Interaction of Axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription
1999; Springer Nature; Volume: 18; Issue: 10 Linguagem: Inglês
10.1093/emboj/18.10.2823
ISSN1460-2075
AutoresMatthew J. Smalley, Elizabeth Sara, Hugh Paterson, Stuart Naylor, David P. Cook, Hiran Jayatilake, Lee G.D. Fryer, Lisa Hutchinson, Michael Fry, Trevor Dale,
Tópico(s)Kruppel-like factors research
ResumoArticle17 May 1999free access Interaction of Axin and Dvl-2 proteins regulates Dvl-2-stimulated TCF-dependent transcription Matthew J. Smalley Matthew J. Smalley Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Elizabeth Sara Elizabeth Sara Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Hugh Paterson Hugh Paterson Section of Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Stuart Naylor Stuart Naylor Oxford Biomedica, Oxford Science Park, Oxford, OX4 4GA UK Search for more papers by this author David Cook David Cook Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Hiran Jayatilake Hiran Jayatilake Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Lee G. Fryer Lee G. Fryer Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Lisa Hutchinson Lisa Hutchinson Signal Transduction Teams, Section of Cell Biology and Experimental Pathology, Toby Robins Breakthrough Breast Cancer Research Centre, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Michael J. Fry Michael J. Fry Signal Transduction Teams, Section of Cell Biology and Experimental Pathology, Toby Robins Breakthrough Breast Cancer Research Centre, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Trevor C. Dale Corresponding Author Trevor C. Dale Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Matthew J. Smalley Matthew J. Smalley Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Elizabeth Sara Elizabeth Sara Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Hugh Paterson Hugh Paterson Section of Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Stuart Naylor Stuart Naylor Oxford Biomedica, Oxford Science Park, Oxford, OX4 4GA UK Search for more papers by this author David Cook David Cook Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Hiran Jayatilake Hiran Jayatilake Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Lee G. Fryer Lee G. Fryer Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Lisa Hutchinson Lisa Hutchinson Signal Transduction Teams, Section of Cell Biology and Experimental Pathology, Toby Robins Breakthrough Breast Cancer Research Centre, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Michael J. Fry Michael J. Fry Signal Transduction Teams, Section of Cell Biology and Experimental Pathology, Toby Robins Breakthrough Breast Cancer Research Centre, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Trevor C. Dale Corresponding Author Trevor C. Dale Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK Search for more papers by this author Author Information Matthew J. Smalley1, Elizabeth Sara1, Hugh Paterson2, Stuart Naylor3, David Cook1, Hiran Jayatilake1, Lee G. Fryer1, Lisa Hutchinson4, Michael J. Fry4 and Trevor C. Dale 1 1Developmental Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK 2Section of Cell and Molecular Biology, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK 3Oxford Biomedica, Oxford Science Park, Oxford, OX4 4GA UK 4Signal Transduction Teams, Section of Cell Biology and Experimental Pathology, Toby Robins Breakthrough Breast Cancer Research Centre, Chester Beatty Laboratories, Institute of Cancer Research, 237 Fulham Road, London, SW3 6JB UK ‡M.J.Smalley, E.Sara and H.Paterson contributed equally to this work *Corresponding author. E-mail: [email protected] The EMBO Journal (1999)18:2823-2835https://doi.org/10.1093/emboj/18.10.2823 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Axin promotes the phosphorylation of β-catenin by GSK-3β, leading to β-catenin degradation. Wnt signals interfere with β-catenin turnover, resulting in enhanced transcription of target genes through the increased formation of β-catenin