o-linked n-acetylglucosaminyltransferaseinhibition...

O-Linked N-Acetylglucosaminyltransferase InhibitionPrevents G2/M Transition in Xenopus laevis Oocytes*

Received for publication, January 16, 2007, and in revised form, February 26, 2007 Published, JBC Papers in Press, February 28, 2007, DOI 10.1074/jbc.M700444200

Vanessa Dehennaut‡§1, Tony Lefebvre§, Chantal Sellier‡, Yves Leroy§, Benjamin Gross¶, Suzanne Walker¶,Rene Cacan§, Jean-Claude Michalski§, Jean-Pierre Vilain‡, and Jean-Francois Bodart‡2

From the ‡Laboratoire de Regulation des Signaux de Division, EA 4020, Universite des Sciences et Technologies de Lille, SN3, IFR147,59655 Villeneuve d’Ascq, France, the §Unite de Glycobiologie Structurale et Fonctionnelle, UMR 8576 du CNRS, IFR147, Batiment C9,Cite Scientifique, 59655 Villeneuve d’Ascq, France, and the ¶Department of Microbiology and Molecular Genetics,Harvard Medical School, Boston, Massachusetts 02115

Full-grown Xenopus oocytes are arrested at the prophase of thefirst meiotic division in a G2-like state. Progesterone triggers mei-otic resumption also called theG2/Mtransition.This event is char-acterized by germinal vesicle breakdown (GVBD) and by a burst inphosphorylation level that reflects activation ofM-phase-promot-ing factor (MPF) and MAPK pathways. Besides phosphorylationandubiquitinpathways, increasingevidencehas suggested that thecytosolic and nucleus-specific O-GlcNAc glycosylation also con-tributes to cell cycle regulation. To investigate the relationshipbetweenO-GlcNAc and cell cycle, Xenopus oocyte, in which mostof theM-phaseregulatorshavebeendiscovered,wasused.Alloxan,an O-GlcNAc transferase inhibitor, blocked G2/M transition in aconcentration-dependent manner. Alloxan prevented GVBD andbothMPFandMAPKactivations, either triggeredbyprogesteroneorbyeggcytoplasm injection.Theadditionofdetoxifyingenzymes(SOD and catalase) did not rescue GVBD, indicating that thealloxan effect did not occur through reactive oxygen species pro-duction. These results were strengthened by the use of a benzox-azolinone derivative (XI), a new O-GlcNAc transferase inhibitor.Conversely, injection of O-(2-acetamido-2-deoxy-D-glucopyrano-sylidene)amino-N-phenylcarbamate, an O-GlcNAcase inhibitor,accelerated the maturation process. Glutamine:fructose-6-phos-phate amidotransferase inhibitors, azaserine and 6-diazo-5-ox-onorleucine, failed to prevent GVBD. Such a strategy appeared tobe inefficient; indeed, UDP-GlcNAc assays in mature and imma-ture oocytes revealed a constant pool of the nucleotide sugar.Finally, we observed that cyclin B2, the MPF regulatory subunit,was associated with an unknown O-GlcNAc partner. The presentwork underlines a crucial role for O-GlcNAc in G2/M transitionand strongly suggests that its function is required for cell cycleregulation.

Immature vertebrates oocytes, arrested at prophase I of mei-osis, can resume their cell cycle, also called maturation, in

response to hormonal stimulation (1, 2). Studies in amphibianXenopus laevis oocyte have greatly contributed to the knowl-edge of the biochemical activities of the key regulatory mole-cules involved in these processes (3).Meiosis entry, analogous to G2/M transition, is promoted by

a cytoplasmic factor called MPF.3 MPF, made up of a catalyticsubunit, Cdk1 (cyclin-dependent kinase 1) (also called Cdc2(cell division cycle 2)), and a regulatory subunit, cyclin B, hasbeen demonstrated to be the universal regulator of mitosis andmeiosis entry. Activation of this cyclin-Cdk complex is con-trolled by phosphorylation and proteolysis (for reviews, seeRefs. 4 and 5). Association between the regulatory subunit andCdk1 requires phosphorylation of Cdk1 on residue Thr161 byCdk-activating kinase (6). Then, to be catalytically active, Cdk1is dephosphorylated on Thr14 and Tyr15 residues by Cdc25, adual specific phosphatase (6, 7). Simultaneously to Cdk1dephosphorylation, cyclin B is phosphorylated (8). Xenopusimmature oocytes, which are synchronized at the diplotenestage of first meiosis, contain large amounts of inactiveMPF, orpre-MPF (4, 5). Inhibition of pre-MPF is provided byMyt1 thatphosphorylates Cdk1 on Thr14 and Tyr15 and then inactivatesthe complex. Pre-MPF can be directly activated byCdc25 injec-tion (9) or through an autoamplification loop. The latter loopinvolves the ability ofMPF to phosphorylate and activateCdc25(10, 11) and does not depend upon protein synthesis (12).Simultaneously to MPF activation, extracellular signal-regu-

