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Research Article

β-Catenin is O-GlcNAc glycosylated at Serine 23:Implications for β-catenin's subcellular localizationand transactivator function

Jacqueline R. Ha, Li Hao, Geetha Venkateswaran, Yu Hao Huang,Elizabeth Garcia, Sujata Persadn

Department of Pediatrics, Faculty of Medicine and Dentistry, University of Alberta, Edmonton, AB, Canada T6G 2E1

a r t i c l e i n f o r m a t i o n

Article Chronology:

Received 10 July 2013Received in revised form26 November 2013Accepted 28 November 2013Available online 14 December 2013

Keywords:

β-CateninO-GlcNAcylationSubcellular localizationTranscriptional function

nt matter & 2013 Elsevier016/j.yexcr.2013.11.021

hor. Fax: þ1 780 492 0723.: sujata.persad@ualberta.c

a b s t r a c t

Background: We have previously reported that β-catenin is post-translationally modified with asingle O-linked attachment of β-N-acetyl-glucosamine (O-GlcNAc). We showed that O-GlcNAcregulated β-catenin's subcellular localization and transcriptional activity.Objective: The objectives of this investigation were to identify the putative O-GlcNAc sites ofβ-catenin and the relevance of identified sites in the regulation of β-catenin's localization andtranscriptional activity.Method: Missense mutations were introduced to potential O-GlcNAc sites of pEGFP-C2-N-Terminal- or pEGFP-C2-Wild Type-β-catenin by site-directed mutagenesis. We determined thelevels of O-GlcNAc-β-catenin, subcellular localization, interaction with binding partners andtranscriptional activity of the various constructs.Results: Serine 23 of β-catenin was determined as a site for O-GlcNAc modification whichregulated its subcellular distribution, its interactions with cellular partners and consequently its

transcriptional activity.Significance: O-GlcNAcylation of Serine 23 is a novel regulatory modification for β-catenin'ssubcellular localization and transcriptional activity. This study is the first report to characterizesite specific regulation of β-catenin by the O-GlcNAc modification.

& 2013 Elsevier Inc. All rights reserved.

Introduction

Beta (β)-catenin is a multifunctional protein that serves a struc-tural role at the adherens junctions [1] and a regulatory functionas a transcriptional co-activator of the wnt/wingless canonicalsignal transduction pathway. Activation of this pathway resultsin stabilization and accumulation of β-catenin in the cytosol, andsubsequent localization of the protein into the nucleus.

Inc. All rights reserved.

a, sujata.persad@med.ualbe

Deregulated Wnt signaling results in increased levels of β-catenin in the nucleus and constitutive transcription of Wnt targetgenes [2–5], some of which are involved in cell proliferation,invasion and metastasis.Under normal conditions, cytosolic levels of β-catenin are tightly

regulated by the ubiquitin-proteasome system, which requires thetargeted phosphorylation of highly conserved serine (Ser) andthreonine (Thr) residues (Ser 33, Ser 37, Thr 41, and Ser 45) at the

rta.ca (S. Persad).

Table 1 – Schematic representation of β-catenin andβ-catenin constructs. (A) Illustration of the protein struc-ture of β-catenin and its three primary domains. (B)Representation of GFP tagged β-catenin constructs usedwithin this study.

1 7811

Armadillo Repeats

“Destruction Box”

CN

GFP

GFP

GFP

GFP

GFP

GFP

GFP

GFP

GFP

GFP

GFP

Wild Type Full Length β-Catenin (WT)

S23G Full Length β-Catenin (S23G)

S33A S37A T41A S45A Mutantβ-Catenin (4M)

S40A Full Length β-Catenin (S40A)

S42A Full Length β-Catenin (S42A)

N-Terminal Wild Type (NT)

S23G N-Terminal Mutant (S23G-NT)

C-Terminal Wild Type (CT)

Armadillo Repeats 1-12 (AF)

Armadillo Repeats 1-6 (A1-6)

Armadillo Repeats 7-12 (A7-12)

TransactivationDomain

2 3 4 5 6 7 8 9 10 11 12

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N-terminal domain of the protein, by glycogen synthase kinase-3beta (GSK-3β) and Casein Kinase (CK)-1 [6,7]. This phosphorylationevent requires β-catenin's association with components of thedestruction complex including axin and the tumor suppressor,adenomatous polyposis coli (APC) [5]. In the presence of Wntsignaling, the destruction complex is inactivated resulting in thecytoplasmic accumulation of β-catenin and its subsequent translo-cation to the nucleus. In the nucleus, β-catenin interacts with the T-cell Factor (TCF)/lymphoid enhancer factor (LEF) family of transcrip-tion factors and activates transcription of Wnt target genes [2–5,7].Interestingly, β-catenin does not use the conventional importinnuclear transport system nor does it contain a nuclear localizationsequence (NLS) [8]. To that end, no mandatory carrier proteins havebeen identified for the transport of β-catenin from the cytoplasminto the nucleus.We have previously shown that β-catenin was post-translationally

modified by O-GlcNAcylation [9]. O-GlcNAcylation involves thesingle O-linked attachment of β-N-acetyl-glucosamine (O-GlcNAc) tothe hydroxyl moiety of serine and threonine residues of proteinsfound in the nucleus and cytoplasm [10]. The nucleo-cytoplasmicenzymes O-GlcNAc-transferase (OGT) and β-N-acetyl-glucosaminidase(O-GlcNAcase, OGA) mediate the addition and removal of O-GlcNAcgroups from proteins, respectively [10]. Our findings were supportedby others [11–13], although, the functional implications and specificsites of O-GlcNAc modification for β-catenin were not established. Ourprevious report demonstrated that increased O-GlcNAcylation ofβ-catenin attenuated its nuclear levels, increased its cytosolic levelsand consequently decreased its transcriptional activity [9]. Moreover,O-GlcNAc of β-catenin was found not to affect the stability of theprotein [9], nor prevent its ability to bind to E-cadherin [11]. TheO-GlcNAc post-translational modification has been recognized to befundamental to the regulation of cellular processes [14] and site-specific modulation of protein function and behaviour including: (1)protein stability/degradation; (2) subcellular localization; (3) protein–protein interactions; and (4) transcription activity (summarized in 10).Hatsell et al. identified Plakoglobin, a homologue of β-catenin,

to be O-GlcNAcylated at Thr 14 near its N-Terminal (NT) ‘destruc-tion box' complex [13]. Plakoglobin and β-catenin share 65%sequence homology at the ARM domain but are divergent at theNT domain with the exception of some highly conserved phos-phorylation sites [13]. Sequence alignment indicated that Thr 14in Plakoglobin was homologous to Ser 23 of β-catenin [13]. In thisstudy we demonstrate that Ser 23 is an important site forO-GlcNAc modification and imparts site-specific functionality forβ-catenin's localization and transactivator function.

