Characterization of Functional Domains within Smad4/DPC4*

(Received for publication, January 16, 1997, and in revised form, March 21, 1997)

Mark P. de Caestecker Dagger , Philip Hemmati , Sarit Larisch-Bloch , Ravi Ajmera , Anita B. Roberts and Robert J. Lechleider

From the Laboratory of Chemoprevention, NCI, National Institutes of Health, Bethesda, Maryland 20892-5055

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Smad proteins are a family of highly conserved, intracellular proteins that signal cellular responses downstream of transforming growth factor-beta (TGF-beta ) family serine/threonine kinase receptors. One of these molecules, Smad4, originally identified as the candidate tumor suppressor gene dpc-4, reconstitutes TGF-beta - and activin-dependent transcriptional responses in Smad4 null cell lines and interacts in a ligand-dependent manner with other Smad family members in both TGF-beta , activin, and bone morphogenetic protein-2/-4 pathways. Here, we used an assay based on the restoration of ligand-dependent transcriptional responses in a Smad4 null cell line to characterize functional domain structures within Smad4. We showed that restoration of TGF-beta -induced transcriptional responses by Smad4 was inhibited by co-transfection with a kinase dead TGF-beta type II receptor and that constitutive activation was blocked with TGF-beta neutralizing antibodies, confirming the essential role of Smad4 in TGF-beta signaling. Using a series of Smad4 mutation, deletion, and Smad1/Smad4 chimera constructs we identified a 47-amino acid deletion within the middle-linker region of Smad4 that is essential for the mediation of signaling responses. In addition, we showed that the NH2-terminal domain of Smad4 augments ligand-dependent activation associated with the middle-linker region, indicating that there is a distinct ligand-response domain within the N terminus of this molecule.


INTRODUCTION

TGF-beta 1 is the prototype for a family of structurally related proteins that mediate a diverse array of biological functions in organisms as disparate as the fruit fly and man. Cloning and characterization of TGF-beta family receptors revealed that these ligands signal through two interacting transmembrane serine/threonine kinase receptors (1). While it is recognized that the ligand binds the type II receptor, which then recruits and transphosphorylates the type I receptor to signal downstream pathways (2), the mechanisms involved in mediating signaling cascades downstream of the type I receptor were, until recently, largely unknown.

Clues from Drosophila genetics have been applied to identify a number of novel mammalian proteins involved in signaling downstream of TGF-beta family members (1). In Drosophila, decapentaplegic (dpp) encodes a TGF-beta superfamily ligand that is essential for dorsal-ventral patterning in developing fruit flies (3). Genetic screening for mutants acting in this pathway identified a novel gene, Mothers against dpp (mad), which encodes an essential cytoplasmic element in the dpp signal transduction pathway (4, 5). Searches for Mad-related proteins in other organisms have revealed an array of conserved homologues in species from Caenorhabditis elegans to man (1). Characteristically these proteins contain highly conserved NH2- and COOH-terminal domains separated by a proline-rich linker and lack any known enzymatic or functional sequence motifs. Five mammalian Mad-related proteins, Smad1 through 5, have been characterized and shown to participate in signaling responses induced by TGF-beta , activin, and BMP2/4.

The rudimentary mechanisms of action of the Smad family proteins are now beginning to be understood. For example, Smad1 is phosphorylated and undergoes nuclear translocation following treatment with BMP2, BMP4 (6, 7), and possibly TGF-beta (8, 9), while Smad2 is phosphorylated and undergoes nuclear translocation in response to TGF-beta and activin (10, 11). This phosphorylation event occurs in association with the activated TGF-beta receptors and results in dissociation of Smad2 from the receptor complex and translocation to the nucleus (11). Recent studies using Xenopus embryos indicate that Smad2 forms a transcriptional complex with the winged-helix transcription factor FAST1 when co-injected with activin mRNA (12), providing a downstream mechanism whereby activin response elements may be activated following ligand-induced phosphorylation of Smad2. Interestingly, Smad3 has also been shown to signal in the TGF-beta pathway, to associate with the activated TGF-beta receptor complex, and to be phosphorylated following activation (13), suggesting that receptor-mediated phosphorylation and subsequent nuclear translocation may represent a more generalized phenomenon. Furthermore, Smad1 displays transcriptional activity in response to BMP4 when fused to a heterologous Gal4 DNA-binding domain (7), indicating that other members of Smad family may act as transcriptional activators once translocated to the nucleus.

