Identification and Characterization of Constitutively Active Smad2 Mutants: Evaluation of Formation of Smad Complex and Subcellular Distribution

Masayuki Funaba and Lawrence S. Mathews

Department of Biological Chemistry University of Michigan Ann Arbor, Michigan 48109-0606


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Smads mediate activin, transforming growth factor ß (TGFß), and bone morphogenetic protein signaling from receptors to nuclei. According to the current model, activated activin/TGFß receptors phosphorylate the carboxyl-terminal serines of Smad2 and Smad3 (SSMS-COOH); phosphorylated Smad2/3 oligomerizes with Smad4, translocates to the nucleus, and modulates transcription of defined genes. To test key features of this model in detail, we explored the construction of constitutively active Smad2 mutants. To mimic phosphorylated Smad2, we made two Smad2 mutants with acidic amino acid substitutions of carboxyl-terminal serines: Smad2–2E (Ser465, 467Glu) and Smad2–3E (Ser464, 465, 467Glu). The mutants enhanced basal transcriptional activity in a mink lung epithelial cell line, L17. In a Smad4-deficient cell line, SW480.7, Smad2–2E did not affect basal signaling; however, cotransfection with full-length Smad4, but not transfection of Smad4 alone, resulted in enhanced basal transcriptional activity, suggesting that the constitutively active Smad2 mutant also requires Smad4 for function. In vitro protein interaction analysis revealed that Smad2–2E bound more tightly to Smad4 than did wild-type Smad2; dissociation constants were 270 ± 66 nM for wild-type Smad2:Smad4 complexes and 79 ± 18 nM for Smad2–2E:Smad4 complexes. Determination of the subcellular localization of Smad2 revealed that a greater percentage of Smad2–2E was localized in the nucleus than wild-type Smad2. These results suggest that Smad2 phosphorylation results in both tighter binding to Smad4 and increased nuclear concentration; those changes may be responsible for transcriptional activation by Smad2.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Members of the transforming growth factor ß (TGFß) family, including activins, TGFßs, and bone morphogenetic proteins (BMPs), are protein growth and differentiation factors that manifest an extraordinary variety of biological activities (1). These proteins signal through the sequential activation of two cell surface receptors, termed type I and type II, both of which are protein serine-threonine kinases (2, 3). Extensive efforts to elucidate the downstream signaling mechanisms led to the discovery of a series of Smad proteins and trials for clarification of Smad signaling mechanisms.

Smads are categorized into three subclasses: receptor-regulated Smads, which include Smad2 and 3 (activin/TGFß receptor regulated) and Smad1, 5, and 8 (BMP receptor regulated); the common-partner Smad (Smad4); and the inhibitory Smads (Smad6 and 7) (2, 3, 4). Receptor complexes activated by appropriate ligands phosphorylate conserved serine residues at the carboxyl terminus of receptor-regulated Smads (5, 6, 7), which promotes association with a common partner Smad, Smad4 (8, 9, 10). The complexes of receptor-regulated Smad and Smad4 translocate into the nucleus and participate in the activation of target gene transcription either by associating with other transcription factors or by direct DNA binding (2–4, 11).

Recently, on the basis of this phosphorylationrelated Smad activation model, detailed studies on Smad signaling mechanism have been conducted. As for receptor-regulated phosphorylation, SARA (for Smad anchor for receptor activation), identified as a Smad2 binding protein, was suggested to facilitate Smad phosphorylation by localization of unphosphorylated Smad in proximity to the activated receptor complexes (12). In addition, several nuclear proteins, including p300, the AP-1 complex, TFE3, vitamin D receptor, Evi-1, c-Ski, and SnoN, have been found to interact with Smads and influence Smad function (13, 14, 15, 16, 17, 18, 19, 20, 21).

The model for phosphorylation-related Smad activation and function is broadly accepted (2, 3, 4), but significant points remain to be verified; for example, it is unclear whether phosphorylation directly influences Smad2 binding to Smad4, or subsequent signaling events. In addition, the current model derives primarily from qualitative data. To explore the quantitative, biochemical basis for this model, in this study we assessed the mechanistic implications of Smad2 phosphorylation. Because it is not currently possible to generate large amounts of stoichiometrically phosphorylated, purified Smad2, we demonstrated that replacement of the phosphorylated serines with acidic amino acids yielded a constitutively active form of Smad2. We have used that mutant to investigate quantitatively the biochemical function of Smad2 phosphorylation.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Identification of Constitutively Active Mutants of Smad2
To identify constitutively active Smad2 mutants, we replaced the carboxyl-terminal serines of Smad2 (SSMS-COOH) with glutamic acid to mimic phosphorylation. These serines were previously identified as ligand-induced phosphorylation sites (5, 6, 7); in particular, the last two serine residues of Smad2 were phosphorylated in response to TGFß stimulation (22, 23). To evaluate transcriptional activities of Smad2–2E (Ser465, 467Glu) and -3E (Ser464, 465, 467Glu) in L17 cells, we used three different luciferase-based reporter genes: 3TP-Lux, which contains regulatory elements from the PAI-1 promoter (24); AR3-Lux, which contains regulatory sequences from the Xenopus Mix.2 gene, and which requires coexpression of the transcription factor FAST1 for ligand induction (25); and SBE4-Lux, which contains optimized Smad-binding regulatory sequences (26).

