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β-Arrestin Promotes Wnt-induced Low Density Lipoprotein Receptor-related Protein 6 (Lrp6) Phosphorylation via Increased Membrane Recruitment of Amer1 Protein*

  • Vítězslav Kříž
    Footnotes
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic

    Department of Cytokinetics, Institute of Biophysics, Academy of Science of the Czech Republic, 612 65 Brno, Czech Republic
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  • Vendula Pospíchalová
    Footnotes
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic
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  • Jan Mašek
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic

    Institute of Molecular Genetics, Academy of Science of the Czech Republic, 142 20 Prague, Czech Republic
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  • Michaela Brita Christina Kilander
    Affiliations
    Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
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  • Josef Slavík
    Affiliations
    Department of Toxicology, Pharmacology, and Immunotherapy, Veterinary Research Institute, 621 00 Brno, Czech Republic
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  • Kristina Tanneberger
    Affiliations
    Nikolaus-Fiebiger-Center, University of Erlangen-Nürnberg, 91054 Erlangen, Germany
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  • Gunnar Schulte
    Footnotes
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic

    Department of Physiology and Pharmacology, Karolinska Institutet, 171 77 Stockholm, Sweden
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  • Miroslav Machala
    Affiliations
    Department of Toxicology, Pharmacology, and Immunotherapy, Veterinary Research Institute, 621 00 Brno, Czech Republic
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  • Alois Kozubík
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic

    Department of Cytokinetics, Institute of Biophysics, Academy of Science of the Czech Republic, 612 65 Brno, Czech Republic
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  • Juergen Behrens
    Footnotes
    Affiliations
    Nikolaus-Fiebiger-Center, University of Erlangen-Nürnberg, 91054 Erlangen, Germany
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  • Vítězslav Bryja
    Correspondence
    To whom correspondence should be addressed: Institute of Experimental Biology, Faculty of Science, Masaryk University, Kotlářská 2, 611 37 Brno, Czech Republic. Tel.: 420-549-49-3291; Fax: 420-541-21-1214
    Affiliations
    Faculty of Science, Institute of Experimental Biology, Masaryk University, 611 37 Brno, Czech Republic

    Department of Cytokinetics, Institute of Biophysics, Academy of Science of the Czech Republic, 612 65 Brno, Czech Republic
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  • Author Footnotes
    * This work was supported by Czech Science Foundation Grants 204/09/0498, 204/09/J030, and GA13-32990S; a European Molecular Biology Organization (EMBO) installation grant; and Ministry of Education Youth and Sport of the Czech Republic Grant MSM0021622430. The collaboration between Masaryk University and Karolinska Institutet is supported by Project KI-MU Grant CZ.1.07/2.3.00/20.0180.
    This article contains supplemental Figs. 1 and 2.
    1 Both authors contributed equally to this work.
    2 Supported by a project co-financed by the European Social Fund and the state budget of Czech Republic “Postdoc I,” Grant CZ.1.07/2.3.00/30.0009.
    3 Supported by Knut and Alice Wallenberg Foundation Grant KAW2008.0149, Swedish Research Council Grants K2008-68P-20810-01-4 and K2008-68K-20805-01-4, and The Swedish Foundation for International Cooperation in Research and Higher Education (STINT).
    4 Supported by Deutsche Forschungsgemeinschaft Grant Be1550/6-1.
Open AccessPublished:November 21, 2013DOI:https://doi.org/10.1074/jbc.M113.498444
      β-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/β-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the β-arrestin function in Wnt/β-catenin signaling. We demonstrate that β-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. β-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that β-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that β-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIβ. Importantly, cells lacking β-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that β-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by β-arrestin- and Dishevelled-associated kinases.

