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• W2109955944 abstract "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. 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. Canonical Wnt signaling causes the sequestration of GSK3 inside endosomal vesicles Protease protection and cryoimmuno-EM show that GSK3 is sequestered inside MVBs The MVB-forming ESCRT components HRS/Vps27 and Vps4 are required for Wnt signaling In pulse-chase experiments overall cellular protein half-life is prolonged by Wnt3a Canonical Wnt signaling plays a crucial role in development, tissue regeneration, stem cells, and cancer (Logan and Nusse, 2004Logan C.Y. Nusse R. The Wnt signaling pathway in development and disease.Annu. Rev. Cell Dev. Biol. 2004; 20: 781-810Crossref PubMed Scopus (4221) Google Scholar, Clevers, 2006Clevers H. Wnt/beta-catenin signaling in development and disease.Cell. 2006; 127: 469-480Abstract Full Text Full Text PDF PubMed Scopus (4491) Google Scholar, MacDonald et al., 2009MacDonald B.T. Tamai K. He X. Wnt/β-Catenin signaling: components, mechanisms, and diseases.Dev. Cell. 2009; 17: 9-26Abstract Full Text Full Text PDF PubMed Scopus (4151) Google Scholar, Angers and Moon, 2009Angers S. Moon R.T. Proximal events in Wnt signal transduction.Nat. Rev. Mol. Cell Biol. 2009; 10: 468-477Crossref PubMed Scopus (3) Google Scholar). A cytoplasmic destruction complex consisting of Glycogen Synthase Kinase 3 (GSK3, which has α and β isoforms), Casein Kinase 1 (CK1), Adenomatous Polyposis Coli (APC), and Axin mediates the phosphorylation of β-Catenin. Phosphorylation targets β-Catenin for polyubiquitinylation and subsequent degradation in proteasomes. In the presence of Wnt, the destruction complex becomes inactivated in ways that are incompletely understood. Wnt triggers signaling by binding to Frizzled and LDL-receptor related protein 6 (LRP6), causing the aggregation of Dishevelled (Dvl) and Axin on the plasma membrane (Bilic et al., 2007Bilic 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.Science. 2007; 316: 1619-1622Crossref PubMed Scopus (692) Google Scholar, Zeng et al., 2008Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. Doble B. Woodgett J. et al.Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions.Development. 2008; 135: 367-375Crossref PubMed Scopus (353) Google Scholar). The key step in the canonical pathway is the inactivation of GSK3, as evidenced by the fact that pharmacological inhibition of this enzyme elicits a typical Wnt signal. The molecular mechanism of GSK3 inhibition remains one of the main open questions in the Wnt field (Wu and Pan, 2010Wu D. Pan W. GSK3: a multifaceted kinase in Wnt signaling.Trends Biochem. Sci. 2010; 35: 161-168Abstract Full Text Full Text PDF PubMed Scopus (603) Google Scholar). Internalization of receptor complexes is an absolute requirement for Wnt signaling (Blitzer and Nusse, 2006Blitzer J.T. Nusse R. A critical role for endocytosis in Wnt signaling.BMC Cell Biol. 2006; 7: 28Crossref PubMed Scopus (176) Google Scholar, Yamamoto et al., 2006Yamamoto H. Komekado H. Kikuchi A. Caveolin is necessary for Wnt-3a-dependent internalization of LRP6 and accumulation of beta-catenin.Dev. Cell. 2006; 11: 213-223Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar). Bilic et al., 2007Bilic 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.Science. 2007; 316: 1619-1622Crossref PubMed Scopus (692) Google Scholar discovered that cytoplasmic particles designated LRP6-signalosomes—containing aggregates of phospho-LRP6, Frizzled, Dvl, Axin, and GSK3—are formed at and under the plasma membrane 15 min after Wnt addition. Activated Wnt receptors recruit Axin and GSK3, which phosphorylates five critical PPPS/TP sequences in the intracellular domain of LRP6 (Zeng et al., 2008Zeng X. Huang H. Tamai K. Zhang X. Harada Y. Yokota C. Almeida K. Wang J. Doble B. Woodgett J. et al.Initiation of Wnt signaling: control of Wnt coreceptor Lrp6 phosphorylation/activation via frizzled, dishevelled and axin functions.Development. 