Smad4 is a Mad-related protein independently identified as the candidate tumor suppressor gene dpc4, deleted or mutated in a proportion of pancreatic (14), breast, ovarian (15), and colorectal tumors (16), with tumor suppressor activity attributable to its participation in TGF-beta receptor-mediated signaling (13, 17). However, while this molecule shares broad sequence similarity with the other Smad family members, it displays significant variance in all three molecular domains (1), suggesting that it may have distinct functional characteristics. For example, while Smad4 has been clearly implicated in both TGF-beta and activin signaling pathways (13, 17), it is phosphorylated only in response to activin (17) and does not associate with activated TGF-beta receptor complexes (11, 13). Smad4 also lacks the COOH-terminal serine residues that are phosphorylated in Smad2 following activation of the TGF-beta receptor complex (11) and are present in all the other mammalian Smad family members. In addition, Smad4 not only forms ligand-dependent heterodimeric complexes with Smad2 following activation of TGF-beta or activin pathways, but also with Smad1 following activation through BMP2/4 receptors (17). These data further support a model in which Smad4 acts in a manner distinct from Smad1, -2, and -3, and is central to signaling pathways involving multiple TGF-beta family ligands. However, the mechanisms regulating ligand-dependent transmission of signaling responses by Smad4 remain largely unknown.

In this study, we employ an assay based on the restoration of ligand-dependent transcriptional responses in a Smad4 null cell line to characterize the functional domains within Smad4 that are essential for the mediation of TGF-beta -dependent signaling responses. Using a series of mutants, chimeras, and deletion constructs, we have identified a 47-amino acid region within the proline-rich middle linker domain of Smad4 which, when deleted, disrupts the ability of the wild-type protein to restore TGF-beta -dependent signal transduction in the Smad4 null cell line. We also show that the NH2-terminal domain of Smad4 enhances TGF-beta -dependent activation associated with the middle-linker region, indicating that there is separate ligand-response domain within the N terminus of this molecule.


MATERIALS AND METHODS

Construction of cDNA Plasmids

Mutant, chimeric, deletion, and COOH-terminal constructs were designed as shown in the Figs. 5, 6, and 8 based on the known sequence similarity domains (8), and cloned into the pCDNA3 expression vector. These were generated by polymerase chain reaction amplification and primer extension with proofreading polymerase, using Smad4 and Smad1 templates. Deletion constructs were sequenced using the dideoxynucleotide method.


Fig. 5. Functional analysis of the COOH-terminal domains of Smad1 and Smad4. A, a schematic of Smad1 and Smad4 truncations based on the known sequence homology domains of the Smad family of proteins. B, Smad C-terminal truncations do not restore signaling responses. MDA-MB468 cells were transfected with the p3TP-Lux reporter along with equal concentrations of the indicated constructs and assayed for luciferase activity after treatment with TGF-beta 1 for 24 h. The results are expressed as the mean (±S.D.) of duplicate luciferase assays, corrected for transfection efficiency, and standardized as fold changes relative to untreated vector alone. The experiment was repeated three times with similar results.
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Fig. 6. Mapping of the activation domain in Smad4. A, schematic of intra-domain Smad4 deletions. B, deletion of residues 274-331 in Smad4 prevents the restoration of TGF-beta -induced signaling responses. MDA-MB468 cells were transfected with the p3TP-Lux reporter along with equal concentrations of the indicated deletion constructs and assayed for luciferase activity after treatment with TGF-beta 1 for 24 h. The results are expressed as the mean (±S.D.) of duplicate luciferase assays, corrected for transfection efficiency, and standardized as fold changes relative to untreated vector alone. The experiment was repeated three times with similar results.
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Fig. 8. Characterization of ligand-responsive domain structures in Smad4. A, schematic of Smad1 and Smad4 domain swap chimeras. B, the Smad4 middle-linker domain restores transcription activity in MDA-MB 468 cells. Cells were transfected with the p3TP-Lux reporter along with equal concentrations of the indicated chimeric constructs and assayed for luciferase activity after treatment with TGF-beta 1 for 24 h. The results are expressed as the mean of duplicate luciferase assays, corrected for transfection efficiency, and standardized as fold changes relative to untreated vector alone. The experiment was repeated three times with similar results.
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Cell Lines and Proliferation Assay

