Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes

Canonical Wnt signaling requires inhibition of Glycogen Synthase Kinase 3 (GSK3) activity, but the molecular mechanism by which this is achieved remains unclear. Here, we report that Wnt signaling triggers the sequestration of GSK3 from the cytosol into multivesicular bodies (MVBs), so that this enzyme becomes separated from its many cytosolic substrates. Endocytosed Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition, endogenous GSK3 activity decreased in the cytosol, and GSK3 became protected from protease treatment inside membrane-bounded organelles. Cryoimmunoelectron microscopy showed that these corresponded to MVBs. Two proteins essential for MVB formation, HRS/Vps27 and Vps4, were required for Wnt signaling. The sequestration of GSK3 extended the half-life of many other proteins in addition to β-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3 phosphorylation sites. We conclude that multivesicular endosomes are essential components of the Wnt signal-transduction pathway.

[1]  R. Nusse,et al.  The Wnt signaling pathway in development and disease. , 2004, Annual review of cell and developmental biology.

[2]  W. Hob.  Catecholamine Secretion from Digitonin-treated Adrenal Medullary Chromaffin Cells , 2001 .

[3]  Jean Gruenberg,et al.  The biogenesis of multivesicular endosomes , 2004, Nature Reviews Molecular Cell Biology.

[4]  T. Sun,et al.  Involvement of Vps33a in the Fusion of Uroplakin‐Degrading Multivesicular Bodies with Lysosomes , 2009, Traffic.

[5]  J. Hurley,et al.  Molecular Mechanism of Multivesicular Body Biogenesis by ESCRT Complexes , 2010, Nature.

[6]  P. Lemaire,et al.  Expression cloning of Siamois, a xenopus homeobox gene expressed in dorsal-vegetal cells of blastulae and able to induce a complete secondary axis , 1995, Cell.

[7]  Xi He,et al.  Wnt/beta-catenin signaling: components, mechanisms, and diseases. , 2009, Developmental cell.

[8]  D. Kimelman,et al.  β-Catenin destruction complex: insights and questions from a structural perspective , 2006, Oncogene.

[9]  Dianqing Wu,et al.  GSK3: a multifaceted kinase in Wnt signaling. , 2010, Trends in biochemical sciences.

[10]  P. Cohen,et al.  Glycogen synthase kinase-3 from rabbit skeletal muscle. , 2005, Methods in enzymology.

[11]  P. Woodman,et al.  ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking. , 2000, Molecular biology of the cell.

[12]  Nam‐Gyun Kim,et al.  Identification of targets of the Wnt pathway destruction complex in addition to β-catenin , 2009, Proceedings of the National Academy of Sciences.

[13]  J. Slot,et al.  Improving structural integrity of cryosections for immunogold labeling , 1996, Histochemistry and Cell Biology.

[14]  B. Deurs,et al.  Rab7: a key to lysosome biogenesis. , 2000, Molecular biology of the cell.

[15]  Chika Yokota,et al.  Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions , 2007, Development.

[16]  Leszek Rychlewski,et al.  ELM server: a new resource for investigating short functional sites in modular eukaryotic proteins , 2003, Nucleic Acids Res..

[17]  Tony Hunter,et al.  Activation of protein kinase C decreases phosphorylation of c-Jun at sites that negatively regulate its DNA-binding activity , 1991, Cell.

[18]  M. Overduin,et al.  The DIX domain targets dishevelled to actin stress fibres and vesicular membranes , 2002, Nature.

[19]  Christof Niehrs,et al.  Wnt Induces LRP6 Signalosomes and Promotes Dishevelled-Dependent LRP6 Phosphorylation , 2007, Science.

[20]  P. De Camilli,et al.  Dynamin at actin tails , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[21]  H. Clevers.  Wnt/beta-catenin signaling in development and disease. , 2006, Cell.

[22]  R. Pagano,et al.  Glycosphingolipids Internalized via Caveolar-related Endocytosis Rapidly Merge with the Clathrin Pathway in Early Endosomes and Form Microdomains for Recycling* , 2003, The Journal of Biological Chemistry.

[23]  S. Cohen,et al.  Hormone receptor topology and dynamics: morphological analysis using ferritin-labeled epidermal growth factor. , 1979, Proceedings of the National Academy of Sciences of the United States of America.

[24]  D. M. Ferkey,et al.  GBP, an Inhibitor of GSK-3, Is Implicated in Xenopus Development and Oncogenesis , 1998, Cell.

[25]  Ajamete Kaykas,et al.  Zebrafish Prickle, a Modulator of Noncanonical Wnt/Fz Signaling, Regulates Gastrulation Movements , 2003, Current Biology.

[26]  M. Boutros,et al.  Cell cycle control of wnt receptor activation. , 2009, Developmental cell.

[27]  R. Holz,et al.  Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. , 1983, The Journal of biological chemistry.

[28]  P. Dolan,et al.  The Low Density Lipoprotein Receptor-related Protein 6 Interacts with Glycogen Synthase Kinase 3 and Attenuates Activity* , 2006, Journal of Biological Chemistry.

[29]  Mariann Bienz,et al.  Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization , 2010, Journal of Cell Science.

[30]  M. Gonzalez-Gaitan,et al.  Directional Delta and Notch trafficking in Sara endosomes during asymmetric cell division , 2009, Nature.

[31]  D. M. Ferkey,et al.  Glycogen Synthase Kinase-3β Mutagenesis Identifies a Common Binding Domain for GBP and Axin* , 2002, The Journal of Biological Chemistry.