Neither wild-type Smad2 (WT-Smad2) nor the mutants affected basal expression of 3TP-Lux and SBE4-Lux (Fig. 1AGo). In addition, cotransfection of Smad4 also had no effect. On the contrary, WT-Smad2 resulted in a 2-fold increase in basal expression of AR3-Lux, and cotransfection of Smad4 resulted in a further increase in AR3-Lux expression (Fig. 1AGo). This induction was dependent on FAST1 expression (data not shown). Smad2–2E and Smad2–3E elevated basal expression of AR3-Lux 21-fold and 13-fold, respectively. Treatment with activin in the presence of either wild-type or mutant Smad2 yielded additional AR3-Lux expression (Fig. 1BGo). The increased basal expression in the presence of the acidic mutants was approximately the same as the amount of activin-induced expression in the presence of WT-Smad2. The absolute activin-induced increase in all cases except for WT-Smad2 was approximately the same, consistent with the activation of endogenous Smads. We also examined other possible phosphorylation-mimic mutants; 432Thr-Ser-Thr, located at the L3 loop of the MH2 domain, were replaced with three glutamic acid residues, because the L3 loop was solvent exposed and protruded from the ß-sandwich core structure (27) and involved in Smad-receptor interaction (28). However, none of the reporter genes responded to this mutant (data not shown).



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Figure 1. Smad2–2E and -3E Are Constitutively Active Mutants in the Expression of AR3-Lux in L17 Cells

A, Effect of Smad2 mutants on 3TP-Lux, FAST1-dependent AR3-Lux, and SBE4-Lux transcriptional responses. L17 cells were transiently transfected with a reporter (3TP-Lux, AR3-Lux or SBE4-Lux), ß-galactosidase, FAST1 for AR3-Lux, and wild-type (WT) or mutant versions of Smad2 together with (hatched bar) or without (solid bar) Smad4. Luciferase activity was normalized to ß-galactosidase activity, and luciferase activity in the cell lysates in the absence of exogenous Smads and FAST1 for each reporter was set to 1. Data were expressed as the mean ± SD of triplicates from a representative experiment. B, Responses to activin by Smad2 mutants on expression of AR3-Lux. L17 cells were transiently transfected with the AR3-Lux reporter, ß-galactosidase, FAST1, Smad4, and WT or mutant versions of Smad2 as indicated. Cells were treated with 2 nM activin A for 16 h, and luciferase activity was measured in cell lysates. Luciferase activity was normalized to ß-galactosidase activity, and luciferase activity in the cell lysates in the absence of exogenous Smad2 and FAST1 was set to 1. Data were expressed as the mean ± SD of triplicates from a representative experiment.

 
Smad4 Requirement for Constitutive Smad2 Activity
To determine whether the constitutively active mutants of Smad2 needed Smad4 to manifest their activity, we conducted transcriptional activation assays in Smad4-deficient SW480.7 cells (29, 30). AR3-Lux expression did not respond to activin stimulation (data not shown), although the reason is not clear. Therefore, we used TGFß as a ligand, because TGFß as well as activin could activate this reporter gene transcription (9, 27). In the absence of Smad4, neither WT-Smad2 nor Smad2–2E transfection affected AR3-Lux expression (Fig. 2Go). Expression of full-length Smad4 increased AR3 expression in response to TGFß stimulation by WT-Smad2 transfection. In the presence of Smad4, Smad2–2E transfection caused enhanced basal expression (Fig. 2Go). In contrast, a Smad4 construct with a small carboxyl-terminal truncation, i.e. deletion of amino acids 516–552 [Smad4(1–515)] failed to increase either basal or TGFß-induced luciferase activity in the presence of either WT-Smad2 or Smad2–2E. These findings suggest that Smad4 is essential for Smad2–2E-induced AR3 transcription.



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Figure 2. Role of Smad4 for Smad2-Induced AR3-Lux Transcription

Smad4-deficient SW480.7 cells were transiently transfected with the AR3-Lux reporter, ß-galactosidase, FAST1, and WT-Smad2 or Smad2–2E together with Smad4 expression constructs as indicated. After 24 h of transfection, cells were treated with 100 pM TGFß1 for 16 h, and luciferase activity was measured in cell lysates. Luciferase activity was normalized to ß-galactosidase activity, and luciferase activity in the cell lysates in the absence of exogenous Smad2 and FAST1 was set to 1. Data were expressed as the mean ± SD of triplicates from a representative experiment.

 
Binding of Smad2 to Smad4
The reporter assays described above revealed that Smad4 was required for enhanced basal transcriptional activity of Smad2–2E. Phosphorylated Smad2 is proposed to associate with Smad4 for transcriptional activation (3, 11). To test that hypothesis, we characterized the Smad2:Smad4 association by two assays, a glutathione-S-transferase (GST)-pull down assay and fluorescent analysis.

The GST-pull down assay revealed that more in vitro translated 35S-Smad2–2E bound to GST-Smad4 than did 35S-WT-Smad2 (Fig. 3BGo). No Smad binding was detected after incubation with GST beads alone. These results suggested that the Smad2–2E:Smad4 interaction was higher affinity than the WT-Smad2:Smad4 interaction.



