Title: The adaptor molecule Disabled-2 links the transforming growth factor beta receptors to the Smad pathway
Abstract: Article1 June 2001free access The adaptor molecule Disabled-2 links the transforming growth factor β receptors to the Smad pathway Barbara A. Hocevar Barbara A. Hocevar Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Abdelkrim Smine Abdelkrim Smine Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Xiang-Xi Xu Xiang-Xi Xu Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA, 30322 USA Search for more papers by this author Philip H. Howe Corresponding Author Philip H. Howe Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Barbara A. Hocevar Barbara A. Hocevar Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Abdelkrim Smine Abdelkrim Smine Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Xiang-Xi Xu Xiang-Xi Xu Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA, 30322 USA Search for more papers by this author Philip H. Howe Corresponding Author Philip H. Howe Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA Search for more papers by this author Author Information Barbara A. Hocevar1, Abdelkrim Smine1, Xiang-Xi Xu2 and Philip H. Howe 1 1Department of Cell Biology, The Lerner Research Institute, Cleveland Clinic Foundation, Cleveland, OH, 44195 USA 2Department of Biochemistry and Winship Cancer Center, Emory University School of Medicine, Atlanta, GA, 30322 USA *Corresponding author. E-mail: [email protected] The EMBO Journal (2001)20:2789-2801https://doi.org/10.1093/emboj/20.11.2789 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Using a genetic complementation approach we have identified disabled-2 (Dab2), a structural homolog of the Dab1 adaptor molecule, as a critical link between the transforming growth factor β (TGFβ) receptors and the Smad family of proteins. Expression of wild-type Dab2 in a TGFβ-signaling mutant restores TGFβ-mediated Smad2 phosphorylation, Smad translocation to the nucleus and Smad-dependent transcriptional responses. TGFβ stimulation triggers a transient increase in association of Dab2 with Smad2 and Smad3, which is mediated by a direct interaction between the N-terminal phosphotyrosine binding domain of Dab2 and the MH2 domain of Smad2. Dab2 associates with both the type I and type II TGFβ receptors in vivo, suggesting that Dab2 is part of a multiprotein signaling complex. Together, these data indicate that Dab2 is an essential component of the TGFβ signaling pathway, aiding in transmission of TGFβ signaling from the TGFβ receptors to the Smad family of transcriptional activators. Introduction The transforming growth factor β (TGFβ) superfamily consists of cytokines that modulate essential cellular functions, such as cellular proliferation, differentiation and apoptosis (Massagué, 1998). Signaling by TGFβ, the prototypic member of the TGFβ superfamily, is initiated following ligand binding to the constitutively active ser/thr kinase type II receptor (TβRII). The type I receptor (TβRI), which also possesses ser/thr kinase activity, is then recruited to TβRII, leading to the formation of an oligomeric complex (Wrana et al., 1994). The subsequent phosphorylation and activation of the type I receptor by the type II receptor leads to further propagation of TGFβ signaling by the Smad family of proteins. The Smads can be divided into three functional groups: the receptor-activated or R-Smads, consisting of Smads 1, 2, 3, 5 and 8; the common-mediator or Co-Smad, Smad 4; and the inhibitory Smads, consisting of Smads 6 and 7 (reviewed in Heldin et al., 1997; Piek et al., 1999; Zhang and Derynck, 1999). The R-Smads are recruited to and phosphorylated by their cognate type I receptors; Smads 1, 5 and 8 transmit signaling by BMP receptors, while Smads 2 and 3 mediate signaling by TGFβ and activin receptors. Following phosphorylation, R-Smads dimerize with Smad4 and translocate to the nucleus, where the complex has been shown to activate gene transcription by binding directly to DNA (Dennler et al., 1998) as well as by interaction with other transcription factors such as FAST1, FAST2, c-Jun and c-Fos (Chen et al., 