Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P 2 to LRP6 phosphorylation
2011; Springer Nature; Volume: 30; Issue: 8 Linguagem: Inglês
10.1038/emboj.2011.28
ISSN1460-2075
AutoresKristina Tanneberger, Astrid S. Pfister, Katharina Brauburger, Jean Schneikert, Michel V. Hadjihannas, Vı́tězslav Křı́ž, Gunnar Schulte, Vı́tězslav Bryja, Jürgen Behrens,
Tópico(s)Skin and Cellular Biology Research
ResumoArticle8 February 2011free access Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation Kristina Tanneberger Kristina Tanneberger Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Astrid S Pfister Astrid S Pfister Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Katharina Brauburger Katharina Brauburger Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Jean Schneikert Jean Schneikert Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Michel V Hadjihannas Michel V Hadjihannas Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Vitezslav Kriz Vitezslav Kriz Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Search for more papers by this author Gunnar Schulte Gunnar Schulte Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Vitezslav Bryja Vitezslav Bryja Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Search for more papers by this author Jürgen Behrens Corresponding Author Jürgen Behrens Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Kristina Tanneberger Kristina Tanneberger Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Astrid S Pfister Astrid S Pfister Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Katharina Brauburger Katharina Brauburger Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Jean Schneikert Jean Schneikert Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Michel V Hadjihannas Michel V Hadjihannas Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Vitezslav Kriz Vitezslav Kriz Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Search for more papers by this author Gunnar Schulte Gunnar Schulte Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden Search for more papers by this author Vitezslav Bryja Vitezslav Bryja Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic Search for more papers by this author Jürgen Behrens Corresponding Author Jürgen Behrens Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany Search for more papers by this author Author Information Kristina Tanneberger1, Astrid S Pfister1, Katharina Brauburger1, Jean Schneikert1, Michel V Hadjihannas1, Vitezslav Kriz2,3, Gunnar Schulte4, Vitezslav Bryja2,3 and Jürgen Behrens 1 1Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Erlangen, Germany 2Faculty of Science, Institute of Experimental Biology, Masaryk University, Brno, Czech Republic 3Department of Cytokinetics, Institute of Biophysics, Academy of Sciences of the Czech Republic, Brno, Czech Republic 4Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden *Corresponding author. Nikolaus-Fiebiger-Center, University Erlangen-Nürnberg, Glückstr. 6, 91054 Erlangen, Germany. Tel.: +49 9131 8529109; Fax: +49 9131 8529111; E-mail: [email protected] The EMBO Journal (2011)30:1433-1443https://doi.org/10.1038/emboj.2011.28 There is a Have you seen? (April 2011) associated with this Article. PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Phosphorylation of the Wnt receptor low-density lipoprotein receptor-related protein 6 (LRP6) by glycogen synthase kinase 3β (GSK3β) and casein kinase 1γ (CK1γ) is a key step in Wnt/β-catenin signalling, which requires Wnt-induced formation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2). Here, we show that adenomatous polyposis coli membrane recruitment 1 (Amer1) (also called WTX), a membrane associated PtdIns(4,5)P2-binding protein, is essential for the activation of Wnt signalling at the LRP6 receptor level. Knockdown of Amer1 reduces Wnt-induced LRP6 phosphorylation, Axin translocation to the plasma membrane and formation of LRP6 signalosomes. Overexpression of Amer1 promotes LRP6 phosphorylation, which requires interaction of Amer1 with PtdIns(4,5)P2. Amer1 