Materials and methods

Constructs and transformation

Recombinant β-catenin was amplified by polymerase chain reac-tion (PCR) using wild type Flag tagged human full lengthβ-catenin in a pcDNA 3.1Zeo vector as a template. The resultingPCR products were subcloned into a green fluorescent protein(GFP) expression vector (pEGFP-C2). Recombinant β-cateninconstructs subcloned into pEGFP-C2 were as follows: wild typeN-terminal [pEGFP-C2 �h-NT] (NT); Serine 23 to Glycine NTmutant [pEGFP-C2 �h-S23G-NT] (S23G-NT); C-terminal [pEGFP-C2 �h-CT] (CT); full length Wild Type [pEGFP-C2 �h-WT] (WT); full

length S23G mutant [pEGFP-C2 �h-S23G] (S23G); Armadillorepeats 1–12 [pEGFP-C2 �h-AF] (AF); Armadillo repeats 1–6[pEGFP-C2 �h-A1-6] (A1-6); Armadillo repeats 7–12 [pEGFP-C2 �h-A7-12] (A7-12); Full Length quadruple mutant [pEGFP-C2 �h-4M](Ser/Thr-Alanine (A): Ser33A, Ser37A, Ser45A,Thr41A (4M); FullLength mutants [pEGFP-C2 �h-S40A] (Ser40A) and [pEGFP-C2 �h-S42A] (Ser42A) [gifts from Dr. D.W. Andrews] and pEGFP-C2(empty vector control). Table 1 shows a list of all constructs used inthis study. β-catenin constructs were introduced into Max EfficiencyDH5α Escherichia coli (New England Biolabs) and positive colonieswere amplified in Luria-Bertani (LB) media containing Kanamycin.Plasmid constructs were purified using Qiagen Plasmid PurificationKit (Qiagen) as per manufacturer's protocols.

Site directed mutagenesis

Site-directed mutagenesis (SDM) was used to introduce missensemutations to potential O-GlcNAc sites in pEGFP-C2-NT or pEGFP-C2-WT β-catenin. Mutants were created using Stratagene XLQuick-Change SDM Kit. Forward and reversed primers were designedcomprising sequences flanking a three-base mutation convertingSer/Thr codons to glycine (G) or alanine (A). The primers used wereas follows: S23G sense 50-GAAAAGCGGCTGTTGGTCACTGGCAGC-30;S23G antisense 50-GCTGCCAGTGACCAACAGCCGCTTTTC-30; ΔS33,37, 45 ΔThr 41 (4M) mutant, Ser40A and Ser42A.

PCR reactions were divided into two sets containing 25 ng of DNAtemplate, 25 mM dNTP mix, 10� Pfu DNA Polymerase reactionbuffer, ddH2O, Pfu Turbo DNA Polymerase and 135 ng of sense andantisense primers separately into each mixture. The mutant plas-mid was generated and amplified following parameters that havebeen optimized for use in the Stragene's QuickChange SDM.

Contrary to the report by Hatsell et al. where Thr 14 wassubstituted to alanine [13], we substituted Ser 23 of β-catenin

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with glycine. The decision to do so was based on the observationthat Ser 23 was encoded by the AGT codon. An alanine substitu-tion required two point mutations (AGT to ACG) to introduce thismissense mutation, whereas a serine to glycine mutation onlyrequired one (AGT to GGT). Glycine substitutions are recognizedto have the same effect as alanine, as they both remove the sidechain beyond the β-carbon of an amino acid residue while notaltering the main-chain conformation [14,15].

Cell culture and transfection of plasmid constructs

Human cancer cell lines, DU145 prostate cancer (CaP), and, U2OSosteosarcoma (American Type Culture Collection) were grown inDulbecco's Modified Eagle's medium (DMEM; Invitrogen) supple-mented with 10% FBS and 1% Penicillin/Streptomycin (Invitrogen).All cells were maintained in 5% CO2 at 37 1C. Cells were grown to70% confluency and transfected with the various plasmid constructs:NT, S23G-NT, CT, AF, A1-6, A7-12, WT, S23G, 4M, S40A, S42A orpEGFP empty vector control. All transfections utilized 3 μg DNAconstruct and Lipofectamine 2000 (Invitrogen) according to manu-facturer's protocols.

PUGNAc treatment

O-(2-Acetamido-2-deoxy-D-gluco-pyranosylidene) amino-N-phe-nylcarbamate (PUGNAc) (Toronto Research Chemicals) is anO-GlcNAc analogue that inhibits O-GlcNAcase. This prevents thecycling of O-GlcNAc on proteins and leads to globally elevatedlevels of O-GlcNAcylation. PUGNAc was dissolved in water to aconcentration of 20 mM, and diluted to a final concentration of100 μM. Prior to PUGNAc treatment, cells were incubated for 18 hwith media containing DMEM 1% FBS [16]. Thereafter, cells weretreated with PUGNAc (100 μM) in the absence of serum for 14 h.

Whole cell lysis and nuclear extracts

Whole cell lysates were obtained by incubating 100 μl of lysisbuffer (NP-40-DOC Buffer: 10 mM Tris–HCl pH 7.5, 1% NP-40, 0.5%Sodium Deoxycolate, 2 mM phenylmethylsulfonyl fluoride(PMSF), 80 ng/ml aprotinin, 40 ng/ml chymostatin, 40 ng/ml anti-pain, 40 ng/ml leupeptin, 40 ng/ml pepstatin) on ice for 10 min.Cellular debris was removed by centrifugation for 5 min at16,000� g. Nuclear and cytosolic extracts were separated usingthe NE-PER Nuclear Cytoplasmic Extraction Kit (ThermoScientific)according to the manufacturer's protocols. Protein concentrationswere determined using Bicinchoninic (BCA) Protein Determina-tion Assay (ThermoScientific).

Immunoblotting

Equivalent protein quantities were resolved by 8% Tricine poly-acrylamide gel electrophoresis. Proteins were transferred ontopolyvinylidene difluoride (PVDF) membrane (Millipore). Mem-branes were blocked in 1% Bovine Serum Albumin (BSA) andincubated with Wheat Germ Agglutinin (WGA)-Horse RadishPeroxidase (HRP) or blocked with 3% BSA and incubated withvarious antibodies at 4 1C overnight. Western blots were

visualized using Western Lightnings Plus-ECL (PerkinElmer, LASInc.). For Western Blot Analysis (WB), antibodies to the followingproteins were included: β-Catenin (Cell Signalling), E-cadherin(Cell Signalling; Santa Cruz Biotechnology Inc.), TCF (Cell Signal-ling), α–β Tubulin (Cell Signalling), Lamin B (Calbiochem), Actin(Santa Cruz Biotechnology Inc.), and GFP (AbCam; Santa CruzBiotechnology Inc.) followed by peroxidase-conjugated secondaryantibodies (GE Healthcare UK Limited). Densitometric analysis wasperformed by IMAGEJ Software. Histograms are representative ofthree or more independent experiments as indicated. Statisticalanalysis was performed by Student's t-test (npo0.05) using Sigma-Plot Software.