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Figure 3. Association of Smad2 with Smad4 by GST-Pull Down Assay

Association of in vitro translated Smad2 and GST-Smad4. A, GST or GST-Smad4 proteins were expressed in BL21 and affinity-purified by glutathione-Sepharose 4B beads. B, 35S-Labeled WT-Smad2 or Smad2–2E protein was first incubated with GST beads preadsorbed with BSA. The flow-through fraction was then reacted with GST beads or GST-Smad4 beads that were preadsorbed with BSA. After the beads were washed, the proteins were resolved by SDS-PAGE, and the 35S-labeled bands were detected by autoradiography.

 
Association of Smad2 with Smad4 was quantified by fluorescent analysis using dansylated Smad4 and purified Smad2. For this purpose, Smad2 and Smad4 proteins expressed in Escherichia. coli were purified to near homogeneity (Fig. 4AGo). Maximum fluorescence intensity of dansylated Smad4 increased in the presence of Smad2 in a dose-dependent manner (Fig. 4Go, B and C), suggesting the detection of Smad2:Smad4 complexes. In contrast, no increase in the fluorescence intensity was observed in the presence of an unrelated protein, BSA (data not shown). When differences of maximum fluorescence intensity between dansylated Smad4 in the presence of Smad2 and that in the absence of Smad2 were plotted as a function of free Smad2 concentration, a one-site binding model provided the best fit for both the WT-Smad2:Smad4 data and the Smad2–2E:Smad4 data. The average Kd values of three independent trials were 270 ± 66 nM (mean ± SE) for WT-Smad2:Smad4 and 79 ± 18 nM for Smad2–2E:Smad4; these Kds were statistically different (P < 0.05), indicating tighter binding of Smad4 to Smad2–2E as compared with WT-Smad2.



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Figure 4. Association of Smad2 with Smad4 by Fluorescence Analysis

A, Purified Smad2 and Smad4 proteins for fluorescence analysis. Protein expressed in E. coli as GST fusion protein was purified to near homogeneity. The proteins were loaded on a 10% SDS-polyacrylamide gel and stained by Coomassie brilliant blue R-250. B, Emission spectrum of dansylated Smad4 in the presence of purified Smad2. Purified Smad4 was labeled with dansyl aziridine. Dansylated Smad4 was mixed with purified Smad2. Excitation was performed at 340 nm, and emission was scanned from 430 nm to 570 nm. C, Titration curve with Smad2 for differences of maximum fluorescence intensity between dansylated Smad4 in the absence of Smad2 and that in the presence of Smad2. To determine the dissociation constant of the Smad2:Smad4 complex, after calculating free Smad2 concentration, one-site binding model, y = a x x/(Kd + x), was applied. The figure shows a plot of the fluorescence responses against total Smad2 concentration, and each point shows average and SE of three independent experiments; to compare data among experiments, y axis data were normalized to show the percentage of the maximum response in each experiment.

 
Subcellular Localization of Smad2
A number of qualitative immunocytochemical analyses have indicated that receptor-regulated Smads accumulated in the nucleus in response to TGFß or BMP stimulation (9, 31, 32, 33), suggesting that phosphorylation of the serine residues at the carboxyl terminus caused nuclear translocation. We examined the distribution of unactivated and activated Smad2 in L17 cells by subcellular fractionation and subsequent immunoblot analysis. Subcellular distribution of lactate dehydrogenase (LDH) as a cytosolic marker and DNA as a nuclear marker confirmed that cross-contamination of cytosol with nucleus was minimal (Fig. 5Go, A and C).



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Figure 5. Subcellular Distribution of Smad2 and Smad4 in L17 Cells

L17 cells were transiently transfected with HA-WT-Smad2 or HA-Smad2–2E with or without Flag-Smad4 expression constructs. After 48 h of transfection, cells were treated with or without 2 nM activin A for 45 min. Cell suspensions were homogenized in 0.25 M sucrose buffer and cytosol was separated from nuclei by centrifugation. A and C, Cross-contamination of each fraction. LDH activity was measured as a cytosol marker, whereas DNA was measured as a nuclear marker. B and D, Twenty-microgram proteins were subjected to Western blot using anti-HA or anti-Flag antibody to detect expressed Smad2 or Smad4.

 
The amount of WT-Smad2 was higher in cytosol than in nucleus (Fig. 5BGo). The amount of Smad2–2E in cytosol was also higher than in nucleus; however, there was relatively more Smad2–2E in the nuclear fraction than WT-Smad2. This effect was independent of exogenous Smad4 expression (Fig. 5BGo). A tendency of higher nuclear localization of Smad2–2E was also detected by immunocytochemical analyses (data not shown). Unlike Smad2, exogenously expressed Smad4 was evenly distributed within the cell, and the distribution was not changed by Smad2–2E expression (Fig. 5BGo). Activin stimulation had quite minimal effect on Smad2 subcellular distribution both in cells transfected with WT-Smad2 and in cells transfected with Smad2–2E (Fig. 5DGo). No remarkable changes in subcellular distribution of Smad2 were observed by immunocytochemical analyses, although Smad2 lacking the amino-terminal region [Smad2(264–467)] was constitutively localized in nucleus (data not shown), consistent with the previous results (34, 35).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
In this study we showed that mimicking receptormediated phosphorylation of Smad2, by mutation of the phosphorylated serines to acidic amino acids, enhanced Smad2’s basal transcriptional activity. Using this constitutively activated Smad2 mutant (Smad2–2E), we explored the molecular mechanisms linking phosphorylation to changes in function. In vitro binding assays revealed that Smad2–2E bound more tightly to Smad4 than did WT-Smad2. In addition, there was relatively more Smad2–2E in the nucleus than WT-Smad2. Together, those two effects likely account for the enhanced basal activity of Smad2–2E, consistent with data from qualitative studies suggesting that phosphorylation-induced Smad2 complex formation with Smad4 and nuclear translocation are related to transcriptional activation (2, 3).