lated kinase-like Xp42mpk1 is phosphorylated and activated(13). Xp42mpk1 belongs to themitogen-activated protein kinase(MAPK) pathway that is turned on byMos oncoprotein synthe-sis in response to progesterone stimulation (14). Once acti-vated, Xp42mpk1 phosphorylates and activates ribosomal S6kinase (p90rsk), which negatively regulates Myt1 (15). Activa-tion of the Mos-Xp42mpk1 pathway has been shown not to berequired for MPF activation but for timely M-phase entrywhen oocytes are stimulated either by progesterone or insu-lin (16–18). Nevertheless, this pathway has been shown to be

* This work was supported in part by the “Centre National de la RechercheScientifique,” the “Association pour la Recherche contre le Cancer,” and the“Universite des Sciences et Technologie de Lille I.” The costs of publicationof this article were defrayed in part by the payment of page charges. Thisarticle must therefore be hereby marked “advertisement” in accordancewith 18 U.S.C. Section 1734 solely to indicate this fact.

1 Recipient of a fellowship from the “Ministere de la Recherche et del’Enseignement.”

2 To whom all correspondence should be addressed. Tel.: 32-0436847; Fax:32-0434038; E-mail: [email protected].

3 The abbreviations used are: MPF, M-phase-promoting factor; O-GlcNAc,O-linked N-acetylglucosamine; PTM, post-translational modification; DON,6-diazo-5-oxonorleucine; GFAT, glutamine:fructose-6-phosphate amido-transferase; GVBD, germinal vesicle breakdown; MAPK, mitogen-activatedprotein kinase; OGT, O-linked N-acetylglucosaminyltransferase; O-Glc-NAcase, O-linked N-acetylglucosaminidase; PUGNAc, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate; ROS, reactiveoxygen species; SOD, superoxide dismutase; WGA, wheat germ agglutinin;MOPS, 4-morpholinepropanesulfonic acid; ALX, alloxan.

THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 282, NO. 17, pp. 12527–12536, April 27, 2007© 2007 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in the U.S.A.

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responsible for S-Phase suppression between meiosis I andmeiosis II, for metaphase II arrest, and for spindle morpho-genesis (17, 19–21).Mitogenic signals orchestrate a regulatory network of pro-

teins mainly through post-translational modifications (PTMs).Although phosphorylation is the molecular mechanism associ-ated with the regulation of cell cycle, it is clearly not the onlypost-translational mechanism involved in cell cycle progres-sion. Recent observations have suggested that the cytosolic andnucleus-specific O-linked N-acetylglucosaminylation (O-Glc-NAc) could be involved in cell cycle progression (22–24).O-GlcNAc is a highly dynamic PTM whose versatility is regu-lated by two enzymes (25): the O-GlcNAc transferase (OGT)that catalyzes the transfer of the GlcNAc moiety from UDP-GlcNAc and O-GlcNAcase that hydrolyzes the GlcNAc resi-due. Although the functions played by this single PTM remainto be determined, several studies tend to demonstrate thatO-GlcNAc is tightly linked to cell cycle regulation. Microinjec-tion of bovine galactosyltransferase inhibited Xenopus oocytesM-phase entry and blocked S- toM-phase transition (26). Slaw-son et al. (27) showed that perturbation of Xenopus oocyteO-GlcNAc levels, either by glucosamine or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate(PUGNAc) treatments, modified maturation kinetics. We pre-viously reported that theG2- toM-phase transitionwas accom-panied with a noticeable increase in O-GlcNAc glycosylation(22). Last, PUGNAc was used to inhibit O-GlcNAcase onsomatic cultured cells; such treated cells progressed throughthe cell cycle more slowly than untreated cells (24). This delaywas more pronounced during the S-phase progression and atthe G2/M boundary. By contrast, inhibition of glutamine:fruc-tose-6-phosphate amidotransferase (GFAT) with 6-diazo-5-oxonorleucine (DON), resulting in low O-GlcNAc level, short-ened S-phase, and G2/M progression. In return, GFATinhibition delayed G1 progression in comparison with controlsor PUGNAc-treated cells. Taken together, these data demon-strate that O-GlcNAc dynamic is a key regulatory PTM in cellcycle progression. The present study pointed out a role forO-GlcNAc glycosylation as a key regulator mechanism for pro-gression fromG2 arrest toM-phase inXenopus oocyte, throughthe regulation of both MPF and MAPK pathways.

