fresh medium of the same composition containing 0.2 mCi of
[35S]Cys and Met (PerkinElmer Life Sciences) for 30 min.
Thereafter they were rinsed twice with HEPES dissociation buffer and
once with chase medium (Dulbecco's modified Eagle's medium containing 100 µg/ml Met and 100 µg/ml Cys), followed by incubation in the chase medium. At the indicated times, cells were rinsed three times
with HEPES dissociation buffer and lysed in RIPA buffer (10 mM sodium phosphate, pH 7.2, 150 mM NaCl, 5 mM EDTA, 1% Nonidet P-40, 0.1% SDS, 1% sodium
deoxycholate) for 30 min at 4 °C. Insoluble material was discarded
after centrifugation at 600 × g for 5 min at 4 °C.
Lysates were immunoprecipitated overnight at 4 °C with anti-HA
antibody (12CA5), and protein A-agarose beads were added for 1 h
at 4 °C. They were then washed three times in RIPA buffer, two times
in 0.5 M LiCl, and two times in deionized water, followed by elution in SDS-PAGE sample buffer.
All samples were subjected to 10% SDS-PAGE. Gels of
32P-labeled samples were blotted to polyvinylidene
difluoride membranes prior to exposure to x-ray film. Gels of
35S-labeled samples were soaked for 30 min in 1 M sodium salicylate prior to drying and exposure to x-ray film.
In Vitro Kinase Assay--
Smad2 and Smad4 proteins were
expressed as GST fusion proteins in Escherichia coli
and extensively purified after GST cleavage by thrombin, as described
previously (26). The proteins were >90% pure on SDS-PAGE. The
purified Smad proteins had appropriate in vitro biological
activities; TGF- receptor complexes phosphorylated Smad2, and Smad4
protein bound to Smad-binding DNA element (26). GST-MEK1(ED), GST-ERK1,
and GST-Elk1 (305-425) were expressed in E. coli and
purified with glutathione-Sepharose beads (Roche Molecular
Biochemicals). The proteins were eluted from the beads with 50 mM Tris-HCl, pH 8.0, containing 10 mM reduced
glutathione, followed by change of elution buffer to 20 mM
HEPES, pH 7.5, 50 mM NaCl, 1 mM EDTA, 1 mM dithiothreitol by means of Centriprep-10 (Millipore,
Bedford, MA).
Five ng/µl of GST-MEK1(ED) and/or 5 ng/µl of GST-ERK1 were
incubated for 25 min at 30 °C in kinase assay buffer (20 mM HEPES, pH 7.5, 10 mM MgCl2, 0.2 mM ATP, 1 mM dithiothreitol). Subsequently, 4 µg of Smad protein or 3 µg of synthetic Smad peptide was incubated for 25 min at 30 °C with 5 µl of the activated kinase (above), in
a total of 30 µl of kinase buffer and 0.5 µCi of
[32P]ATP. To examine the effect of calmodulin on Smad
phosphorylation, 4 µg of Smad2 or GST-Elk1 (305-425) was incubated
for 10 min at 25 °C with calmodulin in kinase buffer containing 0.1 mM CaCl2 or 1 mM EGTA prior to
in vitro kinase reaction with activated ERK1. The calmodulin
was purified from bovine brain (27). On the basis of the dissociation
constants of Smad2 and calmodulin (58 nM),2 sufficient
calmodulin was added to complex with either 50 or 99% of Smad2. The
reaction was terminated by adding 7.5 µl of SDS-PAGE sample buffer.
The 32P-labeled samples were visualized as described above,
except for SDS-PAGE of synthetic peptides where Tricine buffer was used
(28). For Western blotting, anti-Smad2 antibody (S-20; Santa Cruz
Biotechnology, Santa Cruz, CA) or anti-Smad4 antibody (H-552; Santa
Cruz Biotechnology) was used as primary antibody, and bands were
visualized with ECL reagent (Amersham Biosciences).
Two-dimensional Tryptic Peptide Mapping and Phosphoamino Acid
Analysis--
Purified Smad2 was phosphorylated with
[32P]ATP in vitro by activated ERK1, separated
by SDS-PAGE, and transferred to a polyvinylidene difluoride membrane.
Tryptic digestion, two-dimensional phosphopeptide mapping, and
phosphoamino acid analyses were performed as described previously (29,
30).
Mapping of Phosphorylation Sites--
100 µg of purified Smad2
was phosphorylated by activated ERK1 in the presence of
[32P]ATP, followed by digestion with TPCK-trypsin (29).