Isolation and determination of O-GlcNAcylated proteins

O-GlcNAc-β-catenin was determined by one of two methods.O-GlcNAcylated proteins were isolated by precipitation withwheatgerm agarose (WGA) beads (Vector Laboratories) andβ-catenin was identified by WB with anti-β-catenin (Cell Signal-ling), or anti-GFP (AbCam; Santa Cruz Biotechnology Inc.) anti-body. In this method WGA-agarose beads were incubated with100 μg of cell lysate in 200 μl of lysis buffer overnight at 4 1C.Beads were precipitated by centrifugation and washed withphosphate buffered saline (PBS). Samples were then eluted bySDS PAGE sample buffer. Complexes were separated on 8% Tricinepolyacrylamide gels and proteins were immunoblotted with anti-β-catenin or anti-GFP antibodies. Alternatively, levels of O-GlcNA-cylated β-catenin was determined by immunoprecipitation (IP) oflysates with anti-β-catenin or anti-GFP antibodies preabsorbed toProtein A/G agarose beads (Santa Cruz Biotechnology Inc.). Beadswere precipitated by centrifugation. Samples were washed withPBS and eluted by SDS PAGE sample buffer. Isolated complexeswere separated on Tricine polyacrylamide gels and characterizedby WB. O-GlcNAc modified proteins were identified usingWGA-HRP (Vector Laboratories) at a dilution of 1 mg/ml. Eachsample condition (7PUGNAc) was normalized to their internalcontrols to ensure equal IP of the protein of interest. Alterations inO-GlcNAcylation was based on comparison between 7PUGNAcconditions. Where indicated, input was 25% of initial load.

Immunoprecipitation

100 mg of protein lysate were incubated with anti-β-catenin, anti-E-cadherin, anti-TCF or anti-GFP antibodies preabsorbed to ProteinA/G agarose beads. Samples were washed with PBS and eluted bySDS PAGE sample buffer. Isolated complexes were separated onTricine polyacrylamide gels and characterized by WB analysis.

Immunofluorescence

Cells were cultured onto coverslips for immunofluorescence (IF)analysis of the expression and localization of specific proteins.Briefly, cells were washed with PBS and fixed with 4% formalde-hyde subsequently incubated in cold 100% methanol at �20 1C for10 min for cell membrane permeabilization. Cells were stainedwith the relevant primary antibody followed by incubation withAlexaFluors 488 conjugated secondary antibodies (Invitrogen) forvisualization. GFP tagged β-catenin proteins were visualized by

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monitoring GFP fluorescence. Cell nuclei were stained with 40,6-diamidino-2-phenylindole (DAPI). Cells were viewed by CarlZeiss Laser Scanning Microscope and data was analyzed usingLSM510 software.

Luciferase assay for β-catenin/TCF promoter activities

β-Catenin/TCF-induced transcriptional activity was determined byusing a β-catenin/TCF promoter-Luciferase reporter construct,pTOPFlash, by techniques reported previously [17,9] and accord-ing to the manufacturer's instructions (Stratagene). Briefly, a dualluciferase reporter assay was performed whereby cells (DU145)were transfected with experimental plasmid constructs and theTCF promoter/luciferase reporter gene (pTOPFlash) and subse-quently treated with PUGNAc (100 μM) as described above.The TOPFlash-luciferase reporter construct specifically measuresβ-catenin/TCF regulated transcriptional function and is comprisedof a multimeric synthetic β-catenin/TCF-4 binding site upstreamof a Thymidine Kinase (TK) minimal promoter and a Luciferaseopen reading frame [17]. A mutated TCF-Luciferase reporterconstruct (pFOPFlash) served as a negative control for TOPFlashactivity. A control reporter pRL-TK renilla luciferase (Promega)was co-transfected in each sample to serve as an internal controlfor transfection efficiency. Transfections of cells were done withthe use of Lipofectamine2000 (Invitrogen) according to manufac-turer's guidelines. Reporter activity was measured using a lumin-ometer (Fluo Star OMEGA: BMG Labtech).

Quantitative reverse transcription polymerase chainreaction (RT-qPCR) analysis of cyclin D1 and VEGFA mRNAexpressions

β-Catenin/TCF-induced transcriptional activity was also deter-mined by evaluation of the mRNA expressions of two promonentWnt/β-catenin target genes, Cyclin D1 and VEGFA. DU145 cellswere allowed to reach 70% confluency in DMEM 10% FBS media.Cells were transfected with plasmid constructs and subsequentlytreated with PUGNAc (100 μM) as described above. Total RNA wasisolated from DU145 cells using RNeasy Qiagen Kit according tothe manufacturer's instructions and quantified spectrophotome-trically. A 1 μg of total RNA was reverse-transcribed using Super-Scripts II Reverse Transcriptase (Invitrogen) according to themanufacturer's protocol. Quantification of Cyclin D1 and VEGFgene expressions were assessed using Power SYBR Green PCRMaster Mix (Applied Biosystems). Samples were amplified with aprecycling hold at 95 1C, 15 s, 30 cycles of annealing and extensionat 60 1C for 1 min.The primers used are as follows: Cyclin D1-50-CTGGCCATGAACTA-

CCTGGA-30 (sense) and 50-GTCACACTTGATCACTCTGG-30 (anti-sense),VEGF-50-GCAGAATCATCACGAAGTGG-30 (sense) and 50-GCATGGTGA-TGTTGGACTCC-30 (anti-sense), and Glyceraldehyde-3-PhosphateDehydrogenase (GAPDH)-50-ACCTGGTGCTCAGTGTAGCC-30 (sense)and 50-CAATGACCCCTTCATTGACC-30 (anti-sense). The GAPDH geneserved as the endogenous control. Each measurement was performedin triplicate with Rotor-Gene 3000 instrument (Montreal Bio-techInc.) and analysed using ROTOR-GENE-6 Software. Gene expressionwas determined using the relative standard curve method normalizedto GAPDH-binding protein expression. Histograms are reported as a

fold change of control which was set at 1.0. Statistical analysis wasperformed by Student's t-test (npo0.05) using SigmaPlot Software.

Anchorage-independent growth in soft agar

To test anchorage-independent growth on soft agar, we trans-fected recombinant full length GFP-WT or GFP-S23G β-cateninconstructs in DU145 cells and treated the cells with/withoutPUGNAc. 1�104 of these transfected DU145 cells were seededin six-well plates with a bottom layer of 0.7% Bacto agar in DMEMand a top layer of 0.3% Bacto agar in DMEM. Fresh DMEM with orwithout PUGNAc (100 μΜ) was added to the top layer of the softagar. Plates were incubated in a 5% CO2 humidified incubator at37 1C. The culture medium was changed twice a week. After 16days, colonies were stained with 0.005% crystal violet andcolonies with more than 50 cells were scored as positive usingan inverted microscope equipped with a measuring grid.