translocates to the plasma membrane in a PtdIns(4,5)P2-dependent manner after Wnt treatment and is required for LRP6 phosphorylation stimulated by application of PtdIns(4,5)P2. Amer1 binds CK1γ, recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. Fusion of Amer1 to the cytoplasmic domain of LRP6 induces LRP6 phosphorylation and stimulates robust Wnt/β-catenin signalling. We propose a mechanism for Wnt receptor activation by which generation of PtdIns(4,5)P2 leads to recruitment of Amer1 to the plasma membrane, which acts as a scaffold protein to stimulate phosphorylation of LRP6. Introduction The Wnt/β-catenin signalling pathway regulates cell proliferation, differentiation and apoptosis, and has an important role during embryonic development, adult tissue homoeostasis and various diseases including cancer (Lustig and Behrens, 2003; Clevers, 2006). In the absence of extracellular Wnt ligands the levels of cytoplasmic β-catenin are kept low by the action of a multiprotein destruction complex that targets β-catenin for proteasomal degradation. The core components of this complex are the scaffold proteins Axin and its homologue Conductin (Axin2), the tumour suppressor adenomatous polyposis coli (APC) and glycogen synthase kinase 3β (GSK3β), which phosphorylates β-catenin and thereby earmarks it for ubiquitin-mediated degradation in the proteasome (MacDonald et al, 2009). The binding of Wnt ligands to the transmembrane receptors Frizzled (Fz) and low-density lipoprotein receptor-related protein 6 (LRP6) initiates a signalling cascade that results in the inhibition of β-catenin phosphorylation and degradation leading to the activation of β-catenin-dependent transcription (Huang and He, 2008; Angers and Moon, 2009; MacDonald et al, 2009). A key step after Wnt stimulation is the phosphorylation of the intracellular domain (ICD) of LRP6 at five reiterated PPPSPxS motifs and adjacent Ser/Thr clusters (Tamai et al, 2004; Davidson et al, 2005; Zeng et al, 2005; MacDonald et al, 2008; Supplementary Figure S7A). Phosphorylation at the PPPSPxS motifs (e.g., at Ser1490) is mediated by GSK3β, whereas the Ser/Thr clusters (e.g., at Thr1479) are phosphorylated by casein kinase 1γ (CK1γ) (Davidson et al, 2005; Zeng et al, 2005). The phosphorylated PPPSPxS motifs provide docking sites for Axin (Mao et al, 2001; Tamai et al, 2004; Davidson et al, 2005) and can directly inhibit the activity of GSK3β (Cselenyi et al, 2008; Piao et al, 2008; Wu et al, 2009). Phosphorylation of LRP6 by GSK3β requires binding of Dishevelled (Dvl) to Fz, which in turn leads to recruitment of the Axin/GSK3β complex (Zeng et al, 2008). In contrast, CK1γ is constitutively localized to the plasma membrane (Davidson et al, 2005). It was recently suggested that co-clustering of Fz-LRP6 receptors together with Axin and Dvl in so-called LRP6 signalosomes is involved in LRP6 phosphorylation (Bilic et al, 2007). Signalosome formation depends on the ability of Dvl to dynamically polymerize, which might provide a high density of phosphorylation sites for GSK3β and CK1γ (Bilic et al, 2007; Schwarz-Romond et al, 2007a, 2007b). Recent evidence indicates that the Wnt-induced generation of phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) at the plasma membrane is required for LRP6 phosphorylation by GSK3β as well as CK1γ and for signalosome formation (Pan et al, 2008). This process is mediated by Dvl, which binds and activates phosphatidylinositol 4-kinase type IIα (PI4KIIα) and phosphatidylinositol-4-phosphate 5-kinase type Iβ (PIP5KIβ) sequentially acting to produce PtdIns(4,5)P2 (Pan et al, 2008; Qin et al, 2009). Amer1 (APC membrane recruitment 1) was initially described by our group as an APC-binding protein, which can associate with the plasma membrane via two N-terminal PtdIns(4,5)P2-binding domains (Grohmann et al, 2007). Amer1 is identical to the tumour suppressor WTX (Wilms Tumour gene on the X chromosome) mutated in a significant fraction of Wilms tumours (Rivera et al, 2007) and in the inherited disease OSCS (osteopathia striata congenita with cranial sclerosis) (Jenkins et al, 2009). Amer1 is found in complexes with β-catenin and components of the β-catenin destruction machinery such as APC, Axin and β-TrCP, and can block canonical Wnt signalling by inducing proteasomal degradation of β-catenin (Major et al, 2007). Studies in Zebrafish and Xenopus also suggest a negative role of Amer1 in Wnt signalling (Major et al, 2007). Several key players involved in LRP6 phosphorylation have been identified (Fz, Dvl, Axin, GSK3β, CK1γ and PtdIns(4,5)P2), but a coherent picture of their interactions and the sequence of events are still missing. In particular, the mechanism by which Wnt-induced PtdIns(4,5)P2 formation results in LRP6 phosphorylation has remained elusive. In the present study, we identify an unexpected role of Amer1 as a positive regulator of Wnt signalling acting at the LRP6 receptor level by showing that Amer1 links Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation. Results Amer1 is required for Wnt-induced LRP6 phosphorylation To investigate whether Amer1 has a function in Wnt signalling at the receptor level, we knocked down its expression using two different siRNAs and analysed Wnt-induced phosphorylation of LRP6 in plasma membrane fractions by western blotting. We found that Amer1 knockdown prevented LRP6 phosphorylation at Ser1490 and Thr1479 in Wnt3A-treated HEK293T cells (Figure 1A and B; Supplementary Figure S1A). Amer1 knockdown also prevented LRP6 phosphorylation in SW480 colon carcinoma cells (Supplementary Figure S1B). Reciprocally, overexpression of Amer1 stimulated LRP6 phosphorylation at Ser1490 (Figure 1C; Supplementary Figure S1C). Notably, the previously described recruitment of Axin to the plasma membrane after Wnt stimulation was also abolished after knockdown of Amer1 (Figure 1A; Mao et al, 2001). These data show that Amer1 is essential for Wnt-induced LRP6 phosphorylation and Axin translocation to the plasma membrane and point to a positive role of Amer1 in the activation of Wnt signalling at the receptor level. Figure 1.Amer1 is required for Wnt-induced LRP6 phosphorylation and signalosome formation. (A, B) Amer1 is required for LRP6 phosphorylation at Ser1490 (A) and Thr1479 (B). HEK293T cells stably expressing LRP6-EGFP were transfected with the indicated siRNAs and incubated with Wnt3A for 1 h. Membrane fractions were analysed by western blotting. (C) Overexpression of Amer1 promotes LRP6 phosphorylation at Ser1490. HEK293T cells stably expressing VSVG-LRP6 were transiently transfected with EGFP-Amer1 and treated with Wnt3A for 20 min. Note that Amer1 is expressed in two splice variants represented by the two bands on the anti-GFP western blot (cf. Supplementary Figure S3A). In panels (A) and (B), the anti-Amer1 antibody detects only the larger splice variant of endogenous Amer1. (D, E) Amer1 is required for Wnt-induced signalosome formation. (D) Signalosome formation in HeLa cells co-expressing LRP6-EYFP and Fz8-EYFP transfected with the indicated siRNAs. Images show EYFP fluorescence with and without Wnt3A treatment for 1 h. Right-hand panels represent higher magnifications of the boxed regions from the left. Arrowheads point to signalosomes. Scale bar is 10 μm. (E) Quantification of signalosome formation in cells from (D). Error bars indicate s.e.m. from three independent experiments. Download figure Download PowerPoint Amer1 is required for Wnt-induced signalosome formation Given the role of Amer1 in Wnt receptor activation we analysed whether it is involved in signalosome formation. Signalosomes were detected by monitoring the aggregation of YFP-tagged LRP6 and Fz8 after Wnt3A stimulation. Indeed knockdown of Amer1 efficiently reduced formation of LRP6 signalosomes (Figure 1D and E). These results show that Amer1 is required for Wnt-induced signalosome formation and corroborate a role for Amer1 in the activation of the Wnt pathway at the receptor level. Membrane localization of Amer1 through PtdIns(4,5)P2 binding is required for its effect