Axin is a scaffold protein in TGF-β signaling that promotes degradation of Smad7 by Arkadia
2006; Springer Nature; Volume: 25; Issue: 8 Linguagem: Inglês
10.1038/sj.emboj.7601057
ISSN1460-2075
AutoresWei Liu, Hongliang Rui, Jifeng Wang, Shu‐Yong Lin, Ying He, Mingliang Chen, Qinxi Li, Zhiyun Ye, Suping Zhang, Siu Chiu Chan, Ye‐Guang Chen, Jiahuai Han, Sheng‐Cai Lin,
Tópico(s)Liver physiology and pathology
ResumoArticle6 April 2006free access Axin is a scaffold protein in TGF-β signaling that promotes degradation of Smad7 by Arkadia Wei Liu Wei Liu Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Hongliang Rui Hongliang Rui Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, ChinaPresent address: Department of Nephrology, the China-Japan Friendship Hospital, Beijing, China Search for more papers by this author Jifeng Wang Jifeng Wang Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Shuyong Lin Shuyong Lin Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Ying He Ying He Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Mingliang Chen Mingliang Chen Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Qinxi Li Qinxi Li Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Zhiyun Ye Zhiyun Ye Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Suping Zhang Suping Zhang State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China Search for more papers by this author Siu Chiu Chan Siu Chiu Chan Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Ye-Guang Chen Ye-Guang Chen State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China Search for more papers by this author Jiahuai Han Jiahuai Han Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Department of Immunology, the Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Sheng-Cai Lin Corresponding Author Sheng-Cai Lin Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Wei Liu Wei Liu Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Hongliang Rui Hongliang Rui Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, ChinaPresent address: Department of Nephrology, the China-Japan Friendship Hospital, Beijing, China Search for more papers by this author Jifeng Wang Jifeng Wang Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Shuyong Lin Shuyong Lin Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Ying He Ying He Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Mingliang Chen Mingliang Chen Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Qinxi Li Qinxi Li Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Zhiyun Ye Zhiyun Ye Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Suping Zhang Suping Zhang State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China Search for more papers by this author Siu Chiu Chan Siu Chiu Chan Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Search for more papers by this author Ye-Guang Chen Ye-Guang Chen State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China Search for more papers by this author Jiahuai Han Jiahuai Han Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Department of Immunology, the Scripps Research Institute, La Jolla, CA, USA Search for more papers by this author Sheng-Cai Lin Corresponding Author Sheng-Cai Lin Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China Search for more papers by this author Author Information Wei Liu1, Hongliang Rui1, Jifeng Wang2, Shuyong Lin1, Ying He2, Mingliang Chen2, Qinxi Li2, Zhiyun Ye2, Suping Zhang3, Siu Chiu Chan1, Ye-Guang Chen3, Jiahuai Han2,4 and Sheng-Cai Lin 1,2 1Department of Biochemistry, Hong Kong University of Science and Technology, Kowloon, Hong Kong, China 2Key Laboratory of Ministry of Education for Cell Biology and Tumor Cell Engineering, School of Life Sciences, Xiamen University, Fujian, China 3State Key Laboratory of Biomembrane and Membrane Biotechnology, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing, China 4Department of Immunology, the Scripps Research Institute, La Jolla, CA, USA *Corresponding author. Department of Biochemistry, Hong Kong University of Science and Technology, Clear Water Bay, Kowloon, Hong Kong, China. Tel.: +852 2358 7294; Fax: +852 2358 1552; E-mail: [email protected] or [email protected] The EMBO Journal (2006)25:1646-1658https://doi.org/10.1038/sj.emboj.7601057 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info TGF-β signaling involves a wide array of signaling molecules and multiple controlling events. Scaffold proteins create a functional proximity of signaling molecules and control the specificity of signal