Roles of Pathway-Specific and Inhibitory Smads in Activin Receptor Signaling
Jean-Jacques Lebrun1,
Kazuaki Takabe,
Yan Chen and
Wylie Vale
Clayton Foundation Laboratories for Peptide Biology Salk
Institute for Biological Studies La Jolla, California
92037
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ABSTRACT
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Activins and other members of the
transforming growth factor-ß-like superfamily of growth factors
transduce their signals by interacting with two types of receptor
serine/threonine kinases. The Smad proteins, a new family of
intracellular mediators are involved in the signaling pathways of these
receptors, but the initial stages of their activation as well as their
specific functions remain to be defined. We report here that the
pathway-specific Smad2 and 3 can form a complex with the activin
receptor in a ligand-dependent manner. This complex formation is rapid
but also transient. Indeed, soon after their association with the
activin receptor, Smad2 and Smad3 are released into the cytoplasm where
they interact with the common partner Smad4. These Smad complexes then
mediate activin-induced transcription. Finally, we show that the
inhibitory Smad7 can prevent the association of the two
pathway-specific Smads with the activin receptor complex, thereby
blocking the activin signal.
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INTRODUCTION
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Activins, members of the transforming growth factor-ß-like
(TGFß-like) superfamily exert a broad range of biological effects in
various target tissues (1). Members of this family interact with two
different types of receptors (type I and type II), both containing an
extracellular domain, a single transmembrane region, and a large
intracellular domain that includes a serine/threonine kinase domain. An
activin dimer first binds to the type II receptor, a constitutively
phosphorylated kinase, and this in turn recruits the type I receptor
into the complex (2, 3, 4). Subsequently, the type I receptor is
transphosphorylated by the kinase domain of the type II receptor and
signal transduction is initiated. Several proteins have been identified
for their abilities to interact with the type I and type II receptor
complexes. Among these molecules are TRIP-1 (TGFß
receptor-interacting protein-1) (5), farnesyl transferase-
(6),
FKBP-12 (7, 8) and TAK1, a protein involved in the mitogen-activated
protein kinase pathway (9, 10). These molecules interact with
TGFß-like receptor members in the yeast two-hybrid system, yet their
functional importance is not established in the action of TGFß,
activin, or bone morphogenetic proteins (BMPs). Interestingly, FKBP-12
appears to be a regulatory molecule capable of inhibiting TGFß
signaling in the absence of ligand (8, 11). The importance of the
interactions between these molecules and the receptors of the
superfamily, as well as their biological implications, remains
unclear.
Recently, genetic screening for modifiers of dpp
(decapentoplegic) signaling in Drosophila revealed that Mad
(mothers against dpp) proteins are involved in the mechanism of action
of the TGFß-like receptor superfamily (12, 13, 14, 15). Based on sequence
similarities to Drosophila Mad, several mammalian homologs
(Smad 18) have been characterized (15, 16, 17, 18, 19, 20, 21, 22, 23). Smad proteins can
be categorized into three subclasses, the pathway-specific Smads, which
include Smad1, 2, 3, 5, and 8; the common-partner Smad (Smad4 or dpc4);
and the inhibitory Smads (Smads 6 and 7). Smad1 and Smad5 mediate
BMP/dpp signaling pathways (13, 14, 15, 16, 24, 25, 26) while Smad2 and 3 have been
shown to transduce activin/TGFß-signaling pathways (18, 20, 21, 27).
Smad8 mediates ALK2 receptor signaling, for which the ligand remains
unknown (23). Nevertheless, a direct association between a type
I receptor and Smad2 or 3 has not been shown with intact but only with
kinase-deficient receptors. After activation by their respective type I
receptor kinases, the pathway-specific Smads appear to complex with the
common-partner Smad4 (28). Then the pathway-specific Smad/Smad4
complexes translocate to the nucleus where they participate in the
activation of target gene transcription (29). The third group comprises
the inhibitory Smads (Smad6 and Smad7). They lack the C-terminal
phosphorylation motif common to the pathway-specific Smads and do
not appear to be regulated by phosphorylation (30). Smad6 and Smad7
have been shown to inhibit BMP and TGFß receptor signaling (30, 31, 32)
as well as to partially block activin responses in Xenopus
animal cap assays (33). Although results suggest that the Smads are
important intracellular mediators of the TGFß and activin receptor
superfamily, many aspects, including the initial stages of their
activation by the type I receptors, remain unclear.