complexes containing TCF transcription factors. Little is known about how GSK-3β-mediated β-catenin turnover is regulated in response to Wnt signals. We have explored the relationship between Axin and Dvl-2, a member of the Dishevelled family of proteins that function upstream of GSK-3β. Expression of Dvl-2 activated TCF-dependent transcription. This was blocked by co-expression of GSK-3β or Axin. Expression of a 59 amino acid GSK-3β-binding region from Axin strongly activated transcription in the absence of an upstream signal. Introduction of a point mutation into full-length Axin that prevented GSK-3β binding also generated a transcriptional activator. When co-expressed, Axin and Dvl-2 co-localized within expressing cells. When Dvl-2 localization was altered using a C-terminal CAAX motif, Axin was also redistributed, suggesting a close association between the two proteins, a conclusion supported by co-immunoprecipitation data. Deletion analysis suggested that Dvl-association determinants within Axin were contained between residues 603 and 810. The association of Axin with Dvl-2 may be important in the transmission of Wnt signals from Dvl-2 to GSK-3β. Introduction The current model of the Wnt-signalling pathway suggests that in the absence of a Wnt signal, the serine/threonine kinase glycogen synthase kinase-3β (GSK-3β) phosphorylates β-catenin as part of a multiprotein complex which contains the GSK-3β, adenomatous polyposis coli (APC), Axin and β-catenin proteins. Phosphorylation targets β-catenin for ubiquitin-mediated degradation (Aberle et al., 1997; Orford et al., 1997). When the Wnt-signalling pathway is activated via ligand binding to Frizzled receptors, GSK-3β function is inhibited, and β-catenin accumulates and interacts with TCF/LEF-1 transcription factors to activate transcription from promoters containing TCF-binding sites (reviewed in Gumbiner, 1995; Peifer, 1995; Bienz, 1998; Dale, 1998). Although several lines of evidence support the role of Axin-APC-GSK-3β as a β-catenin turnover complex, little is known about the mechanisms by which upstream signals inhibit GSK-3β and/or Axin-GSK-3β function in β-catenin turnover. Genetic epistasis experiments (Hooper, 1994; Siegfried et al., 1994) suggest that GSK-3β down-regulation may be mediated by Dishevelled proteins (Dsh in Drosophila; XDsh in Xenopus; Dvl-1, -2 and -3 in mammalian systems). The Dvl family of proteins have no known enzymatic functions but do have several potential interaction motifs, namely an N-terminal DIX domain, a central PDZ domain and a C-terminal DEP domain (Klingensmith et al., 1994; Sussman et al., 1994; Theisen et al., 1994; Axelrod et al., 1998). Ectopic expression of Dsh activated Wnt-dependent processes in Xenopus and Drosophila systems (Sokol et al., 1995; Yanagawa et al., 1995). In Drosophila cells lines, the DIX and PDZ domains were shown to be required for Armadillo (β-catenin) stabilization, whereas deletion of the XDsh PDZ domain prevented XDsh-dependent axis duplication and generated a dominant negative protein that was able to interfere with endogenous pathways (Yanagawa et al., 1995; Sokol, 1996). The DEP domain was not required for β-catenin stabilization in Drosophila cl-8 cells (Yanagawa et al., 1995), but was required for the establishment of ommatidial polarity during Drosophila eye development (Boutros et al., 1998; Cooper and Bray, 1999). A role for the DEP domain in this process probably reflects its involvement in an alternative Wnt-dependent 'tissue-polarity' pathway that branches downstream of Dsh and involves Rho and Jnk (Strutt et al., 1997; Boutros et al., 1998). Interestingly, Dsh, but not DshΔDEP, was recruited to the cell membrane in a Frizzled-dependent manner when co-injected in Xenopus blastocoelar cells (Axelrod et al., 1998). Addition of soluble Wingless ligand to Drosophila cl-8 cells resulted in Dsh hyperphosphorylation and Armadillo stabilization. Overexpression of Dsh in the Drosophila S2 cell line also