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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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-{alpha} (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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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. 1AGo (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. 1BGo, left panel) and Smad2 (Fig. 1CGo, left panel). Finally, the membrane was stripped and reprobed with an anti-ActRII antibody. As shown in Fig. 1DGo (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).

 
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. 1AGo (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. 1Go, B and C (right panels). The membrane was also stripped and rehybridized with an anti-ActRII antibody. As shown in Fig. 1DGo (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. 1Go, 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. 1CGo, 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. 1AGo, left and right panels) and was therefore, transfected, together with Smad4-flag, in 293 cells stably overexpressing ALK4. As shown in Fig. 2AGo, 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. 2CGo). 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. 3AGo, 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. 3AGo). 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.

 
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. 3BGo, 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. 1Go, 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. 3CGo) and Smad4-flag (Fig. 3DGo) 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. 4Go. 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. 4Go). 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. 4Go). 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. 4Go 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.

 
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. 5Go. 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. 5Go, the activin-induced response on 3TPLux was maximal in the presence of Smad2, 3, and 4, confirming the previous observation (Fig. 4Go). However, this maximized effect of activin on 3TPLux was also fully antagonized by the presence of overexpressed Smad7 (Fig. 5Go). 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. 6Go 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.

 
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. 6Go (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. 6Go, lower panel). Together with Fig. 5Go, 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.

 

    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
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
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Materials
Recombinant activin A was generously supplied by Dr. J. Mather (Genentech, Inc., South San Francisco, CA). Affinity-purified rabbit {alpha}-ALK4 serum () was raised against the carboxyl-terminal end of ALK4 (aa 493–505), and affinity-purified rabbit {alpha}-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 {alpha}-ALK4 () or an {alpha}-myc or an {alpha}-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 {alpha}-ALK4 or {alpha}-myc or {alpha}-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; {alpha}-rabbit Ig-horseradish peroxidase, NA 934 at a 1:4000 dilution or {alpha}-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.


    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. Back

Received for publication July 10, 1998. Accepted for publication September 28, 1998.


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 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
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