Cell proliferation assays

DU145 cells transfected with pEGFP-WT or pEGFP-S23G β-cateninand treated with or without PUGNAc were grown to 70%confluence in a 96-well plate and incubated at 37 1C. Cellproliferation was measured by the modised tetrazolium salt-3-(4-5 dimethylthiozol-2-yl) 2-5 diphenyl-tetrazolium bromide(MTT) assay after 24, 48, 72 and 96 h. For this, 0.025 ml of MTTsolution (5 mg/ml in PBS) was added to each well. After a 2 hincubation at 37 1C, 0.1 ml of the extraction buffer (20% sodiumdodecyl sulphate, 50% dimethyl formamide) was added. After anovernight incubation at 37 1C, the optical densities at 570 nmwere measured using a multiscanner autoreader (DynatechMR5000), with the extraction buffer used as a blank.

Results

β-Catenin is O-GlcNAc modified at its N-terminal domain

In order to determine the putative domain at which β-catenin wasactively cycling O-GlcNAc groups, we transfected the followingGFP-tagged constructs into DU145 prostate cancer (CaP) cells:wild type-N-terminal (NT), wild type-C-terminal (CT), Armadillorepeats 1–12 (AF), Armadillo repeats 1–6 (A1–6), or Armadillorepeats 7–12 (A7–12). Transfected cells were treated with orwithout PUGNAc (100 mM) and O-GlcNAcylation of β-cateninwas evaluated by WGA-agarose precipitation and Western blotanalysis (WB) using anti-GFP antibody (as described in Materialsand methods). Fig. 1A shows that there was a 30% induction ofO-GlcNAcylation of β-catenin at the NT fragment upon PUGNActreatment indicative of active cycling of O-GlcNAc modification atthis domain. On the other hand, although the CT construct wasobserved to have high baseline levels of O-GlcNAcylation, this wasnot induced with PUGNAc treatment. This suggested that therewas no active cycling of O-GlcNAc modification occurring at theCT domain (Fig. 1A). There was no detectable O-GlcNAc modifica-tion of the AF, A1-6 and A1-7 domains of β-catenin in thepresence or absence of PUGNAc (Fig. 1B).

Fig. 1 – N-terminal but NOT the Armadillo and C-terminal domain of β-catenin is O-GlcNAc modified. Whole cell lysates of DU145cells transfected with GFP tagged N-terminal (NT), C-terminal (CT), Armadillo repeats 1–12 (AF), and, Armadillo repeats 1–6 (A1–6)and 7–12 (A7–12) were treated with or without 100 lM PUGNAc, precipitated with WGA-agarose beads, and characterized byWestern blot analysis. (A) There was a 30% induction of O-GlcNAcylation at the NT domain upon PUGNAc treatment, suggesting thepresence of active O-GlcNAc cycling within this region. High baseline levels of O-GlcNAc modification was observed for the CTdomain; however, O-GlcNAcylation at the CT domain was not inducible with PUGNAc treatment. (B) NT domain was O-GlcNAcmodified while there was no O-GlcNAc modification of the Armadillo domain observed. Each group of data is representative of 4 ormore experiments. npo0.05.

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β-Catenin is O-GlcNAc modified at Serine 23 at the NTdomain

Sequence alignment indicated that Thr 14 of plakoglobin washomologous to Ser 23 of β-catenin [13]. To determine whether Ser23 was a potential site for O-GlcNAc modification within the NTdomain, we introduced a Ser 23 to Glycine (G) mutation to theGFP-tagged NT domain (S23G-NT). GFP-S23G-NT and wild typeGFP-NT domains were transfected into DU145 cells and the cellswere treated with or without PUGNAc (100 mM). Fig. 2A showsthat unlike the wild type GFP-NT construct which showedsignificant induction of O-GlcNAcylation upon PUGNAc treatment,there was no induction in the O-GlcNAc modification levels withPUGNAc treatment in the S23G-NT mutant protein (Fig. 2A). Thissuggested that Ser 23 at the NT domain was an important site forO-GlcNAcylation. The presence of baseline levels of O-GlcNAcmodification despite a S23G mutation indicated that alternativesites of O-GlcNAcylation may exist within the NT domain.

In order to assess the functional impact of O-GlcNAcylation atSer 23 on β-catenin, we created a Full length GFP-S23G (S23G) andfull length GFP-Wild Type (WT) β-catenin. These constructs were

then transfected into DU145 cells and the transfected cells weretreated with or without PUGNAc. Alterations in the levels ofO-GlcNAcylation were characterized by WGA-HRP (Fig. 2B) andWGA-precipitation (Fig. 2C). Similar to the NT domain construct,O-GlcNAc levels of full length GFP-WT β-catenin was increased3-fold relative to the untreated group. However, there was nosignificant change in O-GlcNAc modification levels of the fulllength GFP-S23G β-catenin upon PUGNAc treatment compared tothe untreated control (Fig. 2B and C).

NT phosphorylation sites of β-catenin are not O-GlcNAcmodified

The presence of baseline levels of O-GlcNAc modification despite aS23G mutation (Fig. 2A) raised the possibility that other signifi-cant sites of O-GlcNAcylation may exist within the NT domain.Thus, we looked at other potential sites of O-GlcNAc at the NT ofβ-catenin. O-GlcNAcylation has been suggested to competitivelyoccupy the same and/or adjacent sites of phosphorylation [18].Accordingly, we looked at the four phosphorylation sites at the NTdomain destruction box (Ser 33, Ser37, Thr41 and Ser45) and

Fig. 2 – Serine 23 is a site for O-GlcNAc modification. (A) DU145 cells transfected with wild type GFP-NT or mutant GFP taggedSerine 23 to Glycine mutant NT (GFP-S23G-NT) β-catenin constructs treated with or without 100 lM PUGNAc. Levels ofO-GlcNAcylation at the NT domain increased upon treatment with PUGNAc. There was no change in O-GlcNAc levels of S23G-NTwith PUGNAc treatment. (B) and (C) DU145 cells transfected with GFP tagged full length Wild Type (WT) or full length S23G mutantβ-catenin and treated with or without PUGNAc. Levels of O-GlcNAcylation of full length GFP-WT β-catenin increased with PUGNAc.O-GlcNAc levels of full length GFP-S23G mutant β-catenin were not altered upon PUGNAc treatment. Alterations in O-GlcNAcylationwere evaluated in whole cell lysate of DU145 cells by (B) immunoprecipitation with anti-GFP antibody followed by WB withWGA-HRP or (C) precipitation with WGA-agarose beads followed by anti-GFP antibody. Each group of data is representative of4 or more experiments. npo0.05.