In this paper we have analyzed the interactions between ALK4, the
activin receptor type I, and the different Smads, which are involved in
the activin signaling pathway (Smad2, Smad3, Smad4, and Smad7). Using
our type I and type II activin receptor-inducible cell line, KAR6, we
show that activin can specifically induce the association of Smad2 and
Smad3 but not Smad4 with the activin receptor complex. This association
is very rapid and transient and presumably occurs through an
interaction of the pathway-specific Smads with the type I receptor
(ALK4). After Smad2/3 association with ALK4, the pathway-specific Smads
are then released into the cytoplasm where they interact with the
common-partner Smad4 and ultimately modulate transcriptional events.
Finally, we show here that Smad7 is a strong inhibitor of activin
signaling and exerts its effect by preventing the association of the
pathway-specific Smads with the activin receptor. Together, these
results permit a better understanding of the sequence of the first
steps and the involvement of Smad proteins in the transduction of
signals by activin receptor serine kinases.
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RESULTS
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Smad2 and Smad3 Associate with the Activin Receptor Complex upon
Activin Stimulation
Previous reports of cross-linking experiments using radioiodinated
TGFß had revealed that both Smad2 and Smad3 can form a complex with
the kinase-deficient but not the wild type TGFß type I receptor (27, 34). In addition, a N-terminally tagged form of Smad3 could also
associate with the kinase-deficient but not the wild-type TGFß type I
receptor (34). Interestingly, a C-terminally tagged, biologically
inactive form of Smad3 could bind the wild- type TGFß receptor (21, 34). These data suggested a transient association between Smad2 and
Smad3 and the TGFß type I receptor detectable only when using a
kinase-inactive form of the type I receptor with an active Smad or a
wild-type receptor with an inactive form of the Smad (21, 26, 34).
Activin and TGFß type I receptors (ALK4 and ALK5) signal through
Smad2 and Smad3; therefore, a similar mechanism of interaction
between the activin receptor and the Smads was considered possible.
Characterization of interactions between wild-type Smads and receptors
has been difficult because of the fact that transient inordinate
overexpression of both type I and type II receptors leads to
nonspecific, ligand-independent heterodimerization and activation
of the receptors (35, 36). This presumably also could lead to
ligand-independent activation of the Smads. To circumvent that problem,
we previously described the establishment of a stable cell line (KAR6
cells), in which the expression of type II (ActRII) and type I (ALK4)
is under control by a lac operon-switch and can be induced by
isopropyl-ß-D-thiogalactoside (IPTG) (37). In this
system, the levels of overexpressed receptors are much lower than that
observed in transient overexpression systems and, therefore, these
cells are closer to the physiological conditions. We have already
demonstrated that in such a system activin can specifically induce
activin receptor heterodimerization and exert biological effects on
gene transcription and cell proliferation (37). Therefore, we used the
same system to study the possible interactions between the Smads and
the activin receptor complex.
To investigate the capability of Smad2 to interact with the activin
receptors, KAR6 cells were transfected with the cDNAs encoding both
Smad2 N-terminally tagged with a Myc epitope and Smad4 tagged with a
flag epitope. After transfection, the receptors were induced by IPTG
and the cells treated with or without activin before receptor
immunoprecipitation with an antibody against ALK4 and analysis by
Western blotting using an anti-myc monoclonal antibody. As shown in
Fig. 1A
(left panel), in the
absence of ligand, very little Smad2 was found immunoprecipitated with
ALK4. On the other hand, when cells were stimulated for 5 min with
activin, a complex of Smad2 and ALK4 was formed and was clearly
detectable. This is the first demonstration of a ligand-induced
association between the activin receptor complex and Smad2. The
membrane was then rehybridized with an anti-flag antibody (Smad4), but
no detectable signal could be observed (data not shown). These results
indicate that Smad2, but not Smad4, interacts with the activin receptor
complex. A similar experiment was performed in another stable cell line
(KAR13 cells), which overexpresses only ALK4 upon IPTG induction (37).
The results were similar to those observed with KAR6 cells; Smad2, but
not Smad4, could associate with the receptor complex (data not shown),
suggesting that Smad2 association with the activin receptor complex is
probably mediated through an interaction with ALK4 rather than ActRII.