MDA-MB468 is a cell line derived from a patient with breast carcinoma, which has a homozygous deletion of the complete Smad4 coding region (15). A549 is a lung carcinoma cell line with intact Smad4 genes. All cells were obtained from the American Type Culture Collection and maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum and antibiotics. Cellular proliferation was determined by [3H]thymidine incorporation as described previously (18), after treatment with or without 10 ng/ml recombinant human TGF-beta 1 over a 48-h period in medium containing 10% fetal bovine serum.

Receptor Cross-linking

Recombinant human TGF-beta 1 was labeled with 125Iodine using the chloramine-T method (19). Cells were seeded at 60% confluence in 100-mm plates and incubated for 4 h at 4 °C with 100 pM [125I] TGF-beta 1 with or without 100-fold excess of unlabeled TGF-beta 1 competitor. Cross-linking was performed with disuccinimidyl suberate as described previously (20), samples subjected to electrophoresis on a 4-12% gradient sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography.

Plasminogen Activator Inhibitor-1 (PAI-1) and Fibronectin Assays

Cells were seeded in six-well dishes, incubated for 18 h in medium containing 0.2% fetal bovine serum with or without 1 ng/ml TGF-beta 1, and labeled with 50 µCi/ml [35S]methionine over the last 6 h. After metabolic labeling, PAI-1 and fibronectin were extracted from the cell associated extracellular matrix and supernatants, respectively, as described previously (21). Both preparations were subjected to 10% SDS-polyacrylamide gel electrophoresis followed by autoradiography. PAI-1 and fibronectin were identified from their characteristic sizes (45 and 240 kDa) and inducibility by TGF-beta in a TGF-beta -responsive cell line.

Transcriptional Response Assays

MDA-MB468 cells were transiently transfected using the polyamine transfection reagent LT-2 (Pan Vera Co.). Cells were seeded to 50% confluence in six-well plates and incubated in serum-free medium with LT-2, cDNAs, reporter constructs, and empty vector to equalize amounts of DNA. After 6 h, cells were washed and allowed to recover for 24 h in growth medium. Cells were serum starved overnight prior to the addition of TGF-beta 1 for a further 24 h. Luciferase activity was determined in the cell lysate using an assay kit (Analytic Luminescence Laboratory), and a Dynatech Laboratories ML3000 luminometer. Activities were normalized on the basis of beta -galactosidase expression from pSV beta -galactosidase in all luciferase reporter experiments. All experiments were repeated at least three times with similar results.


RESULTS

Smad4 Is an Essential Signaling Intermediate in the TGF-beta Receptor-mediated Pathway

To develop a functional assay for Smad4, we used the MDA-MB468 breast carcinoma cell line that has a homozygous deletion of the complete Smad4 coding region (15). Northern blot analysis shows that these cells express Smad1, Smad2, Smad3, and Smad5 and confirms the absence of Smad4 mRNA expression.2 Receptor cross-linking shows that these cells express types I and II TGF-beta receptors (Fig. 1A), but show no growth inhibitory (Fig. 1B), PAI-1 or fibronectin induction responses to TGF-beta (Fig. 1C). Having established that Smad4 deficiency is associated with a lack of responsiveness to TGF-beta , we performed a series of experiments to characterize the role of this molecule in TGF-beta -dependent signaling responses. Transient transfection with Smad4 and the reporter constructs p3TP-Lux (which contains multiple TGF-beta response elements (21)) or p800-Luc (which contains only the PAI-1 gene promoter (22)), resulted in a clear activation of luciferase reporter activity in response to TGF-beta (Fig. 2A). Ligand-dependent activation of p800-Luc was lower than that of p3TP-Lux, although both were dependent on the dose of Smad4 DNA added to the transfection mix (Fig. 2A) and the concentration of exogenous TGF-beta used to treat the cells (Fig. 2B).