transduction. While many components involved in the TGF-β pathway have been elucidated, little is known about how those components are coordinated by scaffold proteins. Here, we show that Axin activates TGF-β signaling by forming a multimeric complex consisting of Smad7 and ubiquitin E3 ligase Arkadia. Axin depends on Arkadia to facilitate TGF-β signaling, as their small interfering RNAs reciprocally abolished the stimulatory effect on TGF-β signaling. Specific knockdown of Axin or Arkadia revealed that Axin and Arkadia cooperate with each other in promoting Smad7 ubiquitination. Pulse-chase experiments further illustrated that Axin significantly decreased the half-life of Smad7. Axin also induces nuclear export of Smad7. Interestingly, Axin associates with Arkadia and Smad7 independently of TGF-β signal, in contrast to its transient association with inactive Smad3. However, coexpression of Wnt-1 reduced Smad7 ubiquitination by downregulating Axin levels, underscoring the importance of Axin as an intrinsic regulator in TGF-β signaling. Introduction TGF-β signaling plays a wide spectrum of roles from early embryonic development to mature tissues in controlling biological processes, including cell proliferation, differentiation, apoptosis, and cell fate determination (Massague, 2000). TGF-β superfamily members elicit biological responses through a complex cascade of signaling molecules that include receptors, Smads, and importantly the regulatory factors that positively or negatively regulate the signaling system (Shi and Massague, 2003). The TGF-β superfamily can be divided into TGF-β, bone morphogenetic proteins (BMP), and Nodal. Schematically, much has been known about the molecular components involved in TGF-β signaling. TGF-β ligands bind to their receptors that are associated with a family of signal transducers called Smads (R-Smads) (Massague, 1998). TGF-β, BMP, and Nodal receptors make use of different sets of Smads. Upon ligand stimulation, different R-Smads bind to common mediator Smad4 (Co-Smad) to form activated Smad complex that is then translocated into the nucleus to regulate transcription of target genes (Massague and Chen, 2000; Massague and Wotton, 2000). The third subclass of Smads is comprised of inhibitory Smads (I-Smads), consisting of Smad6 and Smad7 in vertebrates. I-Smads inhibit TGF-β signaling through binding to activated type I receptors and competing with R-Smads for receptor interaction, and by recruiting the receptor for degradation (Hayashi et al, 1997; Imamura et al, 1997; Nakao et al, 1997; Hata et al, 1998). Many ubiquitin ligases have been implicated in fine-tuning the levels of TGF-β signaling components including receptors, R-Smads and I-Smads. Smurf-1, an HECT type E3 ligase, was shown to bind to and cause the ubiquitin-mediated degradation of Smad1 and Smad5 (Zhu et al, 1999; Datto and Wang, 2005; Yamashita et al, 2005). In addition, Smurfs are recruited by inhibitory Smad6 and Smad7 to TGF-β and BMP receptors, resulting in the degradation of these receptors (Kavsak et al, 2000; Ebisawa et al, 2001; Suzuki et al, 2002; Murakami et al, 2003). However, a recent report shows that genetic disruption of the Smurf-1 gene does not alter the canonical Smad-mediated TGF-β or BMP signaling, but instead enhances the JNK MAPK cascade (Yamashita et al, 2005). Detailed analysis has shown that ubiquitination and degradation of MEKK2, an upstream kinase of JNK, was impaired in the Smurf1-deficient osteoblasts. Together with the finding that Smurf1 physically interacts with MEKK2, those observations suggest that Smurf is an E3 ligase for MEKK2 and that in Smurf−/− mice accumulated MEKK2 elevates JNK activity, at least in osteoblasts, leading to an age-dependent increase of bone mass seen in the mutant mice. Most recently, Ectodermin, another E3 ligase that possesses a RING finger on its N-terminal region, has been shown to play a crucial role in specification of the ectoderm by limiting the mesoderm-inducing activity of TGF-β by targeting Smad4 to ubiquitination and degradation (Dupont et al, 2005). Arkadia was originally identified through an insertional mutagenesis in mice. Mice with Arkadia disrupted exhibit abnormal formation of the mammalian organizer during early embryogenesis (Episkopou et al, 2001; Niederlander et al, 2001). In Arkadia mutant embryos, anterior structures such as midbrain and forebrains are lost by mid-neurula stages of development. Structurally, it contains a RING finger domain in its C-terminal region that