EXPERIMENTAL PROCEDURES

Animals, Chemicals, and Bioreagents—Adult Xenopusfemales come from the University of Rennes I (France). Tric-aine methane sulfonate was purchased from Sandoz (Levallois-Perret, France). Collagenase A was purchased from RocheApplied Science. Progesterone, picroindigocarmine, alloxan,uracil, azaserine, DON, catalase, superoxide dismutase (SOD),WGA-agarose beads, monoclonal anti-p34cdc2 antibody, andDowex 50WX2-400were purchased fromSigma. PUGNAcwasa kind gift from Prof. Jerome Lemoine (UMR/CNRS 5579, Vil-leurbanne, France). Benzoxazolinone derivative (XI) was syn-thesized in Suzanne Walker’s laboratory (28). Mouse mono-clonal anti-O-GlcNAc (RL-2) was purchased from AffinityBioreagents (Golden, CO); polyclonal anti-�-catenin (H-102),monoclonal anti-Erk2 (D-2), and rabbit polyclonal anti-p90rsk(C21) were from Santa Cruz Biotechnology, Inc. (Santa Cruz,

CA); monoclonal anti-Hsp/Hsc70 (SPA-820) was from Stress-gen (Victoria, Canada); rabbit anti-phospho-Tyr15-Cdc2 andanti-phospho-p44/42MAPK (Erk1/2) were fromCell SignalingTechnology (Danvers, MA); and rabbit polyclonal anti-cyclinB2 (JG103) was a gift fromDr. J. Gannon (ICRF, SouthMimms,UK). Anti-mouse, anti-rabbit horseradish peroxidase-labeledsecondary antibodies and enhanced chemiluminescence werepurchased from GE Healthcare (Saclay, France).Handling of Oocytes—After anesthetizing Xenopus females

by immersion in 1 g/liter MS222 solution (tricaine methanesulfonate), ovarian lobes were surgically removed and placed inND96 medium (96 mM NaCl, 2 mM KCl, 1.8 mM CaCl2, 1 mMMgCl2, 5 mM HEPES-NaOH, pH 7.5). Fully grown stage VIoocytes were isolated, and follicle cells were partially removedby 1 mg/ml collagenase A treatment for 30 min followed by amanual microdissection. Oocytes were stored at 14 °C in ND96medium until experiments.Stimulation and Analysis of G2/M Transition (Meiotic

Resumption) in Xenopus Oocytes—Meiotic resumption wasinduced by incubating oocytes in ND96 medium containing 10�M progesterone or by cytoplasmmicroinjection. Briefly, cyto-plasm of matured oocytes was collected and microinjected inimmature oocytes (50 nl/oocyte) using a positive displacementdigital micropipette (Nichiryo, Tokyo, Japan). GVBD wasscored by the appearance of a white spot at the animal pole ofthe oocyte and confirmed by hemisection of oocyte after heatfixation (100 °C, 5 min). Alternatively, oocytes were fixed over-night in Smith’s fixative, dehydrated, and embedded in paraffinfor cytological studies. 7-�msectionswere stainedwith nuclearred for the detection of nuclei and chromosomes, whereaspicroindigocarmine was used to reveal cytoplasmic structures(21).Oocyte Treatments—Before stimulation of meiotic resump-

tion, oocytes (15–20 oocytes/condition) were incubated over-nightwith alloxan concentrations ranging from1 to 5mM, 5mMuracil, 500 �M Me2SO-solubilized benzoxazolinone derivative,40–80 units of catalase, 150 units of SOD, or 0.1–5 mMhydrogen peroxide. For GFAT inhibition experiments, 100�M DON or 20 �M azaserine were directly injected intooocytes before progesterone treatment. For O-GlcNAcaseinhibition, PUGNAc (concentrations ranging from 100 to400 �M) was also microinjected in immature oocytes beforemeiotic resumption stimulation. 5–10 oocytes were taken atthe end of the experiment respecting the white spot ratio,and stored at �20 °C until further biochemical analysis.Enrichment of O-GlcNAc-bearing Proteins withWGA Immo-

bilized on Agarose Beads—These experiments were performedin two conditions as previously described (29): in smooth con-ditions, which allows the recovery of allO-GlcNAc-modifiedproteins and their associated partners, and in more stringentconditions in which all protein-to-protein interactions arebroken.WGA Enrichment in Smooth Conditions—Batches of 20

immature or matured oocytes were taken and lysed in 200 �l ofhomogenization buffer (60 mM �-glycerophosphate, 15 mMparanitrophenylphosphate, 25 mM MOPS, 15 mM EGTA, 15mMMgCl2, 2mM dithiothreitol, 1mM sodium orthovanadate, 1mM NaF, and protease inhibitors, pH 7.2). After centrifugation