led to hyperphosphorylation and Armadillo stabilization. However, the role of Dsh phosphorylation in signalling is not yet clear, since Dfrz2 overexpression in S2 cells induced phosphorylation of endogenous Dsh without Armadillo stabilization (Yanagawa et al., 1995; Willert et al., 1997). At least one kinase which phosphorylates Dsh has been identified as casein kinase-2 (Willert et al., 1997). Axin was first identified as the product of the murine fused locus (Zeng et al., 1997) and is closely related to the Axil/Conductin protein described recently (Behrens et al., 1998; Yamamoto et al., 1998). Axin contains binding regions for several proteins (Figure 2), namely APC (RGS homology domain), GSK-3β, β-catenin, PP2Ac and Axin itself (DIX domain) (Ikeda et al., 1998; Kishida et al., 1998; Hsu et al., 1999). Three endpoint assays have been used to study Axin function: β-catenin stabilization; TCF-dependent transcription; and axis duplication in Xenopus. Figure 1.Diagrammatic representation of Axin fusion proteins and deletion constructs. Binding sites as defined by Hart et al. (1998), Ikeda et al. (1998) and Hsu et al. (1999) are illustrated as they correspond to the mouse Axin (Form 1 sequence co-ordinates). Form 2 contains a 36 amino acid insert shown by II*. Form 2 was not used in the studies presented here. Download figure Download PowerPoint Overexpression of wild-type Axin or Conductin suppressed an existing Wnt signal. In transcriptional assays, both wild-type Axin and Axin lacking the first 299 residues (including most of the RGS domain) inhibited Wnt-1-stimulated TCF-dependent transcription in 293 cells (Sakanaka et al., 1998). Overexpression of Axin or Conductin alone in APC-mutant SW480 cells reduced the constitutively high levels of β-catenin (Behrens et al., 1998; Nakamura et al., 1998) and resulted in the suppression of TCF-dependent transcription (Sakanaka et al., 1998). Expression of Axin or Conductin in Xenopus dorsal blastomeres prevented formation of the endogenous axis (Zeng et al., 1997; Behrens et al., 1998) in a process that could be relieved by co-expression of β-catenin, but not XWnt-8. This suggested that Axin functioned downstream of Wnt, but upstream of β-catenin (Zeng et al., 1997). Dorsal microinjection of murine Axin mRNA with a C-terminal truncation at residue 724 (lacking the DIX domain and part of the PP2Ac-binding domain) also blocked Xenopus endogenous axis formation (Itoh et al., 1998). Mutants of Axin were also shown to activate downstream Wnt-dependent processes in the absence of a Wnt signal. In contrast to mammalian studies, deletion of the Axin RGS or DIX domains in Xenopus resulted in active proteins able to induce an ectopic ventral axis (Zeng et al., 1997; Itoh et al., 1998). In Neuro 2A cells, deletion of the APC, GSK-3β and β-catenin binding regions from Conductin stabilized β-catenin (Behrens et al., 1998). In this paper we describe the use of a domain-deletion and domain-expression approach to study the relationship between Axin, Dvl-2 and GSK-3β. The results support a function for GSK-3β downstream of Dvl-2 in a mammalian system. Our data more precisely delineate the GSK-3β-binding domain of Axin and suggest a role for the N-terminal region, including the RGS motif, which is independent of APC. We show that the introduction of a mutation into the GSK-3β-binding domain of Axin prevents GSK-3β-Axin interaction and turns Axin into a transcriptional activator. Finally, we demonstrate that Axin and Dvl-2 can direct each other's cellular localization and co-immunoprecipitation data suggest a physical association between the two proteins. Results GSK-3β and Axin interfere with Dvl-2-dependent TCF transcription Expression of Wnt-1 in 293 cells (Sakanaka et al., 1998) and Rat-1 fibroblasts (Young et al., 1998) activated TCF-dependent transcription. To address whether Dvl-2 (the closest of the three mammalian homologues to Drosophila Dishevelled) activated the Wnt signal transduction pathway in