Fig. 1. Smad4 null cells are resistant to TGF-beta . A, TGF-beta receptor cross linking. Cells were incubated with 125I-TGF-beta 1 with or without unlabeled TGF-beta 1 competitor, cross-linking performed with disuccinimidyl suberate as described under "Materials and methods" and samples electrophoresed followed by autoradiography. Positions of types I, II, and III receptors (RI, RII, and RIII) and free TGF-beta are indicated on the left. B, MDA-MB 468 cells are resistant to growth inhibition by TGF-beta . Treatment with 10 ng/ml recombinant TGF-beta 1 over a 48-h period had no effect on [3H]thymidine uptake by subconfluent MDA-MB468 cells, but was significantly inhibited in A549 cells. Data are presented as the mean (±S.D.) [3H]thymidine incorporation in counts/min, measured in triplicate. C, fibronectin and PAI-1 assays. Cells were treated for 18 h with/or without 1 ng/ml TGF-beta 1 in medium containing 0.2% serum and labeled with [35S]methionine over the last 6 h. Fibronectin and PAI-1 were extracted and analyzed as described under "Materials and Methods." Only relevant portions of the autoradiographs are shown.
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Fig. 2. Smad4 restores TGF-beta responsiveness in Smad4 null cells. MDA-MB468 cells were transiently transfected with the indicated constructs along with pSV beta -galactosidase and TGF-beta -responsive reporter constructs. Following transfection, cells were allowed to recover for 24 h prior to the addition of TGF-beta 1 for an additional 24 h. Cell lysate luciferase activity was determined and activities corrected on the basis of beta -galactosidase expression in all luciferase reporter experiments. Results are expressed as the mean (± S.D.) of duplicate luciferase assays from a representative experiment. A, transfection with Smad4 restores transcriptional activation responses to TGF-beta . Cells were transfected with the indicated concentrations of Smad4 and/or empty vector, and two TGF-beta -responsive reporter constructs, p3TP-Lux (left panel) or p800-Luc (right panel). The results are standardized as fold changes relative to the untreated vector, and the experiment repeated three times with similar results. B, transfection with Smad4 restores dose-dependent transcriptional activation responses to TGF-beta . MDA-MB468 cells were transfected using 1 µg of Smad4 DNA/reaction with the indicated reporter constructs and treated with varying concentrations of TGF-beta 1 for 24 h. Results are shown from a representative experiment, repeated twice with similar results.
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To determine if these responses were TGF-beta receptor-dependent, we evaluated reporter gene activation after co-transfection of TGF-beta type I and type II receptors with and without Smad4 (Fig. 3A). Transfection of TGF-beta type I or type II receptors alone had no effect on the transcriptional response to TGF-beta , although co-transfection of Smad4 with the TGF-beta type II receptor caused an apparent ligand-independent activation of the p3TP-Lux reporter gene (Fig. 3A). Importantly, this effect was blocked almost entirely by treatment with TGF-beta neutralizing antibodies (Fig. 3B), indicating that the apparently ligand-independent signaling response seen in cells transfected with both the type II TGF-beta receptor and Smad4 is the result of stimulation by secreted TGF-beta . In addition, while transfection of the kinase inactive TGF-beta type II receptor alone had no effect on the transcriptional response to TGF-beta , co-transfection of the mutant receptor with Smad4 caused a nearly complete loss of ligand-induced signaling (Fig. 4), confirming the receptor specificity of the Smad4 induced response. Furthermore, basal transcriptional activation of the p3TP-Lux reporter with Smad4 was also inhibited by co-transfection with the kinase-inactive type II TGF-beta receptor, indicating that stimulation of endogenous receptors by autocrine or paracrine TGF-beta may account for some of the constitutive activation seen in the absence of additional exogenous ligand.