is responsible for its E3 ligase activity. Based on a genetic crossmating between heterozygous Arkadia and Nodal mice, it was revealed that Arkadia plays a role in Nodal signaling, and that Arkadia depends on Nodal in the induction of nodes. It was later shown that Arkadia enhances TGF-β signaling by physically interacting with, and inducing polyubiquitination and subsequent degradation of, the inhibitory Smad7 (Koinuma et al, 2003). A remarkable feature in signaling transduction is that individual pathways rely on a group of proteins referred to as scaffolds. Major scaffolds include Axin in Wnt/β-catenin signaling (Salahshor and Woodgett, 2005), and JIP-1 in JNK MAP kinase signaling (Yasuda et al, 1999). These proteins are able to bind simultaneously to several components in the same signaling route, facilitating and augmenting specificity during signal transduction, presumably by changing conformation and providing molecular proximity towards one another. In the case of Axin in the Wnt pathway, in the absence of Wnt signal Axin interacts with APC, GSK3β, and casein kinase Iα to promote β-catenin phosphorylation by GSK3β, which leads to the degradation of β-catenin (Zeng et al, 1997; Hart et al, 1998; Peifer and Polakis, 2000; Liu et al, 2002). Whether such a scaffold exists in the TGF-β signaling pathway is unclear. SARA (Smad anchor for receptor activation) that binds to TGF-β receptors as well as Smad2/3 has been suggested to work as a scaffold protein to bring Smad substrates to the receptors and thus facilitate Smad activation (Tsukazaki et al, 1998). However, SARA is a membrane-bound protein mainly located in early endosomes, and is most likely confined to the perimembrane action (Hayes et al, 2002; Di Guglielmo et al, 2003). Here, we describe our finding that Axin is a major scaffold for TGF-β signaling. Remarkably, Axin interacts with not only Smad7 but also Arkadia. We also show that Axin2, which has been shown to be functionally equivalent to Axin (Chia and Costantini, 2005), also interacts with Arkadia and Smad7. Two-step co-immunoprecipitation experiment reveals that Arkadia, Axin, and Smad7 form a ternary complex. Axin enhances TGF-β signaling in an Arkadia-dependent manner. Axin sequesters Smad7 in the cytoplasm, where Arkadia facilitates Smad7 polyubiquitination and degradation. These data all suggest the possibility that Axin may well be a major scaffold in the TGF-β pathway, serving to promote Smad3 phosphorylation in response to TGF-β ligands (Furuhashi et al, 2001), and to downregulate negative factors such as Smad7. Results Identification of Arkadia and Smad7 as novel Axin-interacting proteins In the course to identify new Axin-interacting proteins that may cooperate or antagonize the scaffolding roles of Axin in multiple pathways, we had previously employed a yeast two-hybrid screen using the C-terminus of Axin as a bait, and identified a variety of important factors (Rui et al, 2002). One of the clones such identified (designated as AIP7 for Axin-Interacting Protein 7) encodes an amino-acid sequence corresponding to aa (amino acids) 83–433 of Arkadia (Figure 1A). To test for the interaction in mammalian cells between Axin and Arkadia in vivo, we first raised and affinity-purified the antibody against Arkadia (for characterization of the Arkadia antibody, see Supplementary Figure 1). We then carried out immunoprecipitation using the lysates from HEK293T cells, with the newly raised anti-Arkadia and the anti-Axin C2b antibody as described (Rui et al, 2004). As shown in Figure 1B, Axin was readily detected in the precipitate by anti-Arkadia, and Arkadia detected in the immunoprecipitate of Axin, indicating that Axin indeed interacts with Arkadia at their endogenous levels. Figure 1.Identification of Arkadia as a novel Axin-interacting protein. (A) Yeast two-hybrid screening using Axin C-terminal as a bait was described previously (Rui et al, 2002). AIP7, one of the identified clones, contains a cDNA insert corresponding to aa 83–433 of Arkadia, as diagrammed on the top. AH109 cells cotransformed with pACT2-AIP7 and pGBKT7-AxinCT, but not the others, could grow on Ade−/Leu−/His−/Trp− medium plates. (B) Arkadia interacts with Axin at its endogenous levels. The 293T cells were treated with MG132 (10 μM for 4 h) before harvest. Cell lysates were immunoprecipitated with rabbit anti-Arkadia, rabbit anti-Axin, and control rabbit IgG, respectively, followed by immunoblotting with their respective antibodies as indicated. (C) Axin and Arkadia