The resulting peptides were separated by reverse phase HPLC using a
C18 column (4-60% acetonitrile linear gradient for 60 min). Peptide fractions were collected every 0.375 min and counted for
32P. The 32P-labeled peptides were then
subjected to amino acid sequencing.
Reporter Assays--
Luciferase assays were conducted
essentially as described previously (6, 7). L17 cells were transiently
transfected with various Smad2 constructs (0.5 µg/well except for
titration), FoxH3 (0.5 µg/well), TRII, ALK5, or ALK5(TD) (0.1 µg/well), and MEK1(ED) (0.5 µg/well), together with a reporter
construct (AR3-lux) (0.25 µg/well) and a plasmid expressing
-galactosidase (pCMV--Gal) (0.01 µg/well) in 24-well plates.
Equal amounts of DNA were transfected in each experiment, and adjusted
with pcDNA1 and HA-tagged pcDNA3, and the cells were harvested
40 h after transfection. Luciferase activity was normalized to
-galactosidase activity, and the luciferase activity in the cell
lysate transfected with empty vector was set at 1.
Sequential Immunoprecipitation-Immunoblotting--
COS7 cells
were transfected with HA-tagged Smad2 and FLAG-tagged Smad4, with or
without MEK1*. 48 h after transfection, cells were lysed in TNE
containing 10% (v/v) glycerol and protease inhibitors. After 30 min on
ice, cell debris was removed by centrifugation at 600 × g for 5 min at 4 °C, and the supernatant was
immunoprecipitated overnight at 4 °C with anti-HA antibody (12CA5)
and incubated with protein A-agarose beads for 1 h at 4 °C. The
beads were washed four times with TNE buffer containing 10% (v/v)
glycerol, followed by elution in SDS-PAGE sample buffer. The
immunoprecipitates were subjected to Western blotting with anti-HA
antibody (12CA5) or anti-FLAG antibody (M2; Sigma) as primary antibody,
and the bands were visualized with ECL reagent (Amersham Biosciences).
For calmodulin overexpression, L17 cells transfected with HA-tagged
Smad2 (1 µg/well) and ALK5 (1 µg/well) were co-transfected with
calmodulin expression construct (3 or 6 µg/well) in 6-well plates. At
38 h post-transfection, cells were treated with TGF- (100 pM) for 30 min. For ionomycin treatment, L17 cells were
transfected with HA-tagged Smad2 (1.5 µg/well) and ALK5 (0.5 µg/well). At 36 h post-transfection, cells were incubated with
ionomycin (1 or 2 µM) for 1.5 h, followed by TGF-
(100 pM) for 30 min. Cells were lysed, and HA-Smad2 was
detected as described above.
GST Pull Down Assay--
35S-Labeled Smad2 wild-type
and mutants were translated in vitro with the TNT rabbit
reticulocyte lysate kit (Promega, Madison, WI), and the GST and
GST-fused Smad4 proteins were expressed in E. coli and
purified with glutathione-Sepharose beads according to the
manufacturer's protocol. GST pull down assays were performed as
described previously (6).
Fluorescence Analysis--
The interaction of Smad2 mutants with
calmodulin was evaluated by fluorescence assay as described previously
(6). Various concentrations of purified Smad2 (>90% pure) were mixed
with 140 nM dansylated calmodulin (Sigma) in 20 mM HEPES, pH 7.5, 130 mM KCl in the presence of
0.1 mM CaCl2 or 1 mM EGTA.
Fluorescence was measured with a FluoreMax-2 (Instruments SA Inc.,
Edison, NJ) with excitation at 340 mm, and 5- and 10-nm slits for
excitation and emission, respectively. Emission spectra were recorded
between 430 and 570 nm, and the maximal fluorescence intensity was
measured. The differences in maximum intensity with and without Smad2
were plotted against free Smad2 concentration. A one-site binding model was applied (y = a × x/(Kd + x), where x
is the concentration of free Smad2, and y is the difference
between the fluorescence intensity in the presence and absence of
Smad2, and the dissociation constant was calculated by use of GraphPad
PRISM (GraphPad Software, Inc., San Diego, CA). Free Smad2
concentration was estimated from the following equation: free Smad2
(nM) = total Smad2 (nM) 
total calmodulin (nM) × 
F/F
where
F and F are the difference
in fluorescence intensity at a given Smad2 concentration and at the
highest Smad2 concentration, respectively.
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RESULTS |
Smad2 Is Phosphorylated by Activated ERK1--
To determine
whether Smad2 is phosphorylated in response to activation of the MAP
kinase pathway, we examined Smad2 phosphorylation in response to EGF
stimulation in COS7 cells metabolically labeled with
[32P]orthophosphate. Incorporation of isotope into Smad2
increased rapidly in response to EGF stimulation (Fig.