1996; Labbe et al., 1998; Zhang et al., 1998). An accessory protein has recently been identified, SARA (Smad anchor for receptor activation), which helps recruit Smad2/3 to the activated TβRI (Tsukazaki et al., 1998). SARA binds to unphosphorylated Smad 2/3 and by virtue of its lipid-binding FYVE domain localizes Smad2/3 to the proper subcellular compartment for interaction with TβRI (Tsukazaki et al., 1998). Once phosphorylation of Smad 2/3 has occurred, the SARA–Smad complex dissociates, exposing an intrinsic nuclear import signal on Smad2/3 that results in its accumulation in the nucleus (Xu et al., 2000). Disabled-2 (Dab2) is a signaling molecule that was first identified as DOC-2 (for differentially expressed in ovarian carcinoma) (Mok et al., 1994), and subsequently as p96, a protein whose phosphorylation was stimulated by CSF-1 (Xu et al., 1995). Dab2 contains an N-terminal phosphotyrosine binding domain (PTB) or phosphotyrosine interacting domain (PID) and a C-terminal proline-rich domain (PRD) (Xu et al., 1995), indicative of a function as an adaptor molecule (Pawson and Scott, 1997). We demonstrate here that stable expression of Dab2 functionally complements a TGFβ-signaling mutant cell line, restoring all assayed TGFβ responses including TGFβ-induced Smad2 phosphorylation, Smad nuclear translocation and Smad-dependent transcriptional responses. Sequencing of the Dab2 message expressed by the mutant cell line demonstrates that it harbors a missense mutation in the C-terminal domain of Dab2, which causes a decrease in the stability and steady-state expression of Dab2. Dab2 associates with Smad2 and 3 in a time- and ligand-dependent manner, which is mediated by a direct interaction between the N-terminal PTB domain of Dab2 and the MH2 domain of Smad2. Mutation of a conserved phenylalanine residue in the PTB domain of Dab2 abrogates its ability to complement the mutant cell line, while expression of the PTB domain alone or Dab2 lacking the C-terminal PRD also fails to mediate complementation, demonstrating that both domains are required for TGFβ signaling. Dab2 is also found in association with both TβRI and TβRII, suggesting that it is part of a multiprotein signaling complex. Dab2 thus appears to function as an adaptor molecule, serving to bridge the TGFβ receptor complex to the Smad pathway. Results Dab2 functionally complements the TGFβ-signaling mutant 903 cell line We have previously developed a genetic system consisting of a set of recessive TGFβ-mutant cell lines that could be functionally complemented to identify TGFβ signaling molecules (Hocevar and Howe, 1996). This system utilizes the human fibrosarcoma HT1080-derived parental cell line, BAHgpt, which contains the selectable marker Escherichia coli guanine phosphoribosyltransferase (gpt) linked to the TGFβ-responsive promoter 3TP (Figure 1A). Treatment of the BAHgpt cell line with TGFβ leads to expression of the gpt gene and allows for cell growth in media containing hypoxanthine, aminopterin and thymidine (HAT); conversely, in media containing 6-thioguanine (6TG), TGFβ treatment leads to cell death. Recessive mutant cell lines were generated from the parental cell line following successive rounds of chemical mutagenesis by selection in media containing 6TG and TGFβ. The mutant cell line 903, selected in this manner, has been characterized as being deficient in a variety of TGFβ-stimulated responses (Lee et al., 1997; Hocevar et al., 1999). Transfection of the 903 cell line with a cDNA expression library followed by selection in media containing HAT and TGFβ resulted in the generation of a complemented clone designated 903L2. To identify candidate genes responsible for the functional complementation, analysis of genes differentially expressed by the complemented 903L2 cell line and the mutant 903 cell line was performed using a gene filter array. One such gene identified as having differential expression corresponded to the C-terminal region of the DOC-2 gene, also known as Dab2 and p96. Figure 1.Dab2 expression mediates functional complementation of the 903 TGFβ-signaling mutant cell line. (A) Diagrammatic