Smad1 Recognition and Activation by the ALK1 Group of Transforming Growth Factor-beta Family Receptors*

Ye-Guang ChenDagger and Joan Massagué§

From the Cell Biology Program and Howard Hughes Medical Institute, Memorial Sloan-Kettering Cancer Center, New York, New York 10021

    ABSTRACT
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Abstract
Introduction
References

Two structural elements, the L45 loop on the kinase domain of the transforming growth factor-beta (TGF-beta ) family type I receptors and the L3 loop on the MH2 domain of Smad proteins, determine the specificity of the interactions between these receptors and Smad proteins. The L45 sequence of the TGF-beta type I receptor (Tbeta R-I) specifies Smad2 interaction, whereas the related L45 sequence of the bone morphogenetic protein (BMP) type I receptor (BMPR-I) specifies Smad1 interactions. Here we report that members of a third receptor group, which includes ALK1 and ALK2 from vertebrates and Saxophone from Drosophila, specifically phosphorylate and activate Smad1 even though the L45 sequence of this group is very divergent from that of BMPR-I. We investigated the structural elements that determine the specific recognition of Smad1 by ALK1 and ALK2. In addition to the receptor L45 loop and the Smad1 L3 loop, the specificity of this recognition requires the alpha -helix 1 of Smad1. The alpha -helix 1 is a conserved structural element located in the vicinity of the L3 loop on the surface of the Smad MH2 domain. Thus, Smad1 recognizes two distinct groups of receptors, the BMPR-I group and the ALK1 group, through different L45 sequences on the receptor kinase domain and a differential use of two surface structures on the Smad1 MH2 domain.

    INTRODUCTION
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Abstract
Introduction
References

The Smad family of proteins play a central role in signal transduction by the transforming growth factor-beta (TGF-beta )1 family (1-3). Smad proteins are directly phosphorylated by type I TGF-beta family receptors, an event that induces their accumulation in the nucleus where they activate transcription of specific genes. Smad proteins act as signal transducers for different members of the TGF-beta family, including TGF-beta itself, the activins, and the bone morphogenetic proteins (BMPs). The type I receptors for TGF-beta and activin, which are known as Tbeta R-I and ActR-IB, respectively, signal via Smad2 and its close homolog Smad3. The BMP type I receptors BMPR-IA and BMPR-IB signal via Smad1 and possibly its close homologs, Smad5 and Smad8. Upon receptor-mediated phosphorylation, and on their way to the nucleus, all these Smad proteins associate with Smad4, a member of a separate subclass that is required for the formation of transcriptional complexes. The Drosophila orthologs of BMP (Dpp), BMPR-I (Thickveins), Smad1 (Mad), and Smad4 (Medea) are functionally linked in a similar fashion (4-6).

The maintenance of specificity in this system requires that each member of the type I receptor family be able to discriminate among different groups of Smad proteins. The specific interaction between Smad proteins and type I receptors is determined by two structural elements, namely, the L3 loop in the carboxyl-terminal domain (or MH2 domain) of Smad proteins and the L45 loop in the kinase domain of the receptors (7, 8). Both elements consist of a short amino acid sequence that is highly conserved among Smad proteins or receptors of similar specificity but differs on a few critical residues between functionally distinct Smad proteins or receptors. Thus, the L45 loop sequence of the Tbeta R-I/ActR-IB group (which also includes the orphan receptors ALK7, XTrR-I and Atr-I) is compatible with the L3 loop sequence common to Smad2 and -3, allowing functional interactions between these receptors and Smad proteins. A similar relationship exists between the L45 loop of the BMPR-I group (which also includes Thickveins) and the L3 loop of Smad1, -5, -8, and Mad (see Figs. 1A and 4A for L45 and L3 sequences).