form complex in 293T cells. FLAG-tagged Arkadia and HA-tagged Axin were transfected either alone or together into HEK293T cells. At 32 h post-transfection, cells were treated for 4 h with 10 μM MG132, and were then subjected to immunoprecipitation, followed by Western blotting analysis with anti-HA or anti-FLAG as indicated. (D) GST pulldown assay. GST-Axin-fusion protein was expressed in E. coli cells and was purified as described previously (Rui et al, 2004); it was added to the lysate of MG132-treated 293T cells ectopically expressing FLAG-Arkadia. Note that only GST-Axin but not GST could pull down Arkadia. Experiments were repeated with essentially the same results. Download figure Download PowerPoint We also carried out a reciprocal co-immunoprecipitation experiment using 293T cell lysates that contained ectopically expressed FLAG-tagged Arkadia and HA-tagged Axin (Figure 1C), and GST pulldown experiment (Figure 1D). The results also demonstrate that Axin and Arkadia strongly interact with each other. Since it has been reported that the inhibitory Smad, Smad7, is a substrate of ubiquitin ligase Arkadia (Koinuma et al, 2003), we wondered if Axin might serve as a scaffold to bring I-Smad to the proximity of the E3 ligase for ubiquitination, which in turn facilitates TGF-β signaling. In particular, Smad3 has been shown to interact with Axin (Furuhashi et al, 2001). We cotransfected Axin separately with Smad3, Smad7 as well as other different Smads (indicated in Figure 2A), and carried out co-immunoprecipitation using anti-FLAG and anti-HA, respectively, for Smads and Axin (Figure 2A, left and right panels). Indeed, Axin interacted with Smad7, as strongly as with Smad3 (Figure 2A). In addition, Axin also bound to Smad6, albeit to a lesser extent. Immunoprecipitation using endogenous proteins in 293T cells also indicated that Axin interacts with Smad7 (Figure 2B). Figure 2.Axin interacts with Smad7, and Axin, Arkadia, and Smad7 form a ternary complex. (A) Identification of Smad7 as another novel Axin-interacting protein. FLAG-tagged Smads 1–7 were separately cotransfected with HA-Axin into 293T cells. Reciprocal immunoprecipitation with anti-FLAG and anti-HA was carried out, followed by Western blotting analysis. Smad3, 6, and 7 (marked by asterisks) were co-precipitated with Axin. (B) Endogenous Axin and Smad7 interact with each other in 293T cells. The 293T cell lysate was incubated with goat Smad7 polyclonal antibody (Santa Cruz Biotech.), followed by Western blotting with rabbit AxinC2b polyclonal antibody. Three separate experiments were carried out and similar results were obtained. (C) Two-step co-immunoprecipitation to test for ternary complex formation of Axin, Arkadia, and Smad7. The procedures of two-step co-immunoprecipitation are outlined in the left box. HEK293T cells were transfected with Myc-Smad7, FLAG-ArC937A, and HA-Axin (or untagged Axin as control, marked by asterisk). The first immunoprecipitation was performed with anti-HA antibody. The complex was eluted by using HA peptide, followed by the second step of immunoprecipitation with anti-Myc or control mouse IgG. Protein samples from each step were then subjected to Western blotting analysis separately by using anti-Axin, anti-Myc, and anti-Arkadia antibodies. The experiment was repeated with essentially the same result. (D) Axin increases the interaction affinity of Arkadia for Smad7. FLAG-ArC937A and Myc-Smad7 were cotransfected with or without Axin into 293T cells. Immunoprecipitation was carried out with anti-FLAG, followed by immunoblotting with anti-Myc to detect Smad7, and anti-HA to detect Axin. Increased amount of Myc-Smad7 was co-immunoprecipitated with Arkadia from cells coexpressing Axin. Download figure Download PowerPoint Recently, it has been shown that Axin2/Conductin is functionally equivalent to Axin, at least as far as development is concerned (Chia and Costantini, 2005). This raised a critical question as to if Conductin can also facilitate TGF-β signaling. We generated the expression plasmid and carried out co-immunoprecipitation assay to address, first of all, whether Conductin also interacts with Smad7 and Arkadia. Indeed, Conductin was co-immunoprecipitated with Smad7 and Arkadia, underscoring the importance of Axin/Conductin functional linkage to the TGF-β pathway (see Supplementary Figure 2). Axin, Arkadia, and Smad7 form a ternary complex To further examine whether Axin, Arkadia, and Smad7 could form a ternary complex, we performed a two-step