Wnt Signaling Requires Sequestration
of Glycogen Syntha Kina3死人出殡
inside Multivesicular Endosomes
Vincent F.Taelman,1,3Radoslaw Dobrowolski,1,3Jean-Louis Plouhinec,1Luis C.Fuentealba,1,4Peggy P.Vorwald,1 Iwona Gumper,2David D.Sabatini,2and Edward M.De Robertis1,*
1Howard Hughes Medical Institute and Department of Biological Chemistry,University of California,Los Angeles,Los Angeles,CA 90095-1662,USA
2Department of Cell Biology,New York University School of Medicine,New York,NY10016-6497,USA
3The authors contributed equally to this work
4Prent address:Department of Neurological Surgery,University of California,San Francisco,San Francisco,CA94143-0525,USA *Correspondence:ederobertis@mednet.ucla.edu
DOI10.ll.2010.11.034
SUMMARY
Canonical Wnt signaling requires inhibition of Glycogen Syntha Kina3(GSK3)activity,but the molecular mechanism by which this is achieved remains unclear.Here,we report that Wnt signaling triggers the questration of GSK3from the cytosol into multivesicular bodies(MVBs),so that this enzyme becomes parated from its many cytosolic substrates.Endocytod Wnt colocalized with GSK3 in acidic vesicles positive for endosomal markers. After Wnt addition,endogenous GSK3activity decread in the cytosol,and GSK3became pro-tected from protea treatment inside membrane-bounded organelles.Cryoimmunoelectron micros-copy showed that the corresponded to MVBs. Two proteins esntial for MVB formation,HRS/ Vps27and Vps4,were required for Wnt signaling. The questration of GSK3extended the half-life of many other proteins in addition to b-Catenin, including an artificial Wnt-regulated reporter protein containing GSK3phosphorylation sites.We conclude that multivesicular endosomes are esntial compo-nents of the Wnt signal-transduction pathway. INTRODUCTION
Canonical Wnt signaling plays a crucial role in development, tissue regeneration,stem cells,and cancer(Logan and Nus, 2004;Clevers,2006;MacDonald et al.,2009;Angers and Moon,2009).A cytoplasmic destruction complex consisting of Glycogen Syntha Kina3(GSK3,which has a and b
isoforms), Cain Kina1(CK1),Adenomatous Polyposis Coli(APC),and Axin mediates the phosphorylation of b-Catenin.Phosphoryla-tion targets b-Catenin for polyubiquitinylation and subquent degradation in proteasomes.In the prence of Wnt,the destruction complex becomes inactivated in ways that are incompletely understood.Wnt triggers signaling by binding to Frizzled and LDL-receptor related protein6(LRP6),causing the aggregation of Dishevelled(Dvl)and Axin on the plasma membrane(Bilic et al.,2007;Zeng et al.,2008).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 Wntfield (Wu and Pan,2010).
Internalization of receptor complexes is an absolute require-ment for Wnt signaling(Blitzer and Nus,2006;Yamamoto et al.,2006).Bilic et al.(2007)discovered that cytoplasmic particles designated LRP6-signalosomes—containing aggre-gates of phospho-LRP6,Frizzled,Dvl,Axin,and GSK3—are formed at and under the plasma membrane15min after Wnt addition.Activated Wnt receptors recruit Axin and GSK3,which phosphorylatesfive critical PPPS/TP quences in the intracel-lular domain of LRP6(Zeng et al.,2008;Niehrs and Shen, 2010).A number of mechanisms have been propod to explain the inhibition of GSK3(Kimelman and Xu,2006).For example,the LRP
6tail may act as a direct inhibitor of GSK3(Mi et al.,2006; Clenyi et al.,2008;Piao et al.,2008;Wu et al.,2009).The LRP6PPPSP repeats rve both as substrates and binding sites for GSK3and may act as competitive inhibitors of this enzyme, although at low affinity(K i of1.3310À5M;Clenyi et al.,2008). GSK3has many substrates in addition to b-Catenin,including Dvl,Axin,and APC(Jope and Johnson,2004).This promiscuous enzyme phosphorylates rine or threonine at positionÀ4of sites primed by phosphorylation(S/TXXXS/T[PO3])(Cohen and Frame,2001).We reported that the transcription factor Smad1 is polyubiquitinylated and degraded after GSK3phosphorylation and is stabilized by canonical Wnt signaling,resulting in the inte-gration of BMP and Wnt signaling(Fuentealba et al.,2007).Addi-tional substrates destabilized by GSK3phosphorylation have since been identified(Kim et al.,2009).