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those adjacent to these sites, Ser 40 and Ser 42, as potential sitesof O-GlcNAcylation. We transfected DU145 cells with the follow-ing constructs: quadruple β-catenin mutant (4M¼Ser(S) 33A,S37A, Thr41A and S45A), S40A- or S42A-β-catenin. Transfectedcells were treated with or without PUGNAc and O-GlcNAc wasdetermined by WGA-agarose precipitation and WB with anti-β-catenin antibody. Upon PUGNAc treatment, the 4M, S40A andS42A β-catenin mutants all showed a time dependant increase inO-GlcNAc modification which paralleled the increase in O-GlcNA-cylation of endogenous β-catenin (Fig. 3A–C, respectively).This indicated that the NT phosphorylation sites and the adjacentSer 40 and Ser 42 sites were not involved in the active cycling ofO-GlcNAc groups.

O-GlcNAcylation at Serine 23 regulates the subcellulardistribution of β-catenin

To observe the putative functional significance of the O-GlcNAcmodification of Ser 23 of β-catenin, we carried out IF analysis ofendogenous β-catenin as well as recombinant full length GFP-WTand GFP-S23G β-catenin constructs in DU145 cells in the presence orabsence of PUGNAc treatment. Fig. 4A shows that endogenousβ-catenin as well as GFP-WT and GFP-S23G β-catenin have verysimilar nuclear and cytoplasmic distribution in untreated DU145 cells.However, when cells were treated with 100 mM PUGNAc, both theendogenous and the GFP-WT β-catenin preferentially translocated tothe plasma membrane. In contrast, the subcellular distribution of

Fig. 3 – Ser33, Ser37, Thr41, Ser45, Ser 40 and Ser42 are not sites of O-GlcNAc modification of β-catenin. (A) DU145 cells transfectedwith GFP tagged full length quadruple S33A, S37A, S45A and Thr41A β-catenin mutant (4M) were treated with 100 lM PUGNAc for aset time course. Whole cell lysates were precipitated with WGA-agarose beads and any alterations in O-GlcNAcylation of β-cateninevaluated with anti-β-catenin antibody. O-GlcNAcylation of the 4M mutant increased in a time dependent manner parallel toendogenous β-catenin. Similar results were seen with (B) S40A and (C) S42A mutants. The results observe indicated that S33, S37,Thr41, S45, Ser 40 and S42 were not sites of O-GlcNAc modification. Each group of data is representative of 4 or more experiments.npo0.05.

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GFP-S23G β-catenin mutant was unaltered in the presence of PUGNActreatment compared to the untreated cells. Supplementary Table 1summarizes a quantitative evaluation of the percentage of cells thatshowed altered (plasma membrane) localization of β-catenin (endo-genous & constructs) with PUGNAc treatment.

We further confirmed this observation using the osteosarcomacell line U2OS (Fig. 4B). Similar to DU145 cells, the U2OS cell lineexpressed endogenous β-catenin and low levels of the O-GlcNAcy-lated form of the protein. IF analysis indicated the presence of β-catenin in the cytosol and nucleus in the U2OS cell line whichredistributed preferentially to the plasma membrane upon PUGNActreatment. This was comparable to that in the DU145 cell line.

Collectively, these results suggested that Ser 23 is an importantsite for O-GlcNAc modification of β-catenin which plays a

functional role in regulating the subcellular distribution/localiza-tion of the protein.

O-GlcNAc modification at Serine 23 increases β-catenin'sinteraction with E-cadherin

The observation that O-GlcNAcylation of endogenous and GFP-WTβ-catenin resulted in a preferential localization of the protein tothe plasma membrane, raised the question whether this mod-ification played a role in regulating β-catenin's interaction withone of its major cellular interactor at the cell membrane, namelyE-cadherin. Interaction between β-catenin and E-cadherin wasevaluated by IP of β-catenin with anti-β-catenin antibody or anti-GFP antibody and detection of E-cadherin in the immunoprecipitate

Fig. 4 – Serine 23 regulates β-catenin's subcellular localization. (A) Immunofluorescence analysis of the subcellular localization ofendogenous, full length GFP tagged WT and S23G mutant β-catenin in DU145 cells with or without 100 lM PUGNAc treatment.Endogenous β-catenin was seen to be dispersed throughout the cytoplasm and the nucleus (Panel 1). Upon PUGNAc treatment,β-catenin localized to the plasma membrane. The GFP-WT β-catenin behaved similarly to endogenous β-catenin (Panel 2).However, PUGNAc treatment had no effect on the subcellular distribution of GFP-S23G mutant β-catenin as it remained dispersedthroughout the cell in the presence or absence of PUGNAc (Panel 3). Nucleus (Blue) was stained with DAPI. Endogenous β-cateninwas visualized using Alexafluors 488 secondary antibody to anti-β-catenin primary antibody. GFP-WT and GFP-S23G mutantβ-catenin constructs were followed by their GFP tag. (B) Similar results were seen with the osteosarcoma cell line U2OS. Eachgroup of data is representative of 4 or more experiments. npo0.05.

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with anti-E-cadherin antibody. All experiments were redone withreverse IP (Fig. 5). First, endogenous β-catenin was immunopreci-pitated with anti-β-catenin antibody from whole cell lysates ofDU145 cells and treated with or without PUGNAc. Fig. 5A showsthat there was significant increase in the interaction of endogenousβ-catenin with E-cadherin upon PUGNAc treatment compared tothe untreated cells. A similar increase in interaction upon PUGNAc

treatment was observed between the full length GFP-WT β-cateninand E-cadherin (Fig. 5B). However, no differences in the interactionbetween the full length GFP-S23G β-catenin mutant and E-cadherinwas observed in the presence or absence of PUGNAc treatment(Fig. 5B). WGA-agarose precipitation (Fig. 5C) or WGA-HRP analysis(Fig. 5A) confirmed increased O-GlcNAcylation upon PUGNActreatment of both endogenous and recombinant GFP-WT β-catenin,

Fig. 5 – β-Catenin's interaction with E-cadherin is regulated by O-GlcNAc modification at Serine 23. (A) Immunoprecipitation ofwhole cell lysates from DU145 cells treated with or without PUGNAc showed increased binding of endogenous β-catenin withE-cadherin. Increased β-catenin-E-cadherin interaction was confirmed by reverse immunoprecipitation. Presence ofO-GlcNAcylation was detected by WGA-HRP. (B) Immunoprecipitation of lysate from DU145 cells transfected with full length GFP-WT β-catenin treated with or without PUGNAc resulted in increased binding of full length GFP-WT β-catenin with E-cadherin.However, there was no change in interactions between E-cadherin and the full length GFP-S23G mutant β-catenin in the presenceor absence of PUGNAc. Results were confirmed by reverse immunoprecipitation. Total non-specific IgG antibody served as thecontrol. (C) The same lysates as (B) were used for the detection of the presence of O-GlcNAcylation using WGA-agarose beads.O-GlcNAcylation of GFP-WT β-catenin was induced upon PUGNAc treatment. As previously shown, the O-GlcNAcylation of GFP-S23Gmutant β-catenin was unchanged despite PUGNAc treatment. Each group of data is representative of 4 or more experiments.npo0.05.