co-immunoprecipitation assay (Figure 2C) (Rui et al, 2004). As Arkadia is an E3 ubiquitin ligase, to prevent Arkadia-mediated protein degradation, an E3-defective mutant, ArC937A, was used in protein–protein interaction assay. HEK293T cells were transfected with HA–Axin, Myc-Smad7, and FLAG-ArC937A. As a control, Axin with no tag was transfected. In the first step of immunoprecipitation, anti-HA was used to pull down Axin, and HA peptide (Santa Cruz Biotech.) was used to elute the complex. The eluate was then immunoprecipitated with anti-Myc or control IgG, followed by Western blotting to detect Arkadia. As shown in Figure 2C, Arkadia was present in the final immunoprecipitate but not in the control sample, indicating that Axin, Smad7, and Arkadia are in a ternary complex. The observation that in the presence of overexpressed Axin higher levels of Myc-Smad7 were co-precipitated with FLAG-Arkadia is also consistent with a formation of the ternary complex, and indicates that Axin enhances the interaction of Arkadia with Smad7 (Figure 2D). Determination of interaction regions of Axin with Arkadia and Smad7 The above data indicated that Arkadia and Smad7 each physically interact with Axin. We then determined the amino-acid regions of the three proteins that are required for their mutual interactions. Expression vectors containing wild-type as well as their different truncation mutants are indicated schematically (on the top of each panel, Figure 3). When HA–Axin-M1 alone was transfected, it was not present in the anti-FLAG (ArC937A) immunoco-precipitate (Figure 3A, first lane); however, when cotransfected with FLAG-ArC937A, full-length Axin was strongly co-immunoprecipitated with Arkadia. Axin deletion mutants C1, N2, and M1 retained their ability to form a complex with Arkadia, albeit with lower affinity compared to full-length Axin. However, Axin deletion mutants N1, C2, and ΔAr lost their ability to interact with Arkadia, indicating that the region around aa 507–757 in Axin is critical for Arkadia interaction. Figure 3.Determination of domains for interactions between Axin and Arkadia, Smad7. Structures of deletion mutants of Axin, Arkadia, and Smad7 are shown on the top of each panel. Functional domains of each protein are indicated above the schema and relative positions of the remaining fragment(s) in each deletion mutant are numbered on the left. HEK293T cells were cotransfected with different plasmid constructs as indicated. At 36 h post-transfection, cells were lysed and immunoprecipited with respective antibodies, followed by Western blotting. Of note, none of tagged deletion mutants when expressed alone was immunoprecipitated by the antibody against the other tag epitope. (A) Mapping of the domain in Axin for interaction with Arkadia. Shown on the top are schematic diagrams of the different Axin constructs used. The region around aa 507–757 in Axin is critical for Arkadia interaction. (B) Identification of Axin-binding sites on Arkadia. The RING domain of Arkadia (aa 937–977) is indicated. The ligase-defective mutant of Arkadia, ArC937A, was created by altering cysteine 937 to alanine. The region spanning aa 241–404 of Arkadia is responsible for Axin interaction. (C) Determination of Smad7-binding sites in Axin. Axin-N3 containing the N-terminal 183 aa showed partial Smad7-binding activity as compared to wild-type Axin; Axin-C3 and Axin-M2 that contain different C-terminal regions each also possessed partial activity to bind Smad7. Axin-M4 containing both the N- and C-terminal region retained a Smad7-binding affinity comparable to that of the wild-type Axin. (D) The MH2 domain (aa 260–426) is indicated. Note that both the N- and C-terminal regions of Smad7 are involved in the interaction with Axin. Download figure Download PowerPoint We then cotransfected different deletion mutants of Arkadia with full-length Axin to 293T cells to determine the domain of Arkadia for Axin interaction. As shown in Figure 3B, the N1, C2, and ΔM1 deletion mutants could not interact with Axin, indicating that the region spanning aa 241–404 of Arkadia is critical for Axin interaction. To determine the mutual interaction domains between Axin and Smad7, different constructs of Axin as indicated (on the top of Figure 3C) were cotransfected with Smad7 into 293T cells. The N-terminal region of aa 1–183 weakly interacted with Smad7; C-terminal region of aa 506–757 alone also showed partial binding ability. Of note, as C3 and M2 exhibited similar binding affinity for Smad7, the DIX