OGT Inhibition Blocks M-phase Entry

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at 20,000 � g, supernatants were collected and diluted withphosphate-buffered saline. Samples were then incubated for 90min at 4 °C with 50 �l of WGA-agarose beads. Proteins boundto WGA-beads were collected by centrifugation, washed fourtimes with phosphate-buffered saline, resuspended in 50 �l ofLaemmli buffer, and boiled for 10 min.WGA Enrichment in Stringent Conditions—Briefly, batches

of 20 immature or mature oocytes were lysed in 200 �l ofhomogenization buffer (10 mM Tris-HCl, pH 7.5, 1 mM EDTA,1mMEGTA, 0.5%TritonX-100, and protease inhibitors). Aftercentrifugation at 20,000 � g, supernatants were collected, and800�l of homogenization bufferwas added. Then sampleswereincubated with WGA-agarose beads, and WGA-bound pro-teins were collected, washed four times in 10 mM Tris-HCl, pH7.5, 100 mM NaCl, 0.4% sodium deoxycholate, 0.3% SDS, 0.2%Nonidet P-40, and resuspended in Laemmli buffer.SDS-PAGE and Western Blotting—Proteins (the equivalent

of one oocyte was loaded per lane) were run on a 10% SDS-PAGE for O-GlcNAc, phospho-p42/44 MAPK, Hsp/Hsc70,and �-catenin immunodetection. 17.5% modified SDS-PAGE(18, 30) was also run for phospho-Tyr15-Cdc2, Erk2, cyclin B2,and p90rsk immunodetection, since it allows a better discrimi-nation between active and inactive forms of these proteins, andelectroblotted onto a nitrocellulose sheet. Although the quan-tity of proteins remains rather constant in Xenopus oocyte,equal loading and transfer efficiency were checked using Pon-ceau red staining. Blots were saturated in 5% (w/v) milk in Tris-buffered saline-Tween (15 mM Tris, 140 mM NaCl, 0.05% (v/v)Tween) for 45 min. Primary antibodies were incubated over-night at 4 °C. Mouse monoclonal anti-O-GlcNAc (RL-2),mouse monoclonal anti-Erk2 (D-2), mouse monoclonal anti-Hsc/Hsp70 (SPA-820), rabbit polyclonal anti-phospho-p42/44MAPK, rabbit polyclonal anti-�-catenin (H102), rabbit poly-clonal anti-cyclin B2, and rabbit polyclonal anti-p90rsk wereused at a dilution of 1:1,000. Rabbit polyclonal anti-phospho-Tyr15-Cdc2 antibodies were used at a dilution of 1:750. Thennitrocellulose membranes were washed three times for 10 minin Tris-buffered saline-Tween and incubated with either ananti-mouse horseradish peroxidase-labeled secondary anti-body or an anti-rabbit horseradish peroxidase-labeled second-ary antibody at a dilution of 1:10,000 for �-catenin, Erk2, andphospho-p42/44 MAPK detection, at a dilution of 1:5,000 forcyclin B2, p90rsk, and phospho-Tyr15-Cdc2 detection. Finally,threewashes of 10min eachwere performedwithTris-bufferedsaline-Tween, and the detection was carried out with enhancedchemiluminescence.Measurement of UDP-GlcNAc Pools by High Performance

Anion Exchange Chromatography—Ten oocytes were lysed in 1ml of hypotonic buffer (10 mM Tris/HCl, 10 mM NaCl, 15 mM2-mercaptoethanol, 1 mM MgCl2, and proteases inhibitors, pH7.2). 50 �l of 1 M HCl were then added to the lysate, and themixture was passed through a 1.5-ml Dowex 50WX2-400 col-umn. The columnwas washed with 10ml of bipermuted water.The unbound fraction and washes were collected on ice andadjusted to pH 8.0 with 500 �l of 1 M Tris/HCl. 250 �l of thediluted fraction was injected using a ProPAC-PA1 column (4�250mm) on a Dionex (Jouy en Josas, France) high performanceliquid chromatography system. The elution was achieved as

follows: Tris/HCl (20 mM, pH 9.2) (solution A) for 1 min; elu-tion gradient for 29 min with 85% A and 15% NaCl at 2 M(solution B); plateau of 5 min in the same conditions; 10-minelution gradient until 100%Bwas reached; plateau at 100%B for5 min. The column was then re-equilibrated in 100% A. Theflow rate was 1ml/min. Detection was performed using a Spec-troflow 757 UV spectrophotometer (Kratos-Analytical, Shi-madzu, Champs surMarne, France) at a wavelength of 256 nm.