Fig. 3. Restoration of signaling by Smad4 is TGF-beta receptor dependent. MDA-MB468 cells were transiently transfected using the p3TP-Lux reporter and assayed for luciferase activity after treatment with TGF-beta 1 for 24 h. Results are expressed as the mean (±S.D.) of duplicate luciferase assays, corrected for transfection efficiency and standardized as fold changes relative to untreated vector alone. A, Smad4 signaling is dependent on the level of TGF-beta type II receptor expression. Cells were transfected with equal amounts of the indicated TGF-beta receptors with or without Smad4, and results shown from a representative experiment that was repeated three times with similar results. B, endogenous TGF-beta augments basal reporter gene activation after reconstituting Smad4 expression. Cells were co-transfected with TGF-beta type II receptor and Smad4 and treated for 36 h with 50 µg/ml of a murine monoclonal pan-specific TGF-beta neutralizing antibody (Genzyme Co.) or mouse IgG1 control prior to determining luciferase activity. Results are shown from a representative experiment that was repeated twice with similar results.
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Fig. 4. Dominant negative activity of a kinase inactive TGF-beta type II receptor on the restoration of signaling responses by Smad4. MDA-MB468 cells were transfected with the p3TP-Lux reporter and the indicated constructs prior to treatment with TGF-beta 1. Results are expressed as the mean (±S.D.) of duplicate luciferase assays, corrected for transfection efficiency, and standardized as fold changes relative to untreated vector alone. Data are shown from a representative experiment repeated three times.
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Functional Analysis of COOH-terminal Domains of Smad1 and Smad4

Studies on Smad1 and Smad2 have identified mutations within the COOH-terminal domains which block ligand-dependent phosphorylation (6, 10) and inhibit dorsal mesoderm induction by Smad2 in Xenopus (10). Furthermore missense mutations of a highly conserved glycine residue in the COOH-terminal domain of Drosophila Mad, and C. elegans Sma2, generate null phenotypes in both species (4, 23), indicating that the functional activity of these molecules may be disrupted by specific point mutations within this domain. The corresponding glycine to serine substitution at position 508 in Smad4 (Fig. 5A) did not abolish ligand-dependent augmentation of reporter gene activation, although it significantly reduced the maximal luciferase induction compared with the wild-type protein (Fig. 5B). This suggests that the biological inactivation resulting from mutation of this residue in other Mad-related proteins (4, 10, 23) may result from a decrease in specific transcriptional responses below critical levels rather than a total loss of function. Alternatively, other Mad family members may be completely inactivated by this mutation, while the Smad4 G508S mutation retains some signaling activity.

The COOH-terminal domains of Smad1 (234-465) and Smad4 (266-552) display constitutive transcriptional activity when fused to a heterologous Gal4 DNA-binding domain, while the full-length Smad1 fusion protein demonstrates ligand-dependent transcriptional activation (7). In addition, Lagna et al. (17) have shown a constitutive, ligand-independent activation of p3TP-Lux reporter activity when MDA-MB468 Smad4 null cells are transfected with the 266-552 COOH-terminal Smad4 construct. In our study, however, neither the Smad1 (270-465) nor the Smad4 (322-552) COOH-terminal domain constructs (Fig. 5A) induced constitutive or ligand-dependent transcriptional activation of the reporter gene (Fig. 5B). This indicates that there may be an activation domain between amino acids 266-321 in Smad4, which is involved in the regulation of signaling responses by this protein.

Mapping of a Smad4 Activation Domain (SAD) within the Middle-linker Region of Smad4

To define the activation domain within the middle-linker region of Smad4 further, we created a series of amino acid deletions within this region (Fig. 6A). When these constructs were transfected into MDA-MB468 cells, the Delta M1 (136-181) and Delta M2 (182-227) deletions had similar functional activity to the full-length molecule in restoring TGF-beta -dependent activation of the p3TP-Lux reporter. In contrast, deletion of amino acids 228-273 in Delta M3 substantially reduced this response, while the Delta M4 (274-321) deletion had virtually no signaling activity (Fig. 6b).

Characterization of a Ligand-response Domain in Smad4

Smad1 alone does not restore reporter gene activation in MDA-MB468 cells, nor does it augment Smad4-dependent activity (Fig. 5B; Fig. 7). For this reason, we were able to extend our studies to identify regions of Smad4 that confer ligand-specific responses, by generating a panel of domain-swap chimeras between Smad4 and Smad1 (Fig. 8A). Like the Smad4 G508S mutant, none of the chimeras reconstituted the degree of luciferase expression seen with intact Smad4 (Fig. 8B), but the presence of a Smad4 proline-rich linker domain was associated with reproducible ligand-responsive reporter gene activation, regardless of the amino- or carboxyl-terminal domain structures. Furthermore, while the N-terminal domain of Smad4 had no effect on ligand-dependent transcriptional activity in the 4.1.1 and 4.1.4 chimeras, ligand-dependent activation was dramatically increased when the Smad4 NH2 terminus was associated with the Smad4 middle-linker domain in the 4.4.1 chimera, bringing the level of ligand-dependent activation to within 36 (±7)% of the full-length Smad4 (results from three separate experiments expressed as mean (±S.D.)). This is comparable with the ligand-dependent transcriptional activation seen with the G508S mutation of Smad4 (Fig. 5B), suggesting that this mutation may be functioning like the 4.4.1 chimera, with incomplete activation of response elements in Smad4. These data indicate that there is a ligand-response domain within the N terminus of Smad4 and that this region requires the presence of a Smad4 middle-linker domain to produce ligand-dependent transcriptional activation responses to TGF-beta .