花甲怎么炒才好吃During our investigations on signaling integration,we measured GSK3enzyme activity in Wnt-treated cell extracts (prepared in the prence of Triton X-100)and were surprid tofind that Wnt did not change GSK3activity(data not shown),
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even though in the direct GSK3inhibition model one would have predicted inhibition.How could this b
e?Upon reflection,we real-ized that following ligand binding and endocytosis,growth factor receptors are incorporated into multivesicular endosomes within 15min(Gruenberg and Stenmark,2004).Multivesicular body (MVB)formation is an obligatory step before degradation in lyso-somes can take place(Katzmann et al.,2002).Asfirst 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.,1979).Therefore,Wnt-induced MVB formation would cau GSK3bound to phosphorylated LRP6cytoplasmic tails(and other GSK3substrates such as Axin,APC,b-Catenin,and Dvl)to become questered from its cytosolic substrates by two layers of membrane(e model in Figure7),effectively inhibiting its activity.
In this study,we tested the GSK3questration hypothesis of Wnt signaling.Fluorescence microscopy showed that Wnt signaling caud the relocalization of cytoplasmic GSK3to vesi-cles that colocalized with endocytod xWnt8-Venus protein, and with the MVB and lysosomal markers Rab7and Lyso-Tracker.Wnt signal transduction was blocked by depletion of Hrs/Vps27or expression of dominant-negative Vps4,two proteins esntial for intralumenal vesicle formation in MVBs (Katzmann et al.,2002;Wollert and Hurley,2010).Moreover, Wnt treatment decread cytosolic GSK3activity levels (measured in Digitonin-permeabilized cells),yet full enzyme activity was recovere
d after solubilization of all membranes with Triton X-100.Similarly,Wnt caud endogenous GSK3b to become partially protected from Proteina K digestion,but only in the abnce of Triton X-100.Finally,cryoimmunoelectron microscopy showed that GSK3was indeed translocated from the cytosol into MVBs after Wnt pathway activation.Bioinfor-matic analys revealed that20%of the human proteome contains multiple putative GSK3phosphorylation sites.Total protein half-life was extended by Wnt treatment or GSK3inhibi-tion.The addition of GSK3phosphorylation sites was sufficient to place the stability of a Green Fluorescent Protein(GFP) bionsor under the control of Wnt.We conclude that canonical Wnt signaling questers GSK3inside MVBs,reducing its cyto-solic levels and extending the half-life of many GSK3substrates.
RESULTS
Wnt Caus GSK3Relocalization in Acidic Cytoplasmic Vesicles
Wefirst asked whether Wnt treatment changed the subcellular localization of GSK3.Human293T cells expressing xWnt8-Venus(Mii and Taira,2009)were cultured together with mou 3T3cells transfected with GSK3-RFP.Remarkably,endocy-tod Wnt-Venus and GSK3-RFP accumulated in the same vesicular structures(arrows in Figure1F),while GSK3-RFP levels decread in the cytosol(
Figures1A–1F).Relocalization of endogenous GSK3b was also obrved in the cocultures (Figures S1A–S1C available online).Wnt-Venus puncta were counted in each responding cell(n=80),and56%±9%of them colocalized with GSK3-RFP puncta(e histogram in Fig-ure1C0).Thus,cytosolic GSK3decreas and becomes relocal-ized to the same endosomes as the internalized Wnt ligand.