The reporter gene assays indicated that Smad4 was essential for Smad2-mediated signaling (Fig. 2Go). The findings were consistent with the previous reports; Smad2 and Smad4 synergized on reporter gene transcription (8, 9, 33), but Smad4 lacking the carboxyl terminus activated neither 3TP-Lux (8) nor AR3 gene transcription (9). Because full-length Smad4 but not the truncated Smad4 could form complexes with Smad2 in response to agonist stimulation (36), the current concept that Smad2:Smad4 complexes are the active signaling form has been proposed (2, 3, 11). The tighter binding of Smad2–2E to Smad4, as determined by in vitro binding assays, could thus at least partially explain the elevated basal transcriptional activity. The Kd for the Smad2–2E:Smad4 interaction (~80 nM) is in the same range as that determined for other transcription factor pairs, including nuclear factor{kappa}B (NF{kappa}B) p50-p50 homodimer [~1 µM (37)] and Jun:Fos heterodimers [23–110 nM (38, 39)]. A change in affinity due to Smad2 phosphorylation could thus conceivably alter the amount of active complex.

A greater percentage of Smad2–2E was localized in the nucleus than WT-Smad2, and exogenous Smad4 expression had no effect on the subcellular distribution. These results were consistent with the immunocytochemical observation on nuclear translocation of receptor-regulated Smads by agonist stimulation even in Smad4-deficient cells (9). Because Smad4 was necessary for Smad2–2E-mediated AR3 transcription, nuclear translocation of Smad2 would not be sufficient for the signaling.

Currently, intracellular signaling of activin is indistinguishable from that of TGFß; Smad2 was phosphorylated in response to stimulation by activin as well as TGFß with a peak at 60 min after ligand addition (8, 35), a time at which strongest formation of Smad2:Smad4 complexes was also seen (40). However, nuclear translocation of Smad2 by activin stimulation was less evident than that of Smad3 by activin stimulation or that of Smad2 by TGFß stimulation (41). Although our results that activin had quite minimal effect on subcellular distribution of Smad2 might result from transient overexpression of Smad2, there might be additional activin-regulated steps for Smad2-mediated signaling independent of subcellular distribution.

Mutation of the carboxyl-terminal serines of Smad2 to glutamate resulted in elevated basal expression of AR3-Lux; however, no effect was observed for two other reporter genes, 3TP-Lux and SBE4-Lux. There are several possible explanations for that finding. 1) Smad2 may participate in transcriptional activation of AR3-Lux, but not in the activation of the other promoters. The activin-responsive factor that bound the Mix.2 promoter element in response to activin stimulation in Xenopus embryos (42) contained Smad2, and Smad2 was essential for formation of the complex (43, 44). In contrast, Smad2 did not bind DNA elements from either 3TP-Lux (45) or SBE4-Lux (26). Furthermore, Smad2 had little effect on basal expression of either of those reporter genes (5, 22, 23, 33, 45, 46). 2) Transcription of 3TP-Lux and SBE4-Lux could require additional ligand-induced events, as yet undefined, whereas for AR3-Lux transcription Smad2 carboxyl-terminal phosphorylation could be sufficient. 3) The nuclear concentration of Smad2 required for transcriptional activation of AR3-Lux may be lower than that needed for 3TP-Lux and SBE4-Lux. The EC50 for activin induction of AR3-Lux is lower than that for the other reporter genes (M. Bayram and L. S. Mathews, unpublished), suggesting that AR3-Lux is a more sensitive reporter for Smad-mediated signals.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
cDNA Constructs
The following plasmids were provided: 3TP-Lux (24) by Dr. J. Massagué, SBE4-Lux (26) by Dr. B. Vogelstein, AR3-Lux (25) and Xenopus FAST1 cDNA (43) by Dr. M. Whitman, carboxyl-terminal Flag-tagged human Smad cDNAs (30) by Dr. R. Derynck, Xenopus Smad2 cDNA (47) by Dr. J. M. Graff, HA-pcDNA3 and Flag-pcDNA3 expression vector (48) by Drs. N. Inohara and T. Koseki, human Smad4 subcloned into the vector pGEX-KG using BamHI and EcoRI sites by E. Tang and Dr. K.-L. Guan.

The human Smad2 mutants for expression vectors were constructed by a one-step PCR method. The human Smad2 and human Smad4 cDNAs were subcloned into the EcoRI and XbaI sites of hemagglutinin (HA)-pcDNA3 to produce N-terminal HA-tagged proteins. For GST-Smad2 protein expression, Smad2 cDNA was subcloned into the vector pGEX-2T using SmaI and EcoRI sites.