      Introduction

      Wnt/β-catenin signaling plays a key role in homeostasis and embryonic development. Deregulation of Wnt/β-catenin pathway has been shown to cause pathophysiological conditions such as developmental abnormalities, tumorigenesis, or osteoporosis (
      • Clevers H.
      Wnt/β-catenin signaling in development and disease.
      ,
      • Logan C.Y.
      • Nusse R.
      The Wnt signaling pathway in development and disease.
      ). The Wnt/β-catenin cascade is activated by Wnts, which act as agonists for the class Frizzled (Fzd)
      The abbreviations used are:
      Fzd
      Frizzled
      Apc
      adenomatous polyposis coli
      β-arr
      β-arrestin
      CK1
      casein kinase 1
      CM
      conditioned medium
      DKO
      double knock-out
      Dvl
      Dishevelled
      EGFP
      enhanced green fluorescent protein
      FRAP
      fluorescence recovery after photobleaching
      GSK3β
      glycogen synthase kinase 3β
      ICD
      intracellular domain
      Lrp
      low density lipoprotein receptor-related protein
      MEF
      mouse embryonic fibroblast
      PI4KIIα
      phosphatidylinositol 4-kinase type IIα
      PIP5KIβ
      phosphatidylinositol-4-phosphate 5-kinase type Iβ
      PtdIns
      phosphatidylinositol
      PtdIns(4,5)P2
      phosphatidylinositol 4,5-bisphosphate
      TCL
      total cell lysate.
      receptors (
      • Schulte G.
      • Bryja V.
      The Frizzled family of unconventional G protein-coupled receptors.
      ). When extracellular Wnt is present, it links its receptor Fzd and its co-receptor low density lipoprotein receptor-related protein 5 or 6 (Lrp5/6). Fzds bind Dishevelled (Dvl), which is required for the Wnt-induced phosphorylation of the intracellular domain (ICD) of Lrp5/6 (
      • Bilic J.
      • Huang Y.L.
      • Davidson G.
      • Zimmermann T.
      • Cruciat C.M.
      • Bienz M.
      • Niehrs C.
      Wnt induces LRP6 signalosomes and promotes Dishevelled-dependent LRP6 phosphorylation.
      ,
      • Zeng X.
      • Huang H.
      • Tamai K.
      • Zhang X.
      • Harada Y.
      • Yokota C.
      • Almeida K.
      • Wang J.
      • Doble B.
      • Woodgett J.
      • Wynshaw-Boris A.
      • Hsieh J.C.
      • He X.
      Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via Frizzled, Dishevelled, and Axin functions.
      ). Dvl and two Dvl-associated kinases, phosphatidylinositol 4-kinase type IIα (PI4KIIα) and phosphatidylinositol-4-phosphate 5-kinase type Iβ (PIP5KIβ), which produce phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) in two sequential steps from phosphatidylinositol (PtdIns), were found to be crucial for Lrp5/6 phosphorylation (
      • Pan W.
      • Choi S.C.
      • Wang H.
      • Qin Y.
      • Volpicelli-Daley L.
      • Swan L.
      • Lucast L.
      • Khoo C.
      • Zhang X.
      • Li L.
      • Abrams C.S.
      • Sokol S.Y.
      • Wu D.
      Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation.
      ). PtdIns(4,5)P2 is recognized by Amer1/WTX, which links PtdIns(4,5)P2 production with the machinery phosphorylating Lrp5/6 (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ). Phosphorylated Lrp5/6 subsequently recruits Axin1 (
      • Tamai K.
      • Zeng X.
      • Liu C.
      • Zhang X.
      • Harada Y.
      • Chang Z.
      • He X.
      A mechanism for Wnt coreceptor activation.
      ), which is a key component of the β-catenin destruction complex formed by the scaffolding proteins Axin1 and Apc (adenomatous polyposis coli) and kinases GSK3β (glycogen synthase kinase 3β) and CK1α (casein kinase 1α). As a consequence, phosphorylation and subsequent proteosomal degradation of β-catenin are blocked, β-catenin is stabilized in the cytoplasm, and following translocation into the nucleus it drives transcription of Wnt-responsive genes in cooperation with the Tcf/Lef transcription factors (