mammalian cells, we used a transient-transfection reporter assay to measure TCF-dependent transcription. Fetal human kidney 293 epithelial cells were transfected with plasmids containing multimeric TCF- or mutant TCF-binding sites upstream of a basal c-fos-luciferase reporter gene (TOPFLASH and FOPFLASH; Korinek et al., 1997). As described previously, co-transfection of a dominantly active β-catenin mutant (ΔN-β-catenin) strongly increased transcription from TOPFLASH compared with FOPFLASH (data not shown). Ectopic expression of murine Dvl-2 stimulated TCF-dependent transcription in a concentration-dependent manner up to 50-fold within the range of protein expression levels tested (Figure 1A). Results were similar for hemagglutinin (HA)-tagged Dvl-2 (data not shown). Figure 2.GSK-3β and Axin inhibit Dvl-2 regulation of TCF-dependent transcription. (A) Concentration-dependent activation of transcription by Dvl-2 from a promoter containing multimerized TCF-binding sites. Fold activation is a ratio of expression from luciferase reporter constructs containing promoters with wild-type or mutant TCF-binding sites. Expression of Dvl-2 at each of the concentrations of transfected plasmid is illustrated by the accompanying immunoblot. (B) The effect of GSK-3βHA and mutants on Dvl-2 activated transcription. One hundred nanograms of each plasmid were transfected. The accompanying immunoblot shows the expression of the GSK-3β HA variants in the supernatant (S) or pellet (P) fractions. (C) Axin inhibition of Dvl-2-activated transcription. An immunoblot showing expression from the Flag-tagged Axin and Dvl-2 is shown. Download figure Download PowerPoint Genetic epistasis in Drosophila predicted that zw-3/sgg (GSK-3β) functions downstream of Dsh (Hooper, 1994; Siegfried et al., 1994). However, this has not been demonstrated directly in a mammalian system. We therefore tested whether overexpression of GSK-3β could block Dvl-2-activated, TCF-dependent transcription (Figure 1B). When Dvl-2 and wild-type GSK-3β (GSK-3β wt) were co-expressed, Dvl-2-activated TCF-dependent transcription was suppressed by up to 50%, supporting a role for GSK-3β function downstream from, or parallel and dominant to, that of Dvl-2. Co-expression of Dvl-2 with the kinase-dead GSK-3β K85M mutant had no effect on Dvl-2-activated TCF-dependent transcription, supporting the current model of Wingless signalling that the kinase activity of GSK-3β may be required to inhibit TCF-dependent transcription. When Dvl-2 was co-expressed with a constitutively active mutant variant of GSK-3β, which had the regulatory serine at position 9 mutated to an alanine (GSK-3β S9A), transcription was suppressed by >80%. Phosphorylation of Ser9 by factors such as PKB and p90rsk-1 has been shown to inhibit GSK-3β activity in other systems (Stambolic and Woodgett, 1994; Cross et al., 1995). None of these GSK-3β constructs had any effect on TCF-dependent transcription when expressed alone (Figure 1B). Interestingly, although the wild-type GSK-3β and GSK-3β S9A proteins appeared to be distributed equally between the pellet and supernatant fractions, the GSK-3β K85M was found principally in the supernatant (Figure 1B). As Axin has been shown to inhibit Wnt-1-induced TCF-dependent transcription (Sakanaka et al., 1998), we investigated whether it could also block Dvl-2-activated TCF-dependent transcription. We used form 1 Axin, which lacks the residues 36 amino acids found in form 2 (Figure 2; Zeng et al., 1997). As shown in Figure 1C, full length, Flag-tagged murine Axin [mFlagAx-(1-956)] inhibited Dvl-2-activated, TCF-dependent transcription in a dose-dependent manner, but did not alter basal levels of TCF-dependent transcription (Figure 3B, second bar). To localize the regions of Axin that interfered with TCF-dependent transcription, we expressed a series of Flag-epitope-tagged Axin deletion proteins (Figure 2; results summarized in Table I). With the exception of mFlagAx-(Δ662-723), the truncated Axin