RESULTS

The OGT Inhibitor Alloxan Blocks O-GlcNAc Increase andProgesterone-induced G2/M Transition—In a previous report,we demonstrated that Xenopus oocyte meiotic resumption wasaccompanied with a burst in O-GlcNAc content (22). In orderto understand the biological significance of this glycosylationincrease, we inhibited OGT using the uracil analogue alloxan(ALX) (31). Recently, alloxan has been successfully used for itsOGT-inhibitory effect, in isolated neonatal ventricular car-

FIGURE 1. Meiotic progression is prevented by alloxan, an OGT inhibitor,in Xenopus oocyte. Immature oocytes were treated with increasing amountsof alloxan (lanes 4 – 8). M-phase entry was then induced by progesteronetreatment. A, GVBD was assessed using white spot observation. The histo-gram shows the average GVBD values � S.D. Results are from three or four(asterisks) independent experiments. B, oocytes were homogenized in lysisbuffer, and Western blot analyses were performed using a panel of antibod-ies. O-GlcNAc content of oocytes was tested using an anti-O-GlcNAc-directedantibody (RL-2). Activation of Rsk, Cdc2, and cyclin B2 was assessed asdescribed under “Experimental Procedures.” �-Catenin accumulation wasalso analyzed. Lane 1, immature controls; lane 2, mature controls; lane 3,oocytes treated with 5 mM uracil. Protein mass markers (kDa) are indicated tothe left. WB, Western blot; URA, uracil; Pg, progesterone.




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diomyocytes (32), in intact rat heart (33), and in C2C12 myo-blasts (34). Oocytes were incubated with progesterone to trig-ger G2/M transition, together with increasing amounts ofalloxan, and a dose-dependent inhibition of GVBD wasobserved (Fig. 1A). Used as a negative control, uracil had noeffect onmeiosis resumption induced by hormonal stimulation(third lane). A significant inhibitory effect of alloxan wasobserved for concentrations ranging from 3 to 5 mM, whereaslower doses (1 and 2 mM) had no or little effect on G2/M tran-sition. Prevention of M-phase by alloxan appeared to be corre-lated with an O-GlcNAc level reduction: O-GlcNAc progres-sively decreased from 3 to 5 mM alloxan-treated oocytes tofinally reach an O-GlcNAc pattern similar to that of immatureoocytes (G2-phase). At the biochemical level, oocytes treatedwith these alloxan concentrations did not exhibit the typicalpattern of M-phase-entered oocytes (Fig. 1B, lanes 6–8). Rsk,whose phosphorylation directly depends upon MAPK activity,remained unphosphorylated, in contrast to phosphorylatedforms that shift in electrophoretic mobility in metaphase II-arrested control oocytes; Cyclin B2 remained unphosphoryla-ted (proven by the presence of a doublet of isoforms), and Cdc2phosphorylation on Tyr15 confirmed that MPF heterodimerwas under an inactive form in these oocytes (compare lanes 1, 2,and 8). Accumulation of �-catenin was also examined as amarker of the meiotic process. �-Catenin accumulates duringG2/M transition due to inhibition of GSK3� (glycogen synthasekinase 3�) that phosphorylates �-catenin on a PEST sequence,leading to its degradation by the proteasome system (35). In a

previous work, we reported that �-catenin O-GlcNAc contentwas enhanced after progesterone stimulation (22). As expected,�-catenin was easily detected in oocytes incubated solely withprogesterone or with low concentrations of alloxan (1 and 2mM). Expression of �-catenin was dramatically reduced whenalloxan was used at higher concentrations (Fig. 1B, bottom).

At the cytological level, high concentrations of alloxan pre-vented meiotic spindle formation, as proved by the presence ofan intact germinal vesicle envelope (Fig. 2C). Meiotic spindlesdetected were similar in oocytes treated with uracil (Fig. 2D) tothose examined in control mature oocytes (Fig. 2B).To check for the nontoxicity and reversibility of alloxan

effect, oocytes that were incubated with 5mMALXwere rinsedafter overnight treatment and were allowed to reach the G2/Mtransition in alloxan-free ND96 medium containing progester-one (Fig. 3); in these conditions, more than half of the rinsedoocytes exhibited GVBD (57 � 8.1%) (Fig. 3A), and removal ofalloxan allowed activation of both MAPK and MPF pathways,

FIGURE 2. Alloxan prevents GVBD. Oocytes were fixed, dehydrated, andembedded in paraffin. Sections (7 �m) were stained with nuclear red fordetecting nuclei and chromosomes, whereas picroindigocarmine was usedto reveal cytoplasmic structures. A, immature oocyte showing an intact ger-minal vesicle (GV). B, progesterone-treated oocyte (mature oocyte) with atypical metaphase spindle (MS) and the first polar body (PB). C, alloxan-treated oocyte showing an immature profile. D, uracil-treated oocyte show-ing a mature profile.