      • Behrens J.
      • Jerchow B.A.
      • Würtele M.
      • Grimm J.
      • Asbrand C.
      • Wirtz R.
      • Kühl M.
      • Wedlich D.
      • Birchmeier W.
      Functional interaction of an Axin homolog, conductin, with β-catenin, APC, and GSK3β.
      ,
      • Kimelman D.
      • Xu W.
      β-Catenin destruction complex: insights and questions from a structural perspective.
      ,
      • MacDonald B.T.
      • Tamai K.
      • He X.
      Wnt/β-catenin signaling: components, mechanisms, and diseases.
      ).
      Previous studies by us and others have demonstrated the key role of β-arrestin (β-arr) scaffolding protein in Wnt signaling (for review, see Ref.
      • Schulte G.
      • Schambony A.
      • Bryja V.
      β-Arrestins: scaffolds and signalling elements essential for WNT/Frizzled signalling pathways?.
      ). β-Arrestins were shown to be required for Wnt/β-catenin signaling (
      • Chen W.
      • Hu L.A.
      • Semenov M.V.
      • Yanagawa S.
      • Kikuchi A.
      • Lefkowitz R.J.
      • Miller W.E.
      β-Arrestin1 modulates lymphoid enhancer factor transcriptional activity through interaction with phosphorylated Dishevelled proteins.
      ,
      • Bryja V.
      • Gradl D.
      • Schambony A.
      • Arenas E.
      • Schulte G.
      β-Arrestin is a necessary component of Wnt/β-catenin signaling in vitro and in vivo.
      ) as well as for branches of Wnt signaling, which do not activate β-catenin (collectively referred to as noncanonical Wnt pathways) (
      • Bryja V.
      • Schambony A.
      • Cajánek L.
      • Dominguez I.
      • Arenas E.
      • Schulte G.
      β-Arrestin and casein kinase 1/2 define distinct branches of noncanonical WNT signalling pathways.
      ,
      • Chen W.
      • ten Berge D.
      • Brown J.
      • Ahn S.
      • Hu L.A.
      • Miller W.E.
      • Caron M.G.
      • Barak L.S.
      • Nusse R.
      • Lefkowitz R.J.
      Dishevelled 2 recruits β-arrestin 2 to mediate Wnt5A-stimulated endocytosis of Frizzled 4.
      ,
      • Kim G.H.
      • Her J.H.
      • Han J.K.
      Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements.
      ,
      • Kim G.H.
      • Han J.K.
      Essential role for β-arrestin 2 in the regulation of Xenopus convergent extension movements.
      ). Whereas in noncanonical Wnt signaling β-arrestins regulate signal propagation via their well defined role in the clathrin-mediated receptor endocytosis (
      • Kim G.H.
      • Her J.H.
      • Han J.K.
      Ryk cooperates with Frizzled 7 to promote Wnt11-mediated endocytosis and is essential for Xenopus laevis convergent extension movements.
      ,
      • Kim G.H.
      • Han J.K.
      Essential role for β-arrestin 2 in the regulation of Xenopus convergent extension movements.
      ), the mechanism how β-arrestins control Wnt/β-catenin signaling, which does not depend on the clathrin-mediated endocytosis (
      • Kikuchi A.
      • Yamamoto H.
      • Sato A.
      Selective activation mechanisms of Wnt signaling pathways.
      ), is unclear.
      In the present study we aimed to elucidate the role of β-arrestins in the process of the Wnt/β-catenin pathway activation. We demonstrate that β-arrestin is a novel binding partner of Amer1/WTX/Fam123b (further referred to as Amer1). We show that β-arrestin binds PtdIns(4,5)P2-producing kinases PI4KIIα/PIP5KIβ and that it is required for PtdIns(4,5)P2-controlled membrane dynamics of Amer1 upon Wnt3a stimulation. This function of β-arrestin governs Wnt-induced Lrp6 phosphorylation. We propose that β-arrestins acts as a scaffold, which brings Amer1 physically close to the site of Wnt3a-induced PtdIns(4,5)P2 production.