on LRP6 phosphorylation We next analysed whether LRP6 activation depends on plasma membrane localization of Amer1, which is mediated by two short domains in its N-terminus. These domains bind to PtdIns(4,5)P2 and are characterized by a high proportion of highly conserved lysine residues known for their capacity to interact with PtdIns(4,5)P2 (Kagan and Medzhitov, 2006; Grohmann et al, 2007; Supplementary Figure S2A). Mutation of seven of these lysine residues to alanine (Amer1(7μLys)) abolished interaction with PtdIns(4,5)P2 as well as membrane association of Amer1 (Supplementary Figure S2B and C). Importantly, this mutant was defective in stimulating LRP6 phosphorylation in HEK293T cells (Figure 2A, left panels). Because the Amer1(7μLys) mutant is enriched in the nucleus, this experiment does not rule out a role of Amer1 in the cytoplasm. Fusion of the Amer1(7μLys) mutant to a nuclear export sequence (NES-Amer1(7μLys)) led to cytoplasmic localization of Amer1, but did not restore its ability to stimulate LRP6 phosphorylation (Figure 2A, right panels; Supplementary Figure S2C). Together, these results show that PtdIns(4,5)P2-mediated membrane association of Amer1 is required for its effect on LRP6 phosphorylation and that cytoplasmic localization alone is not sufficient. Figure 2.Membrane localization of Amer1 through PtdIns(4,5)P2 binding is required for its effect on LRP6 phosphorylation. (A) N-terminal lysine mutants of Amer1 (EGFP-Amer1(7μLys), EGFP-NES-Amer1(7μLys)) lacking PtdIns(4,5)P2 binding and membrane association are deficient for LRP6 phosphorylation. HEK293T cells stably expressing VSVG-LRP6 were transiently transfected with EGFP-tagged Amer1 constructs as indicated and treated with Wnt3A for 20 min. For details on the constructs, see Supplementary Figure S2A–C. (B) Effect of specific knockdown of Amer1 splice variants Amer1-S1 and Amer1-S2 on LRP6 phosphorylation. HeLa cells transfected with the indicated Amer1 siRNAs were incubated with Wnt3A for 1 h and endogenous LRP6 was examined by western blotting (WB). Specific RT–PCR for expression of the splice variants is shown below. See also Supplementary Figure S3A–D. In (A) and (B) numbers below the lanes reflect relative levels of phosphorylated LRP6 (pS1490) normalized to LRP6 as determined by densitometry. Experiments were repeated at least three times. Download figure Download PowerPoint Amer1 is expressed in two splice isoforms termed Amer1-S1 or Amer1-S2, which differ by the presence or absence of amino acids 50–326, comprising a large part of the membrane association/PtdIns(4,5)P2-binding domain (Supplementary Figure S3A; Jenkins et al, 2009). While Amer1-S1, which binds to the plasma membrane, stimulated LRP6 phosphorylation, Amer1-S2 lacking the membrane association failed to do so (Supplementary Figure S3B and C). Importantly, specific knockdown of the Amer1-S1 isoform reduced Wnt-induced LRP6 phosphorylation, whereas knockdown of Amer1-S2 had no effect (Figure 2B; Supplementary Figure S3A and D). These data further support the importance of membrane localization of Amer1 for its effect on LRP6 phosphorylation. Wnt induces plasma membrane translocation of Amer1, which requires the formation of PtdIns(4,5)P2 Next, we studied whether activation of Wnt signalling alters the association of Amer1 with the plasma membrane and whether PtdIns(4,5)P2 is involved. Wnt3A treatment induced a rapid increase of endogenous Amer1 in the plasma membrane fraction of HEK293T cells, whereas total amounts of Amer1 in whole cell lysates were not altered (Figure 3A). To determine whether Wnt-induced association of Amer1 with the plasma membrane depends on the formation of PtdIns(4,5)P2 we knocked down PI4KIIα, which was shown to be essential for PtdIns(4,5)P2 synthesis after Wnt stimulation (Pan et al, 2008). Wnt-induced plasma membrane recruitment of Amer1 was strongly reduced when PI4KIIα was knocked down by two different siRNAs (Figure 3B; Supplementary Figure S4). Neomycin binds PtdIns(4,5)P2 and can block its interaction