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respectively. Induction of O-GlcNAcylationwith PUGNAc treatment wasnot observed for GFP-S23G β-catenin (Fig. 5C). pEGFP empty vector andtotal non-specific IgG antibody served as the internal controls.

Collectively these results suggest that O-GlcNAc modification atSer23 of β-catenin is important in regulating its interaction withE-cadherin.

Fig. 6 – Nuclear localization of β-catenin is regulated by O-GlcNAcylation at Serine 23. (A) Nuclear lysates of DU145 cells transfectedwith full length GFP-WT or GFP-S23G mutant β-catenin and treated with or without 100 lM PUGNAc were characterized by WBAnalysis. Levels of GFP-WT β-catenin decreased within the nucleus upon PUGNAc treatment. No change in localization wasobserved in GFP-S23G mutant β-catenin. The presence of Lamin B ensured an enriched nuclear fraction. Cytosolic fractions wereprecipitated for O-GlcNAc-β-catenin with WGA-agarose beads to ensure the presence of O-GlcNAcylation. (B) β-catenin's interactionwith TCF is regulated by O-GlcNAc modification at Ser 23. Nuclear lysates of DU145 cells transfected with full length GFP-WT orGFP-S23G mutant β-catenin and treated with or without 100 lM PUGNAc were immunoprecipitated with anti-TCF antibody.Alterations in GFP-WT and GFP-S23G interactions with TCF were characterized by WB Analysis using anti-GFP antibody. GFP-WTβ-catenin-TCF interactions decreased upon PUGNAc treatment. No change was observed in GFP-S23G mutant β-catenin-TCFinteractions. The presence of O-GlcNAcylation was confirmed through WGA-agarose precipitation. Each group of data isrepresentative of 4 or more experiments. npo0.05.

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O-GlcNAcylation at Serine 23 regulates β-catenin nuclearlocalization and its interaction with TCF

The redistribution of β-catenin to the plasma membrane wasassociated with correspondingly lower nuclear levels of β-catenin(Fig. 4). We carried out WB analysis of nuclear lysates from DU145cells transfected with GFP-WT or GFP-S23G full length β-cateninconstructs treated with or without PUGNAc. Our results showedthat there was a significant decrease in the nuclear levels ofGFP-WT β-catenin upon PUGNAc treatment (Fig. 6A). However,nuclear levels of GFP-S23G mutant β-catenin remained unaltered

in the presence and absence of PUGNAc treatment (Fig. 6A).O-GlcNAc modification of β-catenin was confirmed through WGA-agglutinin precipitation of the cytosolic fraction.

Further, IP of nuclear lysates with anti-TCF antibody showed thatthe decrease in nuclear β-catenin was associated with a concomitantdecrease in the interaction between TCF and GFP-WT β-catenin withPUGNAc treatment (Fig. 6B). This was in agreement with our previousreport which demonstrated that increased O-GlcNAcylation decreasedthe interactions between TCF and endogenous β-catenin [9]. Interest-ingly, the interaction between GFP-S23G β-catenin and TCF wasunaltered in the presence of PUGNAc treatment compared to control.

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This suggested that O-GlcNAc modification at Ser 23 plays a role inregulating the nuclear availability of β-catenin and its interactionwith TCF both of which are important components relevant to thetranscriptional function of the protein.

O-GlcNAcylation at Serine 23 decreases the transcriptionalactivity of β-catenin

In order to determine the importance of Ser 23 in the transcrip-tional function of β-catenin, we measured TOPFlash-luciferasereporter activity in the DU145 cell lines transfected with GFP-WT orGFP-S23G β-catenin (Fig. 7A). Our data shows that PUGNAc-treatmentresulted in a significant decrease in the TOPFlash activity in cellsoverexpressing GFP-WT β-catenin. However, there was no change inTOPFlash activity upon PUGNAc treatment of the GFP-S23G trans-fected DU145 cells compared to those that were untreated. Interest-ingly, the baseline TOPFlash activity of the S23G mutant β-cateninexpressing cells in the absence of PUGNAc, was increased relative toGFP-WT β-catenin expressing DU145 cells.

To further ascertain the significance of Ser 23 in the transcrip-tional function of β-catenin we looked at β-catenin's transactivationof wnt target genes. Specifically, we looked at the transcriptionalregulation of vascular endothelial growth factor A (VEGFA) andcyclin D1. RT-qPCR analysis indicated significant decreases for bothVEGFA (�40%) and cyclin D1 (�40%) mRNA copy number uponPUGNAc treatment of GFP-WT β-catenin transfected DU145 cells(Fig. 7B and C). In contrast, mRNA copy number of VEGFA and cyclinD1 in DU145 cells transfected with the GFP-S23G mutant β-cateninwas not altered upon PUGNAc treatment compared to untreatedcells (Fig. 7B and C).

Collectively, these results highlight the significant role of Ser 23for the transcriptional activity of β-catenin.

O-GlcNAcylation at Serine 23 of β-catenin alters the rateof proliferation of DU145 Cells

Since O-GlcNAc of β-catenin effectively reduced cyclin D1 levelswe determined whether this had any effect on the rate ofproliferation of cells. We used the MTT assay for evaluation ofcell proliferation. The Tetrazolium dye reduction in the MTT assayis dependent on NAD(P)H-dependent oxidoreductase enzymeslargely in the cytosolic compartment of the cell. Therefore,reduction of MTT and other tetrazolium dyes increases withcellular metabolic activity due to elevated NAD(P)H flux. Restingcells that are viable but metabolically quiet reduce very little MTT.In contrast, rapidly dividing cells exhibit high rates of MTTreduction. Our results show that treatment with PUGNAc ofDU145 cells transfected with pEGFP (empty vector) or pEGFP-β-catenin-WT resulted in significant decrease in the rate ofproliferation (Fig. 7D). On the other hand, treatment with PUGNAcof DU145 cells transfected with pEGFP-β-catenin-S23G mutanthad no effect on the rate of proliferation of the cells.

O-GlcNAcylation at Serine 23 of β-catenin results indecreased anchorage-independent growth of DU145 cells

We evaluated anchorage independent growth using Soft Agarassay with DU145 cells transfected with pEGFP-WT or pEGFP-S23G β-catenin and treated with or without PUGNAc. Our resultsindicate that untreated pEGFP-β-catenin-WT transfected DU145

cells formed a significant number of colonies on soft agar whichdecreased upon PUGNAc treatment indicating a decreased trans-formed/tumorigenic phenotype (Fig. 7E). Untreated pEGFP-S23Gβ-catenin-transfected DU145 cells formed greater numbers ofcolonies on soft agar compared to the untreated pEGFP-WTβ-catenin-transfected DU145 cells. However, treatment with PUG-NAc of DU145 cells transfected with pEGFP-β-catenin-S23-G hadno effect on the number of colonies (Fig. 7E).