1A, lanes 2 and
3), and pretreatment with MEK1 inhibitor PD98059 effectively
blocked Smad2 phosphorylation (Fig. 1A, lanes 4 and 5). These results were consistent with previous studies
(31, 32). Because EGF activates the MEK-ERK pathway, we checked whether
this kinase phosphorylates Smad2 in vitro.
32P-Labeled Smad2 was indeed detected upon co-incubation
with ERK1 in the presence of activated MEK1 (Fig. 1B),
suggesting that activated ERK1 was responsible for the phosphorylation
of Smad2 in intact cells. 32P-Labeled Smad4 was not
detected following incubation with activated ERK1 (Fig. 1B).
Tryptic peptide mapping of 32P-labeled Smad2 revealed
several spots that migrated toward the cathode (Fig. 1C,
left panel). This tryptic digestion pattern differs from
that of Smad2 phosphorylated by TGF- receptor complexes, because the
latter migrates toward the anode (26, 33-35). Phosphoamino acid
analysis of 32P-labeled Smad2 showed that ERK1
phosphorylates Ser and Thr, but not Tyr, residues (Fig. 1C,
right panel).
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Fig. 1.
Smad2 phosphorylation by activated ERK1.
A, Smad2 phosphorylation in response to EGF stimulation.
COS7 cells were transiently transfected with HA-tagged Smad2 without or
with MEK1 inhibitor PD98059 prior to the addition of EGF. HA-Smad2 was
recovered by immunoprecipitation from 32P-labeled cells,
followed by SDS-PAGE and autoradiography. B, in
vitro phosphorylation of purified Smad2 and Smad4 by MEK1 and
ERK1. Purified Smad2 or Smad4 were incubated with activated MEK1 and
ERK1, or activated ERK1 alone, in the presence of
[32P]ATP, followed by SDS-PAGE and autoradiography.
C, tryptic digest of ERK1-phoshorylated Smad2 resolved by
two-dimensional peptide mapping (left) and phosphoamino acid
analysis of 32P-labeled Smad2 (right).
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To identify the Smad2 sites phosphorylated by ERK1,
32P-labeled Smad2 was digested with trypsin, and the
resulting peptides were separated by HPLC (Fig.
2A, upper). There
were two peaks of radioactivity (Fig. 2A, lower).
Amino acid sequencing indicated that peak a was the peptide
beginning 183HIEILT, and peak b was the
N-terminal peptide. Kretzschmar et al. (32) have suggested
that the linker region of Smad2 is phosphorylated by the Ras pathway,
and they proposed that Thr220 and Ser245,
Ser250, and Ser255 were possible
phosphorylation sites. The phosphorylation of peak a peptide
by ERK1 is consistent with that prediction. There is also an ERK site
(PX(S/T)P) located near the N terminus of Smad2 (PF8TP). To establish whether it is this Thr that is
phosphorylated by activated ERK1, an in vitro kinase assay
was performed using synthetic Smad peptides as substrate. Smad2 (2-21)
was phosphorylated by ERK1 in a manner dependent on activated MEK1, and
the residue phosphorylated was a Thr (Fig. 2B). As there is
only one Thr in this peptide, namely Thr8, this result
confirms that the predicted ERK site is phosphorylated. Smad4 (78-88)
was not phosphorylated by activated ERK1, and Smad1 (4-23) was only
weakly phosphorylated; the latter has an 11SP sequence that
is phosphorylated by proline-directed protein kinases including ERK
(36).
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Fig. 2.
Smad2 sites phosphorylated by ERK1.
A, HPLC fractionation of a tryptic digest of
32P-labeled Smad2 phosphorylated by ERK1. Absorbance at 216 (upper) and radioactivity (lower) are shown.
Peaks a and b were the two fractions
subjected to amino acid sequencing. B, in vitro
phosphorylation of synthetic peptides of Smad by MEK1 and ERK1
(upper) and phosphoamino acid analysis of Smad2 (2-21)
peptide phosphorylated by activated ERK1 (lower).
C, phosphorylation of HA-tagged wild-type (WT)
Smad2 or a mutant lacking all five potential phosphorylation sites in
the MH1 and linker region (T8V/T220V, 3SA; VA) in response
to EGF stimulation in COS7. D, in vitro
phosphorylation of wild-type (WT) Smad2 and Smad2(VA) by
activated ERK1.