FIGURE 3. Alloxan is not toxic, and its effect can be reversed. Immatureoocytes were first incubated overnight with 5 mM ALX, and then meioticresumption was stimulated (Pg). 16 h after progesterone addition, alloxan-containing medium was removed (rinse), and oocytes were incubated inalloxan-free medium containing progesterone. A, GVBD was assessed usingwhite spot observation. The histogram shows the average GVBD values � S.D.Results are from four independent experiments. B, oocytes were homoge-nized in lysis buffer, and Western blot analyses were performed. O-GlcNAccontent of oocytes; activation of Rsk, MAPK, and cyclin B2; Cdc2, and �-cate-nin accumulation were assessed as described under “Experimental Proce-dures.” Protein mass markers (kDa) are indicated to the left. WB, Western blot;Pg, progesterone.




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accumulation of �-catenin, and increase in O-GlcNAc level(Fig. 3B, compare lanes 3 and 4).Since alloxan could interfere with any enzyme using uridine

and exert unrelated effects, a benzoxazolinone derivative (XI),another OGT inhibitor (referred to as compound number 5 inRef. 28), has been tested for its ability to block M-phase entry.Prior to hormonal stimulation, oocytes were incubated with 5mM alloxan (Fig. 4, lane 3) or 500 �M benzoxazolinone deriva-tive (lane 4) or without any inhibitor (lane 2). As for alloxan, thebenzoxazolinone derivative blocked G2/M transition; MPF andMAPK pathways were not activated, and the O-GlcNAc leveldid not increase after stimulation with progesterone.M-phase Entry Inhibition Induced by Alloxan Does Not

Depend upon Reactive Oxygen Species Formation—Free radi-cals have been reported to presumably control cell cycle (e.g.through Cdc25 regulation) (36). When in solution, alloxan is inequilibrium with its reduction product, dialuric acid, whichgenerates superoxide radicals (O2

. ) through a redox cycle (for areview, see Ref. 37). These superoxide radicals are used by SODto generate hydrogen peroxide (H2O2). Within cells, hydrogenperoxidemolecules sustain the Fenton reaction in the presenceof Fe2� ions and are then splinted into two hydroxyl radicals(OH�).

To counteract potentially alloxan-mediated reactive oxygenspecies (ROS) generation, alloxan-treated oocytes were incu-bated with SOD and catalase (which produces H2O and 1⁄2 O2fromH2O2) either 1 h prior to the progesterone addition (Fig. 5,lanes 4 and 6) or overnight prior to progesterone addition (Fig.5, lanes 8 and 10). In these conditions, the presence of the twodetoxifying enzymes did not prevent the action of alloxan atconcentrations ranging from 3 to 5mM: 1) GVBD did not occurfollowing progesterone stimulation (Fig. 5A); 2)O-GlcNAc lev-els did not increase; and 3) Rsk, p42MAPK, and cyclin B2 phos-phorylation patterns, as well as the �-catenin accumulationprofile, were typical of oocytes arrested in G2 (Fig. 5B).Finally, oocytes were also incubated in increasing concentra-

tions of hydrogen peroxide (H2O2; concentrations rangingfrom 0.1 to 5 mM). Although losing their pigmentation for 3, 4,

FIGURE 4. OGT inhibitor benzoxazolinone derivative (XI) prevents meioticresumption. Immature oocytes were treated with either 500 �M benzoxazolin-one derivative (XI) or 5 mM ALX or used as a vehicle control with 0.5% Me2SO.M-phase entry was then induced by progesterone treatment. A, GVBD wasassessed based on white spot observation. The histogram shows the averageGVBD values � S.D. Results are from three or four (asterisks) independent exper-iments. B, oocytes were homogenized in lysis buffer, and Western blot analyseswere performed. O-GlcNAc content of oocytes and activation of Rsk and cyclin B2were assessed as described under “Experimental Procedures.” Protein massmarkers (kDa) are indicated to the left. WB, Western blot; Pg, progesterone; DMSO,dimethylsulfoxide.

FIGURE 5. Incubation of antioxidant enzymes does not reverse alloxaneffect. Immature oocytes were treated with different concentrations ofalloxan (lanes 3–10). To counteract the potentially alloxan-mediated ROS gen-eration, alloxan-treated oocytes were incubated with SOD and catalase eitherfor 1 h prior to progesterone incubation (lanes 4 and 6) or overnight (O/N)prior to progesterone incubation (lanes 8 and 10). Lanes 1 and 2 correspond toimmature and mature controls, respectively. A, GVBD was assessed usingwhite spot observation. The histogram shows the average GVBD values � S.D.Results are from four independent experiments. B, oocytes were homoge-nized in lysis buffer, and Western blot analyses were performed. O-GlcNAccontent of oocytes; activation of Rsk, Cdc2, and cyclin B2; and �-catenin accu-mulation were assessed as described under “Experimental Procedures.” Pro-tein mass markers (kDa) are indicated to the left. WB, Western blot; Pg,progesterone.