proteins had a reduced ability to interfere with Dvl-2-activated transcription when compared with the wild type. An N-terminal Axin deletion, lacking the APC-binding region [mFlagAx-(351-956)], was a weak inhibitor of Dvl-2-activated transcription. This result contrasted with a similar N-terminal deletion of Axin (residues 1-288, including part of the RGS domain), which resulted in a protein with an increased ability to interfere with Wnt-1-dependent transcription in 293 cells (Sakanaka et al., 1998). mFlagAx-(351-956) showed no significant transcriptional activation when expressed alone (Figure 3A, second bar), in contrast to studies reported by Itoh et al. (1998) where a deletion of the RGS domain generated a dominantly active protein in Xenopus studies. A C-terminally deleted Axin lacking the DIX domain [mFlagAx-(1-810)] had little or no effect on TCF-dependent transcription, neither interfering with HA-Dvl-2-activated transcription nor acting as a transcriptional activator (Figure 3B, bars three to five; Table I). In contrast, C-terminal deletions (of residues 725-992 and 812-992 of form 2 Axin, both including the DIX domain) did not interfere with the ability of Axin to prevent endogenous axis formation in Xenopus (Itoh et al., 1998). Figure 3.TCF-dependent transcriptional regulation by truncated Axin proteins with and without the L521P mutation. (A) N-terminal truncation, mFlagAx-(351-956). (B) C-terminal truncation mFlagAx-(1-811). Fold activation as described in the legend to Figure 1. Expression of transfected constructs is shown by immunoblot against Flag epitope. Download figure Download PowerPoint Table 1. Summary of activity of Axin proteins Construct Activity Activation Dvl-signal inhibition Co-localization FlagAx-(1-956) − strong yesb FlagAx-(1-956)-L521P +++ nt nt FlagAx-(351-956) − weak yesb FlagAx-(351-956)-L521P ++++ nt nt FlagAx-(602-956) − − yesb FlagAx-(811-956) − − no FlagAx-(867-956) − − nt FlagAx-(1-810) − nt yes FlagAx-(1-810)-L521P ++ nt yes FlagAx-(1-477) + − no FlagAx-(368-701) + − nt FlagAx-(368-701)-L521P − nt no FlagAx-(501-560) ++++ − nt FlagAx-(501-560)-L521P − − nt FlagAx-(560-622) − −a nt FlagAx-(622-701) − − nt FlagAx-(196-354) − −a nt FlagAx-(Δ662-723) − strong yes Activation assays: −, no activation relative to TCF only control; +, 2- to 5-fold activation; ++, 5- to 10-fold activation; +++, 12- to 20-fold activation; ++++, 20- to 40-fold activation. Two methods were used to allow cross-comparison. First, activation was compared relative to Δ-Nβ-catenin, which was used as a positive control in most TCF-transcription assays. Secondly, protein expression from activating constructs was cross-compared within a single TCF-transcription assay (data not shown). a Co-operated with Dvl-2 to increase TCF-dependent activity. Co-localization: b Assayed with both HA-Dvl2 and HA-Dvl2-CAAX. nt, not tested. Two regions of Axin behaved as activators of TCF-dependent transcription in our assays, mFlagAx-(1-477) and mFlagAx-(368-701) (Figure 4A; Table I). The N-terminal mFlagAx-(1-477) region lacked all recognized binding sites except for the RGS domain and might therefore function by titrating APC. The central mFlagAx-(368-701) region lacked both the APC-binding region and the DIX domain but contained the GSK-3β- and β-catenin-binding regions. The mFlagAx-(1-810) protein, which contained both the mFlagAx-(1-477) and mFlagAx-(368-701) regions, might have been expected to be a stronger activator than either alone. However, this was not the case (Table I), possibly because mFlagAx-(1-810) contained all the regions implicated in β-catenin turnover and may have retained residual ability to prevent β-catenin stabilization. Figure 4.Activation of TCF-dependent transcription by regions of Axin. (A) FlagAx-(368-701)-dependent activation of TCF-dependent transcription. Immunoblot analysis revealed a band of the expected size at 64 kDa, together with a smaller band at 50 kDa. (B) FlagAx-(501-560) strongly activated TCF-dependent