The level of expression of both Smad4-flag and Smad2-myc was measured
by Western blotting and revealed no difference between the different
samples for Smad4 (Fig. 1B
, left panel) and Smad2 (Fig. 1C
, left panel). Finally, the membrane was stripped and reprobed
with an anti-ActRII antibody. As shown in Fig. 1D
(left
panel), no detectable complex between the two receptors was
observed in the absence of ligand but after stimulation of the cells
with activin, this complex was rapidly formed, confirming what was
previously described with the anti-ALK4 antibody. In aggregate, these
results indicate that both receptor heteromerization and association
with Smad2 are very rapid events and are likely to represent an early
step in the activin signaling pathway.

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Figure 1. Activin-Induced Association of Smad2 and Smad3 with
the Activin Receptor Complex
KAR6 cells, overexpressing ALK4 and ActRII, were transfected with
both Smad2-myc (left panels) or Smad3-myc (right
panels) and Smad4-Flag. Cells were stimulated (+) or not (-)
with 1 nM of activin for 5 min, lysed, immunoprecipitated
with an anti-ALK4 antibody, and analyzed by Western blotting with an
anti-myc (9E10) monoclonal antibody (A). The arrows on the
right indicate the position of Smad2-myc and Smad3-myc as well
as of the heavy chains of the immunoglobulins (IgG). B, 1/25 of the
previous experiment was immunoprecipitated and revealed by Western blot
with an anti-Flag antibody to indicate the presence of the Smad4-Flag.
C, Similarly, another fraction of the initial experiment was used to
control the presence of Smad2-myc and Smad3-myc. D, Finally, panel A
was stripped and subsequently reprobed with an anti-ActRII antibody to
monitor the presence of the type II activin receptor (ActRII).
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Since both Smad2 and Smad3 can mediate the biological effects of
TGFß and activin (18, 20, 21, 27), we also analyzed the mechanism of
activation of Smad3. KAR6 cells were transfected with both Smad3-myc
and Smad4-flag cDNAs. Cells were inducted with IPTG to overexpress ALK4
and ActRII and stimulated for 5 min with activin before being lysed.
Cell lysates were immunoprecipitated with an anti-ALK4 antibody and
analyzed by Western blotting with an anti-myc antibody. As Shown in
Fig. 1A
(right panel), low levels of Smad3 were observed to
associate with the activin receptor complex in the absence of ligand.
Nevertheless, when cells were stimulated by activin, the amount of
Smad3 associated with ALK4 increases. This indicates that similar to
Smad2, activin can rapidly induce the association of Smad3 with the
activin receptor complex. The levels of overexpressed Smad3-myc and
Smad4-flag were similar in all lanes as shown in Fig. 1
, B and C
(right panels). The membrane was also stripped and
rehybridized with an anti-ActRII antibody. As shown in Fig. 1D
(right panel), activin stimulation of the cells rapidly
induced the formation of an heteromeric complex between the two
receptors in correlation with Smad3 association with the receptor
complex.
The level of Smad2/Smad3 associated with the activin receptor
complex is rather small compared with the total amount of Smad2/3
overexpressed (Fig. 1
, A and C, left and right panels). This
is probably due to the transient nature of this association between
Smad2/3 and the receptor but also to the fact that the activin
receptors are limiting in this system. Indeed, only a limited number of
type I and type II activin receptors are stably overexpressed in these
cells (37), while, on the other hand, the Smads are transiently
transfected and expressed at high levels (Fig. 1C
, left and right
panels).
Kinetics of Association between Smad2/3, Smad4, and ALK4
Because the activin receptor heteromerization is a transient event
(37), we analyzed the kinetics of association between the
pathway-specific Smads (Smad2 and 3) and the receptor. To address this
issue, Smad2-myc and Smad4-flag cDNAs were transfected into KAR6 cells.