Fig. 7. Smad1 does not interfere with TGF-beta responses in Smad4 null cells. MDA-MB468 cells were transfected with the p3TP-Lux reporter along with equal concentrations of the indicated Smad1 and Smad4 constructs and assayed for luciferase activity after treatment with TGF-beta 1 for 24 h. The results are expressed as the mean of duplicate luciferase assays, corrected for transfection efficiency, and standardized as fold changes relative to untreated vector alone. The experiment was repeated three times with similar results.
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DISCUSSION

Smad4/DPC4 is a member of the Smad family of intracellular signaling proteins and appears to be central to signaling pathways involving multiple TGF-beta family ligands (17). Despite this, the mechanisms regulating the transmission of signaling responses by this molecule are largely unknown. In these studies, we characterize separate activation and ligand-response domains within Smad4 using a functional assay based on the restoration of TGF-beta responsiveness in a Smad4 null cell line by transient transfection with Smad4.

We have shown that restoration of cellular responses to TGF-beta by Smad4 in MDA-MB468 cells is dependent on TGF-beta receptor function, as reporter gene activation is inhibited by co-transfection with a kinase-inactive TGF-beta type II receptor mutant shown previously to exert a dominant negative effect on TGF-beta -induced transcriptional responses in other systems (24). Furthermore, augmentation of basal reporter gene activation seen following co-transfection of Smad4 and the wild-type TGF-beta type II receptor, and its reversal following incubation of the transfected cells with TGF-beta neutralizing antibodies, indicates that restoration of cellular responses following transfection with Smad4 is dependent both on the level of receptor expression and the degree of autocrine or paracrine stimulation of this pathway resulting from the secretion of TGF-beta into the culture medium. In the same manner, basal reporter gene activation following transfection with Smad4 alone could be inhibited by co-transfection with a kinase inactive TGF-beta type II receptor, indicating that stimulation of endogenous receptors by secreted TGF-beta could account for some of the variability in basal activation of this system. This also has implications for other assay systems. For example, the apparently ligand-independent activation of a PAI-1 reporter construct seen following co-transfection of Smad3 and Smad4 into Smad4 null SW480 cells (13) could result from the effects of endogenous TGF-beta on this cell line. Indeed these authors showed that constitutive reporter gene activation associated with transfection of Smad3 and Smad4 was reduced when their cells were co-transfected with a dominant negative TGF-beta type II receptor construct, indicating that the observed basal reporter gene activation is likely to have resulted from stimulation of the TGF-beta receptor signaling pathway by endogenous TGF-beta .

Having characterized this assay for Smad4, we have used it to identify functional domains within the Smad4 molecule that are involved in regulating TGF-beta -induced transcriptional responses. Comparison of our initial studies, demonstrating that a 322-552 COOH-terminal Smad4 construct had no effect on constitutive or ligand-dependent activation of the p3TP-Lux reporter gene (Fig. 5), with those of Lagna et al. (17) demonstrating constitutive p3TP-Lux activation in the same cell line following transfection with a 266-552 Smad4 construct (17), indicates that there may be critical residues within the intervening amino acids 266-321 in Smad4 that are required to activate signaling responses by this protein. We have delineated this region further with the demonstration that deletion of amino acid residues 274-321 in Smad4 prevents the restoration of TGF-beta responsiveness by this molecule in our assay system (Fig. 6). While it is possible that this is an indirect effect resulting from conformational disruption of other domain structures within Smad4, taken together with data derived using the Smad4 COOH-terminal constructs, these results suggest that there is an SAD within the COOH-terminal portion of the middle-linker region of Smad4 that is necessary for this molecule to transduce signaling responses downstream from TGF-beta receptors. This domain of Smad4 shows minimal sequence similarity with the other Smad family members, and it is of interest that a COOH-terminal Smad1 construct 234-465, corresponding to residues 270-552 in Smad4, does not restore constitutive activation of the p3TP-Lux reporter in this assay system (17), indicating that the localization of the SAD between residues 274 and 322 in Smad4 may be unique to this particular Smad family member.