An important question left open by previous studies is about the Smad specificity of a third group of type I receptors. This group includes ALK1, ALK2, and Drosophila Saxophone. Saxophone is essential for dorsal closure of the Drosophila embryo and is believed to mediate Dpp signals along with Thickveins (9-12). ALK1 (also known as TSR-I) is highly expressed in vascular endothelial cells (13). Inactivating mutations of ALK1 in humans cause hereditary hemorrhagic telangiectasia (14). Hereditary hemorrhagic telangiectasia is an autosomal dominant disorder characterized by epithelial vascular dysplasia with a high propensity to hemorrhage in the nasal and gastrointestinal mucosa. ALK2 (also known as ActR-I or Tsk7L) is a broadly expressed receptor that can bind BMPs, activin, and, under certain conditions, TGF-beta in vitro (1-3, 13, 15-19). However, ALK2 does not mediate activin responses like those mediated by ActR-IB (20). In Xenopus, the activity of ALK2 (21) is akin to that of BMPR-I (22, 23), because both receptors signal ventral mesoderm induction, and defects in ventral mesoderm formation caused by a dominant-negative ALK2 construct can be rescued by overexpression of Smad8 (24).

In this study, we have investigated the Smad specificity of this third group of type I receptors. We were intrigued by the fact that the L45 sequence of these receptors is very different from that of the Tbeta R-I group or the BMPR-I group (see Fig. 1A). Nonetheless, we observed that ALK1 and ALK2, like BMPR-I, recognize and activate Smad1. This was paradoxical because Tbeta R-I, whose L45 sequence is much closer to that of BMPR-I, does not recognize Smad1. Insights from the crystal structure of the Smad MH2 domain allowed us to address this paradox.

    EXPERIMENTAL PROCEDURES

R1B/L17, COS-1, and HepG2 cells were maintained as described previously (8, 25). Mouse embryonic P19 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and 2 mM glucose. Mutagenesis of Smad proteins and receptors was performed by polymerase chain reaction using appropriate oligonucleotides, and verified by DNA sequencing. Tbeta R-I(LA) contains a replacement of the L45 sequence ADNKDNGTW with the sequence SDMTSRHSS. Smad2(H1-1) contains the mutations A323S, V325I, and M327N, and Smad2(HL1) contains the mutations A323S, V325I, M327N, R427H, and T430D. Other constructs have been reported previously (7, 8).

Activation of the 3TP-luciferase reporter (20) and Mix2 A3-luciferase reporter (26) by receptors were analyzed in R1B/L17 cells as described previously (8, 25). To measure the activity of a XVent2-luciferase reporter (27), P19 cells were transfected with this construct, and the constitutively active forms of type I receptors, using Lipofectin (Life Technologies, Inc.) according to manufacturer's instructions. Luciferase activity was measured 40 h after transfection.

Metabolic labeling of transfected cells with [32P]orthophosphate or [35S]Met/Cys was performed as described previously (8, 25). Immunoprecipitates with monoclonal anti-Flag M2 antibody (IBI, East Kodak) were visualized by SDS-polyacrylamide gel electrophoresis followed by autoradiography. Subcellular localization of Smad proteins was examined in HepG2 cells transfected with N-terminally Flag-tagged Smad proteins and the constitutively active receptors. Immunofluorescence was carried out as described previously (8). At least 200 cells were scored per assay. The percentage of Flag-positive cells with predominantly or exclusively nuclear immunofluorescence is plotted in the figures.

    RESULTS AND DISCUSSION

ALK1 and ALK2 Transduce BMP-like Signals-- To investigate the signaling specificity of human ALK1 and ALK2, we used transcriptional assays that discriminate between BMP-like signaling and TGF-beta /activin-like signaling. Since the natural ligands for ALK1 and ALK2 have not been conclusively defined, we generated ALK1 and ALK2 mutant constructs [ALK1(QD) and ALK2(QD)] containing a Gln to Asp mutation in the penultimate residue of the regulatory domain (GS domain), which activates type I receptors in a ligand-independent manner (28-30). The corresponding mutant forms of the BMP type I receptors, BMPR-IA(QD) and BMPR-IB(QD), activate luciferase expression when cotransfected with the XVent2-Luc reporter construct into mouse P19 cells (Fig. 1B). XVent2-Luc contains a BMP-responsive region from the Xenopus Vent.2 gene driving expression of luciferase (27) and can be activated via Smad1 but not Smad2 (8). ALK1(QD) and ALK2(QD) were also able to activate XVent2-Luc, whereas the constitutively active TGF-beta receptor construct, Tbeta R-I(TD), had no effect (Fig. 1B).