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and 5 mM H2O2, oocytes nevertheless underwent GVBD, asshown by hemisection along the animal-vegetal axis after heatfixation (Fig. 6A; data not shown). Thus, G2/M progression wasnot impaired by H2O2. Staining with anti-O-GlcNAc, anti-Rsk1, anti-phospho-MAPK, anti-cyclin B2, anti-phospho-Tyr15-Cdc2, and anti-�-catenin showed that oocyte incubationwith hydrogen peroxide did not prevent progesterone-inducedG2/M transition (Fig. 6B).Alloxan Blocks O-GlcNAc Glycosylation and G2/M Transi-

tion Induced by Egg Cytoplasm Injection—G2/M transitioninduced by progesterone depends upon protein synthesis.However, when metaphase II-arrested oocyte (or egg) cyto-plasm is injected into immature recipient oocytes, it triggersG2/M transition through the MPF autoamplification loop,independently of protein synthesis (12). In comparison withprogesterone-treated oocytes, cytoplasm-injected oocytesexhibited no decrease in O-GlcNAc content following 3 mMalloxan incubation (Fig. 7B, top). They also exhibited a typicalpattern of phosphorylation/dephosphorylation and �-catenin

accumulation similar to those of M-phase oocytes (Fig. 7B): 1)cdc2 Tyr15 was dephosphorylated; 2) Rsk and cyclin B2 werephosphorylated; and 3) �-catenin was accumulated in identicalproportion to the cytoplasm-injected control oocyte alone.Complete inhibition for GVBD and activation of bothMPF andMAPK pathways were obtained for 4 and 5 mM concentration(Fig. 7, A and B). Alloxan similarly blocks GVBD, MAPK, andMPF pathways activations in progesterone-treated oocytes andin cytoplasm-injected oocytes. It must be noted that comparedwith the first experiment in which progesterone was directlyadded to trigger maturation, the alloxan concentration neededto inhibit M-phase entry was higher (4 mM for egg cytoplasminjection versus 3 mM for hormonal stimulation).O-GlcNAcase Inhibition Accelerates Xenopus Oocyte M-phase

Entry—Because twoOGT inhibitors were tested and preventedXenopus oocyte M-phase entry (Figs. 1, 2, 4, and 7), we thenchecked the effect of O-GlcNAcase inhibition on the matura-tion process.To this end, increasing amounts of PUGNAc, an inhibitor of

O-GlcNAcase (38), were microinjected in Xenopus oocytes priorto hormonal stimulation (Fig. 8). PUGNAc caused a slight accel-eration in maturation kinetics (p � 0.05 for 400 �M PUGNAc);

FIGURE 6. Hydrogen peroxide does not prevent Xenopus oocyte meioticresumption. Immature oocytes were incubated with increasing amounts ofhydrogen peroxide (H2O2) overnight before progesterone treatment. A, GVBDwas assessed using white spot observation. The histogram shows the averageGVBD values � S.D. Results are from four independent experiments. B, oocyteswere homogenized in lysis buffer, and Western blot analyses were performed.O-GlcNAc content of oocytes; activation of Rsk, Cdc2, and cyclin B2; and�-catenin accumulation were assessed as described under “ExperimentalProcedures.” Protein mass markers (kDa) are indicated to the left. WB, Westernblot; Pg, progesterone.

FIGURE 7. Alloxan-mediated OGT inhibition also prevents cytoplasm-in-duced meiotic resumption. Immature oocytes were incubated with ALX over-night before stimulation of meiotic resumption. Meiotic resumption was inducedby injection of mature oocyte cytoplasm into immature oocyte. A, GVBD wasassessed using white spot observation. The histogram shows the average GVBDvalues � S.D. Results are from three independent experiments. B, oocytes werehomogenized in lysis buffer, and Western blot analyses were performed.O-GlcNAc content of oocytes; activation of Rsk, Cdc2, and cyclin B2; and�-catenin accumulation were assessed as described under “ExperimentalProcedures.” Protein mass markers (kDa) are indicated to the left. WB, Westernblot; cyto, injected cytoplasm.