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We next expressed a mutant form of Smad2, referred to as
Smad2(VA), that has Thr8 and Thr220
changed to Val and Ser245, Ser250, and
Ser255 changed to Ala (see Fig. 4A), and
examined its phosphorylation in response to EGF stimulation in intact
cells and by activated ERK1 in vitro. 32P
labeling of Smad2(VA) did not increase in response to EGF treatment in
COS7 cells (Fig. 2C) nor did incubation of purified
Smad2(VA) with activated ERK1 give rise to phosphorylation (Fig.
2D). These data confirm that the residues mutated in
Smad2(VA) include all the sites phosphorylated by ERK1, both in
vivo and in vitro.
Phosphorylation of Smad2 by ERK Increases Its Transcriptional
Activity--
Next we explored the effect of ERK-dependent
phosphorylation on the transcriptional activity of Smad2. For this
purpose we used a reporter gene, FoxH3-dependent AR3-lux,
that contains three copies of an activin-responsive element from
Mix.2, fused to luciferase (17). Transfection of type II
(TRII) and type I (ALK5) TGF- receptor into a mink lung
epithelial cell line did not affect luciferase expression, whereas
transfection of a constitutively active type I TGF- receptor
(ALK5(TD)) (21) increased luciferase expression 7.5-fold. Transfection
of a constitutively active form of MEK1, MEK1(ED) (15), had little
effect on transcriptional activity by itself. In addition,
co-transfection of MEK1(ED) with TRII or ALK5 also did not affect
luciferase expression. However, when it was co-transfected with
ALK5(TD) there was a further increase in luciferase expression. This
suggests that the MEK1-ERK pathway synergizes with the TGF- pathway
in promoting transcription of AR3. We next checked the effect of the
MEK1-ERK pathway on Smad2-mediated AR3 transcription. Co-transfection
of MEK1(ED) increased wild-type Smad2-induced luciferase activity (Fig.
3B). Expression of a
constitutively active Smad2, Smad2(2E) that has the TGF- receptor
kinase phosphorylation sites Ser465 and Ser467
replaced by Glu (6), resulted in 15-fold elevation of basal luciferase
expression. Co-transfection of MEK1(ED) caused a further increase in
luciferase activity. These results suggest that the MEK1-ERK pathway
promotes Smad2-mediated AR3 transcription and that modulation of Smad2
by the MEK1-ERK pathway is independent of regulation via TGF-.
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Fig. 3.
Enhancement of TGF-
receptor or Smad2-mediated AR3-lux transcription by the MEK1-ERK
pathway. A, L17 cells were transiently transfected with
AR3-lux, -galactosidase, FoxH3, and TGF- receptor isoform,
together with (hatched bar) or without (solid
bar) constitutive MEK1(ED) (see "Experimental Procedures").
Data are expressed as mean ± S.D. of triplicates from a
representative experiment. B, effect of MEK1(ED) on
wild-type (WT) Smad2 and constitutive (Smad2(2E))-mediated
AR3-lux transcription. Data are mean ± S.D. of triplicates from a
representative experiment.
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To further explore the role of phosphorylation of Smad2 by ERK1 in
signaling, we introduced mutations at possible sites of phosphorylation
by ERK1 and by activin and TGF- receptor complexes; both
non-phosphorylatable (Thr 
Val and Ser Ala) and
phosphorylation-mimicking (Thr and Ser Asp or Glu) substitutions
were made (see Refs. 34 and 35 and Fig.
4A). Transfection with
wild-type Smad2 elevated basal expression of AR3-lux 24-fold (Fig.
4B). This increase was greater than that shown in Fig.
3B, presumably because of the higher proportion of
Smad2-encoding DNA in the transfection mixture. Although AR3
transcription was not affected by the T8V/T220V mutant and the
T220V, 3SA mutant, the T8V/T220V, 3SA triple mutant greatly decreased
expression. On the other hand, substitution of the possible ERK sites
with Asp increased expression of the reporter.
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Fig. 4.
Modulation of Smad2-mediated AR3-lux
transcription by mutation of possible phosphorylation sites.
A, schematic representation of sites phosphorylated by
activated ERK1 and receptor complexes for activin and TGF-.
B, L17 cells transiently transfected with AR3-lux,
-galactosidase, FoxH3, and Smad2 mutant. Data are the mean ± S.D. of triplicates from a representative experiment. C,
AR3-lux transcription as a function of amount of transfected DNA,
comparing Smad2(2E) with Smad2(T8, 220D, 3SD-2E). The figure gives the
percentage of the normalized luciferase activity obtained with a given
amount of transfected DNA to that obtained with 0.5 µg of
DNA/well.