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Fig. 1.   A, the L45 loop sequences of the type I receptors. The residues that are distinct among different groups are boxed, and the variant residues within a group are italicized. B, XVent2-luciferase reporter was transfected into P19 cells with or without the active forms of receptors as indicated. 24 h after transfection, cells were treated with or without 1 nM BMP2 for 20 h, and subsequently, luciferase activity was determined. C, R1B/L17 cells were transfected with 3TP-luciferase reporter and the indicated receptors. Luciferase activity was measured 48 h later. D, R1B/L17 cells were transfected with A3-luciferase reporter, Xenopus FAST-1 and the indicated receptors. Luciferase was measured 48 h later. Data are the average of three or more assays ± SD.

We did similar experiments using two TGF-beta /activin-responsive reporters, 3TP-Lux and A3-Luc. 3TP-lux contains TGF-beta /activin-response elements from plasminogen activator inhibitor-1 and collagenase (20). A3-Luc contains three copies of the activin response element from the Xenopus Mix.2 gene (26). Both reporters are activated by TGF-beta or activin signals mediated by Smad2 (31-33) or Smad3 (34-36).2 Both reporters were activated by the TGF-beta receptor mutant Tbeta R-I(TD) and the activin receptor mutant ActR-IB(TD) in transfected R1B/L17 mink lung cells (Fig. 1, C and D). ALK1(QD) and ALK2(QD), like BMPR-IB(QD), failed to activate 3TP-lux and A3-Luc (Fig. 1, C and D). Thus, the activation pattern of these three reporter constructs suggests that the signaling specificity of ALK1 and ALK2 is similar to that of the BMP type I receptors BMPR-IA and IB.

Smad Specificity of ALK1 and ALK2-- These results raised the possibility that ALK1 and ALK2 may be able to phosphorylate and activate Smad1 but not Smad2. To investigate this possibility, receptor constructs were cotransfected with N-terminally Flag-tagged Smad1 or Smad2 into R1B/L17 cells, and Smad phosphorylation was determined by anti-Flag immunoprecipitation from the [32P]orthophosphate-labeled transfectants. The basal phosphorylation of Smad1 and Smad2 observed in the absence of receptor activity in these cells (Fig. 2A, left lanes) is caused by MAP kinases acting on phosphorylation sites unrelated to the receptor-mediated phosphorylation sites (37).3 ALK1(QD) and ALK2(QD) increased the phosphorylation of Smad1 but not that of Smad2, which is similar to the effect of BMPR-IB(QD) and opposite to the effect of Tbeta R-I(TD) (Fig. 2A). These results were consistent with ALK1 and ALK2 acting as specific activators of Smad1 but not Smad2.


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Fig. 2.   A, phosphorylation of Smad proteins was examined in R1B/L17 cells by transient transfection with N-terminally Flag-tagged Smad constructs and the active forms of receptors. 40 h later, cells were metabolically labeled with [32P]orthophosphate. Anti-Flag immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and autography. Protein expression was verified in parallel by anti-Flag immunoprecipitation from [35S]Met/Cys-labeled cells. B, nuclear translocation of Smad proteins was determined in HepG2 cells. Flag-tagged Smad constructs and the indicated receptors were cotransfected into HepG2 cells. Immunostaining was processed 48 h later first with mouse anti-Flag and second with fluorescein isothiocyanate-conjugated goat anti-mouse antibodies. About 200-300 fluorescence-positive cells were scored per condition. The percentage of Flag-positive cells with predominantly or exclusively nuclear immunofluorescence is plotted.