2008; 135: 367-375Crossref PubMed Scopus (353) Google Scholar, Niehrs and Shen, 2010Niehrs C. Shen J. Regulation of Lrp6 phosphorylation.Cell. Mol. Life Sci. 2010; 67: 2551-2562Crossref PubMed Scopus (139) Google Scholar). A number of mechanisms have been proposed to explain the inhibition of GSK3 (Kimelman and Xu, 2006Kimelman D. Xu W. β-Catenin destruction complex: insights and questions from a structural perspective.Oncogene. 2006; 25: 7482-7491Crossref PubMed Scopus (490) Google Scholar). For example, the LRP6 tail may act as a direct inhibitor of GSK3 (Mi et al., 2006Mi K. Dolan P.J. Johnson G.V.W. The low density lipoprotein receptor-related protein 6 interacts with glycogen synthase kinase 3 and attenuates activity.J. Biol. Chem. 2006; 281: 4787-4794Crossref PubMed Scopus (80) Google Scholar, Cselenyi et al., 2008Cselenyi C.S. Jernigan K.K. Tahinci E. Thorne C.A. Lee L.A. Lee E. LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin.Proc. Natl. Acad. Sci. USA. 2008; 105: 8032-8037Crossref PubMed Scopus (162) Google Scholar, Piao et al., 2008Piao S. Lee S.H. Kim H. Yum S. Stamos J.L. Xu Y. Lee S.J. Lee J. Oh S. Han J.K. et al.Direct inhibition of GSK3β by the phosphorylated cytoplasmic domain of LRP6 in Wnt/β-Catenin signaling.PLoS ONE. 2008; 3: e4046Crossref PubMed Scopus (161) Google Scholar, Wu et al., 2009Wu G. Huang H. Garcia Abreu J. He X. Inhibition of GSK3 phosphorylation of β-Catenin via phosphorylated PPPSPXS motifs of Wnt coreceptor LRP6.PLoS ONE. 2009; 4: e4926Crossref PubMed Scopus (163) Google Scholar). The LRP6 PPPSP repeats serve both as substrates and binding sites for GSK3 and may act as competitive inhibitors of this enzyme, although at low affinity (Ki of 1.3 × 10−5 M; Cselenyi et al., 2008Cselenyi C.S. Jernigan K.K. Tahinci E. Thorne C.A. Lee L.A. Lee E. LRP6 transduces a canonical Wnt signal independently of Axin degradation by inhibiting GSK3's phosphorylation of β-catenin.Proc. Natl. Acad. Sci. USA. 2008; 105: 8032-8037Crossref PubMed Scopus (162) Google Scholar). GSK3 has many substrates in addition to β-Catenin, including Dvl, Axin, and APC (Jope and Johnson, 2004Jope R.S. Johnson G.V. The glamour and gloom of glycogen synthase kinase-3.Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). This promiscuous enzyme phosphorylates serine or threonine at position −4 of sites primed by phosphorylation (S/TXXXS/T[PO3]) (Cohen and Frame, 2001Cohen P. Frame S. The renaissance of GSK3.Nat. Rev. Mol. Cell Biol. 2001; 2: 769-776Crossref PubMed Scopus (1298) Google Scholar). We reported that the transcription factor Smad1 is polyubiquitinylated and degraded after GSK3 phosphorylation and is stabilized by canonical Wnt signaling, resulting in the integration of BMP and Wnt signaling (Fuentealba et al., 2007Fuentealba L.C. Eivers E. Ikeda A. Hurtado C. Kuroda H. Pera E.M. De Robertis E.M. Integrating patterning signals: Wnt/GSK3 regulates the duration of the BMP/Smad1 signal.Cell. 2007; 131: 980-993Abstract Full Text Full Text PDF PubMed Scopus (416) Google Scholar). Additional substrates destabilized by GSK3 phosphorylation have since been identified (Kim et al., 2009Kim N.G. Xu C. Gumbiner B.M. Identification of targets of the Wnt pathway destruction complex in addition to β-catenin.Proc. Natl. Acad. Sci. USA. 2009; 106: 5165-5170Crossref PubMed Scopus (71) Google Scholar). During our investigations on signaling integration, we measured GSK3 enzyme activity in Wnt-treated cell extracts (prepared in the presence of Triton X-100) and were surprised to find that Wnt did not change GSK3 activity (data not shown), even though in the direct GSK3 inhibition model one would have predicted inhibition. How could this be? Upon reflection, we realized that following ligand binding and endocytosis, growth factor receptors are incorporated into multivesicular endosomes within 15 min (Gruenberg and Stenmark, 2004Gruenberg J. Stenmark H. The biogenesis of multivesicular endosomes.Nat. Rev. Mol. Cell Biol. 2004; 5: 317-323Crossref PubMed Scopus (583) Google Scholar). Multivesicular body (MVB) formation is an obligatory step before degradation in lysosomes can take place (Katzmann et al., 2002Katzmann D.J. Odorizzi G. Emr S.D. Receptor downregulation and multivesicular-body sorting.Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1019) Google Scholar). As first discovered for EGF receptor, the topology is such that the cytoplasmic side of the plasma membrane corresponds to the lumen of the small MVB vesicles (McKanna et al., 1979McKanna J.A. Haigler H.T. Cohen S. Hormone receptor topology and dynamics: Morphological analysis using ferritin-labeled epidermal growth factor.Proc. Natl. Acad. Sci. USA. 1979; 76: 5689-5693Crossref PubMed Scopus (161) Google Scholar). Therefore, Wnt-induced MVB formation would cause GSK3 bound to phosphorylated LRP6 cytoplasmic tails (and other GSK3 substrates such as Axin, APC, β-Catenin, and Dvl) to become sequestered from its cytosolic substrates by two layers of membrane (see model in Figure 7), effectively inhibiting its activity. In this study, we tested the GSK3 sequestration hypothesis of Wnt signaling. Fluorescence microscopy showed that Wnt signaling caused the relocalization of cytoplasmic GSK3 to vesicles that colocalized with endocytosed xWnt8-Venus protein, and with the MVB and lysosomal markers Rab7 and LysoTracker. Wnt signal transduction was blocked by depletion of Hrs/Vps27 or expression of dominant-negative Vps4, two proteins essential for intralumenal vesicle formation in MVBs (Katzmann et al., 2002Katzmann D.J. Odorizzi G. Emr S.D. Receptor downregulation and multivesicular-body sorting.Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1019) Google Scholar, Wollert and Hurley, 2010Wollert T. Hurley J.H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.Nature. 2010; 464: 864-869Crossref PubMed Scopus (527) Google Scholar). Moreover, Wnt treatment decreased cytosolic GSK3 activity levels (measured in Digitonin-permeabilized cells), yet full enzyme activity was recovered after solubilization of all membranes with Triton X-100. Similarly, Wnt caused endogenous GSK3β to become partially protected from Proteinase K digestion, but only in the absence of Triton X-100. Finally, cryoimmunoelectron microscopy showed that GSK3 was indeed translocated from the cytosol into MVBs after Wnt pathway activation. Bioinformatic analyses revealed that 20% of the human proteome contains multiple putative GSK3 phosphorylation sites. Total protein half-life was extended by Wnt treatment or GSK3 inhibition. The addition of GSK3 phosphorylation sites was sufficient to place the stability of a Green Fluorescent Protein (GFP) biosensor under the control of Wnt. We conclude that canonical Wnt signaling sequesters GSK3 inside MVBs, reducing its cytosolic levels and extending the half-life of many GSK3 substrates. We first asked whether Wnt treatment changed the subcellular localization of GSK3. Human 293T cells expressing xWnt8-Venus (Mii and Taira, 2009Mii Y. Taira M. Secreted Frizzled-related proteins enhance the diffusion of Wnt ligands and expand their signaling range.Development. 2009; 136: 4083-4088Crossref PubMed Scopus (148) Google Scholar) were cultured together with mouse 3T3 cells transfected with GSK3-RFP. Remarkably, endocytosed Wnt-Venus and GSK3-RFP accumulated in the same vesicular structures (arrows in Figure 1F ), while GSK3-RFP levels decreased in the cytosol (Figures 1A–1F). Relocalization of endogenous GSK3β was also observed in these cocultures (Figures S1A–S1C available online). Wnt-Venus puncta were counted in each responding cell (n = 80), and 56% ± 9% of them colocalized with GSK3-RFP puncta (see histogram in Figure 1C′). Thus, cytosolic GSK3 decreases and becomes relocalized to the same endosomes as the internalized Wnt ligand.Figure S1Wnt8-Venus or CA-LRP6 Sequester GSK3 in Dynamin-Dependent Signalosomes; GSK3 Mutated in Its Axin-Binding Site Can Accumulate in MVBs, but Catalytically Inactive DN-GSK3 Does not, Related to Figure 1Show full caption(A) Control 3T3 cells (minus Wnt) stained with an anti-GSK3β antibody.