with proteins (Gabev et al, 1989; Pilot et al, 2006). Pre-treatment of cells with neomycin abolished Wnt-induced membrane association of Amer1 and at the same time strongly reduced LRP6 phosphorylation and Wnt-induced recruitment of Axin to the plasma membrane (Figure 3C). Together, these data demonstrate that Amer1 is translocated to the plasma membrane in a PtdIns(4,5)P2-dependent manner after Wnt stimulation. Figure 3.Wnt induces plasma membrane translocation of Amer1, which requires the formation of PtdIns(4,5)P2. (A) Wnt3A induces plasma membrane translocation of Amer1. HEK293T cells stably expressing LRP6-EGFP were incubated with Wnt3A for 1 h and then subjected to subcellular fractionation. Cytoplasmic fractions (C), membrane fractions (M) and whole cell lysates (WCL) were analysed by western blotting. α-Tubulin and LRP6 were used to mark cytoplasmic and membrane fractions, respectively. (B) Knockdown of PI4KIIα prevents Wnt-induced plasma membrane recruitment of Amer1. siRNA transfected HeLa cells were incubated with Wnt3A for 1 h and membrane fractions were analysed by western blotting. (C) Neomycin abolishes Wnt-induced membrane translocation of Amer1. HEK293T cells stably expressing VSVG-LRP6 were treated with 10 mM neomycin for 30 min before incubation with Wnt3A plus neomycin for 1 h and membrane fractions were analysed by western blotting. (D, E) Wnt decreases membrane dynamics of Amer1. HEK293 cells transiently transfected with EGFP-Amer1 were subjected to FRAP analysis. (D) Typical example of EGFP-Amer1 distribution and changes in EGFP fluorescence before and after bleaching. Scale bar is 10 μm. (E) Statistical analysis of FRAP experiments using cells pre-stimulated with PBS (control) or Wnt3A with or without 10 mM neomycin as indicated (N, number of cells analysed per condition). The graphs show mean values and s.e.m., and the best fitting curve model, which was used for calculation of the mobile pool of EGFP-Amer1 (% of fluorescence recovered) and of the recovery halftime (T1/2). Download figure Download PowerPoint Wnt3A decreases membrane dynamics of Amer1 In order to test for effects of Wnt stimulation on the membrane dynamics of Amer1, we employed fluorescence recovery after photobleaching (FRAP) methodology. Selected membrane regions of EGFP-Amer1-expressing cells were bleached by a laser pulse and the recovery of EGFP fluorescence was analysed for 150 s (see Figure 3D for a typical experiment). Pre-incubation with Wnt3A decreased the mobile pool of Amer1 from ∼74% (95% confidence interval: 73.1–75.1%) to 55% (95% confidence interval: 54.4–56.7%) and at the same time increased the halftime required for recovery of Amer1 from 13 to 18 s (Figure 3E; Supplementary Table). This indicates that Wnt signalling generates an immobile pool of Amer1 at the plasma membrane that does not readily exchange with neighbouring or cytoplasmic Amer1. Interestingly, pre-treatment of cells with neomycin restored recovery in the presence of Wnt3A suggesting that Wnt-induced changes in the mobility of membrane Amer1 are dependent on PtdIns(4,5)P2 (Figure 3E). Amer1 is required for PtdIns(4,5)P2-induced LRP6 phosphorylation We previously found that treatment of cells with ionomycin which activates phospholipase C and thereby leads to breakdown of PtdIns(4,5)P2 resulted in release of Amer1 from the plasma membrane (Varnai and Balla, 1998; Grohmann et al, 2007). Interestingly, ionomycin treatment abolished LRP6 phosphorylation after Wnt treatment (Figure 4A). In line, inhibition of PtdIns(4,5)P2 formation by knockdown of PI4KIIα prevents LRP6 phosphorylation after Wnt stimulation (Pan et al, 2008; Figure 4B). To analyse whether this is due to loss of Amer1 from the plasma membrane we asked whether tethering of Amer1 to the membrane independently of PtdIns(4,5)P2 would be able to restore LRP6 phosphorylation. Indeed, reduced LRP6 phosphorylation after knockdown of PI4KIIα was prevented by Amer1 when fused to the transmembrane domain from the low-density lipoprotein (LDL) receptor (Figure 4B; Zeng et al, 2005). Recent data demonstrate that carrier-mediated transfer of exogenous PtdIns(4,5)P2 lipids into cells enhances Wnt-induced LRP6 phosphorylation (Pan et al, 2008). We found that knockdown of Amer1 abolished the stimulation of LRP6 phosphorylation by PtdIns(4,5)P2 (Figure 4C). These data demonstrate that Amer1 mediates the effect of PtdIns(4,5)P2 on LRP6 phosphorylation. Figure 4.Amer1 is required for PtdIns(4,5)P2-induced LRP6 phosphorylation. (A) Ionomycin treatment prevents Wnt-induced LRP6 phosphorylation. HEK293T cells stably expressing VSVG-LRP6 were treated with Wnt3A in the presence or absence of 10 μM ionomycin for 30 min. (B) Amer1ΔN (amino acids 207–1135) linked to the transmembrane domain of the LDL receptor (RFP-TMD-Amer1ΔN) rescues phosphorylation of endogenous LRP6 after knockdown of PI4KIIα. HeLa cells stably expressing RFP or RFP-TMD-Amer1ΔN were transfected with the indicated siRNAs and incubated with Wnt3A for 1 h. The numbers below the lanes reflect relative levels of phosphorylated LRP6 (pS1490) normalized to LRP6 as determined by densitometry. Data are representative of four independent experiments. (C) Amer1 is required for LRP6 phosphorylation stimulated by PtdIns(4,5)P2. HEK293T cells stably expressing VSVG-LRP6 and transfected with the indicated siRNAs were treated with PtdIns(4,5)P2 for 10 min before Wnt3A conditioned medium was added for another 20 min (left). Quantification of LRP6 phosphorylation from four independent experiments (right). Statistical analysis was done using an unpaired Student's t-test. The P-value reflects statistically significant differences. The black bars represent the experiment shown on the left. Download figure Download PowerPoint Amer1 recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6 Our data show that Amer1 is required for Wnt-induced Axin translocation to the plasma membrane (see Figure 1A). Therefore, Amer1 might stimulate LRP6 phosphorylation through recruitment of Axin (Zeng et al, 2005, 2008). It is possible, however, that membrane association of Axin is a consequence rather than a cause of increased LRP6 phosphorylation induced by Amer1 because the phosphorylated PPPSPxS motifs in the cytoplasmic domain of LRP6 can serve as docking sites for Axin (Mao et al, 2001; Tamai et al, 2004; Davidson et al, 2005). To rule out this possibility, we treated cells with LiCl in order to inhibit GSK3β-mediated LRP6 phosphorylation and monitored Wnt-induced Axin translocation to the plasma membrane. LiCl treatment efficiently inhibited LRP6 phosphorylation but had no effect on Axin recruitment (Figure 5A). This shows that phosphorylation of LRP6 is not required for the association of Axin with the plasma membrane after Wnt treatment, suggesting that Amer1 promotes Axin translocation independently of prior LRP6 phosphorylation. We therefore analysed whether Amer1 can directly recruit Axin and the associated GSK3β. Amer1 formed endogenous complexes with Axin as shown by immunoprecipitation (Supplementary Figure S5A). In agreement with previous reports, Axin was diffusely distributed in the cytoplasm in a dotty pattern when exogenously expressed in MCF-7 cells (e.g., Schwarz-Romond et al, 2005; Figure 5B). In contrast, Axin was localized to the plasma membrane when Amer1 was expressed (Figure 5B; Supplementary Figure S5B). Similarly, endogenous Conductin was redistributed by Amer1 to the plasma membrane in SW480 colon carcinoma cells (Supplementary Figure S5C). Axin and Conductin were not redirected to the plasma membrane by Amer1(7μLys) mutants, indicating that membrane association of Amer1 is required (Supplementary Figure S5B and C). Importantly, in the presence but not in the absence of Axin Amer1 was also able to recruit GSK3β to the plasma membrane (Figure 5B). In line, GSK3β co-immunoprecipitated with Amer1 in the presence of wild-type Axin but not in the presence of a mutant that lacks GSK3β binding (AxinL396Q; Zeng et al, 2008), indicating that Axin proteins link GSK3β to Amer1 (Figure 