To confirm that ALK1(QD) or ALK2(QD) specifically activated Smad1, we determined the ability of these constructs to induce nuclear accumulation of Smad proteins. Consistent with the above results, expression of ALK1(QD) or ALK2(QD) induced nuclear accumulation of Flag-Smad1 but not Flag-Smad2, which again is similar to the effect of BMPR-IB(QD) and opposite the effect of Tbeta R-I(TD) (Fig. 2B). Under these conditions, nuclear localization of Flag-Smad proteins was observed in only a fraction of the cells. The incompleteness of this response may be because of limitations in the ability of the receptors to quantitatively activate the overexpressed Smad proteins. In untransfected cells, TGF-beta induces a quantitative translocation of endogenous Smad2 and -3 into the nucleus.3

We repeated these assays using a Smad1 construct [Smad1(AAVA)] that contains three Ser to Ala mutations in the C-terminal sequence SSVS (38). These serines are the sites phosphorylated by BMPR-I, and their mutation to alanine prevents receptor-mediated phosphorylation and activation of Smad1 by BMP (38). The Smad1(AAVA) mutant failed to accumulate in the nucleus when cotransfected with ALK1(QD) or ALK2(QD) vectors (data not shown). Therefore, ALK1 and ALK2 can mimic the ability of BMP type I receptors to specifically phosphorylate and activate Smad1, leading to BMP-like transcriptional responses.

Determinants of Specificity in ALK1 and ALK2-- The sequence of the L45 loop in the kinase domain of Tbeta R-I determines TGF-beta signaling activity (39). Furthermore, the L45 loop of Tbeta R-I and BMPR-I specifies the choice of Smad proteins by these receptors in the cell (8). Five of nine residues in this sequence are conserved between Tbeta R-I and BMPR-I (see Fig. 1A). Thus, the three nonconserved residues are responsible for the different Smad specificity of these two receptors. Mutant Tbeta R-I and BMPR-I constructs with these residues swapped show a switch in their ability to recognize and activate Smad1 and Smad2 (8). The L45 loop is conserved between ALK1 and ALK2 but is very divergent between these receptors and Tbeta R-I or BMPR-I (see Fig. 1A). Because the Smad specificity of ALK1 and ALK2 is similar to that of BMPR-I receptors, we investigated whether this specificity is determined by the L45 sequence of ALK1 and ALK2 despite their lack of similarity to the BMPR-I L45 sequence. To this end, we generated a mutant Tbeta R-I [Tbeta R-I(LA)] containing the L45 sequence SDMTSRHSS, which corresponds to the ALK2 L45 sequence. The signaling specificity of this construct was compared with those of the wild type Tbeta R-I and the previously described mutant Tbeta R-I(LB) containing the L45 sequence of BMPR-I (8). Tbeta R-I(LA) was similar to Tbeta R-I(LB) and BMPR-I in its ability to induce nuclear accumulation preferentially of Smad1 (Fig. 3A) and its pattern of activation of XVent2-Luc (Fig. 3B) and 3TP-Lux (Fig. 3C). These results suggest that the ability of the BMPR-I L45 sequence to specify an interaction with Smad1 is shared by the very divergent L45 sequence of ALK1 and -2 but not by the closely related L45 sequence of Tbeta R-I.


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Fig. 3.   A, Tbeta R-I(LA) induces nuclear translocation of Smad1 but not Smad2. N-terminally Flag-tagged Smad1 or Smad2 were cotransfected into HepG2 cells with wild type or mutant Tbeta R-I, or wild type BMPR-IB, as well as their corresponding type II receptors. Cells were treated with 1 nM TGF-beta 1 or 5 nM BMP2 for 1 h and then processed for anti-Flag immunostaining. 200-300 fluorescence-positive cells were scored. The percentage of Flag-positive cells with predominantly or exclusively nuclear immunofluorescence is plotted. B and C, Tbeta R-I(LA) activates the BMP-responsive XVent-Luc reporter but not the TGF-beta -responsive 3TP-lux reporter. XVent-luciferase and 3TP-luciferase reporters were transfected into P19 or R1B/L17 cells, respectively, with wild type or mutant Tbeta R-I as well as Tbeta R-II. Cells were then incubated with 1 nM BMP2 (B) or 0.5 nM TGF-beta (T) for 1 day, and luciferase activity was determined. Data are the average of three or more assays ± SD.