(B) Coculture with 293T secreting Wnt8-Venus caused the relocalization of endogenous GSK3β in 3T3 cells that endocytosed Wnt. Arrows indicate Wnt8-Venus endosomes that sequestered endogenous GSK3β. Cytosolic levels of GSK3β were decreased with respect to control 3T3 cells minus Wnt, but the depletion was less compelling than that observed with GSK3-RFP in Figures 1A–1F.(C) A 293T cell strongly secreting Wnt8-Venus surrounded by 3T3 cells endocytosing the protein (arrows). Note that Wnt-Venus and GSK3β overlap in endosomes.(D–F) The GSK3 relocalization caused by CA-LRP6 is Dynamin-dependent. The homogenous cytoplasmic localization of GSK3-RFP became vesicular and concentrated close to the nucleus upon CA-LRP6 transfection. When cotransfected with DN-Dynamin, GSK3-RFP translocated to the membrane instead, and could not be further internalized into intracellular LRP6 signalosomes (arrows).(G–I) Wild-type GSK3-RFP colocalized with CA-LRP6-GFP signalosomes in 3T3 cells.(J–L) GSK3-RFP mutated so that its Axin-binding site was inactivated, GSK3-F291L (Ferkey and Kimelman, 2002Ferkey, D.M., and Kimelman, D. (2002). Glycogen synthase kinase 3b mutagenesis identifies a common binding for GBP and Axin. J. Biol. Chem. 277, 16147–16152.Google Scholar), was still sequestered from the cytoplasm by CA-LRP6-signalosomes (arrows). Colocalization was reduced in comparison to wild-type GSK3-RFP. This result indicates that binding of GSK3 to Axin is not essential for its localization within Wnt signaling endosomes.(M–O) Catalytically inactive DN-GSK3-RFP does not associate with CA-LRP6-GFP. This suggests that only active GSK3 molecules associate with CA-LRP6-induced MVBs via binding to its own substrates. To ensure a proper phosphorylation of CA-LRP6 by endogenous GSK3 and its subsequent internalization, DN-GSK3-RFP was transfected in relatively low concentrations in this experiment.View Large Image Figure ViewerDownload Hi-res image Download (PPT) (A) Control 3T3 cells (minus Wnt) stained with an anti-GSK3β antibody. (B) Coculture with 293T secreting Wnt8-Venus caused the relocalization of endogenous GSK3β in 3T3 cells that endocytosed Wnt. Arrows indicate Wnt8-Venus endosomes that sequestered endogenous GSK3β. Cytosolic levels of GSK3β were decreased with respect to control 3T3 cells minus Wnt, but the depletion was less compelling than that observed with GSK3-RFP in Figures 1A–1F. (C) A 293T cell strongly secreting Wnt8-Venus surrounded by 3T3 cells endocytosing the protein (arrows). Note that Wnt-Venus and GSK3β overlap in endosomes. (D–F) The GSK3 relocalization caused by CA-LRP6 is Dynamin-dependent. The homogenous cytoplasmic localization of GSK3-RFP became vesicular and concentrated close to the nucleus upon CA-LRP6 transfection. When cotransfected with DN-Dynamin, GSK3-RFP translocated to the membrane instead, and could not be further internalized into intracellular LRP6 signalosomes (arrows). (G–I) Wild-type GSK3-RFP colocalized with CA-LRP6-GFP signalosomes in 3T3 cells. (J–L) GSK3-RFP mutated so that its Axin-binding site was inactivated, GSK3-F291L (Ferkey and Kimelman, 2002Ferkey, D.M., and Kimelman, D. (2002). Glycogen synthase kinase 3b mutagenesis identifies a common binding for GBP and Axin. J. Biol. Chem. 277, 16147–16152.Google Scholar), was still sequestered from the cytoplasm by CA-LRP6-signalosomes (arrows). Colocalization was reduced in comparison to wild-type GSK3-RFP. This result indicates that binding of GSK3 to Axin is not essential for its localization within Wnt signaling endosomes. (M–O) Catalytically inactive DN-GSK3-RFP does not associate with CA-LRP6-GFP. This suggests that only active GSK3 molecules associate with CA-LRP6-induced MVBs via binding to its own substrates. To ensure a proper phosphorylation