Smad3-myc appears to be more difficult to detect by immunoblot analysis
in KAR6 cells, as compared with Smad2-myc (Fig. 1A
, left and
right panels) and was therefore, transfected, together with
Smad4-flag, in 293 cells stably overexpressing ALK4. As shown in Fig. 2A
, a complex between Smad2 and ALK4 was
observed after 5 min of stimulation by activin. This association was
sustained up to 15 min and then rapidly decreased, returning to basal
levels at 30 min. A fraction of each sample was immunoprecipitated and
analyzed by Western blot with an anti-myc antibody and showed that
similar amounts of Smad2-myc were present in all lanes (Fig. 2C
). This
indicates that the association between Smad2 and ALK4 is a very rapid
event occurring as early as 5 min after ligand stimulation and very
transient event, disappearing after 15 min. Smad3-Myc cDNA was
transfected in 293 cells overexpressing ALK4 and the kinetic of
association between Smad3 and ALK4 was assessed by Western blotting as
described above for Smad2. As shown in Fig. 3A
, Smad3, like Smad2, associates very
rapidly with the activin receptor, reaching its maximum at 15 min.
Then, similar to that observed with Smad2, Smad3 is released to the
cytoplasm (Fig. 3A
). This time course of association between Smad2/3
and ALK4 followed the activin-induced ALK4-ActRII heteromerization
(37). This suggests that Smad2 and Smad3 association with ALK4 is an
early event after receptor heteromerization, in activin signaling.

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Figure 2. Kinetics of Association between Smad2, Smad4, and
ALK4
KAR6 cells were transfected with both Smad2-myc and Smad4-Flag. Cells
were stimulated with 1 nM of activin for the indicated
period of time before being lysed. Half of the reaction was
immunoprecipitated with an anti-ALK4 antibody and analyzed by Western
blotting with the anti-myc monoclonal antibody (A). The arrows
on the right indicate the position of Smad2-myc. For the other
half of the reaction, the lysates were immunoprecipitated with an
anti-myc antibody and analyzed by Western blotting with the anti-Flag
monoclonal antibody (B). The arrows on the right
indicate the position of Smad4-flag and the heavy chains of the IgG. C
and D, 1/25 of the reaction was immunoprecipitated and revealed by
Western blot with an anti-myc antibody or an anti-flag antibody,
respectively, to ensure the presence of equal amounts of both Smad2-myc
and Smad4-flag.
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Figure 3. Kinetics of Association between Smad3, Smad4, and
ALK4
Human embryonic kidney cells (293) stably overexpressing ALK4 were
transfected with the cDNAs encoding Smad3-myc and Smad4-flag. Cells
were stimulated with 1 nM of activin for the indicated
period of time before being lysed. Half of the reaction was
immunoprecipitated with an anti-ALK4 antibody and analyzed by Western
blotting with the anti-myc monoclonal antibody (A). The arrows
on the right indicate the position of Smad3-myc. For the other
half of the reaction, the lysates were immunoprecipitated with an
anti-myc antibody and analyzed by Western blotting with the anti-Flag
monoclonal antibody (B). The arrows on the right
indicate the position of Smad4-flag and the heavy chains of the IgG. C
and D, 1/25 of the reaction was immunoprecipitated and revealed by
Western blot with an anti-myc antibody or an anti-flag antibody,
respectively, to ensure the presence of equal amount of both Smad2-myc
and Smad4-flag.
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In the same experiment we also investigated the kinetics of association
of the common-partner Smad4 with Smad2 and the activin receptor
complex. Both Smad2 and Smad3 have been shown to associate with the
common-partner Smad4 (34), but it is not clear whether this association
takes place while Smad2 and 3 are still bound to ALK4 or after they are
released to the cytoplasm. As mentioned before, no association between
Smad4 and ALK4 was detected in our previous experiments. Therefore, we
considered the hypothesis that the Smad2/Smad4 and Smad3/Smad4
heterodimer formation occurs after Smad2/3 have been released from the
receptor into the cytoplasm. Indeed, this would be consistent with our
observation of a very rapid and transient association between Smad2 and
the activin receptor. Cell lysates were immunoprecipitated with an
anti-myc antibody (Smad2) and analyzed by Western blot with an
anti-flag (Smad4) antibody. As shown in Fig. 3B
, no complexes between
Smad2 and Smad4 preexisted in the absence of ligand. However, activin
stimulation resulted in the oligomerization of the two Smads but only
after a 30 min period. This suggests that the Smad2/Smad4
heterodimerization takes place considerably after the association of
Smad2 with ALK4. Together with the observations in Fig. 1
, these data
suggest that Smad2 rapidly associates with the receptor complex after
ligand stimulation and is then released to the cytoplasm where it
interacts with Smad4. Control experiments were performed to verify that
the levels of expression of both Smad2-myc (Fig. 3C
) and Smad4-flag
(Fig. 3D
) were similar in all samples. A parallel experiment was
performed with both Smad3 and Smad4 and yielded the same results (data
not shown).