Role of the alpha -Helix 1 in Smad Recognition of ALK1 and ALK2-- In light of this, we wondered whether Smad1 might recognize ALK1 and -2 and BMPR-I by different mechanisms. The ability of Smad1 and Smad2 to recognize BMPR-I and Tbeta R-I, respectively, is determined by the sequence of the L3 loop (7). As inferred from the crystal structure of the Smad4 MH2 domain, the L3 loop protrudes from the surface of this domain (40). The sequence of the L3 loop is identical within the Smad2/3 and Smad1/5/8 subgroups, but differs at two critical positions between these two subgroups (Fig. 4, A and B). Swapping these two residues between Smad1 and Smad2 switches their ability to interact with specific receptors (7). Thus, a Smad2 mutant containing the two Smad1-specific residues in the L3 loop [Smad2(L1) construct] is preferentially activated by BMPR-I (7) (Fig. 5A). Using this approach, we determined that ALK1(QD) and ALK2(QD) are relatively weak inducers of nuclear accumulation of Smad2(L1) even though they induce nuclear accumulation of Smad1 (Fig. 5A). These results indicate that the L3 sequence alone is not sufficient to specify Smad1 recognition by ALK1 or ALK2.


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Fig. 4.   A, alpha -helix 1 and L3 sequences of receptor-regulated Smad proteins. Structural elements (arrows, beta -strands; rounded boxes, alpha -helices) correspond to the Smad4 MH2 domain (40). Subtype-specific residues are boxed. The corresponding sequences of Smad4 is also shown. Numbering of the last residue in each sequence corresponds to the Smad species not in the parentheses. B, a close-up view of the Smad4 MH2 structure showing the L3 loop (yellow) with subtype-specific residues (red) and the alpha -helix 1 (purple) with subtype-specific residues (green). The insert shows a frontal view of the location of the L3 loop and helix 1 of each MH2 monomer in the crystallographic trimer.


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Fig. 5.   A, activation of mutant Smad proteins as measured by nuclear accumulation in HepG2 cells. Flag-tagged Smad constructs were cotransfected with the active forms of receptors as indicated, and cells were analyzed for Flag immunostaining. 200-300 fluorescence-positive cells were scored per condition. B, phosphorylation of Smad proteins was examined in HepG2 cells by transient transfection with N-terminally Flag-tagged wild type and mutant Smad2 constructs and the active forms of receptors. 40 h later, cells were metabolically labeled with [32P]orthophosphate. Anti-Flag immunoprecipitates were analyzed by SDS-polyacrylamide gel electrophoresis and autoradiography. Protein expression was verified in parallel by anti-Flag western immunoblotting. B, BMPR-IB(QD); A1, ALK1(QD); A2, ALK2(QD). The number underneath the phosphorylation gel indicates the relative band intensity quantitated by a PhosphorImager.

We searched Smad MH2 domains for other regions containing subtype-specific residues, that is, residues that would be different between Smad1 and -2 but conserved between Smad1, -5, -8, and Mad or between Smad2 and -3. The region immediately preceding the C-terminal phosphorylation sites contains four such residues (7). However, introduction of these mutations into Smad2, along with the L3 loop mutations, did not improve the ability of ALK1(QD) or ALK2(QD) to induce nuclear accumulation of the resulting construct (Fig. 5A, Smad2(LC1) construct). Another region with subtype-specific residues is a sequence corresponding to alpha -helix 1, which is a surface structure in Smad4 (40). The corresponding sequence is conserved between Smad1 and Smad2 except for two residues that are subtype-specific (Fig. 4A). These residues in Smad4 (Glu-374 and Arg-378) are separated by one turn of the alpha -helix and are exposed to solvent in the vicinity of the L3 loop (40) (Fig. 4B). Because of their properties and location, we investigated whether these residues play a role as determinants of the interaction of Smad1 and ALK1 and -2. A Smad2 mutant containing these two residues from Smad1 [Smad2(H1-1)] did not respond to BMPR-IB(QD), ALK1(QD) or ALK2(QD) in the nuclear accumulation assay (Fig. 5A). However, a Smad2 construct containing the two alpha -helix 1 residues and the two L3 loop residues from Smad1 [Smad2(HL1)] underwent a similar extent of nuclear accumulation when cotransfected with ALK1(QD), ALK2(QD) or BMPR-IB(QD) (Fig. 5A). These results suggest a critical role of alpha -helix 1 in Smad1 recognition by ALK1 and ALK2.