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Smad2(2A) (with C-terminal Ser465 and Ser467
replaced by Ala) elevated basal expression of luciferase 21-fold,
whereas the expression induced by the T8V/T220V mutant of
Smad2(2A) was limited to 12-fold. In addition, the T220V, 3SA mutant
and the T8V/T220V, 3SA triple mutant of Smad2(2A) further decreased
expression to 4- and 2-fold, respectively. In contrast, mutation of the
same residues to Asp increased expression 42- to 47-fold. These
results, that the Val/Ala mutants of the ERK sites decreased and that
the Asp mutants increased AR3 transcription, were basically similar to
those observed when the C-terminal sequence was wild-type, although the
inhibitory effects of the T8V/T220V mutant and the T220V, 3SA
mutant were distinct.
The inhibitory effect of non-phosphorylatable mutants of ERK sites was
most striking in the presence of Ser-Glu-Met-Glu at the C
terminus (2E). Smad2(2E) elevated basal luciferase expression 162-fold,
whereas all three Val/Ala mutants of Smad2(2E) clearly decreased
expression below 40-fold, indicating that AR3 transcriptional responses
due to different Val/Ala mutants are qualitatively different with
different C-terminal changes. Although there were no differences between the Asp mutants of Smad2(2E) and Smad2(2E) itself, this was no
doubt because both gave maximal expression. Titration of the amount of
transfected DNA suggested that Smad2(T8V/T220V, 3SD-2E) was more potent
than Smad2(2E) (Fig. 4C). All these data point to the
conclusion that phosphorylation of Smad2 by the MEK1-ERK pathway
promotes AR3 transcription and that phosphorylation by both activated
ERK and activin/TGF- receptor complexes is necessary for
maximal Smad2 activation.
Smad2 Protein Is Stabilized by ERK Phosphorylation--
To explore
the mechanism by which phosphorylation of Smad2 by the MEK1-ERK pathway
stimulates signaling, we tested the effect of activating the MEK1-ERK
pathway on the amount of exogenously expressed Smad2 in COS7 cells.
Western blot analysis revealed that the amount of Smad2 protein was
increased by expression of another constitutively active MEK1, MEK1*,
with amino acid residues 32-51 deleted and Ser218 and
Ser222 replaced by Ala (15) (Fig.
5A). Co-transfection of an ERK
phosphatase, HVH2 (16), with MEK1* resulted in a decrease in Smad2
protein (Fig. 5A). These results suggest that the MEK1-ERK
pathway increases the amount of Smad2 protein. To determine whether the
increase is because of increased protein stability, we performed
pulse-chase experiments. These showed that the half-life of Smad2 was
prolonged by the presence of MEK1* (Fig. 5B), suggesting
that ERK phosphorylation stabilizes Smad2 protein. We then examined the
level of Smad2 protein for various ERK phosphorylation site mutants.
Fig. 5C reveals that protein levels were decreased in the
phosphorylation-defective mutants and increased in the
phosphorylation-mimicking mutants. This tendency was also observed in
the mutants with S465A/S467A or S465E/S467E at their C terminus
(data not shown).
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Fig. 5.
Increased stability of Smad2 phosphorylated
by activated ERK1. A, Western blots of COS7 cells
co-transfected with HA-tagged Smad2 and constitutively active MEK1* or
ERK phosphatase, HVH2. B, SDS-PAGE analysis of COS7 cells
transfected with HA-tagged Smad2 with or without MEK1* and labeled with
[35S]methionine for 30 min followed by chase with cold
methionine and cysteine (upper). The relative amounts of
35S-labeled Smad2 to total labeled Smad2 were plotted
against chase time (mean ± S.D.; lower). C,
Western blots of lysates of cells transfected with HA-tagged Smad2
mutants bearing substitutions that either remove possible
phosphorylation sites or mimic their phosphorylation.
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Because the formation of Smad2·Smad4 complexes is essential for
Smad2-mediated signaling (6, 37-39), the association of Smad2 and
Smad4 was examined by sequential immunoprecipitation and
immunoblotting. Complexes of wild-type Smad2 with Smad4 were below
detection limits in the absence of MEK1* (Fig.
6A, upper); co-transfection of MEK1* resulted in significant levels of Smad4 in the
immunoprecipitates, suggesting that the formation of Smad2·Smad4 complexes is promoted by stimulation of the MEK1-ERK pathway. In
contrast, even in the absence of MEK1*, Smad4 formed complexes with
Smad2(T8D/T220D, 3SD), whereas in this case MEK1* co-transfection did
not increase complex formation. The observed increases in Smad2·Smad4
complex formation correlate with increases in AR3 transcription (Figs.
3B and 4B).
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Fig. 6.