Transcriptional Effects
To assess the importance of the interaction between the Smads and
the activin receptor, we evaluated a biological consequence of such
interactions. IPTG-induced KAR6 cells were transfected with 3TPLux, an
activin-responsive reporter construct, together with different
combination of Smads, as indicated in Fig. 4
. Activin stimulation of IPTG-induced
KAR6 cells resulted in an 8-fold induction of the 3TPLux luciferase
activity. When coexpressed with the common-partner Smad4, the
pathway-specific Smad2 and 3 generated an increase in the
activin-induced 3TPLux response (Fig. 4
). This is in agreement with the
current model of action of the Smad proteins, where both the
pathway-specific Smad and the common-partner Smad4 are needed to form a
complex to transmit the signal. The Smad3/4 effect is stronger than the
Smad2/4 effect (4-fold vs. 2-fold higher than the control
transfected with the expression vector alone). The significance of this
observation is not known and remains to be determined. It might reflect
a more important role for Smad3 in the mediation of the activin signal
in this cell line or be due to different levels of expression between
Smad2 and Smad3. Alternatively, the artificial 3TPLux promotor
construct contains several Smad3/Smad4 binding sites and might be
naturally more responsive to Smad3 than to Smad2. Interestingly, when
both Smad2 and 3 were transfected, the activin-induced signal was
increased to 6-fold more than the control (Fig. 4
). These results
suggest that Smad2 and Smad3 are synergistic and that both are required
to transmit the maximum activin signal, consistent with that observed
previously for TGFß (21).

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Figure 4. Smad2/3 and 4 Stimulate the Activin-Induced
Transcriptional Activity of the 3TPLux Reporter Construct
KAR6 cells were transfected with different combinations of Smad
proteins as indicated on Fig. 4 and the 3TPLux reporter construct as
well as the CMV-ß-galactosidase vector. Cells were induced by IPTG to
stimulate the overexpression of ALK4 and ActRII and stimulated (+) or
not (-) with 500 pM activin for 16 h before being
lysed. The luciferase activity was normalized to the relative
ß-galactosidase values and represent means and SDs of
three independent experiments.
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Smad7 Blocks the Activin-Induced 3TPLux Activity in the Presence of
Overexpressed Smad2/3/4
Two vertebrate inhibitory Smads have been characterized so
far, Smad6 and Smad7, which block TGFß-, activin-, and BMP-signaling
pathways. Indeed, Smad6 was shown to block BMP and TGFß actions (31),
while Smad7 can block the TGFß (30) and also partly limit
activin signaling in the Xenopus animal cap assay (33).
Additionally, Smad7 was also shown to inhibit the 3TPLux activity
induced by a constitutively active ALK4 (32). To verify the ability of
Smad7 to block activin responses in our system, IPTG-induced KAR6 cells
were transfected with the 3TPLux reporter construct and different
combinations of Smads, as indicated in Fig. 5
. In the presence of overexpressed
Smad7, the activin response on the 3TPLux reporter construct was
totally blocked. This indicates that Smad7 can fully inhibit the
ligand-dependent activin transcriptional signal in this cell line. As
shown in Fig. 5
, the activin-induced response on 3TPLux was maximal in
the presence of Smad2, 3, and 4, confirming the previous observation
(Fig. 4
). However, this maximized effect of activin on 3TPLux was also
fully antagonized by the presence of overexpressed Smad7 (Fig. 5
).
These results indicate that Smad7 is a powerful inhibitor of activin
signaling.

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Figure 5. Smad7 Blocks the Activin-Induced 3TPLux Activity in
the Presence or the Absence of Smad2/3 and 4
KAR6 cells were transfected with different combinations of Smad
proteins as indicated on Fig. 6 together with the 3TPLux reporter
construct and the CMV-ß-galactosidase vector. Cells were induced by
IPTG to stimulate the overexpression of ALK4 and ActRII and stimulated
(+) or not (-) with 500 pM of activin for 16 h before
being lysed. The luciferase activity was normalized to the relative
ß-galactosidase values and represent means and SDs of
three independent experiments.