      DISCUSSION

      In the present study we show for the first time that β-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of membrane recruitment and of the dynamics of Amer1. We propose that β-arrestin2 functionally links Fzd-associated (Fzd-Dvl-PI4KII-PIP5KI) and Lrp5/6-associated (Amer1-Axin-GSK3β-CK1γ) complexes, which are both required for the efficient downstream signaling induced by Wnt3a.
      Phosphorylation of Lrp5/6 represents a key event required for downstream signaling leading to the stabilization of β-catenin and subsequent Tcf/Lef-driven transcription (
      • Tamai K.
      • Zeng X.
      • Liu C.
      • Zhang X.
      • Harada Y.
      • Chang Z.
      • He X.
      A mechanism for Wnt coreceptor activation.
      ). The current model of Lrp5/6 phosphorylation pinpointed the key role of PtdIns(4,5)P2 as the required signal mediators, which transduce signal between Fzd/Dvl and Lrp6. Two Dvl-associated kinases, PI4KIIα and PIP5KIβ, which together in two sequential steps produce PtdIns(4,5)P2, were found to be crucial for Lrp5/6 phosphorylation (
      • Pan W.
      • Choi S.C.
      • Wang H.
      • Qin Y.
      • Volpicelli-Daley L.
      • Swan L.
      • Lucast L.
      • Khoo C.
      • Zhang X.
      • Li L.
      • Abrams C.S.
      • Sokol S.Y.
      • Wu D.
      Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation.
      ). We have shown recently that the scaffold protein Amer1 (
      • Grohmann A.
      • Tanneberger K.
      • Alzner A.
      • Schneikert J.
      • Behrens J.
      Amer1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.
      ), also known as WTX (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ), which was first described as the negative regulator of the Wnt signaling (
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ), facilitates Wnt3a-induced Lrp6 phosphorylation (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ). Amer1 is a PtdIns(4,5)P2-binding protein, which associates with PtdIns(4,5)P2 produced locally in the membrane. As a consequence of PtdIns(4,5)P2 production by Dvl-associated kinases Amer1 recruits Lrp6 kinases in the proximity of ICD of Lrp6 (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ). Phosphorylation of Lrp6 receptors then takes place in the five-times itinerated PPP(S/T)PXS motives in the Lrp6 ICD. Several kinases including GSK3β, CK1γ, MAPKs, cyclin-dependent kinase, or G protein-coupled receptor kinases phosphorylating Lrp6 have been identified (
      • Chen M.
      • Philipp M.
      • Wang J.
      • Premont R.T.
      • Garrison T.R.
      • Caron M.G.
      • Lefkowitz R.J.
      • Chen W.
      G protein-coupled receptor kinases phosphorylate LRP6 in the Wnt pathway.
      ,
      • Červenka I.
      • Wolf J.
      • Mašek J.
      • Krejci P.
      • Wilcox W.R.
      • Kozubík A.
      • Schulte G.
      • Gutkind J.S.
      • Bryja V.
      Mitogen-activated protein kinases promote WNT/β-catenin signaling via phosphorylation of LRP6.
      ,
      • Krejci P.
      • Aklian A.
      • Kaucka M.
      • Sevcikova E.
      • Prochazkova J.
      • Masek J.K.
      • Mikolka P.
      • Pospisilova T.
      • Spoustova T.
      • Weis M.
      • Paznekas W.A.
      • Wolf J.H.
      • Gutkind J.S.
      • Wilcox W.R.
      • Kozubik A.
      • Jabs E.W.
      • Bryja V.
      • Salazar L.
      • Vesela I.
      • Balek L.
      Receptor tyrosine kinases activate canonical WNT/β-catenin signaling via MAP kinase/LRP6 pathway and direct β-catenin phosphorylation.
      ,
      • Davidson G.
      • Wu W.
      • Shen J.
      • Bilic J.
      • Fenger U.
      • Stannek P.
      • Glinka A.
      • Niehrs C.
      Casein kinase 1γ couples Wnt receptor activation to cytoplasmic signal transduction.
      ,
      • Davidson G.
      • Shen J.
      • Huang Y.L.
      • Su Y.
      • Karaulanov E.
      • Bartscherer K.
      • Hassler C.
      • Stannek P.
      • Boutros M.
      • Niehrs C.
      Cell cycle control of Wnt receptor activation.
      ,
      • Zeng X.