Cell Culture and cDNA Transfection
The L17 cells, a derivative of the mink lung epithelial cell line (Mv1Lu) provided by Dr. J. Massagué, were cultured and transfected as described previously (49). SW480.7 colon carcinoma cell line was provided by Dr. E. J. Stanbridge (29). The cells were cultured in DMEM with 10% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin. For transient transfection, cells in 24-well plates or in 10-cm cell culture dishes were transfected by diethylaminoethyl-dextran method.

Reporter Assay
Luciferase assays were basically conducted as described previously (50). L17 cells and SW480.7 cells were transiently transfected with various Smad constructs and FAST1 together with a reporter construct (3TP-Lux, AR3-Lux or SBE4-Lux) and a plasmid expressing ß-galactosidase (pCMV-ßGal). In each experiment, equal amounts of DNA were transfected, which was achieved by adjusting empty vector (pcDNA1 and pcDNA3). For basal induction experiments, L17 cells were harvested at 40 h after transfection. For ligand stimulation experiments, at 24 h after transfection, cells were treated with 2 nM activin A (provided by Dr. T. K. Woodruff and by the NIH Hormone Distribution Program, NIDDK) or 100 pM TGFß1 (Becton Dickinson and Co., Franklin Lakes, NJ) for 16 h. Luciferase activity was normalized to ß-galactosidase activity, and luciferase activity in the cell lysates in the absence of exogenous Smads, FAST1, and ligand was set to 1.

GST-Pull Down Assay
The GST and GST-fused Smad4 proteins were expressed in E. coli and purified by use of glutathione-Sepharose beads (Amersham Pharmacia Biotech. Arlington Heights, IL) according to the manufacturer’s protocol. Purified GST or GST-Smad4 bound to glutathione-Sepharose beads was preadsorbed with 0.5 mg/ml BSA, 1 mM EDTA to prevent nonspecific binding with beads for 3 h at 4 C. 35S-labeled WT-Smad2 or Smad2–2E proteins, which were translated in vitro by use of the TNT rabbit reticulocyte lysate kit (Promega Corp., Madison, WI), were then loaded on a GST-Sepharose column preequilibrated with binding buffer [50 mM Tris-HCl (pH 7.4), 120 mM NaCl, 2 mM EDTA, 0.1% NP-40, 1 mM phenylmethylsulfonyl fluoride (PMSF)]) for 1 h at 4 C. The flow-through was loaded on a glutathione-Sepharose column bound to 3 µg of GST or GST-Smad4 and incubated for 1.5 h at 4 C. The columns were washed four times with binding buffer. Specifically bound proteins dissolved in SDS-PAGE sample buffer were separated by SDS-PAGE (10% gel) and visualized by fluorography.

Fluorescence Analysis
For detection of Smad binding by fluorescence analyses, Smad proteins were highly purified from E. coli expressed as GST-fused proteins (90% < purity). The purified Smad proteins had appropriate in vitro biological activities; Smad2 was phosphorylated by TGFß receptor complexes and bound to calmodulin in a calcium-dependent manner, and Smad4 protein bound to SBE4 (M. Funaba and L. S. Mathews, in preparation). A preliminary trial showed that Xenopus Smad2 expression construct yielded more protein than human Smad2 expression construct. Therefore, we expressed Xenopus Smad2 protein. Because of high homology of human Smad2 with Xenopus Smad2 (98% identity), these two proteins are expected to behave similarly on binding with human Smad4.

The purified Smad4 protein (8 µM) was labeled with 1 µl of 200 mM dansyl aziridine (Molecular Probes, Inc., Eugene, OR) at the thiol sites for 2 h at room temperature. One microliter of 1 M DTT was then added to consume excess thiol-reactive reagent. The conjugate and free thiol-reactive reagents were separated by use of a Sephadex G-25 spin column (Roche Molecular Biochemicals, Indianapolis, IN). Fluorescence measurements were performed by using 40 nM dansylated Smad4 in 20 mM HEPES (pH 7.5), 200 mM NaCl, 1 mM CaCl2, and 1 mM DTT in the presence of various concentrations of Smad2. The measurements were taken with an FluoreMax-2 (Instruments SA Inc., Edison, NJ) with excitation wavelength set at 340 nm and band width of 10 nm for both excitation and emission wavelengths. Emission scans were recorded between 430 and 570 nm and fluorescent intensity of maximum emission was measured. To determine the dissociation constant (Kd), difference of maximum intensity at each concentration of Smad2 from that of Smad4 without Smad2 was plotted against the free Smad2 concentration. A one-site binding model was applied (y = a x x/(Kd + x) where x is the concentration of free Smad2 and y is the difference between the fluorescence intensity in the presence and absence of Smad2) and the Kd was calculated by use of Graph Pad PRISM (GraphPad Software, Inc., San Diego, CA). Free Smad2 concentration was estimated from the following equation: free Smad2 (nM) = total Smad2 (nM) - F/F{infty} x 40 where F and F{infty} are the difference of the fluorescence intensity at the Smad2 concentration and at the highest Smad2 concentration, respectively. Binding experiments were conducted three times using three different lots of proteins. The comparison of Kd values between WT-Smad2:Smad4 and Smad2–2E:Smad4 was evaluated by Student’s t test, and differences of P < 0.05 were considered statistically significant.