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Smad7 Inhibits the Binding of Smad2 to the Activin Receptor
We then examined the mechanism of action of this inhibitory
effect of Smad7 on the activin response. Smad7 could either interfere
with the heterodimerization between the pathway-specific Smads and the
common-partner Smad4, or could prevent the association of these
pathway-specific Smads with the type I receptor. Indeed, Smad7 binds
directly to the type I activin receptor ALK4 once activated by the type
II receptor (Y. Chen and W. W. Vale, unpublished results). To test
the latter hypothesis, Smad2 and Smad4 were cotransfected in KAR6 cells
with or without the inhibitory Smad7. After activin stimulation of the
cells, the association between Smad2 and ALK4 was observed by Western
blotting. As shown in Fig. 6
(upper
panel), activin stimulation of the cells resulted in the formation
of a complex between Smad2 and the activin receptor. However, in the
presence of Smad7, no complex between Smad2 and ALK4 was detected after
ligand stimulation of the cells, despite equal levels of overexpressed
Smad2 in all samples (Fig. 6
, lower panel). Together with
Fig. 5
, these results indicate that Smad7 prevents the interaction of
the pathway-specific Smad2 with the activin receptor complex,
resulting in the inhibition of the effects of activin on gene
transcription.

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Figure 6. Inhibition of the Smad2/ALK4 Interaction by Smad7
KAR6 cells were transfected with Smad2-myc and Smad4-Flag (20
µg each) in the absence or the presence of 40 µg of Smad7 cDNA.
Cells were then induced by IPTG and stimulated or not with 1
nM of activin for 10 min. Cells were then lysed,
immunoprecipitated with an anti-ALK4 antibody, and analyzed by Western
blotting with the anti-myc monoclonal antibody (upper
panel). The arrows on the right indicate the
position of Smad2-myc (Smad2). Lower panel, 1/25 of the
previous experiment was immunoprecipitated and revealed by Western blot
with an anti-myc antibody to ensure the presence of equal amount of
Smad2-myc (Smad2) in all lanes.
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DISCUSSION
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With the discovery of the newly described Smad
family of proteins, improved understanding of the cellular actions of
receptor serine kinases has been achieved. Distinct subclasses of Smad
proteins can modulate alone or in combination with activin and TGFß
responses in a positive or negative way. The two pathway-specific Smads
(Smad2 and Smad3) as well as the common-partner Smad (Smad4) transduce
activin and TGFß signals. Key events in the activation of
transcription of activin and TGFß target genes include
phosphorylation of Smad2 and Smad3 within their C-terminal SSXS motifs
(38, 39), complex formation of Smad2/3 with Smad4 (34), nuclear
translocation of these heteromeric complexes, and association with
transcription factors in the nucleus (40). While the pathway-specific
Smads are essential mediators of activin signaling, the initial stages
of their activation remained unclear. Associations between wild-type
Smads and activin receptors had not been reported. The fact that these
associations were only detected when using functionally inactive Smads
and receptors suggest that this association is very transient or
requires the presence of an intermediate protein (21, 27, 34).
We report here that activin can specifically induce the
association of Smad2 and Smad3 but not the common-partner, Smad4, with
the activin receptor complex. This demonstrates ligand-induced
association of Smad2 or Smad3 with the activin receptor. This is in
agreement with recent observations for Smad5 and BMP receptors (41).
Indeed, BMP-2 stimulation of HEK-293 cells transiently transfected with
type I and II BMP receptors and Smad5 results in the association of
Smad5 with the receptor complex after 15 min of stimulation. We
previously reported that the ligand-induced heteromerization between
the type I and type II activin receptors is detectable as early as 1
min after ligand stimulation, reaching its maximum after 5 min (37). We
show here that the interaction of the pathway-specific Smads with the
activin receptor is also rapid, detectable as early as 5 min after
stimulation by the ligand, and represents a very early intracellular
event in the activin-signaling pathway after receptor oligomerization
and transphosphorylation. Collectively, these data are consistent with
the hypothesis that the association of pathway-specific Smads with the
type I receptor serine kinase receptor is an early step after receptor
heteromerization in TGFß superfamily signal transduction.