Association of Smad2 and Smad4.
A, the interaction of HA-tagged Smad2(WT) or of Smad2(D), a
mutant mimicking all five potential ERK1 phosphorylation sites (T8,
220D, 3SD), with FLAG-tagged Smad4 examined by immunoprecipitation
(IP) followed by immunoblotting (Blot) in COS7
cells. B, in vitro association of
35S-labeled Smad2(WT), Smad2(D), Smad2(2E), or Smad2(D-2E),
a mutant mimicking phosphorylation by ERK1 and activin/TGF-
receptors (T8, 220D, 3SD-2E), with Smad4 examined by GST-pull down
assay (see "Experimental Procedures").
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To determine whether the increase in Smad2·Smad4 complex formation
following activation of the MEK1-ERK pathway is because of an increase
in the affinity of Smad2 for Smad4, their in vitro association was examined by GST-pull down assay. The results in Fig.
6B show that in vitro translated
35S-labeled Smad2(T8D/T220D, 3SD) bound to GST-Smad4 beads
with similar efficiency to 35S-wild-type Smad2. Moreover,
in agreement with our previous study (6), more Smad2(2E) than wild-type
Smad2 bound to GST-Smad4. Smad2(T8D/T220D, 3SD-2E) also bound to
GST-Smad4 more efficiently than did wild-type Smad2, but no difference
was seen between Smad2(2E) and Smad2(T8D/T220D,3SD-2E). These data
suggest that phosphorylation of Smad2 by the MEK1-ERK pathway does not
directly affect its affinity for Smad4.
Calmodulin Blocks Smad2 Phosphorylation by ERK1 and Decreases Smad2
Protein--
We have shown that calmodulin associates physically with
Smad2 (7), and the primary calmodulin-binding site has been mapped to
the N-terminal basic amphiphilic
-helix,3 which overlaps
with the ERK sites. It was therefore of interest to examine the effect
of calmodulin on the phosphorylation of Smad2 by ERK1. Neither
activated MEK1 nor activated ERK1 phosphorylated calmodulin (data not
shown). Smad2 was incubated with activated ERK1 in the presence and
absence of calmodulin. As shown in Fig. 7, A and B,
calmodulin significantly inhibited Smad2 phosphorylation by ERK1,
although it had no effect on phosphorylation of a well characterized
target of ERK, Elk (40). The inhibitory effect was abolished when the
reactions were carried out in the presence of EGTA to prevent
calmodulin binding (7).
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Fig. 7.
Effects of calmodulin on Smad2
phosphorylation. Smad2 peptide (A) or Smad2 protein
(B) incubated with activated ERK1 in the presence of
[32P]ATP and 0.1 mM CaCl2 or 1 mM EGTA are shown. , without calmodulin; + and ++, 50 and
99%, respectively, of the Smad2 complexed with calmodulin on the basis
of the calculated dissociation constant of 58 nM. GST-Elk1
(305-425) was used as a positive control for phosphorylation by
ERK1.
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We next examined the effect of Smad2 phosphorylation on calmodulin
binding. Because Smad2 could not be stoichiometrically phosphorylated
by ERK1 in vitro, we purified various Smad2 mutants that
have acidic amino acids substitutions at their phosphorylation sites
(Fig. 4A). Interaction of these proteins with calmodulin was
assayed fluorimetrically by binding to dansylated calmodulin. All the
mutants bound with similar affinity (Fig.
8), suggesting that there is little
effect of ERK phosphorylation on the binding of calmodulin to
Smad2.
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Fig. 8.
Association of calmodulin with Smad2.
In vitro association of Smad2(WT) (A), Smad2(T8,
220D, 3SD) (B), Smad2(2E) (C), or Smad2(T8, 220D,
3SD-2E) (D) with calmodulin is shown (see "Experimental
Procedures"). Dissociation constants of calmodulin and the various
Smad2 constructs were estimated as follows: for Smad2(WT), 58 nM; for Smad2(T8, 220D, 3SD), 86 nM; for
Smad2(2E), 108 nM; and for Smad2(T8, 220D, 3SD-2E), 110 nM.
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Because calmodulin blocks ERK phosphorylation of Smad2, and because
phosphorylation results in stabilization, we expected that calmodulin
would decrease Smad2 protein levels. As predicted, calmodulin
overexpression decreased the amount of Smad2 protein in a
dose-dependent manner (Fig.