Subcellular Fractionation
L17 cells were transiently transfected with HA-Smad2 and Smad4-Flag expression constructs. For activin stimulation experiments, at 48 h after transfection, cells were treated with 2 nM activin A for 45 min. The entire subcellular fractionation procedure was performed at 4 C. Cells were washed with HEPES dissociation buffer three times and suspended in isotonic buffer [20 mM Tris-HCl (pH 7.4), 0.25 M sucrose, 1 mM EDTA, 1 mM Na3VO4, 1 mM PMSF, 1% aprotinin], followed by homogenization with a Dounce homogenizer (25 strokes) and a Potter-Elvehjem homogenizer (15 strokes). After centrifugation at 900 x g for 7 min, the supernatant was further centrifuged at 100,000 x g for 1 h. The supernatant was referred to as a cytosolic fraction. The pellet after the first centrifugation was washed with isotonic buffer and resuspended in isotonic buffer. After ultrasonication, the resuspension was centrifuged at 100,000 x g for 30 min. The supernatant was referred to as a nuclear fraction. Protein concentrations from each fraction were measured by the bicinchoninic acid method (51) after concentration by the sodium deoxycholate-trichloroacetic acid method (52). Protein contents in cytosolic fractions and in nuclear fractions were comparable among cell treatments.

Five percent of the protein in each fraction was subjected to Western blot analyses for examining expression of Smad2 and Smad4. To check cross-contamination during homogenization, LDH activity as a cytosolic marker and DNA content as a nuclear marker were measured by the methods of Storrie and Madden (53) and Labarca and Paigen (54), respectively.


    ACKNOWLEDGMENTS
 
We thank Dr. Teresa Woodruff and NIH Hormone Distribution Program, NIDDK, for providing activin A, and Drs. Rik Derynck, Jon Graff, KunLiang Guan, Naohiro Inohara, Takeyoshi Koseki, Joan Massagué, Eric Stanbridge, Eric Tang, Bert Vogelstein, and Malcolm Whitman for providing plasmids and cell line. We also thank Cole Zimmerman and Taju Kariapper for stimulating discussion and Akira Abe for suggestions for fluorescence analysis.


    FOOTNOTES
 
Address requests for reprints to: Masayuki Funaba, Azabu University School of Veterinary Medicine, 1–17-71 Fuchinobe, Sagamihara 229-8501, Japan. E-mail: funaba{at}azabu-u.ac.jp

This work was supported in part by American Cancer Society Grant RPG-98–352-01-TBE (to L.S.M.).

Received for publication February 8, 2000. Revision received June 16, 2000. Accepted for publication July 11, 2000.