representation of the genetic complementation approach. The parental cell line BAHgpt expresses the E.coli gpt gene driven by the 3TP promoter. In media containing 6TG, TGFβ treatment leads to cell death, indicative of TGFβ responsiveness. Mutant cell lines that do not respond to TGFβ maintain growth in media containing 6TG plus TGFβ. (B) Transfection of Dab2 into 903 cells restores TGFβ responsiveness. Parental BAHgpt, and mutant 903, 903L2 and 903 cells transfected with human wild-type Dab2 (903WTDab2) were plated into media containing 6TG (30 μM) and 6TG (30 μM) plus 5 ng/ml TGFβ. After 5 days, cells were fixed with methanol and stained with hematoxylin to visualize growth. (C and D) Dab2 expression restores TGFβ-mediated induction of the 3TPLux (C) and SBE-Luc (D) reporter constructs. BAHgpt, 903, 903L2 and 903WTDab2 cells were transiently transfected with 3TPLux or SBE-Luc and SV40-RL as a control for transfection efficiency. Cells were treated with TGFβ for 18 h, followed by lysis and determination of luciferase activity. Luciferase activity is expressed as a ratio of 3TPLux luciferase activity divided by the SV40-RL activity. Shown is the mean ± SD of duplicates from a representative experiment. Open bars represent untreated cells while black bars denote TGFβ treatment. Download figure Download PowerPoint To assess whether expression of Dab2 was capable of functionally complementing the mutant phenotype of the 903 cell line, human Dab2 was stably introduced into the 903 cell line. Restoration of TGFβ signaling was first tested by growth of the cells in media containing 6TG and 6TG plus TGFβ (Figure 1B). In the absence of TGFβ, 6TG in the media has no effect on cell growth; however, in the presence of TGFβ, cells that are responsive to TGFβ express gpt and die in media containing 6TG (Figure 1A and B). As shown in Figure 1B, all the cell lines tested grow in media containing 6TG alone, while the parental cell line BAHgpt, the original complemented clone 903L2 and a pool of 903 cells stably expressing wild-type Dab2 (903WTDab2) die in media containing 6TG plus TGFβ. These results thus indicate that Dab2 alone can mediate restoration of the TGFβ-responsive phenotype. To further characterize the restoration of TGFβ responsiveness mediated by Dab2 in the 903 mutant, we first performed transient transfection analysis using the luciferase reporter construct 3TPLux, which has been used to measure TGFβ responsiveness of a variety of cell lines (Wrana et al., 1992; Carcamo et al., 1994). As shown in Figure 1C, treatment of BAHgpt cells results in a 4- to 5-fold induction of the 3TPLux reporter, which is deficient in the 903 cell line. Analysis of 903L2 and 903WTDab2 cells reveals restoration of TGFβ-stimulated luciferase induction, without alteration of the basal luciferase level. These results are consistent with the ability of Dab2 to restore regulation of the gpt gene in response to TGFβ (Figure 1B). Since the Smad family of proteins have been shown to be important intracellular mediators of the TGFβ signaling pathway, we next assayed whether Dab2 expression could influence the transcriptional activity of the Smad proteins. To assess this, we utilized a luciferase construct (p6SBE-Luc) that contains a promoter consisting of six tandem copies of the Smad-binding element (SBE) designed to monitor Smad-dependent transcriptional activity (Zawel et al., 1998). Transient transfection of the SBE-Luc reporter followed by TGFβ treatment demonstrates that Dab2 expression mediates restoration of TGFβ-stimulated SBE-Luc induction to the mutant 903 cell line (Figure 1D), suggesting that Dab2 may play a role in TGFβ-mediated Smad-dependent signaling. The 903 cell line expresses a mutant form of Dab2 that exhibits decreased protein stability The gene filter analysis utilized indicated that Dab2 mRNA was down-regulated, but not absent in the mutant 903 cell line. To expand this observation, we determined whether Dab2 protein levels were also decreased in the mutant cell line by western analysis. We find that the mutant 903 cells express less steady-state Dab2 protein than parental BAHgpt cells, which