domain of Axin (aa 757–832) is dispensable for Smad7 interaction. These results demonstrate that Axin requires both its N-terminal region (aa 1–183) and C-terminal region of aa 507–757 for maximal interaction with Smad7. As shown in Figure 3D, among all the Smad7 mutants, only M1 that lacks both the N- and C-terminal regions failed to interact with Axin. Smad7 therefore appears to possess two domains for interaction with Axin, with either N- or MH2 domains alone capable of forming complex with Axin. It is interesting to note that both Axin and Smad7 require two regions for their interaction. Axin colocalizes with Arkadia and Smad7 Next, we asked if Axin is colocalized in the cell with Arkadia and Smad7. We transfected FLAG-tagged Arkadia into COS-7 cells alone or together with Axin. When expressed alone, Arkadia was mainly localized in the nucleus in the absence of TGF-β (Figure 4A), in agreement with a previous report (Koinuma et al, 2003); when the cells were treated with TGF-β, Arkadia appeared to translocate into the cytoplasm and was distributed in the whole cell. In contrast, when coexpressed with Axin, even in the absence of TGF-β, Arkadia was translocated into the cytoplasm and was colocalized with Axin (Figure 4A, lower two panels). Figure 4.Axin is constitutively colocalized with Arkadia or Smad7 and induces cytoplasmic translocation of Smad7. (A) Axin colocalized with Arkadia. COS7 cells were transfected with FLAG-Arkadia with or without Axin. Cells treated with or without TGF-β then fixed and stained as described in Materials and methods. Anti-FLAG staining for Arkadia (red), anti-Axin for Axin (green), and nuclear staining by Hoechst were performed. (B) Axin induces cytoplasmic translocation of Smad7 and colocalized with Smad7 in the cytoplasm. Compared to expression of Smad7 alone, in Axin-coexpressing cells, great majority of Smad7 was seen in the cytoplasm, indicating that Axin caused translocation of Smad7 into the cytoplasm. Most representative results from three rounds of staining are shown. Download figure Download PowerPoint Next, we performed similar experiments to see if Axin and Smad7 are also colocalized in the absence or presence of TGF-β. In the cells that were transfected with Smad7 alone, Smad7 was localized mainly in the nucleus when untreated (Figure 4B) (Itoh et al, 1998). In cells treated with TGF-β, Smad7 underwent nucleocytoplasmic shuttling to become mostly cytoplasmically localized, in accordance with the previous report (Itoh et al, 1998). When coexpressed with Axin, Smad7 was almost exclusively distributed in the cytoplasm and colocalized with Axin regardless of TGF-β treatment. These data suggest that Axin sequesters Smad7 in the cytoplasm. Axin enhances TGF-β signaling through Arkadia It was previously shown that Axin plays a positive role in TGF-β signaling by promoting Smad3 phosphorylation (Furuhashi et al, 2001). Our current finding suggested that Axin might regulate TGF-β signaling by an additional mean through complex formation with Arkadia and Smad7. To test this, we transfected the TGF-β-responsive luciferase reporter (12 × CAGA-Lux) in different combinations with Axin, AxinΔAr (incapable of interacting with Arkadia), Arkadia, and ArkadiaΔC (without the RING domain) into HepG2 and Mv1Lu/L17 cells. As expected, Axin and Arkadia each enhanced both basal and TGF-β-stimulated reporter activity in HepG2 and Mv1Lu/L17 cells (Figure 5A and B) (Furuhashi et al, 2001; Koinuma et al, 2003). When Arkadia and Axin were both expressed, a further increase of the reporter activity was observed in both of the cell lines. However, the Axin mutant AxinΔAr that lacks Arkadia-binding domain (and is hence also unable to bind Smad3 and is weak for Smad7 interaction) failed to enhance TGF-β signaling. Interestingly, ArkadiaΔC had a strong dominant-negative effect on Axin-induced TGF-β signaling. Of note, because Mv1Lu/L17 cells are null for TGF-β type I receptor (TβRI), a constitutively active TGF-β type I receptor (caTβRI) was cotransfected into Mv1Lu/L17 cells instead of treating cells with TGF-β ligand (Figure 5B). Figure 5.Axin enhances TGF-β-dependent transcriptional activity through Arkadia. 12 × CAGA-Lux reporter was cotransfected with different Axin or Arkadia constructs into HepG2, Mv1Lu/L17, and HEK293 cells as indicated. Cells were either treated with TGF-β (A, C) or cotransfected with caTβRI (B). Variation in transfection efficiency among samples was less than 10%
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