Transfection of constitutively active LRP6lacking its extracel-lular domain,designated CA-LRP6,caus a potent Wnt signal (Tamai et al.,2004).CA-LRP6cytoplasmic signalosome forma-tion(Bilic et al.,2007)required Dynamin and caud a striking re-localization of GSK3b from the cytosol into prominent cyto-plasmic puncta(Figures1G–1L0and Figures S1D–S1O). Treatment of3T3cells with LysoTracker,a dye that becomes concentrated in acidic organelles such as MVBs and lysosomes, showed that endogenous GSK3b relocated to the compart-ments(Figure1G–1L0).In addition,GSK3b 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 fud to the LRP6cytoplasmic tail(DIX> Ctail-GFP),which triggers a very strong Wnt signal(Metcalfe et al.,2010),also formed signalosomes colocalizing with acidic vesicles that questered cytosolic GSK3b(Figures S2P–S2Aa).
额头上的痣
CA-LRP6signalosomes colocalized with the late endosomal marker Rab7-GFP(Figures1M–1R)in62%±7%of the GSK3 puncta,indicating that GSK3-RFP relocated to MVBs or lyso-somes(Bucci et al.,2000).About40%of GSK3-RFP vesicles colocalized with Vps4-GFP(e Figures3L–3N0),a marker of thefinal stages of intralumenal vesicle formation in endosomes (Bishop and Woodman,2000;Gruenberg and Stenmark,2004). In untransfected3T3cells the number and size of endogenous GSK3b puncta incread after treatment with Wnt3a conditioned medium(Figures1S–1U)in cells permeabilized with Digitonin (which facilitates the visualization of intracellular organelles; Bishop and Woodman[2000]).
Taken together,the results strongly support the hypothesis that Wnt signaling caus the relocalization of cytosolic GSK3to the endosomal compartment.
GSK3Is Sequestered inside MVBs
To determine whether Wnt3a treatment questers GSK3kina activity from cytosol,endogenous cytosolic kina activity was measured in untransfected L cells permeabilized with Digitonin through the incorporation of phosphate from[g32P]-ATP into a phospho-glycogen syntha peptide substrate(Ryves et al., 1998).Digitonin solubilizes cholesterol-rich patches in the plasma membrane,l
eaving MVBs and other intracellular organ-elles intact(Dunn and Holz,1983).Addition of Wnt3a for4hr decread cytosolic GSK3activity levels by66%±5%(Fig-ure2A,lanes1and2),and the missing GSK3activity was recov-ered when all membranes were dissolved with0.1%Triton X-100 (Figure2A,compare lanes2and4).
The gold standard to determine the localization of a protein inside a membrane-bounded compartment is the protea protection assay.GSK3protein became protected from Proteina K digestion(Vanlandingham and Ceresa,2009)after Wnt3a treatment(Figure2B,compare lanes3and4)in untrans-fected L cells permeabilized with Digitonin.This Wnt-dependent protea protection of GSK3was eliminated when membranes were solubilized with0.1%Triton X-100(Figure2B,lanes4 and6).As a negative control,we ud a-tubulin,which is not contained in vesicular organelles and was not protected from Proteina K digestion(Figure2B).The experiments strongly Cell143,1136–1148,December23,2010ª2010Elvier Inc.1137
suggest that GSK3becomes questered within membrane-bounded organelles upon Wnt treatment.
The relocalization of GSK3to multivesicular endosomes in Wnt3a-treated cells was visualized by cry
oimmunoelectron microscopy.In untransfected 3T3cells treated with control conditioned medium,endogenous GSK3b 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-LRP6and GSK3-GFP,an anti-GFP antibody revealed colloidal Gold particles in MVBs (Figure 2E).In some cas Gold particles were obrved 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 abnce 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
caus the formation of giant multivesicular endosomes contain-ing large numbers of intralumenal vesicles (Wegener et al.,2010).The 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,GSK3b translocated from the cytoplasm into the interior of Rab5-QL multivesicular endosomes (arrows,77%±9%colocal-ization)when CA-LRP6was coexpresd,but not in its abnce.Cytosolic depletion of GSK3-GFP was very clear (compare Figures 2J and 2M).海上神鹰
Taken together,the results demonstrate that the GSK3enzyme becomes translocated from the cytosol into membrane-bounded multivesicular endosomes when Wnt signals.