transcription, whereas co-expression of GSK-3β reversed the induction. Immunoblots show GSK-3HA and FlagAx-(501-560) expression. Fold activation is as described in the legend to Figure 1. (C) Stabilization of endogenous β-catenin following transfection of FlagAx-(501-560) into Neuro 2A cells. (i) Flag detection; (ii) β-catenin detection. Download figure Download PowerPoint Expression of the conserved RGS [mFlagAx-(196-354)] or DIX domains [mFlagAx-(903-956)], or expression of the DIX plus PP2Ac-binding domains [mFlagAx-(602-956)] failed to activate or interfere with HA-Dvl-2-activated TCF-dependent transcription at a range of expression levels (Table I). No single recognizable domain of Axin was found to be sufficient to interfere with Dvl-2-activated transcription. However, two proteins were found to activate transcription when expressed alone, the central region of Axin, containing the GSK-3β- and β-catenin-binding domains, being the most active. A 59 amino acid region from Axin activates TCF-dependent transcription To localize the central region of Axin that activated TCF-dependent transcription, we made three epitope-tagged deletion proteins based on secondary structure predictions. Expression of a 59 amino acid region [mFlagAx-(501-560)] containing a putative coiled-coil motif (SMART program; Schultz et al., 1998), strongly activated TCF-dependent transcription (Figure 4B). Expression of the β-catenin-binding region mFlagAx-(560-622) and mFlagAx-(622-701) failed to activate TCF-dependent transcription and did not interfere with Dvl-2-induced transcription (Table I). As an independent confirmation that mFlagAx-(501-560) was acting to stimulate TCF-dependent transcription through endogenous components of the Wnt-signalling pathway, we expressed the protein in Neuro 2A cells and demonstrated that cells expressing mFlagAx-(501-560) accumulated high levels of nuclear β-catenin (Figure 4C; Behrens et al., 1998). The mFlagAx-(501-560) sequence is contained within a conserved GSK-3β-binding region in rat Axin, as defined by Ikeda et al. (1998), corresponding to the murine Axin residues 478-562. This contains no consensus GSK-3β phosphorylation sites, yet GSK-3β kinase activity has been shown to be required for binding to Axin (Ikeda et al., 1998). When lysates of cells expressing mFlagAx-(501-560) were immunoprecipitated with anti-Flag antibodies, endogenous GSK-3β was co-precipitated with mFlagAx-(501-560), showing that the 59 residues of Axin were sufficient for GSK-3β interaction (Figure 5A). One mechanism by which mFlagAx-(501-560) could function as an activator of TCF-dependent transcription could be the titration of GSK-3β from endogenous Axin complexes, thus preventing β-catenin turnover. A prediction from this model would be that mFlagAx-(501-560) activation of TCF-dependent transcription should be titrated by GSK-3β. Indeed, increasing HA-GSK-3β levels inhibited mFlagAx-(501-560)-induced TCF-dependent transcription in co-transfection experiments, suggesting that mFlagAx-(501-560) activation of TCF-dependent transcription was dependent on its interaction with GSK-3 (Figure 4B). Figure 5.The L521P mutation prevents GSK-3β association with Axin. (A) Immunoprecipitation of mFlagAx-(501-560) wild-type L521 or mutant P521 proteins; probed for endogenous GSK-3β. The top band in the third and fourth lanes (upper panel) is mouse immunoglobulin used in the immunoprecipitation. (B) Immunoprecipitation of wild-type L521 or mutant P521 variants of both mFlagAx-(1-956) and mFlagAx-(1-956) proteins; probed for endogenous GSK-3β. (C) Regulation of TCF-dependent transcription by mFlagAx-(501-560) and mFlagAx-(501-560)-L521P. (D) Activation of TCF-dependent transcription by mFlagAx-(1-956)-L521P. Fold activation is as described in the legend to Figure 1. Download figure Download PowerPoint To confirm that the ability of mFlagAx-(501-560) to activate TCF-dependent transcription was dependent on its ability to bind GSK-3β, a leucine→proline