To verify that both alpha -helix 1 and the L3 loop are required for Smad1 recognition by ALK1 and 2, wild type and mutant Smad2 constructs were transfected into HepG2 cells with the constitutively active forms of receptors, and phosphorylation of Smad2 was determined. Consistent with the pattern of nuclear accumulation, coexpression of BMPR-IB(QD) increased the phosphorylation level of Smad2(L1) and Smad2(HL1), but not wild type Smad2, whereas coexpression of ALK1(QD) and ALK2(QD) only enhanced phosphorylation of Smad2(HL1) (Fig. 5B).

From these results, we conclude that two groups of Smad1-activating receptors coexist in vertebrates and possibly Drosophila, the BMPR-I group and the ALK1 group. A similar conclusion was recently reached by others using a different BMP reporter assay (41). Furthermore, the present work shows that the recognition and activation of Smad1 by ALK1 and ALK2 is specified by the L45 loop on the receptors and the L3 loop together with the alpha -helix 1 on Smad1 (Fig. 6). It is likely that Saxophone recognizes Mad in a similar fashion because the L45 sequences of Saxophone and the L3 sequence and alpha -helix 1 sequence of Mad are very similar or identical to those of their counterparts in vertebrates. It is important to note that the structural elements identified here and previously (7, 8), although playing a major role in dictating the specificity of the recognition between type I receptors and Smad proteins, may be only part of larger regions mediating the association between these proteins. Additionally, the correct assembly of receptor complexes and Smad protein complexes and/or the possible intervention of "adaptor" molecules might be important for the association of receptors and Smad proteins.


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Fig. 6.   Structural motifs in the MH2 domains of Smad1 and Smad2 that determine the specificity of interactions with representative members of three groups of type I receptors. Smad1 recognition by ALK1 and ALK2 requires a structure, the alpha -helix 1, which is not essential for Smad1 interaction with BMPR-I (8). Smad proteins are represented as homotrimers based on the Smad4 MH2 structure (40). See text for additional details.

The BMPR-I group and the ALK1 group recognize Smad1 through related but different mechanisms which involve different L45 sequences on the receptor kinase domain, and a differential use of two surface structures on the Smad1 MH2 domain. Smad1 recognition and activation by these two mechanisms might be qualitatively different in ways that cannot be discerned by the present assays. In a given cellular context, such differences might make one receptor more suitable than the other as an activator of the Smad1 pathway, or both receptors could activate Smad1 in complementary ways or with different response outcomes.

    ACKNOWLEDGEMENTS

We thank Yigong Shi and Nikola Pavletich for insightful discussions and for the structure image. We thank Christof Niehrs for XVent2-Luc, the Genetics Institute for generously providing BMP, and Roger S. Lo for critically reading the manuscript.

    FOOTNOTES

* This work was supported by National Institutes of Health Grant CA34610 (to J. M.) and a Cancer Center grant to the Memorial Sloan-Kettering Cancer Center.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Research Associate of the Howard Hughes Medical Institute.

§ Investigator of the Howard Hughes Medical Institute. To whom correspondence should be addressed: Box 116, Memorial Sloan-Kettering Cancer Ctr., 1275 York Ave., New York, NY 10021. Tel.: 212-639-8975; Fax: 212-717-3298; E-mail: j-massague{at}ski.mskcc.org.

The abbreviations used are: TGF-beta , transforming growth factor beta ; BMP, bone morphogenetic protein.