Wnt Signaling Requires the ESCRT Machinery
The molecular machinery that forms endosomal intralumenal vesicles has been well characterized (Wollert and Hurley,2010).Several endosomal sorting complexes required
for
Figure 1.GSK3b Is Translocated to Acidic Vesicles upon Wnt Signaling
(A–F)In coculture experiments,xWnt8-Venus (green)creted by 293T cells caud the relocalization of GSK3b -RFP (red)in 3T3cells that endocytod Wnt.Arrows in the enlargement in (F)indicate xWnt8-Venus endosomes that quester GSK3-RFP.In controls lacking xWnt8-Venus,GSK3-RFP localization was cyto-plasmic (D and E);asterisks indicate untransfected control 293cell nuclei.
(G–L 0)GSK3b antigen relocalized to LysoTracker-positive endosomes upon activation of Wnt pathway by CA-LRP6in 3T3cells;note that endogenous GSK3b was depleted from cytoplasm.
(M–R 0)Rab7-GFP (green)and GSK3b -RFP (red)colocalized in CA-LRP6signalosomes (in 62%±7%of GSK3-positive puncta,n =80HeLa cells);note GSK3questration from cytosol.
(S–U)Endogenous GSK3b puncta (white)were larger and more numerous after 4hr of Wnt3a addition to untransfected 3T3cells permeabilized with Digitonin.Data are reprented as mean ±SEM.Also e Figure S1and Figure S2.
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俄罗斯人体模特microsoft账户登录transport,or ESCRTs,have been identified through yeast genetics (designated Vacuolar Protein Sorting,Vps,mutants)and biochemistry (Katzmann et al.,2002).The GSK3questra-tion hypothesis predicts that ESCRT proteins esntial for vesicle invagination should be required for Wnt signaling.There-fore,we tested two ESCRT proteins esntial for vesicle forma-tion (Figure 3A).
HRS/Vps27(hepatocyte growth factor regulated tyrosine-kina substrate)initiates formation of the ESCRT-0complex.Depletion of HRS by siRNA blocked the accumulation of b -Cat-enin obrved after 2hr of Wnt3a treatment in 293T cells,whereas control scrambled siRNA had no effect (Figure 3B,lanes 1–4).Total GSK3levels incread by about 70%when HRS was depleted (Figure 3B compare lanes 1and 3,three inde-pendent experiments;e also Figure S3),suggesting that GSK3is normally partially degraded by the endosomal machinery;however,Wnt addition did not significantly change GSK3levels in the experiments.We were expecting a decrea in
total
Figure 2.Wnt Signaling Caus Sequestration of GSK3inside Multivesicular Endosomes
(A)GSK3kina activity was decread by 66%±5%by Wnt3a treatment and was recovered after membrane solubilization with 0.1%Triton X-100in Digitonin-permeabilized L cells.LiCl inhibition shows that the radioactive assay was specific for GSK3.Data are from two independent experiments using untransfected L cells.GSK3b and a -tubulin provide loading controls.
(B)GSK3b becomes protected from Proteina K after Wnt3a treatment,but only in the abnce of Triton X-100(lanes 3–6).Similar results were obtained in five independent experiments (untransfected L cells).All samples were permeabilized with Digitonin,which caus leakage of 37%of the endogenous GSK3(three independent experiments).
(C and D)Cryoimmunoelectron microscopy demonstrating relocation of endogenous GSK3b into MVBs (arrows)after Wnt3a treatment in untransfected 3T3cells.(E–G)GSK3-GFP localized in MVBs (white arrows)in CA-LRP6transfected HeLa cells but was cytosolic in control cells lacking CA-LRP6transfection.(H–J)Rab5-QL-DsRed forms giant endosomes (arrows),whereas GSK3b -GFP remains uniformly distributed in the cytosol (n =100).
(K–M)In the prence of CA-LRP6,GSK3-GFP is translocated inside Rab5-QL giant multivesicular endosomes (e arrows)in 77%±9%,n =80,of cotransfected cells.Note that GSK3becomes depleted from cytosol.Data are reprented as mean ±SEM.
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GSK3levels in western blots becau MVBs are usually targeted to the lysosome and degradation.Many reasons might explain this result.For example GSK3may have a long half-life,it may be replenished by translational regulation,or GSK3may 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.,2009).Despite the lack of change in total levels,the relocation of GSK3from the cytosol into MVBs is clearly documented in the figures above,providing the basis for the questration model for Wnt signaling pre-nted in Figure 7
.