is restored in the 903WTDab2 cell line (Figure 2A). To assess whether this decrease in steady-state expression level is due to increased Dab2 protein turnover, we performed metabolic labeling of the BAHgpt and 903 cells to follow newly synthesized Dab2. Immunoprecipitation with a monoclonal antibody to Dab2, but not control IgG, reveals that Dab2 protein is readily detected in BAHgpt cells following a 4 h labeling period, while Dab2 is barely detectable in the 903 cells (Figure 2B). Figure 2.Mutant Dab2 exhibits decreased protein stability. (A) Steady-state levels of Dab2 are decreased in mutant 903 cells. Fifty micrograms of total cellular protein from BAHgpt, 903 and 903WTDab2 cells were subjected to western analysis utilizing a monoclonal antibody to Dab2 (α-p96). (B) De novo Dab2 synthesis is decreased in mutant 903 cells. BAHgpt and 903 cells were labeled with [35S]methionine for 4 h prior to lysis and immunoprecipitation with either non-immune mouse IgG or a monoclonal antibody to Dab2 (α-p96). Dab2 levels were visualized by autoradiography. (C, D and E) Mutant Dab2 is less stable than WTDab2. Pulse–chase analysis (C) was performed on COS7 cells transiently transfected with Flag-tagged WT or Mut Dab2 as described in Materials and methods. Following immunoprecipitation with anti-Flag antibody, [35S]Dab2 was visualized by autoradiography. Analysis of steady-state levels of WT or Mut Dab2 (D) was performed by western analysis utilizing anti-Flag antibody. (E) Levels of Dab2 proteins in (C) were determined by densitometric scanning and analysis with NIHImage software. Levels are expressed as a percentage of protein remaining at the various time points, with levels at time 0 designated as 100%. Download figure Download PowerPoint To determine whether the Dab2 mRNA expressed by the 903 cell line harbors any mutations that could affect its stability, we cloned the mutant gene by RT–PCR and compared its sequence with wild-type (WT)Dab2. The Dab2 mRNA expressed by the mutant 903 cell line was found to contain a single nucleotide change of G to A, resulting in an amino acid substitution of Ser to Asn at amino acid 634 in the C-terminal domain of Dab2. To assess whether this mutation affects the stability of the Dab2 protein in vivo, Flag-tagged WT and mutant Dab2 were transiently transfected into COS7 cells and pulse–chase analysis performed. As shown in Figure 2C, WTDab2 expression is readily detected following a 30 min labeling and remains stable for 6 h. In contrast, initial lower levels of mutant Dab2 are expressed, followed by a decrease in levels by as early as 1 h post-chase. Steady-state levels of transfected Dab2 protein determined by western analysis show an accumulation of WT, but not mutant Dab2 throughout the time course (Figure 2D). Also readily apparent is the appearance of slower migrating forms of WTDab2 over time, absent from MutDab2, which may reflect phosphorylation of 634Ser (Figure 2C and D). Analysis of Dab2 levels indicates that MutDab2 exhibits a half-life of ∼10 h, while WTDab2 has a half-life of ∼20 h (Figure 2E). Taken together, these results demonstrate that the mutant 903 cells express lower de novo and steady-state levels of Dab2, which can be attributed in part to the 634Ser to Asn mutation. Dab2 restores endogenous TGFβ-mediated responses to the mutant 903 cell line To further characterize the restoration of TGFβ responsiveness mediated by Dab2 in the 903 cell line, we next assessed the effect of Dab2 expression on TGFβ-mediated induction of the extracellular matrix proteins fibronectin and PAI-1. As shown in Figure 3A and B, the 903 cell line fails to induce PAI-1 expression following TGFβ stimulation, but while the basal level of fibronectin is decreased in the mutant cells, TGFβ can still cause an increase in fibronectin expression. Analysis of 903WTDab2 cells demonstrates that TGFβ treatment induces the expression of PAI-1 to the extent observed in BAHgpt cells, while both basal and TGFβ-stimulated levels of fibronectin are increased by Dab2 transfection. Similarly, in BAHgpt cells that stably overexpress Dab2 (BAHgptWTDab2), TGFβ-stimulated