Discussion

We previously reported that β-catenin is O-GlcNAcylated and thatO-GlcNAc modification inversely regulated its nuclear localizationand transcriptional function [9]. The O-GlcNAc modification occursin response to external stimuli and is a highly dynamic modifica-tion moiety turning over at a higher rate than the protein [19,20].A systematic analysis of the three main domains of β-catenin (NT, CTand central ARM domains) indicated that the only domain at whichthere was active cycling O-GlcNAc modification of β-catenin was atthe NT domain. Using a mutational approach we identified Ser 23 atthe NT as a specific and important site of O-GlcNAc modification andfurther showed that O-GlcNAcylation of Ser 23 is an essentialregulator of β-catenin's subcellular distribution and transcriptionalactivity. Specifically, O-GlcNAc modification at Ser 23 resulted in there-localization of β-catenin from the nucleus to mainly the plasmamembrane (Fig. 4). This re-localization was associated with increasedinteraction of β-catenin with E-cadherin (Fig. 5A and B), decreasedβ-catenin-TCF interaction (Fig. 6), and, decreased transcriptionalactivity and wnt target gene expression (Fig. 7).The close proximity of Ser 23 to the destruction box, a sequence

of amino acids that are critical for regulating the stability andtranscriptional functions of the protein, brings interesting func-tional prospects for the O-GlcNAc modification. It raises thepossibility that the NT domain may play a primary role inregulating the stabilization and nuclear localization of β-cateninthrough phosphorylation and O-GlcNAc modification, respec-tively, as the armadillo and CT domains of β-catenin were notactively modified by cycling O-GlcNAc groups (Fig. 1). This isparticularly interesting as little is currently known about themolecular mechanism(s) that regulate the shuttling of β-cateninbetween the cytoplasmic/plasma membrane and nuclear com-partments; β-catenin lacks the classical NLS and does not use theimportin nuclear transport system [7].The flexibility of the NT and CT domains enable differential

regulation of the armadillo domain for β-catenin's various bindingpartners. The NT domain's role in modulating localization andtranscriptional activity of β-catenin can therefore be achievedthrough its direct influence on the binding of its interactorswithin the armadillo domain. To this end, experiments deliveringtruncated armadillo domain void of the NT into the nucleusrevealed an absence of β-catenin-TCF complexes, suggesting thatthe NT influences the gene transcription and chromatin remodel-ing functions of β-catenin [21,22]. Moreover, the presence ofCT-Armadillo interactions was shown to enhance the stability ofβ-catenin by preventing its binding to the axin scaffolding complex;however, the impact of the CT domain upon β-catenin signaling atthe level of the NT destruction box is yet unknown [23,24].As the interactions within the armadillo domain are largely

dependent on ionic charge modulated by phosphorylation events,

Fig. 7 – O-GlcNAcylation of β-catenin decreased its transcriptional function. (A) The relative β-catenin/TCF activities (TOPFlash-luciferasereporter) in response to PUGNAc treatment. DU-145 cells were transiently co-transfected with full length GFP-WT or GFP-S23G mutantβ-catenin and TOPFlash using EXGEN-500 transfection reagent. Figure shows that β-catenin/TCF activity is significantly reduced withPUGNAc treatment in the DU145 cells transfected with full length GFP-WT. FOPFlash activity served as a negative control. However,TOPFlash activity in the DU145 cells transfected with full length GFP-S23G mutant β-catenin was unaltered with PUGNAc treatmentcompared to the untreated cells. Interestingly, the baseline TOPFlash activity of the S23G mutant β-catenin construct transfected cells(in the absence of PUGNAc) was significantly higher than the GFP-WT transfected cells. (B) and (C) S23G mutation maximizes Wnt targetgene expression. RNAwas extracted from DU145 cells transfected with full length GFP-WT or GFP-S23G mutant β-catenin and treated withor without 100 lM PUGNAc. pEGFP empty vector served as the control. RT-qPCR was performed on 1 lg of total RNA extract. mRNA copynumber of (B) Cyclin D1 and (C) VEGFA decreased for GFP-WTand control conditions upon PUGNAc treatment. GFP-S23Gmutant β-catenintransfected cells showed elevated levels of (B) Cyclin D1 and (C) VEGFA mRNA expression relative to GFP-WT β-catenin transfected cells.Moreover, GFP-S23G mutant β-catenin transfected cells demonstrated no change in mRNA copy number of Cyclin D1 and VEGFA in thepresence or absence of PUGNAc treatment. Each set of data is representative of 4 or more experiments. npo0.05. (D) Treatment withPUGNAc of DU145 cells transfected with GFP-Empty vector or GFP-WT-β-catenin slows down the rate of proliferation of the cells. There isno alteration in the rate of proliferation with PUGNAc treatment of DU145 cells transfected with full length GFP-S23G mutant β-catenin.npo0.05. (E) Treatment with PUGNAc reduced the anchorage independent growth of DU145 cells transfected with full length GFP-WTβ-catenin. DU145 cells transfected with full length GFP-S23G mutant β-catenin showed greater number of colonies in soft agar but therewas no change in the number of colonies upon treatment with PUGNAc. npo0.05.

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it is likely that site occupancy of O-GlcNAc at Ser 23 (with a Stokesradius 4–5 times larger than a phosphate [25]) may induceconformational changes of the protein to hinder or facilitate itsinteractions with molecular chaperones. Our results show that inDU145 CaP cells, O-GlcNAcylation of β-catenin increased itsinteraction with E-cadherin at the plasma membrane (Fig. 5Aand B). This is suggestive of a putative role of O-GlcNAc modifica-tion of β-catenin in promoting the stabilization of the adherensjunctions. We also noted that O-GlcNAc of β-catenin decreased itslevels within the nucleus (Fig. 6A) and concomitantly decreased itsinteraction with TCF (Fig. 6B) resulting in the attenuation of itstransactivator function (Fig. 7). However, the O-GlcNAc modification-associated effects observed with GFP-WT β-catenin were dramati-cally prevented by the introduction of a missense mutation S23G toβ-catenin. This suggested that Ser 23 was a key site that regulatedβ-catenin's interactions with E-cadherin and TCF, two critical cellularintertactors of β-catenin in its role as an adherens junction stabilizerand transcription/transactivator function, respectively. The full impli-cation of the effect of such alterations in O-GlcNAcylation of β-cateninis unclear at present but it can be speculated that both phenomenawill effectively support a tumor suppressive role of O-GlcNAcylationfor β-catenin and therefore, potentially have functional relevance indecreasing the metastatic potential of cancer cells.

