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Clayton Foundation Laboratories for Peptide Biology Salk Institute for Biological Studies La Jolla, California 92037
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(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 1–8) 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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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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(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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, 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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. 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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(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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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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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.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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-ALK4 serum () was raised against the carboxyl-terminal end
of ALK4 (aa 493–505), and affinity-purified rabbit -ActRII serum
() was raised against the carboxyl-terminal end of ActRII (aa
482–494).
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 manufacturer’s
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.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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