    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Zimmerman CM, Mathews LS, Activin receptors, their mechanism of action. In: Muttukrishna S, Ledger W (eds) Inhibins, Activins, Follistatin in Human Reproductive Physiology. Imperial College Press, London, in press
  2. Heldin CH, Miyazono K, ten Dijke P 1997 TGF-ß signalling from cell membrane to nucleus through SMAD proteins. Nature 390:465–471[CrossRef][Medline]
  3. Massagué J 1998 TGF-ß signal transduction. Annu Rev Biochem 67:753–791[CrossRef][Medline]
  4. Whitman M 1998 Smads and early developmental signaling by the TGFß superfamily. Genes Dev 12:2445–2462[Free Full Text]
  5. Macías-Silva M, Abdollah S, Hoodless PA, Pirone R, Attisano L, Wrana JL 1996 MADR2 is a substrate of the TGFß receptor and its phosphorylation is required for nuclear accumulation and signaling. Cell 87:1215–1224[Medline]
  6. Liu X, Sun Y, Constantinescu SN, Karam E, Weinberg RA, Lodish HF 1997 Transforming growth factor ß-induced phosphorylation of Smad3 is required for growth inhibition and transcriptional induction in epithelial cells. Proc Natl Acad Sci USA 94:10669–10674[Abstract/Free Full Text]
  7. Kretzschmar M, Liu F, Hata A, Doody J, Massagué J 1997 The TGF-ß family mediator Smad1 is phosphorylated directly and activated functionally by the BMP receptor kinase. Genes Dev 11:984–995[Abstract]
  8. Lagna G, Hata A, Hemmati-Brivanlou A, Massagué J 1996 Partnership between DPC4 and SMAD proteins in TGF-ß signalling pathways. Nature 383:832–836[CrossRef][Medline]
  9. Liu F, Pouponnot C, Massagué J 1997 Dual role of the Smad4/DPC4 tumor suppressor in TGFß-inducible transcriptional complexes. Genes Dev 11:3157–3167[Abstract/Free Full Text]
  10. Hata A, Lagna G, Massagué J, Hemmati-Brivanlou A 1998 Smad6 inhibits BMP/Smad1 signaling by specifically competing with the Smad4 tumor suppressor. Genes Dev 12:186–197[Abstract/Free Full Text]
  11. Derynck R, Zhang Y, Feng XH 1998 Smads: transcriptional activators of TGF-ß responses. Cell 95:737–740[Medline]
  12. Tsukazaki T, Chiang TA, Davison AF, Attisano L, Wrana JL 1998 SARA, a FYVE domain protein that recruits Smad2 to the TGFß receptor. Cell 95:779–791[Medline]
  13. Feng XH, Zhang Y, Wu RY, Derynck R 1998 The tumor suppressor Smad4/DPC4 and transcriptional adaptor CBP/p300 are coactivators for smad3 in TGF-ß-induced transcriptional activation. Genes Dev 12:2153–2163[Abstract/Free Full Text]
  14. Hua X, Liu X, Ansari DO, Lodish HF 1998 Synergistic cooperation of TFE3 and smad proteins in TGF-ßinduced transcription of the plasminogen activator inhibitor-1 gene. Genes Dev 12:3084–3095[Abstract/Free Full Text]
  15. Janknecht R, Wells NJ, Hunter T 1998 TGF-ß-stimulated cooperation of Smad proteins with the coactivators CBP/p300. Genes Dev 12:2114–2119[Abstract/Free Full Text]
  16. Kurokawa M, Mitani K, Irie KM, Takahashi T, Chiba S, Yazaki Y, Matsumoto K, Hirai H 1998 The oncoprotein Evi-1 represses TGFß signaling by inhibiting Smad3. Nature 394:92–96[CrossRef][Medline]
  17. Shen X, Hu PP, Liberati NT, Datto MB, Frederick JP, Wang XF 1998 TGFß-induced phosphorylation of Smad3 regulates its interaction with coactivator p300/CREB-binding protein. Mol Biol Cell 9:3309–3319[Abstract/Free Full Text]
  18. Wong C, Rougier-Chapman EM, Frederick JP, Datto MB, Liberati NT, Li JM, Wang XF 1999 Smad3-Smad4 and AP-1 complexes synergize in transcriptional activation of the c-Jun promoter by transforming growth factor ß. Mol Cell Biol 19:1821–1830[Abstract/Free Full Text]
  19. Zhang Y, Feng XH, Derynck R 1998 Smad3 and Smad4 cooperate with c-Jun/c-Fos to mediate TGF-ß-induced transcription. Nature 394:909–913[CrossRef][Medline]
  20. Yanagisawa J, Yanagi Y, Masuhiro Y, Suzawa M, Watanabe M, Kashiwagi K, Toriyabe T, Kawabata M, Miyazono K, Kato S 1999 Convergence of transforming growth factor-ß and vitamin D signaling pathways on SMAD transcriptional coactivators. Science 283:1317–21[Abstract/Free Full Text]
  21. Luo K, Stroschein SL, Wang W, Chen D, Martens E, Zhou S, Zhou Q 1999 The Ski oncoprotein interacts with the Smad proteins to repress TGFß signaling. Genes Dev 13:2196–2206[Abstract/Free Full Text]
  22. Abdollah S, Macías-Silva M, Tsukazaki T, Hayashi H, Attisano L, Wrana JL 1997 TßRI phosphorylation of Smad2 on Ser465 and Ser467 is required for Smad2-Smad4 complex formation and signaling. J Biol Chem 272:27678–27685[Abstract/Free Full Text]
  23. Souchelnytskyi S, Tamaki K, Engstrom U, Wernstedt C, ten Dijke P, Heldin CH 1997 Phosphorylation of Ser465 and Ser467 in the C terminus of Smad2 mediates interaction with Smad4 and is required for transforming growth factor-ß signaling. J Biol Chem 272:28107–28115[Abstract/Free Full Text]
  24. Wrana JL, Attisano L, Carcamo J, Zentella A, Doody J, Laiho M, Wang XF, Massagué J 1992 TGFß signals through a heteromeric protein kinase receptor complex. Cell 71:1003–1014[Medline]
  25. Hayashi H, Abdollah S, Qiu Y, Cai J, Xu YY, Grinnell BW, Richardson MA, Topper JN, Gimbrone Jr MA, Wrana JL, Falb D 1997 The MAD-related protein Smad7 associates with the TGFß receptor and functions as an antagonist of TGFß signaling. Cell 89:1165–1173[Medline]