Figure 3.Components of the ESCRT Machinery Are Required for Wnt Signaling
(A)HRS and Vps4are proteins required for intralumenal vesicle formation in MVBs.GSK3is indicated in red.(B)HRS siRNA inhibits Wnt3a-induced accumulation of b -Catenin.
(C)TCF-Lucifera reporter gene assays showing that Wnt signaling requires HRS (brackets).
(D–E 0)HRS is required for questration of GSK3-RFP in CA-LRP6signalosomes (78%±8%,n =150).
(F)Wnt signaling induced by CA-LRP6mRNA in Xenopus animal caps was blocked by HRS morpholino (brackets).(G–I)Induction of condary axis in Xenopus embryos by CA-LRP6mRNA (80pg)requires HRS (neural tissue visualized with Sox2probe).
(J and K)Expression of Vps4-EQ,but not Vps-WT,inhibited signaling by Wnt3a or CA-LRP6(brackets)in 293T cells.
(L–N 0)CA-LRP6signalosomes questered endogenous GSK3b and partly colocalized with Vps4-WT-GFP,a multivesicular endosome marker (43%±5%of vesicles,arrows,in n =80cells).
(O–Q 0)Overexpression of Vps4-EQ-GFP inhibits questration of endogenous GSK3b in CA-LRP6signalosomes.(R–T)Head formation in axes induced by CA-LRP6was inhibited by Vps4-EQ ,but not by Vps4-WT mRNA.
Data are reprented as mean ±SEM.Also e Figure S3and Figure S4.
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The requirement of HRS for Wnt signal transduction was demonstrated directly in the cultures by measuring the expression of the SuperTopFlash-Lucifera reporter,which contains multiple TCF-binding sites(Figure3C,brackets). In addition we obrved that relocalization of GSK3into vesicle-like structures in CA-LRP6transfected cells was blocked by HRS siRNA and was not affected by control scrambled siRNA (Figures3D–3E0).In animal cap explants the activation of TCF-Lucifera by CA-LRP6mRNA was blocked by HRS anti-n morpholino(Figure3F,brackets).In Xenopus embryos, injection of CA-LRP6mRNA into a single ventral blastomere at the eight-cell stage caud complete axis duplications,which were eliminated by coinjection of HRS MO(Figures3G–3I and Figure S4N).HRS depletion did not affect cell viability or prolifer-ation until neurula stage(Figures S4A–S4M).The effects of HRS MO could be partially rescued by human HRS mRNA(Figures4C and4O).
老师再见了Vps4is an ATPa required for the disasmbly of ESCRT-III complexes,the last step in the pinching off of intralumenal vesi-cles in multivesicular endosomes(Gruenberg and Stenmark, 2004).A point mutation in the ATPa site(Vps4-EQ)creates a potent dominant-negative form that inhibits MVB formation (Bishop and Woodman,2000).Vps4-EQ blocked canonical Wnt3a signaling(Figure3J,brackets).The control in this exper-iment was Vps4-WT,which differs by only one amino acid yet was without effect in TCF-Lucifera assays.Vps4-EQ also in-hibited the transcriptional effects of CA-LRP6(Figure3K, brackets).Vps4-EQ cotransfection inhibited the relocalization of endogenous cytosolic GSK3b into CA-LRP6signalosomes (Figures3L–3Q).In Xenopus embryos,Vps4-EQ,but not Vps4-WT,coinjected with CA-LRP6mRNA into a ventral blastomere inhibited the formation of complete condary axes containing head structures(Figures3R–3T and Figure S4O).Importantly, we also tested the requirement of the ESCRT machinery for axis induction by Siamois,a homeobox gene activated by Wnt signaling.We found that Vps4-EQ mRNA was unable to inhibit Siamois condary axes(e Figures4Ba–4Da and Figure S5A). This epistatic experiment placed the requirement for Vps4early in the Wnt pathway,upstream of its transcriptional effector Siamois.
The experiments show that two components of the ESCRT machinery,HRS/Vps27and Vps4,are required for canonical Wnt signaling.