2 F. Liu and J. Massagué, unpublished work on A3-Luc.

3 M. Kretzschmar, J. Doody, and J. Massagué, unpublished work.

    REFERENCES
Top
Abstract
Introduction
References

  1. Attisano, L., and Wrana, J. L. (1998) Curr. Opin. Cell Biol. 10, 188-194[CrossRef][Medline] [Order article via Infotrieve]
  2. Heldin, C.-H., Miyazono, K., and ten Dijke, P. (1997) Nature 390, 465-471[CrossRef][Medline] [Order article via Infotrieve]
  3. Massagué, J. (1998) Annu. Rev. Biochem. 67, 753-791[CrossRef][Medline] [Order article via Infotrieve]
  4. Das, P., Maduzia, L., Wang, H., Finelli, A., Cho, S. H., Smith, M., and Padgett, R. (1998) Development 125, 1519-1528[Abstract/Free Full Text]
  5. Hudson, J., Podos, S., Keith, K., Simpson, S., and Fergusson, E. (1998) Development 125, 1407-1420[Abstract/Free Full Text]
  6. Wisotzkey, R., Mehra, A., Sutherland, D., Dobens, L., Liu, X., Dohrmann, C., Attisano, L., and Raftery, L. (1998) Development 125, 1433-1445[Abstract/Free Full Text]
  7. Lo, R. S., Chen, Y. G., Shi, Y. G., Pavletich, N., and Massagué, J. (1998) EMBO J. 17, 996-1005[Abstract/Free Full Text]
  8. Chen, Y. G., Hata, A., Lo, R. S., Wotton, D., Shi, Y., Pavletich, N., and Massagué, J. (1998) Genes Dev. 12, 2144-2152[Abstract/Free Full Text]
  9. Brummel, T., Twombly, V., Marques, G., Wrana, J. L., Newfeld, S., Attisano, L., Massagué, J., O'Connor, M. B., and Gelbart, W. M. (1994) Cell 78, 251-261[Medline] [Order article via Infotrieve]
  10. Nellen, D., Affolter, M., and Basler, K. (1994) Cell 78, 225-237[Medline] [Order article via Infotrieve]
  11. Penton, A., Chen, Y., Staehling-Hampton, K., Wrana, J. L., Attisano, L., Szidonya, J., Cassill, J. A., Massagué, J., and Hoffmann, F. M. (1994) Cell 78, 239-250[Medline] [Order article via Infotrieve]
  12. Xie, T., Finelli, A. L., and Padgett, R. W. (1994) Science 263, 1756-1759[Medline] [Order article via Infotrieve]
  13. Attisano, L., Cárcamo, J., Ventura, F., Weis, F. M. B., Massagué, J., and Wrana, J. L. (1993) Cell 75, 671-680[Medline] [Order article via Infotrieve]
  14. Johnson, D. W., Berg, J. N., Baldwin, M. A., Gallione, C. J., Marondel, I., Yoon, S. J., Stenzel, T. T., Speer, M., Pericak-Vance, M. A., Diamond, A., Guttmacher, A. E., Jackson, C. E., Attisano, L., Kucherlapati, R., Porteous, M. E., and Marchuk, D. A. (1996) Nat. Genet. 13, 189-195[Medline] [Order article via Infotrieve]
  15. Ebner, R., Chen, R.-H., Lawler, S., Zioncheck, T., and Derynck, R. (1993) Science 262, 900-902[Medline] [Order article via Infotrieve]
  16. ten Dijke, P., Yamashita, H., Ichijo, H., Franzén, P., Laiho, M., Miyazono, K., and Heldin, C.-H. (1994) Science 264, 101-104[Medline] [Order article via Infotrieve]
  17. ten Dijke, P., Yamashita, H., Sampath, T. K., Reddi, A. H., Estevez, M., Riddle, D. L., Ichijo, H., Heldin, C.-H., and Miyazono, K. (1994) J. Biol. Chem. 269, 16985-16988[Abstract/Free Full Text]
  18. Liu, F., Ventura, F., Doody, J., and Massagué, J. (1995) Mol. Cell. Biol. 15, 3479-3486[Abstract]
  19. Rosenzweig, B. L., Imamura, T., Okadome, T., Cox, G. N., Yamashita, H., ten Dijke, P., Heldin, C.-H., and Miyazono, K. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 7632-7636[Abstract]