  26. Zawel L, Dai JL, Buckhaults P, Zhou S, Kinzler KW, Vogelstein B, Kern SE 1998 Human Smad3 and Smad4 are sequence-specific transcription activators. Mol Cell 1:611–617[Medline]
  27. Shi Y, Hata A, Lo RS, Massagué J, Pavletich NP 1997 A structural basis for mutational inactivation of the tumour suppressor Smad4. Nature 388:87–93[CrossRef][Medline]
  28. Lo RS, Chen YG, Shi Y, Pavletich NP, Massagué J 1998 The L3 loop: a structural motif determining specific interactions between SMAD proteins and TGF-ß receptors. EMBO J 17:996–1005[Abstract/Free Full Text]
  29. Goyette MC, Cho K, Fasching CL, Levy DB, Kinzler KW, Paraskeva C, Vogelstein B, Stanbridge EJ 1992 Progression of colorectal cancer is associated with multiple tumor suppressor gene defects but inhibition of tumorigenicity is accomplished by correction of any single defect via chromosome transfer. Mol Cell Biol 12:1387–1395[Abstract]
  30. Zhang Y, Feng X, We R, Derynck R 1996 Receptor-associated Mad homologues synergize as effectors of the TGF-ß response. Nature 383:168–172[CrossRef][Medline]
  31. Liu F, Hata A, Baker JC, Doody J, Carcamo J, Harland RM, Massagué J 1996 A human Mad protein acting as a BMP-regulated transcriptional activator. Nature 381:620–623[CrossRef][Medline]
  32. Hoodless PA, Haerry T, Abdollah S, Stapleton M, O’Connor MB, Attisano L, Wrana JL 1996 MADR1, a MAD-related protein that functions in BMP2 signaling. Cell 85:489–500[Medline]
  33. Nakao A, Imamura T, Souchelnytskyi S, Kawabata M, Ishisaki A, Oeda E, Tamaki K, Hanai J, Heldin CH, Miyazono K, ten Dijke P 1997 TGF-ß receptor-mediated signalling through Smad2, Smad3 and Smad4. EMBO J 16:5353–5362[Abstract/Free Full Text]
  34. Baker JC, Harland RM 1996 A novel mesoderm inducer, Madr2, functions in the activin signal transduction pathway. Genes Dev 10:1880–1889[Abstract]
  35. Lo RS, Massagué J 1999 Ubiquitin-dependent degradation of TGF-ß-activated Smad2. Nat Cell Biol 1:472–478[CrossRef][Medline]
  36. Hata A, Lo RS, Wotton D, Lagna G, Massagué J 1997 Mutations increasing autoinhibition inactivate tumour suppressors Smad2 and Smad4. Nature 388:82–87[CrossRef][Medline]
  37. Sengchanthalangsy LL, Datta S, Huang DB, Anderson E, Braswell EH, Ghosh G 1999 Characterization of the dimer interface of transcription factor NF{kappa}B p50 homodimer. J Mol Biol 289:1029–1040[CrossRef][Medline]
  38. Pernelle C, Clerc FF, Dureuil C, Bracco L, Tocque B 1993 An efficient screening assay for the rapid and precise determination of affinities between leucine zipper domains. Biochemistry 32:11682–11687[Medline]
  39. Patel LR, Curran T, Kerppola TK 1994 Energy transfer analysis of Fos-Jun dimerization and DNA binding. Proc Natl Acad Sci USA 91:7360–7364[Abstract]
  40. Lebrun JJ, Takabe K, Chen Y, Vale W 1999 Roles of pathway-specific and inhibitory Smads in activin receptor signaling. Mol Endocrinol 13:15–23[Abstract/Free Full Text]
  41. Shimizu A, Kato M, Nakao A, Imamura T, ten Dijke P, Heldin CH, Kawabata M, Shimada S, Miyazono K 1998 Identification of receptors and Smad proteins involved in activin signaling in a human epidermal keratinocyte cell line. Genes Cells 3:125–134[Abstract/Free Full Text]
  42. Huang HC, Murtaugh LC, Vize PD, Whitman M 1995 Identification of a potential regulator of early transcriptional responses to mesoderm inducers in the frog embryo. EMBO J 14:5965–5973[Abstract]
  43. Chen X, Rubock MJ, Whitman M 1996 A transcriptional partner for MAD proteins in TGF-ß signalling. Nature 383:691–696[CrossRef][Medline]
  44. Chen X, Weisberg E, Fridmacher V, Watanabe M, Naco G, Whitman M 1997 Smad4 and FAST-1 in the assembly of activin-responsive factor. Nature 389:85–89[CrossRef][Medline]
  45. Yingling JM, Datto MB, Wong C, Frederick JP, Liberati NT, Wang XF 1997 Tumor suppressor Smad4 is a transforming growth factor ß-inducible DNA binding protein. Mol Cell Biol 17:7019–7028[Abstract]
  46. Le Dai J, Turnacioglu KK, Schutte M, Sugar AY, Kern SE 1998 Dpc4 transcriptional activation and dysfunction in cancer cells. Cancer Res 58:4592–4597[Abstract]
  47. Graff JM, Bansal A, Melton DA 1996 Xenopus Mad proteins transduce distinct subsets of signals for the TGFß superfamily. Cell 85:479–487[Medline]
  48. Inohara N, Koseki T, Chen S, Wu X, Núñez G 1998 CIDE, a novel family of cell death activators with homology to the 45 kDa subunit of the DNA fragmentation factor EMBO J 17:2526–2533[Abstract/Free Full Text]
  49. Willis SA, Zimmerman CM, Li L, Mathews LS 1996 Formation and activation by phosphorylation of activin receptor complexes. Mol Endocrinol 10:367–379[Abstract]
  50. Zimmerman CM, Kariapper MS, Mathews LS 1998 Smad proteins physically interact with calmodulin. J Biol Chem 273:677–680[Abstract/Free Full Text]
  51. Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:76–85[Medline]
  52. Bensadoun A, Weinstein D 1976 Assay of proteins in the presence of interfering materials. Anal Biochem 70:241–250[Medline]
  53. Storrie B, Madden EA 1990 Isolation of subcellular organelles. Methods Enzymol 182:203–225[Medline]
  54. Labarca C, Paigen K 1980 A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102:344–352[Medline]