9A). Treatment with ionomycin, an ionophore that activates calmodulin by stimulating calcium influx
(41), also decreased Smad2 protein levels (Fig. 9B). We
showed previously that overexpression of calmodulin inhibits activin/TGF--induced transcription, whereas inhibiting calmodulin enhances it (7). In view of the present results, this inhibitory effect
may be because of calmodulin binding to Smad2 and blocking its
ERK-dependent phosphorylation, so reducing its stability
and hence the overall level of Smad2 protein.
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Fig. 9.
Effect of calmodulin on Smad2 protein
level. Shown are HA-tagged Smad2 levels in L17 cells transfected
with ALK5 and treated with TGF-, either (A)
co-transfected with calmodulin or (B) treated with
ionomycin.
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DISCUSSION |
Our findings have revealed that Smad2 is phosphorylated by ERK1
and that this results in enhanced transcription of an
activin/TGF--responsive DNA element. They have also shown that the
enhanced Smad2-mediated signaling is because of increased levels of
Smad2 protein, as a result of stabilization of the protein, and that
this in turn leads to increased formation of active signaling complexes
with Smad4. These observations suggest that there is cross-talk between the activin/TGF- and ERK signaling pathways at the level of Smad2 activation and imply that phosphorylation of Smad2 by both activated ERK1 and activin/TGF- receptor complexes is essential for maximal transcriptional activity.
Previous studies have addressed potential cross-talk between the
activin/TGF- and MAP kinase signaling pathways, but no clear consensus has emerged with respect to the functional consequences of
these interactions. For example, activation of MAP kinase negatively regulated activin/TGF--induced cell growth inhibition and apoptosis (14, 32), whereas activin/TGF- signaling and MAP kinase pathways synergized during Xenopus mesoderm induction (9, 42, 43) and
fibronectin synthesis in fibrosarcoma cells (11). In addition, oncogenic Ras and TGF- collaborated in epithelial-fibroblastoid conversion and invasion of epithelial tumor cells (10). These results
suggest that the functional relationship between activin/TGF- signaling and MAP kinase pathway depends on specific cellular context.
In fact, the ability of TGF- to cause invasive growth was not
inhibited by activation of ERK signaling, but the apoptotic effects of
TGF- was blocked in Madin-Darby canine kidney cells (44). A
molecular explanation for the synergistic effects of ERK on Smad2
signaling revealed in this study may explain the relationship between
activin/TGF- signaling and the MAP kinase pathway during early
Xenopus embryogenesis and during metastasis of epithelial
tumor cells. This is also consistent with the recent results of Oft
et al. (45) showing that both Ras and Smad2 signaling are
essential for epithelial to mesenchymal transition and that metastasis
is driven by sequential elevation of Ras and Smad2 expression.
In agreement with previous studies (31, 32), we found that Smad2 was
phosphorylated in response to EGF stimulation. An in vitro
kinase assay revealed that activated ERK1 could phosphorylate Smad2 but
not Smad4, and tryptic peptide mapping yielded a different pattern from
that induced by TGF- receptor complexes (26, 33-35). Brown et
al. (46) have also reported that transfection of constitutively active MEK kinase 1, a component of the JNK pathway, induces Smad2 phosphorylation outside the C-terminal Ser-Ser-Met-Ser site.
Although it remains uncertain whether Smad2 phosphorylation by ERK1 and JNK occurs at the same sites, these results suggest that Smad2 phosphorylation is regulated not only by receptors for activin and
TGF- but also by the MAP kinase family.
Our data on the effects of ERK phosphorylation on
Smad2-dependent transcription are consistent with findings
by others. de Caestecker et al. (31) showed that activation
of receptor tyrosine kinases by EGF or hepatocyte growth factor
activated Smad2-dependent gene expression. In addition,
activated MEK kinase 1 enhanced Smad2-dependent gene
transcription (46), and co-transfection of JNK with Smad3 and Smad4
increased basal expression of a CAGA12-lux reporter gene
(46) whose CAGA elements bind complexes of Smad3 and Smad4 (47).
Furthermore activin and MAP kinase-coupled signals synergized during
early embryogenesis in Xenopus (48, 49). Because the MAP
kinase family is large, with a complex and incompletely understood set
of regulators and targets (50), we cannot state unequivocally which
members of the MAP kinase family phosphorylate Smad2 in
vivo. Nevertheless our data suggest that phosphorylation of Smad2
by activated ERK1 is necessary for maximum transcription of
Smad2-dependent genes.
Ser residues (Ser465 and Ser467) at the C
terminus of Smad2 are phosphorylated in response to TGF- stimulation
(34, 35), and a Smad2 mutant with Ala substituted for these serines has
been reported to associate stably with TGF- receptor complexes and reduce transcription of a TGF--responsive reporter gene, 3TP-lux. This finding was taken as indicating that the mutant protein acted in a
dominant negative manner (33). However, in our hands Smad2(2A) and
wild-type Smad2 had comparable transcriptional activity with AR3-lux as
reporter gene, suggesting that the Smad2(2A) mutant does not have a
dominant negative effect at least when AR3-lux is the reporter.