of CA-LRP6 by endogenous GSK3 and its subsequent internalization, DN-GSK3-RFP was transfected in relatively low concentrations in this experiment. Transfection of constitutively active LRP6 lacking its extracellular domain, designated CA-LRP6, causes a potent Wnt signal (Tamai et al., 2004Tamai K. Zeng X. Liu C. Zhang X. Harada Y. Chang Z. He X. A mechanism for Wnt coreceptor activation.Mol. Cell. 2004; 13: 149-156Abstract Full Text Full Text PDF PubMed Scopus (440) Google Scholar) . CA-LRP6 cytoplasmic signalosome formation (Bilic et al., 2007Bilic 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.Science. 2007; 316: 1619-1622Crossref PubMed Scopus (692) Google Scholar) required Dynamin and caused a striking relocalization of GSK3β from the cytosol into prominent cytoplasmic puncta (Figures 1G–1L′ and Figures S1D–S1O). Treatment of 3T3 cells with LysoTracker, a dye that becomes concentrated in acidic organelles such as MVBs and lysosomes, showed that endogenous GSK3β relocated to these compartments (Figure 1G–1L′). In addition, GSK3β colocalized with Dvl, Axin, and LysoTracker, as well as the PI3P probe FYVE-GFP, when Wnt signaling was activated by overexpression of Dvl (Figures S2A–S2O). Remarkably, a protein consisting of the DIX domain of Dvl fused to the LRP6 cytoplasmic tail (DIX > Ctail-GFP), which triggers a very strong Wnt signal (Metcalfe et al., 2010Metcalfe C. Mendoza-Topaz C. Mieszczanek J. Bienz M. Stability elements in the LRP6 cytoplasmic tail confer efficient signalling upon DIX-dependent polymerization.J. Cell Sci. 2010; 123: 1588-1599Crossref PubMed Scopus (81) Google Scholar), also formed signalosomes colocalizing with acidic vesicles that sequestered cytosolic GSK3β (Figures S2P–S2Aa). CA-LRP6 signalosomes colocalized with the late endosomal marker Rab7-GFP (Figures 1M–1R) in 62% ± 7% of the GSK3 puncta, indicating that GSK3-RFP relocated to MVBs or lysosomes (Bucci et al., 2000Bucci C. Thomsen P. Nicoziani P. McCarthy J. van Deurs B. Rab7: a key to lysosomes biogenesis.Mol. Biol. Cell. 2000; 11: 467-480Crossref PubMed Scopus (804) Google Scholar). About 40% of GSK3-RFP vesicles colocalized with Vps4-GFP (see Figures 3L–3N′), a marker of the final stages of intralumenal vesicle formation in endosomes (Bishop and Woodman, 2000Bishop N. Woodman P. ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking.Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (213) Google Scholar, Gruenberg and Stenmark, 2004Gruenberg J. Stenmark H. The biogenesis of multivesicular endosomes.Nat. Rev. Mol. Cell Biol. 2004; 5: 317-323Crossref PubMed Scopus (583) Google Scholar). In untransfected 3T3 cells the number and size of endogenous GSK3β puncta increased after treatment with Wnt3a conditioned medium (Figures 1S–1U) in cells permeabilized with Digitonin (which facilitates the visualization of intracellular organelles; Bishop and Woodman, 2000Bishop N. Woodman P. ATPase-defective mammalian VPS4 localizes to aberrant endosomes and impairs cholesterol trafficking.Mol. Biol. Cell. 2000; 11: 227-239Crossref PubMed Scopus (213) Google Scholar). Taken together, these results strongly support the hypothesis that Wnt signaling causes the relocalization of cytosolic GSK3 to the endosomal compartment. To determine whether Wnt3a treatment sequesters GSK3 kinase activity from cytosol, endogenous cytosolic kinase activity was measured in untransfected L cells permeabilized with Digitonin through the incorporation of phosphate from [γ32P]-ATP into a phospho-glycogen synthase peptide substrate (Ryves et al., 1998Ryves W.J. Fryer L. Dale T. Harwood A.J. An assay for glycogen synthase kinase 3 (GSK-3) for use in crude cell extracts.Anal. Biochem. 1998; 264: 124-127Crossref PubMed Scopus (67) Google Scholar). Digitonin solubilizes cholesterol-rich patches in the plasma membrane, leaving MVBs and other intracellular organelles intact (Dunn and Holz, 1983Dunn L.A. Holz R.W. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells.J. Biol. Chem. 1983; 258: 4989-4993Abstract Full Text PDF PubMed Google Scholar). Addition of Wnt3a for 4 hr decreased cytosolic GSK3 activity levels by 66% ± 5% (Figure 2A , lanes 1 and 2), and the missing GSK3 activity was recovered when all membranes were dissolved with 0.1% Triton X-100 (Figure 2A, compare lanes 2 and 4). The gold standard to determine the localization of a protein inside a membrane-bounded compartment is the protease protection assay. GSK3 protein became protected from Proteinase K digestion (Vanlandingham and Ceresa, 2009Vanlandingham P.A. Ceresa B.P. Rab7 regulates late endocytic trafficking downstream of multivesicular body biogenesis and cargo sequestration.J. Biol. Chem. 2009; 284: 12110-12124Crossref PubMed Scopus (284) Google Scholar) after Wnt3a treatment (Figure 2B, compare lanes 3 and 4) in untransfected L cells permeabilized with Digitonin. This Wnt-dependent protease protection of GSK3 was eliminated when membranes were solubilized with 0.1% Triton X-100 (Figure 2B, lanes 4 and 6). As a negative control, we used α-tubulin, which is not contained in vesicular organelles and was not protected from Proteinase K digestion (Figure 2B). These experiments strongly suggest that GSK3 becomes sequestered within membrane-bounded organelles upon Wnt treatment. The relocalization of GSK3 to multivesicular endosomes in Wnt3a-treated cells was visualized by cryoimmunoelectron microscopy. In untransfected 3T3 cells treated with control conditioned medium, endogenous GSK3β was found almost exclusively in the cytosol, whereas in Wnt3a-treated cells a substantial fraction was found inside MVBs (Figures 2C and 2D). In HeLa cells cotransfected with CA-LRP6 and GSK3-GFP, an anti-GFP antibody revealed colloidal Gold particles in MVBs (Figure 2E). In some cases Gold particles were observed on the cytoplasmic surface of vesicles fusing with multivesicular endosomes (Figure 2F), as well as within the small vesicles that fill MVBs (arrows with asterisks in Figure 2F), supporting the topology shown in Figure 7. In the absence of CA-LRP6, Gold-labeled GSK3-GFP was located in the cytosol (Figure 2G). To confirm the cryoimmuno localization results in a quantitative way, we resorted to an activated mutant of Rab5. Rab5-Q79L causes the formation of giant multivesicular endosomes containing large numbers of intralumenal vesicles (Wegener et al., 2010Wegener C.S. Malerod L. Pedersen N.M. Prodiga C. Bakke O. Stenmark H. Brech A. Ultrastructural characterization of giant endosomes induced by GTPase-deficient Rab5.Histochem. Cell Biol. 2010; 133: 41-55Crossref PubMed Scopus (81) Google Scholar). These MVBs are so large that the outer membrane (outlined by Rab5-QL-DsRed) can be readily distinguished from its internal contents by light microscopy. As shown in Figures 2H–2M, GSK3β translocated from the cytoplasm into the interior of Rab5-QL multivesicular endosomes (arrows, 77% ± 9% colocalization) when CA-LRP6 was coexpressed, but not in its absence. Cytosolic depletion of GSK3-GFP was very clear (compare Figures 2J and 2M). Taken together, these results demonstrate that the GSK3 enzyme becomes translocated from the cytosol into membrane-bounded multivesicular endosomes when Wnt signals. The molecular machinery that forms endosomal intralumenal vesicles has been well characterized (Wollert and Hurley, 2010Wollert T. Hurley J.H. Molecular mechanism of multivesicular body biogenesis by ESCRT complexes.Nature. 