      • Tamai K.
      • Doble B.
      • Li S.
      • Huang H.
      • Habas R.
      • Okamura H.
      • Woodgett J.
      • He X.
      A dual-kinase mechanism for Wnt co-receptor phosphorylation and activation.
      ); however, the relative contribution of individual kinases is still a matter of debate.
      Based on our data we propose a model of β-arrestin and Amer1 function in the Wnt3a-induced phosphorylation of Lrp6 (schematized in Fig. 8A). The model is based on our finding that β-arrestin can be either present in complex with Dvl or with Amer1. The interaction of Amer1 and β-arrestin takes place near the membrane and most importantly requires PtdIns-P2 and possibly other membrane lipids or membrane itself.
      Figure thumbnail gr8
      FIGURE 8Model: role of β-arrestin in Lrp6 phosphorylation. A, the data presented in FIGURE 1, FIGURE 2, FIGURE 3, FIGURE 4, FIGURE 5, FIGURE 6, FIGURE 7 support a model, where β-arrestin acts as a scaffold, which brings Amer1 close to the site of PIP2 production. A Wnt ligand activates pathway via Frizzled (FZD)/Dvl, which subsequently leads to the activation of PI4KIIα/PIP5KIβ kinases. We propose that the activation of Dvl and the initial production of PIP2 allow translocation of β-arrestin and PI4KIIα/PIP5KIβ toward Amer1-based Lrp6 phosphorylation complex composed of Amer1, Axin, and Lrp6-phosphorylating kinases CK1 and GSK3β. Local production of PIP2 then stabilizes the Lrp6 phosphorylation complex and feeds the phosphorylation process. B, the FoldIndex prediction shows that Amer1 is largely an intrinsically disordered protein (in red) where the domains required for binding of key proteins/metabolites required for Lrp6 phosphorylation do not overlap.
      The interaction of β-arrestin with Dvl seems to have higher affinity than the interaction of β-arrestin with Amer1 or PI4KII/PIP5KI kinases. As a consequence, overexpression of Dvl is able to efficiently disrupt the binding of β-arrestin with Amer1 and PI4KII/PIP5KI (see Figs. 2 and 7). It is known that the Wnt signaling cascade is induced by binding of Wnt to Fzd. This interaction subsequently triggers by an yet unidentified mechanism involving Dvl the activation of PtdIns(4,5)P2-producing kinases PI4KII and PIP5KI (
      • Pan W.
      • Choi S.C.
      • Wang H.
      • Qin Y.
      • Volpicelli-Daley L.
      • Swan L.
      • Lucast L.
      • Khoo C.
      • Zhang X.
      • Li L.
      • Abrams C.S.
      • Sokol S.Y.
      • Wu D.
      Wnt3a-mediated formation of phosphatidylinositol 4,5-bisphosphate regulates LRP6 phosphorylation.
      ).
      The production of PtdIns(4,5)P2 is required and further promotes the interaction of Amer1 and β-arrestin. We speculate that following the activation, Dvl undergoes a conformational change (induced either by posttranslational modifications or by recruitment of other proteins), which breaks the β-arrestin/Dvl interaction and allows the formation of the β-arrestin·Amer1 and β-arrestin·PI4KII·PIP5KI complexes. β-Arrestin thus acts as a switch, which translocates PtdIns(4,5)P2-producing kinases from Dvl toward the Lrp6-phosphorylating complex. This allows efficient phosphorylation of Lrp6 fed by the local production of PtdIns(4,5)P2 associated with β-arrestin. Indeed, direct measurements of PtdInsP and PtdInsP2 showed (i) a Wnt3a-induced increase in these PtdInsPs and (ii) an increase in the steady-state levels of PtdInsP in β-arrestin1/2 DKO MEFs. These data suggest that the defects in the dynamic Wnt-induced phosphorylation of PtdIns are compensated by increase in the level of PtdInsP. Of note, similar phenotype (lack of Wnt-induced dynamics accompanied by increased steady-state activation) has been observed in β-arrestin1/2 DKO MEFs for the levels of TOPFLASH activity and of phosphorylated Dvl (
      • Bryja V.
      • Gradl D.
      • Schambony A.
      • Arenas E.
      • Schulte G.
      β-Arrestin is a necessary component of Wnt/β-catenin signaling in vitro and in vivo.
      ).