induction of fibronectin is increased; however, TGFβ-mediated PAI-1 induction does not change (Figure 3A and B). These results thus show that Dab2 can restore TGFβ-mediated induction of PAI-1 to the mutant cells and can augment TGFβ-stimulated levels of fibronectin in parental and mutant cells. Figure 3.Dab2 expression restores TGFβ-mediated responses to mutant 903 cells. (A and B) Dab2 expression alters TGFβ-mediated induction of the endogenous genes fibronectin (A) and PAI-1 (B). BAHgpt, BAHgpt cells stably transfected with Dab2 (BAHgptWTDab2), 903 and 903WTDab2 cells were examined for [35S]fibronectin (FN) secreted to the media (A) or [35S]PAI-1 deposited to the extracellular matrix (B) following TGFβ treatment, as described in Materials and methods. FN and PAI-1 are indicated by the arrows. (C) Dab2 expression restores TGFβ-stimulated Smad2 phosphorylation to the mutant 903 cell line. BAHgpt, BAHgptWTDab2, 903 and 903WTDab2 cells were untreated or treated with 5 ng/ml TGFβ for 1 h as indicated. Phosphorylated Smad2 was identified by western analysis using an antibody specific only for the TGFβ-stimulated phosphorylated form of Smad2 (anti-phospho Smad2 465/467; Upstate Biotechnology). (D) Equivalent expression of cellular Smad2 in BAHgpt, BAHgptWTDab2, 903 and 903WTDab2 cells was confirmed by western analysis using an anti-Smad2 antibody (Zymed Laboratories). (E and F) Dab2 restores TGFβ-stimulated Smad2 (E) and Smad3 (F) nuclear accumulation in 903 cells. Nuclear proteins were isolated from BAHgpt, 903 and 903WTDab2 cells untreated or treated with TGFβ for 1 h, as described in Materials and methods. The accumulation of Smad2 and Smad3 in the nucleus was confirmed by western analysis using anti-Smad2 (Transduction Laboratories) and anti-Smad3 antibodies (Zymed Laboratories). The positions of Smad2 and Smad3 are indicated by the arrows. Download figure Download PowerPoint Since the transcriptional activity of both Smad2 and Smad3 is dependent on their phosphorylation by TβRI (Macías-Silva et al., 1996; Liu et al., 1997), we wished to determine whether TGFβ-mediated Smad phosphorylation is deficient in the 903 mutant cells. As shown in Figure 3C, TGFβ treatment leads to efficient phosphorylation of Smad2 in BAHgpt and BAHgptWTDab2 cells, as shown by western analysis, with an antibody that specifically recognizes TGFβ-stimulated phosphorylation sites on Smad2. While phosphorylation of Smad2 can not be detected following TGFβ treatment of 903 cells, re-introduction of Dab2 restores TGFβ-mediated Smad2 phosphorylation. This is not due to a lack of expression of Smad2 protein in 903 cells, since Smad2 levels appear to be comparable in BAHgpt and 903 cells (Figure 3D). Following phosphorylation by TβRI, Smad2 and -3 translocate to the nucleus. To assess whether the nuclear accumulation of the Smads is also deficient in mutant 903 cells, nuclear preparations derived from BAHgpt, 903 and 903WTDab2 cells stimulated with TGFβ for 1 h were analyzed for the presence of Smad2 and Smad3 (Figure 3E and F). Treatment of BAHgpt cells with TGFβ results in nuclear accumulation of Smad2 and Smad3, while in mutant 903 cells the levels of nuclear Smad2 and Smad3 do not change following TGFβ stimulation. Re-introduction of WTDab2 into 903 cells restores TGFβ-stimulated Smad2 and Smad3 nuclear accumulation (Figure 3E and F). Taken together, these results suggest that Dab2 plays a role in mediating TGFβ-stimulated Smad phosphorylation, which allows for nuclear accumulation and subsequent stimulation of Smad-dependent transcriptional responses. The PTB and PRD domains of Dab2 are both required for TGFβ signaling The Dab2 protein can be divided into three functional domains: the N-terminal PTB domain, a middle linker region and a C-terminal PRD. To assess the contribution of the individual domains of Dab2 in TGFβ signaling, constructs were engineered of the various domains of Dab2, including a point mutation in the PTB domain (F166VDab2) that has been shown to abrogate PTB domain function (Borg et al., 1996; Dho et al., 1998) (Figure 4A). These constructs were stably transfected into mutant 903 