The association of Smad2/3 with ALK4 was observed in cell lines
overexpressing either ALK4 and ActRII (KAR6) or ALK4 alone (KAR13),
suggesting that the association of Smad2 and Smad3 with the activin
receptor complex is likely to be mediated through the type I receptor.
In addition, Smad2 activation by the TGFß receptor complex is
mediated by the kinase domain of the type I TGFß receptor (27). Also
the phosphorylation of the type I receptor for TGFß is required for
interaction between a kinase-deficient type I and Smad2 and 3 (34).
This is consistent with the current model of action of the receptor of
this family in which the type I receptor appears to be the signaling
unit and binds and phosphorylates the pathway-specific Smads. We
could not detect any ligand-induced phosphorylation of Smad2/3 or
of the activin receptors (type I and II) in our system, presumably due
to the low level of receptor overexpression in these cells (37) and to
the transient nature of the association between Smad2/3 and the
receptor.
The transient nature of the interaction between the pathway-specific
Smads and the receptor also provides more insight concerning the
signaling events occurring in the activation of the Smad proteins and
their oligomerization with the common-partner Smad4. We report here the
temporal sequence of this heterodimerization in regard to receptor
heteromerization and Smad2 and 3 association with this receptor
complex. Our results suggest that Smad4 does not associate with the
receptor complex but forms a complex with the pathway-specific Smads
only after they are released from the receptor. It is possible that the
release of Smad2 and Smad3 from the receptor is triggered by the
phosphorylation of their C-terminal SSXS motifs by the kinase domain of
the type I receptor. It is the C-terminally phosphorylated forms of the
pathway-specific Smads that are proposed to associate with Smad4 in the
cytoplasm before transport to the nucleus where they modulate
transcription.
The effect of Smad3/4 or Smad2/4 on activation of transcription
was also examined by cotransfecting the cells with the 3TPLux reporter
construct and after the luciferase response to activin. Our results
indicate that both Smad2 and Smad3 can increase the activin-induced
transcriptional activity in KAR6 cells, when coexpressed with the
common-partner Smad4. The combined effects of transfection of Smad2 and
Smad3 on transcription are greater than that of either Smad alone as
was previously observed in other systems (21, 34).
Finally, the effects of the inhibitory Smad7 were also examined in this
system. We found that Smad7 can totally block the activin-induced
3TPLux response in KAR6 cells and can also block the activin response
in cells overexpressing Smad2/3 and 4 by preventing the
ligand-dependent association of the pathway-specific Smads with the
activin receptor complex.
In summary, both Smad3 and Smad2 interact with the activin
receptor complex upon activin stimulation, most probably by
functionally interacting with the type I receptor (ALK4). This
indicates that the pathway-specific Smad2 and Smad3 are early
intracellular downstream components of activin receptor signal
transduction. These two Smads are then released to the cytoplasm where
they interact with the common-partner Smad4. The Smad oligomers can
translocate to the nucleus where they exert their effects on the
transcription of target genes. In addition, in the presence of the
inhibitory Smad7, the complex formation between the pathway-specific
Smads and the receptor is prevented, which in turn blocks the activin
signal. These results permit a better understanding of the first steps
in the mechanism of action of the activin receptor and provide a good
model for the interactions occurring between the type I receptor and
the different Smads, leading to activation or inhibition of the activin
responses.
 |
MATERIALS AND METHODS
|
---|
Materials
Recombinant activin A was generously supplied by Dr. J. Mather
(Genentech, Inc., South San Francisco, CA). Affinity-purified rabbit
-ALK4 serum () was raised against the carboxyl-terminal end
of ALK4 (aa 493505), and affinity-purified rabbit
-ActRII serum
() was raised against the carboxyl-terminal end of ActRII (aa
482494).