mutation was introduced into the putative hydrophobic interface of the coiled-coil domain at position 521. An analogous mutation was previously shown to interfere with coiled-coil interactions between yeast MATa1 and MATα2 proteins (Johnson et al., 1998). In mFlagAx-(501-560), this substitution prevented GSK-3β binding (Figure 5A) and also changed the mobility and stability of the protein (Figure 5A and C). The P521 variant of mFlagAx-(501-560) migrated at a size consistent with its theoretical molecular weight both in transfected cells and in in vitro translations (data not shown). The L521P mutation did not affect the stability of the other Flag-Axin proteins into which it was introduced (Figure 5B; also Figure 3A and B). mFlagAx-(501-560)-L521P was unable to activate TCF-dependent transcription (Figure 5C), possibly because of its lower levels of expression than the wild-type equivalent or due to its inability to bind GSK-3β. We favour the latter model principally for two reasons. First, mFlagAx-(501-560) could activate transcription by up to 20-fold when cells were transfected with as little as 5 μg DNA per well (one-tenth of the smallest amount shown in Figure 5C). Secondly, when the L521P mutation was introduced into the construct covering the central region of Axin, which contained the GSK-3β- and β-catenin-binding regions [mFlagAx-(368-701)], the protein failed to activate TCF-dependent transcription and lost the activating potential of its parent (Table I). However, unlike mFlagAx-(501-560)-L521P, there was no gross difference in the levels of expression of mFlagAx-(368-701)-L521P and its parent protein (data not shown). The L521P mutation transforms full-length Axin into a transcriptional activator Next we investigated the effect of the L521P mutation in other Axin constructs. Full-length Axin did not activate TCF-dependent transcription, but unexpectedly, the introduction of L521P into full-length Myc- or Flag-tagged murine Axin [mFlagAx-(1-956)] transformed it into a transcriptional activator (Figure 5D). An N-terminal deletion lacking the RGS domain but containing the L521P mutation [mFlagAx-(351-956)-L521P] and a C-terminal deletion construct, which removed the DIX domain and contained the L521P mutation [mFlagAx-(1-810)-L521P], were also activators of TCF-dependent transcription, whereas the parent proteins were inactive (Figure 3A and B). Immunoprecipitation experiments on lysates from cells transiently transfected with mFlagAx-(351-956), mFlagAx-(351-956)-L521P, mFlagAx-(1-810) or mFlagAx-(1-810)-L521P demonstrated that, as with mFlagAx-(501-560)-L521P, the mutation prevented mFlagAx-(351-956)-L521P and mFlagAx-(1-810)-L521P from interacting with endogenous GSK-3β (Figure 5B). Proteins with wild-type and mutant GSK-3β-binding sites were equally stable. In summary, we identified an activating function of Axin that does not require the RGS domain, GSK-3β binding or the DIX domain. We also demonstrated that the PP2Ac-binding region plus DIX domain [mFlagAx-(602-956)] were not sufficient to substitute for this activity (Table I). The data suggest that this activity is a function of the region lying between residues 351 and 810, and may require the interaction of multiple regions. Subcellular localization of full-length Axin and Axin mutants MDCK cells were microinjected with Flag-Axin constructs and the cells stained for expression after 2 h. At this time, full-length Flag-Axin [mFlagAx-(1-956)] was principally localized to a perinuclear region where it appeared to form irregular, non-vesicular masses (Figure 6A). To some extent this was dependent on the density of the cells and the time after microinjection, since Axin was also found localized adjacent to the plasma membrane in more densely packed cells and after longer periods of expression (data not shown). There was no cross-reactivity of the anti-Flag antibody or second antibody with endogenous or non-Flag-ta
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