cells and assessed for their ability to rescue the mutant phenotype. As shown in Figure 4C and D, expression of WTDab2 restores TGFβ-stimulated 3TPLux and SBE-Luc reporter induction to 903 cells, while expression of F166VDab2, Del-PRD or the PTB domain alone fails to do so. The inability of the various constructs to restore TGFβ responsiveness was not due to differences in expression of the constructs, as verified by western analysis (Figure 4B). Expression of MutDab2 (S634N) results in restoration of ∼50% of the TGFβ-stimulated luciferase induction elicited by WTDab2, suggesting that constitutive overexpression is able to partially compensate for the decreased protein stability of MutDab2 (Figure 2). We were unable to test the ability of the PRD alone to restore TGFβ responsiveness as we could not generate a stable cell line that expressed this construct. Consistent with the reporter construct data, introduction of F166VDab2, PRD-Del and PTB constructs into 903 cells failed to restore other TGFβ-mediated responses as well, including PAI-1 induction, Smad2 phosphorylation, and Smad2 and Smad3 nuclear accumulation (data not shown). Taken together, these results indicate that both the PTB domain and PRD of Dab2 are required for restoration of TGFβ signaling in the mutant 903 cells. Figure 4.Restoration of TGFβ signaling requires both the PRD and PTB domain of Dab2. (A) Diagrammatic representation of various constructs of Dab2. Depicted are full-length and deletion constructs of Dab2 containing the N-terminal PTB domain and C-terminal PRD (gray shading). The asterisk designates the S634N mutation present in mutant 903 Dab2 (Mut-Dab2) and the introduced F166V mutation in full-length WTDab2 (F166V-Dab2). (B) Expression levels of various Flag-tagged Dab2 constructs from stable cell lines were determined by western analysis with α-Flag antibody (M2; Sigma). Lysate from the original mutant 903 cell line was used as a control (Cont.). (C and D) TGFβ-stimulated transcriptional activation requires both the PRD and PTB domain of Dab2. BAHgpt, 903 and 903 cells stably expressing the various forms of Dab2 described above were transiently transfected with the reporter constructs 3TPLux (C) and SBE-Luc (D), and SV40-RL as a control for transfection efficiency. After 24 h, cells were untreated (open bars) or treated (black bars) with TGFβ for 18 h. Following lysis, luciferase activity was determined and expressed as the ratio of specific luciferase activity divided by the SV40-RL activity. Shown is the mean ± SD of duplicates from a representative experiment. Download figure Download PowerPoint Dab2 associates with Smad2 and Smad3 We next wished to assess whether the restoration of TGFβ responsiveness mediated by Dab2 was due to a direct interaction of Dab2 with the Smad proteins. To determine this, full-length WTDab2 was translated in vitro and incubated with bacterially expressed Smad1, Smad2, Smad3 and Smad4 glutathione S-transferase (GST) fusion proteins. Figure 5A demonstrates that while the GST moiety alone fails to precipitate Dab2, GST–Smad2 and GST–Smad3 bind to and precipitate full-length Dab2. Dab2 characteristically appears as a doublet, which represents the use of a second initiator methionine present in the sequence (Xu et al., 1995). GST–Smad1 and GST–Smad4, however, fail to interact with Dab2 (Figure 5A). To more fully characterize the interaction of Dab2 with the Smad proteins, we determined which functional domain of the Smad proteins interacts with Dab2. Incubation of in vitro translated WTDab2 with GST constructs constituting the MH1 and MH2 domains of Smad2 reveals that Dab2 preferentially binds to the MH2 domain, with negligible binding to the MH1 domain (Figure 5A). To map which domain in Dab2 mediates the binding to Smad2 and Smad3, constructs bearing these motifs (Figure 4A) were in vitro translated and tested for interaction with GST fusion proteins as above. Both constructs that contain the PTB domain, PTB and Del-PRD, are capable of interacting with Smad2 and Smad3, while the construct bearing only the PRD fails to do so. The same preference for binding to the MH2 