The “Yin Yang” hypothesis suggests that the relationshipbetween O-GlcNAcylation and O-phosphorylation is as a binaryregulatory system for proteins. Van Noort et al., identifiedβ-catenin to be phosphorylated at Ser 23 by GSK3β [26]. Thisfinding indicated that phosphorylation and O-GlcNAcylationmodifications were in direct interplay at this site, suggesting apossible cross regulation of β-catenin's function by these twomodifications. However, these authors also stated that the phos-phorylation of Ser 23 did not influence β-catenin's interactionswith its cellular interactors or its function as a transcriptionalactivator [26]. Our data, on the other hand, supports a role forO-GlcNAcylation of Ser 23 in regulating β-catenin's subcellularlocalization and its interactions with its cellular partners. We cantherefore hypothesize that O-GlcNAcylation and phosphorylationmay work hand in hand to regulate the function of β-catenin, withO-GlcNAc regulating its localization and phosphorylation regulat-ing the stabilization of its cellular levels.

APC, in its role to export β-catenin from the nucleus, sustains theefficiency of the destruction complex, by controlling the release andrecruitment of β-catenin [10]. Given that the interaction betweenAPC and β-catenin was found to be unaltered upon O-GlcNAcmodification [9], we can speculate that O-GlcNAcylation at Ser 23acts as a regulatory switch for stabilizing β-catenin from degrada-tion within the cytoplasm. This is supported by observations bySayat et al., who demonstrated that the stability of β-catenin wasnot altered upon O-GlcNAc modification [9]. Specifically, β-catenin'sincreased localization to and restriction within the cytoplasm/plasma membrane may be regulated through its interaction withAPC at the destruction complex. A study by Ikeda et al. showed thatthe presence of β-catenin modulated the phosphorylation of APC byGSK3β within the Axin scaffold [27]. Specifically, β-catenin did notaffect the activity of GSK3β, but enhanced/stimulated APC phos-phorylation. Based on this argument, we propose that O-GlcNAcyla-tion of Ser 23 can putatively restrict GSK3β phosphorylation of APC,impeding adequate delivery of β-catenin to the destruction complexand hence its phosphorylation. Therefore, as stated by Mondouxet al., GSK3β would be the likely candidate to regulate β-catenin

through mechanisms that involve this interplay between phosphor-ylation and O-GlcNAcylation [28]. In effect, the unaltered export ofβ-catenin from the nucleus, together with the hindered degradationof β-catenin at the destruction complex upon its O-GlcNAcylation atSer 23, will result in increased levels of the protein within thecytoplasmic/plasma membrane compartments. How the APC-β-catenin interaction may be affected at the level of the destructioncomplex by the O-GlcNAc modification is currently unknown andbeing investigated.The decrease in β-catenin-TCF complexes alongside the attenua-

tion in TOPFlash activity and mRNA expressions of wnt targetgenes (Cyclin D1 and VEGFA) with increased O-GlcNAcylation islikely the direct consequence of the decreased levels of β-cateninwithin the nucleus. Yet, a consistent observation was the elevatedTOPFlash activity and elevated expressions of Cyclin D1 andVEGFA mRNA with the introduction of the S23G mutation. It isunlikely that this increased transcriptional output is due to anyalterations in TCF itself as TCF is not modified by O-GlcNAcylationnor altered in its nuclear levels [9]. TCF/LEF transcription factorsare actively repressed by transcriptional co-repressors such asGroucho/TLE proteins. The binding of β-catenin with DNA-boundTCF/LEF within the nucleus displaces Grouch/TLE proteins andrecruits an array of co-activator proteins including BCL9. As such,this brings into question the ability of O-GlcNAc to regulateβ-catenin's co-activator interactions by altering its affinity to alter-nate transcriptional co-activators. The ability of post translationalmodifications to have such an effect has been shown by Brembecket al. who demonstrated that phosphorylation of Tyrosine 142 ofβ-catenin by c-Met acted as a molecular switch that transformed theadhesive form of β-catenin into one that preferentially bound toBCL9-2 and dramatically decreased wnt target gene transcription[29]. It is then quite possible that O-GlcNAcylation may limit thecapacity of β-catenin to alleviate TCF repression through its inabilityto compete with TCF-Groucho/TLE complexes. By introducing S23Gmutation this inhibitory effect is removed allowing for more efficienttransactivation activity. The question of how O-GlcNAc modificationmay affect TCF-β-catenin interaction and TCF-Groucho competitionat the level of the transcriptional machinery still remains to beanswered.In line with the changes of the transcriptional function of

β-catenin upon O-GlcNAc modification and reflective of thechanges observed with cyclin D1, we also observed significantreduction in the rate of cell growth/proliferation in DU145 cellsoverexpressing WT-β-catenin upon treatment with PUGNAc.Cyclin D1 is a proto-oncogene that is overexpressed in manycancers and plays a role in cell proliferation through activation ofcyclin-dependent kinases. Further, we also noted a significantdecrease in anchorage independent growth upon PUGNAc treat-ment of DU145 cells overexpressing WT-β-catenin. This is indica-tive of a decrease in the tumorigenic potential of the cells withO-GlcNAc. Interestingly, these alterations in rate of proliferationand anchorage-independent growth were not observed withPUGNAc treatment of DU145 cells transfected with the S23G-β-catenin indicating that Ser 23 is an important O-GlcNAc mod-ification site of β-catenin that functionally regulates the oncogenicpotential of the protein. It should be pointed out that PUGNActreatment of the cells would also cause global O-GlcNAcylation ofseveral proteins which would have an impact on these functionalparameters. However, the fact that there is no alteration in the rateof cell proliferation and anchorage independent growth with

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PUGNAc treatment of DU145 cells transfected with the S23G-β-catenin highlights that O-GlcNAc of β-catenin at Ser 23 plays asignificant and specific role in regulating the transformed/tumori-genic phenotype of cells.In conclusion, our investigation demonstrated that Ser 23 is an

important site for O-GlcNAcylation and that O-GlcNAc modifica-tion at Ser 23 played a central role in mediating β-catenin'ssubcellular localization by altering its binding capacity to itsmajor cellular interactors. Specifically, our study suggests thatthe regulatory effect of O-GlcNAcylation of Ser 23 may hinge ondifferentially altering β-catenin's affinity for its cellular interac-tors. Although the molecular mechanism underlying this phe-nomenon remains to be established, we propose a tripartitesystem for the regulation of β-catenin's function, where cellularβ-catenin exists in a phosphorylated, O-GlcNAcylated, or unmo-dified state, which in turn modulates the various intracellularpools of β-catenin (plasma membrane, cytosolic, nuclear) center-ing on the post-translationally modified status of Ser 23 at the NTof the protein.

Acknowledgments

This project was supported by funds from Canadian Institute ofHealth Research, Cancer Research Society of Canada and The HairMassacure and Stollery Children's Hospital Research Foundation to SP.

Appendix A. Supplementary materials

Supplementary data associated with this article can be found inthe online version at http://dx.doi.org/10.1016/j.yexcr.2013.11.021.

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