Immunoprecipitations
KAR6 and 293 cells were incubated in starvation medium (without
FCS) and stimulated with 1 nM of activin at the indicated
times. Cells were then lysed in 1 ml of lysis buffer (50 mM
Tris-HCl, pH 7.5; 150 mM NaCl; 10% glycerol; 0.5% NP-40)
containing protease inhibitors (1 mM phenylmethylsulfonyl
fluoride; 1 µg/ml pepstatin A; 2 µg/ml leupeptin; 5 µg/ml
aprotinin) for 15 min at 4 C. The insoluble material was discarded
after centrifugation at 13,000 rpm for 15 min. Cell lysates were
immunoprecipitated overnight at 4 C with an
-ALK4 () or an
-myc or an
-flag antibody and 30 µl of protein A agarose beads
or protein G agarose (for myc and flag) (10% in lysis buffer). Samples
were then washed three times in 1 ml of washing buffer (50
mM Tris-HCl, pH 7.5; 2 mM EDTA; 150
mM NaCl; 10% glycerol) and eluted in 20 µl of SDS
loading buffer (20% glycerol; 10% ß-mercaptoethanol; 4.6% SDS;
0.125 M Tris-HCl pH 6.8).
Western Blot Analysis
Proteins were separated on a 7.5% polyacrylamide gel,
transferred onto nitrocellulose, and incubated with either an
-ALK4
or
-myc or
-flag antibody (at 0.5 µg/ml), overnight at 4 C.
After incubation, the membranes were washed twice for 15 min in washing
buffer (50 mM Tris-HCl, pH 7.6; 200 mM NaCl;
0.05% Tween 20) and incubated with a secondary antirabbit antibody
coupled to peroxidase (Amersham, Arlington Heights, IL;
-rabbit
Ig-horseradish peroxidase, NA 934 at a 1:4000 dilution or
-mouse Ig-horseradish peroxidase) for 1 h at room temperature.
Then, the membranes were washed four times for 15 min in washing
buffer, and immunoreactivity was normalized by chemiluminescence
(Amersham, ECL kit, RPN 2106) according to the manufacturers
instructions.
Membrane Stripping
After the first round of immunodetection, membranes were
stripped for 15 min at 55 C in stripping buffer (100 mM
2-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCL, pH 6.7) and
were washed for several hours in washing buffer before used again for
immunodetection.
3TPLux Transcription Assay
Cells were grown in complete medium (RPMI, 10%
FCS). For transfections, 2 x 107 cells were
resuspended in 500 µl of HEPES dissociation buffer before
electroporation (Bio-Rad Gene Pulser, Bio-Rad Laboratories, Richmond,
CA; 960 µFarads, 0.22 kV) with 20 µg of 3TPLux plasmid (kindly
provided by Dr. Joan Massagué) and 10 µg of cytomegalovirus
(CMV)-ß-galactosidase plasmid and 20 µg of the indicated Smad
cDNAs. The cells were then resuspended in 20 ml of complete medium and
incubated at 5% CO2, 37 C overnight. The following day,
cells were plated at 106 cells/ml in the starvation medium
(RPMI with no FCS) in the presence of IPTG (5 mM), and were
treated or not with activin, at the indicated concentrations for
24 h before being harvested. Then, cells were lysed in 100 µl of
lysis buffer (1% Triton X-100; 15 mM MgSO4; 4
mM EGTA; 1 mM dithiothreitol; 25 mM
glycylglycine) for 30 min on ice, and the luciferase activity of each
lysate was measured and normalized to the relative ß-galactosidase
activity.
 |
ACKNOWLEDGMENTS
|
---|
The authors are thankful to Joan Massague for
providing the 3TPLux reporter construct and to Joan Vaughan for the
preparation and purification of the antiactivin receptor antibodies. We
also thank Dr. S. Ali for critical reading of this manuscript and
Genentech Inc. for providing recombinant activin.
 |
FOOTNOTES
|
---|
Address requests for reprints to: Wylie Vale, Ph.D., The Clayton Foundation Laboratories for Peptide Biology, The Salk Institute for Biological Studies, 10010 North Torrey Pines Road, La Jolla, California 92037
This work was supported by NICHD Program Project Grant HD-13527, the
Adler Foundation, the Medical Research Council of Canada (J.J.L.), and
the Concern Foundation (J.J.L.). W.V. is a Foundation for Medical
Research, Inc., Senior Investigator. J.J.L. was a Fellow of the
Adler Foundation and is now an Medical Research Council scholar. K.T.
is supported by the Yoshida Scholarship Foundation.
1 Present address: McGill University, Molecular Endocrinology
Laboratory, Royal Victoria Hospital, 687 Pine Avenue West, H3A 1A1,
Montreal, Quebec, Canada. 
Received for publication July 10, 1998.
Accepted for publication September 28, 1998.
 |
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