domain over the MH1 domain of Smad2 is also maintained with the PTB-containing constructs, as observed for the full-length protein (Figure 5A), which is not due to differences in expression of the GST fusion proteins as visualized by Coomassie Blue staining (Figure 5B). Figure 5.Dab2 associates with Smad2 and Smad3 in vitro. (A) Dab2 interacts with the Smads in vitro. Full-length WTDab2 or the various deletion constructs depicted in Figure 4A were synthesized in vitro using [35S]methionine. Equal amounts of 35S-labeled reaction products were incubated with bacterially expressed GST constructs corresponding to GST protein alone (GST), full-length GST fusion proteins of Smad1, Smad2, Smad3 and Smad4, or GST fusion proteins of the MH1 and MH2 domains of Smad2. Following extensive washing, bound proteins were analyzed by SDS–PAGE, subjected to fluorography and visualized by autoradiography. An aliquot (10%) of 35S-labeled reaction product input is shown in the control lane (Cont.). (B) Equal loading of the bacterially expressed GST fusion proteins utilized in (A) is demonstrated by Coomassie Blue staining of the gel following SDS–PAGE. Download figure Download PowerPoint To test whether an interaction between the Smad proteins and Dab2 takes place in vivo, COS7 cells were transfected with the various Smads and Dab2 in the absence or presence of the activated form of TβRI (TβRIAct). Immunoprecipitation of the Smad proteins followed by western analysis for Dab2 reveals that although Dab2 can associate with Smad2 and Smad3 in the absence of TGFβ signaling, this association increases in the presence of TβRIAct. In contrast, Dab2 does not associate with Smad4 or Smad7 in either the absence or presence of TGFβ signaling, although the proteins are expressed at a comparable level (Figure 6B). Additionally we find that F166VDab2 is still able to associate with Smad2 and its association is stimulated in the presence of TβRIAct (Figure 6A). Figure 6.Dab2 associates with Smad2 and Smad3 in vivo in a ligand-dependent manner. (A and B) Smad2 and Smad3 interact with Dab2 in vivo. COS7 cells were transiently transfected with Flag-tagged WT or F166V Dab2 and various Smads with or without the activated form of TβRI (TβRIAct). After 48 h, lysates were prepared and (A) immunoprecipitated with antibodies specific to each Smad [Smad2 and Smad3 (Zymed Laboratories), Smad4 and Smad7 (Santa Cruz)]. The presence of Dab2 in the complex was confirmed by western analysis with a monoclonal antibody to Dab2 (α-p96). Expression of Dab2 and the Smads (B) was determined by western analysis with anti-Flag antibody (M2; Sigma). Download figure Download PowerPoint To test whether endogenous Dab2 interacts with Smad2, parental BAHgpt cells were treated with TGFβ for 1 h and lysates were immunoprecipitated with Smad2 antibody. Analysis of the immunoprecipitates for the presence of Dab2 reveals that the association of endogenous Dab2 with endogenous Smad2 is stimulated by TGFβ treatment (Figure 7A). To address the time course of association of Dab2 with Smad2 and Smad3 following TGFβ stimulation, we treated BAHgpt cells that stably overexpress WTDab2 with TGFβ for various times, followed by immunoprecipitation with Smad2 or Smad3 antibodies or incubation of lysates with the GST–MH2 domain of Smad2. Western analysis of the precipitated proteins reveals that the association of Dab2 with Smad2 and Smad3 is transiently stimulated following TGFβ treatment, reaching a peak at 1 h and returning to baseline by 24 h (Figure 7B). The association of Dab2 with the GST–MH2 domain fusion protein also follows similar kinetics, while the steady-state levels of Dab2 do not change over the course of TGFβ treatment (Figure 7B, lower panel). Together, these results indicate that the in vivo association of Dab2 with the R-Smads Smad2 and Smad3 occurs in a ligand- and time-dependent manner. Figure 7.Endogenous Dab2 associates with endogenous Smad2 and Smad3. (A) Dab2 interacts with Smad2 in a time- and ligand-dependent manner. BAHgpt cells were treated with TGFβ for 1 h followed by immunoprecipitation with anti-Smad2 (Transduction Laborat