Transfection with the full-length Smad4 induces a much lower basal activation of the p3TP-Lux reporter gene than the 266-552 construct (17). This suggests that the signaling response associated the SAD is reversibly inhibited by either direct or indirect interaction with structures upstream of residue 266 in the full-length Smad4. Interestingly, Baker and Harland (25) describe a constitutively active NH2-terminal truncation of Smad2 184-467, corresponding to residues 160-552 in Smad4, that may result from the deletion of a similar inhibitory component in the NH2 terminus of this molecule. Our data indicate that this type of inhibitory domain is also located within the NH2 terminus of Smad4, as none of the segmental deletions upstream of the SAD and within the middle-linker domain of Smad4 Delta M1, Delta M2, and Delta M3 (137-273) were constitutively active in our assay system (Fig. 6). On this basis, ligand-activated signal transduction by Smad4 may result from dissociation of intra- or extramolecular inhibitory structures from the SAD, themselves under the regulation of a ligand-responsive region within the NH2 terminus of Smad4.

Experiments using the Smad1/Smad4 domain swap chimeras further characterize the activation and ligand-responsive domains in Smad4. Thus, while the proline-rich linker domain of Smad4 is essential for the transduction of TGF-beta signaling responses by the Smad1/Smad4 chimeras, results with the 1.4.4 and 1.4.1 chimeras show that the activity of this region is only weakly enhanced by the NH2-terminal domain of Smad1. In contrast, TGF-beta responses are significantly enhanced when the Smad1 NH2 terminus is swapped for the corresponding NH2-terminal domain of Smad4 in the 4.4.1 chimera (Fig. 8). This indicates that there is a ligand-response domain located within the NH2 terminus of Smad4 and that the ligand-dependent regulatory activity of this domain requires the presence of the Smad4 middle-linker region.

Taken together, these data suggest a model for Smad4 functional domains that is reminiscent of that of other signaling molecules. In the absence of ligand, the NH2 terminus and/or middle-linker domains of Smad4 may obscure the SAD located at the extreme COOH terminus of the middle-linker domain, either by direct blockade of this region or by conformational interference. Following ligand-activation, there may be unfolding of the molecular structure resulting in exposure of the SAD, allowing it to interact with other signaling partners. This event is not associated with phosphorylation of Smad4 (17), but may result from intermolecular conformational interference with NH2-terminal domain structures following association between Smad4 and other, activated Smad family members (17). Identification of specific activation domains within the N terminus and how they interact with the rest of the molecule will help elucidate this mechanism.


FOOTNOTES

*   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    Recipient of a Welcome Trust Advanced Training Fellowship from the United Kingdom. To whom correspondence should be addressed: Laboratory of Chemoprevention, NCI, Bldg. 41, Rm. C629, 41 Library Dr., MSC 5055, Bethesda, MD 20892-5055. Tel.: 301-495-2012; Fax: 301-496-8395; E-mail: decaestm{at}dce41.nci.nih.gov.
1   The abbreviations used are: TGF-beta , transforming growth factor-beta ; BMP, bone morphogenetic protein; PAI-1, plasminogen activator inhibitor-1; SAD, Smad4 activation domain.
2   R. J. Lechleider, unpublished data.

ACKNOWLEDGEMENTS

We thank Scott Kern for advice regarding dpc4 null cell lines and other members of the Laboratory of Chemoprevention for their helpful advice and criticisms. Jeff Wrana and Lilliana Attisano generously provided the TGF-beta receptor and p3TP-Lux constructs. The Smad4 expression construct was a kind gift from Seong-Jin Kim, and p800-Luc was provided by D. Luskutoff. Catherine Bonham and China Eng provided technical assistance. We also thank Drs. Shin-Geon Choi, Young-Suk Yi, and Yong-Seok Kim for assistance with the preparation of synthetic oligonucleotides


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