2010; 464: 864-869Crossref PubMed Scopus (527) Google Scholar). Several endosomal sorting complexes required for transport, or ESCRTs, have been identified through yeast genetics (designated Vacuolar Protein Sorting, Vps, mutants) and biochemistry (Katzmann et al., 2002Katzmann D.J. Odorizzi G. Emr S.D. Receptor downregulation and multivesicular-body sorting.Nat. Rev. Mol. Cell Biol. 2002; 3: 893-905Crossref PubMed Scopus (1019) Google Scholar). The GSK3 sequestration hypothesis predicts that ESCRT proteins essential for vesicle invagination should be required for Wnt signaling. Therefore, we tested two ESCRT proteins essential for vesicle formation (Figure 3A). HRS/Vps27 (hepatocyte growth factor regulated tyrosine-kinase substrate) initiates formation of the ESCRT-0 complex. Depletion of HRS by siRNA blocked the accumulation of β-Catenin observed after 2 hr of Wnt3a treatment in 293T cells, whereas control scrambled siRNA had no effect (Figure 3B, lanes 1–4). Total GSK3 levels increased by about 70% when HRS was depleted (Figure 3B compare lanes 1 and 3, three independent experiments; see also Figure S3), suggesting that GSK3 is normally partially degraded by the endosomal machinery; however, Wnt addition did not significantly change GSK3 levels in these experiments. We were expecting a decrease in total GSK3 levels in western blots because MVBs are usually targeted to the lysosome and degradation. Many reasons might explain this result. For example GSK3 may have a long half-life, it may be replenished by translational regulation, or GSK3 may have a long time of residency in MVBs induced by Wnt signaling before lysosomal degradation takes place (Wnt might even affect the rate of MVB processing globally). In addition there is recent evidence that intralumenal MVB vesicles may be recycled back into the cytosol by “back-fusion” to the late endosome-limiting membrane (Falguieres et al., 2009Falguieres T. Luyet P. Gruenberg J. Molecular assemblies and membrane domains in multivesicular endosome dynamics.Exp. Cell Res. 2009; 315: 1567-1573Crossref PubMed Scopus (86) Google Scholar). Despite the lack of change in total levels, the relocation of GSK3 from the cytosol into MVBs is clearly documented in the figures above, providing the basis for the sequestration model for Wnt signaling presented in Figure 7. The requirement of HRS for Wnt signal transduction was demonstrated directly in these cultures by measuring the expression of the SuperTopFlash-Luciferase reporter, which contains multiple TCF-binding sites (Figure 3C, brackets). In addition we observed that relocalization of GSK3 into vesicle-like structures in CA-LRP6 transfected cells was blocked by HRS siRNA and was not affected by control scrambled siRNA (Figures 3D–3E′). In animal cap explants the activation of TCF-Luciferase by CA-LRP6 mRNA was blocked by HRS antisense morpholino (Figure 3F, brackets). In Xenopus embryos, injection of CA-LRP6 mRNA into a single ventral blastomere at the eight-cell stage caused complete axis duplications, which were eliminated by coinjection of HRS MO (Figures 3G–3I and Figure S4N ). HRS depletion did not affect cell viability or proliferation until neurula stage (Figures S4A–S4M). The effects of HRS MO could be partially rescued by human HRS mRNA (Figures 4C and 4O ).Figure 4β-Catenin Is Required for GSK3 Localization in Multivesicular EndosomesShow full caption(A and B) β-Catenin siRNA inhibited GSK3β relocalization in CA-LRP6 signalosomes (in 86% ± 7% of transfected HeLa cells, n = 300).(C) HRS MO blocks the induction of TCF reporter expression (brackets) by β-Catenin mRNA (four injections of 80 pg) in Xenopus animal cap explants, and this was partially rescued by human HRS mRNA.(D–F′) Endogenous phospho-β-Catenin" @default.
• W2109955944 created "2016-06-24" @default.
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• W2109955944 creator A5044680754 @default.
• W2109955944 creator A5046419533 @default.
• W2109955944 creator A5051430111 @default.
• W2109955944 creator A5051690000 @default.
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• W2109955944 date "2010-12-01" @default.
• W2109955944 modified "2023-10-10" @default.
• W2109955944 title "Wnt Signaling Requires Sequestration of Glycogen Synthase Kinase 3 inside Multivesicular Endosomes" @default.
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• W2109955944 doi "https://doi.org/10.1016/j.cell.2010.11.034" @default.
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