[32]  N. Pedersen,et al.  Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5 , 2009, Histochemistry and Cell Biology.

[33]  M. Wehrli,et al.  Endocytic trafficking of Wingless and its receptors, Arrow and DFrizzled-2, in the Drosophila wing. , 2006, Developmental biology.

[34]  P. Lawrence,et al.  Distribution of the wingless gene product in drosophila embryos: A protein involved in cell-cell communication , 1989, Cell.

[35]  A. Eychène,et al.  GSK-3-mediated phosphorylation enhances Maf-transforming activity. , 2007, Molecular cell.

[36]  P. Cohen,et al.  The renaissance of GSK3 , 2001, Nature Reviews Molecular Cell Biology.

[37]  P. Greengard,et al.  GSK-3-selective inhibitors derived from Tyrian purple indirubins. , 2003, Chemistry & biology.

[38]  Sudha Singh,et al.  Rab5‐mediated endosome–endosome fusion regulates hemoglobin endocytosis in Leishmania donovani , 2003, The EMBO journal.

[39]  S. Emr,et al.  Receptor downregulation and multivesicular-body sorting , 2002, Nature Reviews Molecular Cell Biology.

[40]  B. Wang,et al.  Shaggy/GSK3 antagonizes Hedgehog signalling by regulating Cubitus interruptus , 2002, Nature.

[41]  M. Taira,et al.  Secreted Frizzled-related proteins enhance the diffusion of Wnt ligands and expand their signalling range , 2009, Development.

[42]  Brian P Ceresa,et al.  Rab7 Regulates Late Endocytic Trafficking Downstream of Multivesicular Body Biogenesis and Cargo Sequestration* , 2009, Journal of Biological Chemistry.

[43]  M. Komada,et al.  Balanced ubiquitylation and deubiquitylation of Frizzled regulate cellular responsiveness to Wg/Wnt , 2010, The EMBO journal.

[44]  J. Abreu,et al.  Inhibition of GSK3 Phosphorylation of β-Catenin via Phosphorylated PPPSPXS Motifs of Wnt Coreceptor LRP6 , 2009, PloS one.

[45]  I. Weissman,et al.  Wnt proteins are lipid-modified and can act as stem cell growth factors , 2003, Nature.

[46]  Hans Clevers,et al.  The Xenopus Wnt effector XTcf-3 interacts with Groucho-related transcriptional repressors , 1998, Nature.

[47]  M. Lindsay,et al.  Localization of phosphatidylinositol 3‐phosphate in yeast and mammalian cells , 2000, The EMBO journal.

[48]  Akira Kikuchi,et al.  Caveolin Is Necessary for Wnt-3a-Dependent Internalization of LRP6 and Accumulation of β-Catenin , 2006 .

[49]  E. Robertis,et al.  Regulation of ADMP and BMP2/4/7 at Opposite Embryonic Poles Generates a Self-Regulating Morphogenetic Field , 2005, Cell.

[50]  Christof Niehrs,et al.  Regulation of Lrp6 phosphorylation , 2010, Cellular and Molecular Life Sciences.

[51]  Harold E. Varmus,et al.  Glycogen synthase kinase-3 and dorsoventral patterning in Xenopus embryos , 1995, Nature.

[52]  Xi He,et al.  A mechanism for Wnt coreceptor activation. , 2004, Molecular cell.

[53]  H. Bellen,et al.  Internalization is required for proper Wingless signaling in Drosophila melanogaster , 2006, The Journal of cell biology.

[54]  J. Gruenberg,et al.  Molecular assemblies and membrane domains in multivesicular endosome dynamics. , 2009, Experimental cell research.

[55]  R. Moon,et al.  Analysis of the Signaling Activities of Localization Mutants of β-Catenin during Axis Specification in Xenopus , 1997, The Journal of cell biology.

[56]  F. McCormick,et al.  Differential Regulation of Glycogen Synthase Kinase 3β by Insulin and Wnt Signaling* , 2000, The Journal of Biological Chemistry.

[57]  C. Cruciat,et al.  Requirement of Prorenin Receptor and Vacuolar H+-ATPase–Mediated Acidification for Wnt Signaling , 2010, Science.

[58]  T. Dale,et al.  An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts. , 1998, Analytical biochemistry.

[59]  J. Slot,et al.  Cryosectioning and immunolabeling , 2007, Nature Protocols.

[60]  Randall T. Moon,et al.  Proximal events in Wnt signal transduction , 2009, Nature Reviews Molecular Cell Biology.

[61]  Nam-Chul Ha,et al.  Direct Inhibition of GSK3β by the Phosphorylated Cytoplasmic Domain of LRP6 in Wnt/β-Catenin Signaling , 2008, PloS one.

[62]  R. Jope,et al.  The glamour and gloom of glycogen synthase kinase-3. , 2004, Trends in biochemical sciences.

[63]  Ethan Lee,et al.  LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin , 2008, Proceedings of the National Academy of Sciences of the United States of America.

[64]  Andrew M. Jenkinson,et al.  Ensembl 2009 , 2008, Nucleic Acids Res..

[65]  R. Nusse,et al.  BMC Cell Biology BioMed Central Research article , 2006 .

[66]  E. Robertis,et al.  Integrating Patterning Signals: Wnt/GSK3 Regulates the Duration of the BMP/Smad1 Signal , 2007, Cell.

[67]  P. Cohen,et al.  Glycogen synthase kinase-3 from rabbit skeletal muscle. Separation from cyclic-AMP-dependent protein kinase and phosphorylase kinase. , 1980, European journal of biochemistry.