Kretzschmar et al. (32) have reported that Smad3 mutants
with Ser Ala or Thr Val substitutions at four potential ERK sites in the linker region (corresponding to the residues changed in
Smad2; see Fig. 4A) increased basal expression of AR3-lux in Ras transformed cells. This result was taken to suggest that
phosphorylation of Smad3 by the Ras pathway inhibits Smad3-mediated
signaling, a view that contrasts with our findings, as well as those of
others (31, 44, 51). Kretzschmar et al. (32) interpreted the stimulatory effect of EGF or hepatocyte growth factor on transcription of a TGF--responsive reporter gene (31) as pointing to a general stimulation of transcription by the Ras pathway. However, the present
study has shown that amino acid substitutions in Smad2 that either
mimic or inhibit ERK1 phosphorylation increase and decrease
transcription, respectively. Differences between Smad2 and Smad3 may be
responsible for the different result obtained. Thus, although the two
proteins are closely related structurally, and both are thought to be
activin/TGF- signal mediators, Smad3 has been observed to inhibit
Smad2·Smad4-mediated transcription of a reporter gene from the
goosecoid promoter (52). It is also possible that
differences between the cell types used in the experiments may account
for the divergent observations.
Our data suggest that Smad2 that has been phosphorylated by ERK1 has
higher transcriptional activity primarily because it has greater
stability and forms a greater number of complexes with Smad4. The
increase in complex formation is presumably a simple mass action
effect, because a Smad2 substitution mimicking phosphorylation by
activated ERK1 did not alter the affinity of the resulting protein for
Smad4. Brown et al. (46) have shown similarly that
stimulation of the JNK pathway enhances the Smad2-Smad4 interaction in
endothelial cells. It is known that activation of the MAP kinase
pathway affects protein stability; the half-life of c-Myc protein
markedly increased in response to stimulation of the Ras pathway (53),
and phosphorylation of Ser62 by ERK (54) was required for
the Ras-induced stabilization of c-Myc (55).
In previous work we showed that calmodulin interacts with Smad2 in a
calcium-dependent manner and that the N-terminal helix of
Smad2 is the primary calmodulin binding region (7). The present results
show that phosphorylation of Smad2 by ERK1 is blocked by interaction
with calmodulin. Scherer and Graff (8) have also shown that binding of
calmodulin to Smad2 inhibits subsequent ERK2-dependent
phosphorylation of Smad2. It is possible that binding of calmodulin
changes the conformation of Smad2 such that it is less efficiently
phosphorylated. Alternatively, it may sterically block the relevant
phosphorylation sites.
Earlier we demonstrated that overexpression of calmodulin inhibited
activin/TGF--responsive gene transcription and that inhibition of
calmodulin activity stimulated transcription (7). Calmodulin overexpression also blocked Smad2-dependent morphogenesis
in Xenopus (8). Interestingly, Wicks et al. (56)
have shown that inhibition of TGF--mediated transcriptional
activation can result from phosphorylation of Smad2 by
calmodulin-dependent kinase II and consequent inhibition of its
nuclear translocation. In the present study, overexpression of
calmodulin, or ionomycin treatment, decreased the level of Smad2
protein in L17 cells. Hence the inhibition of activin/TGF- signaling
by increased calmodulin activity may be at least partly because of
reduced phosphorylation of Smad2 via the ERK pathway and its resulting
instability. This, therefore, could be an additional mechanism by which
calmodulin regulates Smad2-mediated signaling.
The present study examined the mechanistic basis for regulation of
Smad2 signaling by the ERK pathway and by calmodulin binding. Our
findings suggest that the ERK pathway positively regulates Smad2
signaling by phosphorylating Smad2 and that calmodulin negatively regulates Smad2 activation by inhibiting this phosphorylation. The
rigorous control of Smad2 activity, a signal mediator for activin and
TGF-, by these other major intracellular signaling pathways may
explain some of the diverse biological activities of activin and
TGF- in development, normal physiology, and pathology.
,
TOP
ABSTRACT
INTRODUCTION
To whom correspondence should be addressed: Azabu University
School of Veterinary Medicine, 1-17-71 Fuchinobe, Sagamihara 229-8501, Japan. Tel.: 81-42-754-7111 (ext. 276); Fax: 81-42-754-9930; E-mail: funaba@azabu-u.ac.jp.