物业管家岗位职责b-Catenin Is Required for GSK3Sequestration
While investigating the requirements for GSK3questration,we made an unexpectedfinding:the translocation of GSK3into CA-LRP6signalosomes and its depletion from the cytosol were inhibited by b-Catenin siRNA(Figures4A and4B and Fig-ure S3C).Using SuperTopFlash reporter in animal caps,we found that HRS MO blocked signaling by microinjected b-Cate-nin mRNA and was partially rescued by human HRS mRNA coinjection(Figure4C).We also noticed that endogenous b-Cat-enin,especially its GSK3-phosphorylated ,targeted for degradation),colocalized with CA-LRP6-GFP in signalosomes (Figures4D–4F).Taken together,the results suggest that b-Catenin is required for the questration of GSK3in multivesic-ular endosomes.
We next asked whether b-Catenin overexpression was suffi-cient to trigger GSK3questration in endosomes.Stabilized b-Catenin-GFP(in which its three GSK3sites were mutated into alanines)accumulated in the nucleus as expected but also in cytoplasmic vesicle-like structures,which questered GSK3-RFP(Figures4G–4I).Wild-type b-Catenin-GFP accumu-lated inside giant Rab5-QL MVBs(Figures4J–4L)and in Lyso-Tracker-positive vesicles(Figures S3D–S3F).Injection of b-Cat-enin mRNA into a ventral cell caus twinning in Xenopus embryos.Coinjection of b-Catenin mRNA with HRS-MO or Vps4-EQ showed that MVB formation was
required for b-Catenin axis induction in Xenopus(Figures4M–4R and Figures S4P and S4Q).
This new function of b-Catenin in questering GSK3in MVBs occurs upstream of the well-established transcriptional role of b-Catenin and Tcf3in Wnt signaling(Clevers,2006).Depletion of b-Catenin with MO generated ventralized Xenopus embryos lacking all neural structures(Figures4S and4T).Although GSK3inhibition by DN-GSK3b mRNA(a dominant-negative catalytically inactive form)greatly expanded neural structures, this effect was blocked by b-Catenin or xTcf3depletion(Figures 4U–4W).However,a construct fusing the transactivation domain of Xenopus b-Catenin to DN-xTcf3was able to signal in b-Cate-nin-depleted embryos(Figure4X).In condary axis induction assays,the b-Catenin-DN-xTcf3fusion was active even when MVB formation was inhibited by Vps4-EQ mRNA microinjection (Figures4Y–4Aa and Figure S5B).The experiments show that the classical transcriptional role of b-Catenin/Tcf3is distinct from its new function in facilitating GSK3questration.
We conclude that b-Catenin protein is required and sufficient to cau GSK3questration in acidic cytoplasmic endosomes. It has been propod that the entire destruction complex(which includes b-Catenin)is recruited to phosphorylated LRP6recep-tors(Zeng et al.,2008).Once b-Catenin levels begin to ri during Wnt signaling,b-Catenin would enhance the signal,form-ing a feed-forward loop
by facilitating GSK3questration. Sequestration of GSK3Regulates Protein Half-Life Phosphorylation of b-Catenin or Smad1by GSK3caus recog-nition by E3polyubiquitin ligas and protein degradation(Logan and Nus,2004;Fuentealba et al.,2007).To investigate whether GSK3questration caus the stabilization of other proteins in addition to b-Catenin and Smad1,wefirst designed computer algorithms to identify potential phosphate-primed GSK3sites in the human proteome(Figure5A)and whether they were evolutionary conrved.A large number of proteins (20%of the proteome)were found to contain three or more concutive potential GSK3sites(Table S1and Table S3),signif-icantly more than expected from random distribution(Table S1). The included many novel putative GSK3targets with known regulatory functions(Table S2).
To determine whether Wnt/GSK3signaling regulates overall protein stability,we carried out radioactive pul-cha experi-ments with35S methionine in untransfected293T cells.Cultured cells were radioactively labeled for30min,washed,and then treated with Wnt3a or control conditioned medium containing a4-fold excess of unlabeled methionine.As shown in Figure5B, the total half-life of labeled cellular proteins incread from9.3to Cell143,1136–1148,December23,2010ª2010Elvier Inc.1141