  20. Cárcamo, J., Weis, F. M. B., Ventura, F., Wieser, R., Wrana, J. L., Attisano, L., and Massagué, J. (1994) Mol. Cell. Biol. 14, 3810-3821[Abstract]
  21. Armes, N. A., and Smith, J. C. (1997) Development 124, 3797-3804[Abstract/Free Full Text]
  22. Graff, J. M., Bansal, A., and Melton, D. A. (1996) Cell 85, 479-487[Medline] [Order article via Infotrieve]
  23. Suzuki, A., Chang, C., Yingling, J. M., Wang, X. F., and Hemmati-Brivanlou, A. (1997) Dev. Biol. 184, 402-405[CrossRef][Medline] [Order article via Infotrieve]
  24. Chen, Y., Bhushan, A., and Vale, W. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 12938-12943[Abstract/Free Full Text]
  25. Chen, Y. G., Liu, F., and Massagué, J. (1997) EMBO J. 16, 3866-3876[Abstract/Free Full Text]
  26. Chen, X., Rubock, M. J., and Whitman, M. (1996) Nature 383, 691-696[CrossRef][Medline] [Order article via Infotrieve]
  27. Candia, A. F., Watabe, T., Hawley, S. H., Onichtchouck, D., Zhang, Y., Derynck, R., Niehrs, C., and Cho, K. W. (1997) Development 124, 4467-4480[Abstract/Free Full Text]
  28. Wieser, R., Wrana, J. L., and Massagué, J. (1995) EMBO J. 14, 2199-2208[Abstract]
  29. Attisano, L., Wrana, J. L., Montalvo, E., and Massagué, J. (1996) Mol. Cell. Biol. 16, 1066-1073[Abstract]
  30. Hoodless, P. A., Haerry, T., Abdollah, S., Stapleton, M., O'Connor, M. B., Attisano, L., and Wrana, J. L. (1996) Cell 85, 489-500[Medline] [Order article via Infotrieve]
  31. Macias-Silva, M., Abdollah, S., Hoodless, P. A., Pirone, R., Attisano, L., and Wrana, J. L. (1996) Cell 87, 1215-1224[Medline] [Order article via Infotrieve]
  32. Liu, F., Pouponnot, C., and Massagué, J. (1997) Genes Dev. 11, 3157-3167[Abstract/Free Full Text]
  33. Nakao, A., Imamura, T., Souchelnytskyi, S., Kawabata, M., Ishisaki, A., Oeda, E., Tamaki, K., Hanai, J., Heldin, C. H., Miyazono, K., and ten Dijke, P. (1997) EMBO J. 16, 5353-5362[Abstract/Free Full Text]
  34. Zhang, Y., Feng, X.-H., Wu, R.-Y., and Derynck, R. (1996) Nature 383, 168-172[CrossRef][Medline] [Order article via Infotrieve]
  35. Liu, X., Sun, Y., Constantinescu, S. N., Karam, E., Weinberg, R. A., and Lodish, H. F. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 10669-10674[Abstract/Free Full Text]
  36. Chen, Y., Lebrun, J. J., and Vale, W. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 12992-12997[Abstract/Free Full Text]
  37. Kretzschmar, M., Doody, J., and Massagué, J. (1997) Nature 389, 618-622[CrossRef][Medline] [Order article via Infotrieve]
  38. Kretzschmar, M., Liu, F., Hata, A., Doody, J., and Massagué, J. (1997) Genes Dev. 11, 984-995[Abstract]
  39. Feng, X. H., and Derynck, R. (1997) EMBO J. 16, 3912-3922[Abstract/Free Full Text]
  40. Shi, Y., Hata, A., Lo, R. S., Massagué, J., and Pavletich, N. P. (1997) Nature 388, 87-93[CrossRef][Medline] [Order article via Infotrieve]
  41. Macías-Silva, M., Hoodless, P. A., Tang, S. J., Buchwald, M., and Wrana, J. L. (1998) J. Biol. Chem. 273, 25628-25636[Abstract/Free Full Text]


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