      The known properties of Amer1 make the scenario schematized in Fig. 8A sterically possible. The individual regions of Amer1, which interact with PtdIns(4,5)P2 (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ), APC (
      • Grohmann A.
      • Tanneberger K.
      • Alzner A.
      • Schneikert J.
      • Behrens J.
      Amer1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.
      ), Axin (
      • Tanneberger K.
      • Pfister A.S.
      • Kriz V.
      • Bryja V.
      • Schambony A.
      • Behrens J.
      Structural and functional characterization of the Wnt inhibitor APC membrane recruitment 1 (Amer1).
      ), and β-arrestin (this study) do not overlap (Fig. 8B). The N-terminal part of Amer1 recognizes PtdIns(4,5)P2 and central regions interact with Lrp6 and Axin, whereas the very C-terminal region is responsible for the interaction with β-arrestin. Moreover, Amer1 is, based on the computer predictions, an intrinsically disordered protein with the lack of clearly defined secondary structure (Fig. 8B). This feature allows Amer1 to act as scaffold and to exist in numerous conformations depending on the individual binding partners.
      Amer1 has a dual role in Wnt/β-catenin signaling. It was first identified as a negative regulator of Wnt/β-catenin signaling, which interacts with the components of the destruction complex (Apc, Axin, GSK3β, CK1) (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ,
      • Grohmann A.
      • Tanneberger K.
      • Alzner A.
      • Schneikert J.
      • Behrens J.
      Amer1 regulates the distribution of the tumor suppressor APC between microtubules and the plasma membrane.
      ,
      • Major M.B.
      • Camp N.D.
      • Berndt J.D.
      • Yi X.
      • Goldenberg S.J.
      • Hubbert C.
      • Biechele T.L.
      • Gingras A.C.
      • Zheng N.
      • Maccoss M.J.
      • Angers S.
      • Moon R.T.
      Wilms tumor suppressor WTX negatively regulates WNT/β-catenin signaling.
      ). Amer1 was identified only recently also as a positive regulator of the Wnt/β-catenin signaling, which acts at the level of Lrp6 phosphorylation (
      • Tanneberger K.
      • Pfister A.S.
      • Brauburger K.
      • Schneikert J.
      • Hadjihannas M.V.
      • Kriz V.
      • Schulte G.
      • Bryja V.
      • Behrens J.
      Amer1/WTX couples Wnt-induced formation of PtdIns(4,5)P2 to LRP6 phosphorylation.
      ). It is of interest that its positive role seems to be limited to Amer1 and does not apply to related Amer2 (
      • Pfister A.S.
      • Tanneberger K.
      • Schambony A.
      • Behrens J.
      Amer2 protein is a novel negative regulator of Wnt/β-catenin signaling involved in neuroectodermal patterning.
      ), which lacks the C-terminal sequence (
      • Boutet A.
      • Comai G.
      • Schedl A.
      The WTX/Amer1 gene family: evolution, signature and function.
      ) required for the interaction with β-arrestin.
      In summary, in the present study we provide the so far missing molecular mechanism utilized by β-arrestin to positively regulate Wnt/β-catenin signaling. According to our data β-arrestin acts in the Wnt/β-catenin pathway via Amer1, which is a protein conserved only in vertebrates. This raises the possibility that the function of β-arrestin in the Wnt/β-catenin pathway evolved in parallel and will be limited to vertebrates. This is in agreement with the lack of Wnt/β-catenin-related phenotypes in the Drosophila β-arrestin homologue kurtz and the Caenorhabditis elegans homologue arr-1 mutants. Phosphorylation of Lrp6 via the β-arrestin/Amer1 pathway thus represents a mechanism for the efficient and tightly controlled activation of the Wnt/β-catenin pathway that evolved in vertebrates.

      Acknowledgments

      We thank P. De Camilli for PI4KII antibody and PI4KII/PIP5K plasmids; R. J. Lefkowitz for A1CT/A2CT antibodies, β-arrestin, and Dvl2 plasmids; M. Maurice, Randall Moon, V. Kořínek, J. Kukkonen, and S. Yanagawa for plasmids; and K. Souček for the NB4 cell line.

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