5C). Together, these data suggest that Amer1 stimulates LRP6 phosphorylation by recruiting the Axin/GSK3β complex. In support of this, a C-terminal deletion mutant of Amer1 that retains Axin/Conductin binding (Amer1(2–601)) stimulated LRP6 phosphorylation whereas a mutant lacking the Axin/Conductin-binding region (Amer1(2–530)) failed to do so (Figure 5D; Supplementary Figure S6A–C). Figure 5.Amer1 recruits Axin and GSK3β to the plasma membrane and promotes complex formation between Axin and LRP6. (A) Inhibition of LRP6 phosphorylation by LiCl does not prevent Wnt-induced Axin translocation to the plasma membrane. HEK293T cells stably expressing EGFP-LRP6 were incubated with 50 mM LiCl for 30 min before Wnt3A treatment for 1 h and membrane fractions were analysed by western blotting. (B, C) Amer1 associates with GSK3β and recruits it to the plasma membrane via the interaction with Axin. (B) MCF-7 cells were co-transfected as indicated above the panels. Expressed proteins were detected by CFP and YFP fluorescence and anti-Flag immunofluorescence. Scale bar is 20 μm. (C) GSK3β co-immunoprecipitates with EGFP-Amer1 in the presence of Flag-Axin but not Flag-AxinL396Q, which is defective in GSK3β binding. (D) The binding of Amer1 to Axin/Conductin is required for its effect on LRP6 phosphorylation. HEK293T cells stably expressing VSVG-LRP6 were transiently transfected with EGFP or EGFP-tagged Amer1 mutants as detailed in Supplementary Figure S6A–C. The numbers below the lanes reflect relative levels of phosphorylated LRP6 (pS1490) normalized to LRP6 as determined by densitometry. Data are representative of four independent experiments. (E, F) Amer1 interacts with LRP6. (E) Co-immunoprecipitation of LRP6 with EGFP-Amer1 upon transient transfection of HEK293T cells stably expressing VSVG-LRP6. (F) Co-immunoprecipitation of endogenous Amer1 and LRP6 from lysates of HEK293T cells. Immunoprecipitations were performed with mouse anti-Amer1 or control IgG antibodies. (G) Amer1 links Axin to LRP6. VSVG-LRP6 stably expressed in HEK293T cells co-immunoprecipitated with Flag-Axin in the presence but not in the absence of EGFP-Amer1. (H) Amer1 interacts with CK1γ. Co-immunoprecipitation of endogenous CK1γ and Amer1 from lysates of SW480 cells. Immunoprecipitations were performed with mouse anti-Amer1 or control IgG antibodies. As CK1γ levels in lysates were very low, immunoprecipitation with anti-CK1γ antibodies is shown. Download figure Download PowerPoint Next, we analysed whether Amer1 binds to LRP6 by co-immunoprecipitation experiments. We found that Amer1 and LRP6 form complexes after overexpression and at the endogenous level (Figure 5E and F). Amer1 also co-immunoprecipitated with LRP6 lacking the extracellular domain (LRP6ΔE(1–4); Supplementary Figure S7A and B). Immunoprecipitation with serial LRP6 C-terminal deletion mutants (Davidson et al, 2005) showed that Amer1 interacts with a fragment retaining the membrane proximal PPPSPxS motif and flanking Ser/Thr clusters (LRP6ΔE(1–4)Δ87; Supplementary Figure S7A and B). Deletion of the PPPSPxS motif abolished the interaction (LRP6ΔE(1–4)Δ127). Amer1 did not interact with a fragment consisting only of the PPPSPxS motif and Ser/Thr clusters, indicating that these motifs are not sufficient for Amer1 binding (LDLRΔN-miniC; Supplementary Figure S7A and B). Moreover, alanine substitutions of serines and threonines in the PPPSPxS motifs (LRP6m10; Zeng et al, 2005) did not affect Amer1 binding to LRP6 (data not shown). Together, these data demonstrate that Amer1 binds close to the signalling motifs phosphorylated by GSK3β and CK1γ, but that phosphorylation of these motifs is not required for Amer1 binding. Deletion analysis of Amer1 demonstrated that both central and C-terminal parts interact with LRP6, suggesting that there are multiple LRP6 interaction sites in Amer1 (data not shown). Axin is suggested to form a complex with LRP6 (Mao et al, 2001; Tamai et al, 2004; Davidson et al, 2005). Because A
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