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indeed, the time requested for 50% of oocytes to undergo GVBD(GVBD50) was reached earlier in PUGNAc-injected oocytesthan in water-injected controls (Fig. 8A), and O-GlcNAc levelswere enhanced comparedwith injection of water (Fig. 8B, com-pare lanes 3, 4, and 5 with lane 2). These data reinforced thoseobtained with alloxan and benzoxazolinone derivative. Alto-gether, these results demonstrated that the O-GlcNAc levelinterfered with Xenopus oocyte M-phase entry.GFAT Inhibition Fails to Prevent M-phase Entry in Xenopus

Oocytes—In somatic cells, the UDP-GlcNAc pool directlydepends upon glucose concentration (39). UDP-GlcNAc isgenerated through the hexosamine biosynthetic pathway. Thehexosamine biosynthetic pathway is finely regulated by the keyenzyme, GFAT. Inhibition of GFAT leads to a decrease in theUDP-GlcNAc pool and consequently to a decrease in O-Glc-NAc glycosylation. In somatic cells, such an effect may beobtained using DON and azaserine, which are well knownGFAT inhibitors. Both were used in an attempt to preventG2/M transition induced by progesterone in Xenopus oocytes.When injected in immature oocytes prior to progesteronetreatment, DON and azaserine have no effect on hormonalstimulation-inducedM-phase entry (Fig. 9A). This lack of effectof GFAT inhibitors was previously observed by Slawson et al.(27), who used DON to tentatively modify the GVBD rate. Inour experiments, no delay in GVBD kinetics was observedbetween azaserine orDON-injected oocytes comparedwith the

control ones (water-injected). GVBD50 was similar to control,whatever the conditions used to inhibit GFAT (data notshown). GVBD percentages in oocytes injected either with aza-serine or DON exhibited no significant differences from con-trol oocytes (Fig. 9A). At a biochemical level, cyclin B2, Rsk, andMAPK were not phosphorylated in immature G2-blockedoocytes (Fig. 9B). Upon stimulation by progesterone, cyclin B2,MAPK, and Rsk were phosphorylated, and changes in electro-phoreticmobility were observed for cyclin B2 and Rsk (Fig. 9B).In oocytes injected with either azaserine or DON, we observedthe typical mature oocyte pattern of phosphorylation and�-catenin accumulation (Fig. 9B).GFAT Inhibition Does Not Significantly Impair O-GlcNAc

and UDP-GlcNAc Contents in Xenopus Oocytes—Strikingly,neither azaserine nor DON prevented the progesterone-in-duced O-GlcNAc level increase that goes with M-phase entry(Fig. 9). Increasing concentrations of azaserine (20, 60, and 100

FIGURE 8. O-GlcNAcase inhibitor PUGNAc causes an acceleration in GVBDkinetics. Increasing amounts of PUGNAc were injected into immatureoocytes. We also injected water as a control. Oocyte recovery was permittedovernight before progesterone treatment. A, for each condition, maturationkinetic was performed, and GVBD50 was calculated. The histogram representsthe average values � S.D. of the relative time to GVBD50 for PUGNAc-treatedoocytes compared with water-injected controls (considered as 1 for normal-ization). Results are from three independent experiments. B, oocytes werehomogenized in lysis buffer, and Western blot analyses were performed.O-GlcNAc was assessed as described under “Experimental Procedures.” Pro-tein mass markers (kDa) are indicated to the left. WB, Western blot; Pg,progesterone.

FIGURE 9. GFAT inhibition does not prevent progesterone-induced meioticresumption. Immature oocytes were injected with GFAT inhibitors, DON or aza-serine (AZA), overnight before stimulation of meiotic resumption. Controloocytes were water-injected. A, GVBD was assessed using white spot observa-tion. The histogram shows the average GVBD values � S.D. Results are from sixindependent experiments. B, oocytes were homogenized in lysis buffer, andWestern blot analyses were performed. O-GlcNAc content of oocytes; activationof Rsk, Cdc2, and cyclin B2; and �-catenin accumulation were assessed asdescribed under “Experimental Procedures.” Protein mass markers (kDa) are indi-cated to the left. WB, Western blot; Pg, progesterone.




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�M) and DON (100, 300, and 500 �M) were tested, but no effecton G2/M transition orO-GlcNAc levels was observed (data notshown). To understand these results in apparent contrast withour previous observations, the UDP-GlcNAc pool of each con-dition was assayed using high performance anion exchangechromatography (Fig. 10); no significant changes in the UDP-GlcNAc pools were observed between the different conditions,even in the presence of theGFAT inhibitors. These latter obser-vations suggest that during G2/M transition, oocytes run forO-GlcNAc on an existing UDP-GlcNAc pool and did notrequire UDP-GlcNAc synthesis for the O-glycosylationprocesses.Cyclin B2 Is Associated with an O-GlcNAc Partner—Since

MPF is the universal key regulator of M-phase entry, we exam-

ined the putative glycosylation of cyclin B2 during oocyte mat-uration (Fig. 11). Xenopus oocytes extracts were enriched onWGA-beads using two different conditions (29); enrichmentswere performed in smooth conditions (Fig. 11A) to preserveprotein/protein interactions and in stringent conditions to dis-sociate complexes (Fig. 11B). Bound proteins were separated bySDS-PAGE and blotted using an anti-cyclin B2 antibody. Hsc/Hsp70 were used as a positive control, since several papersrelated their O-GlcNAc modification (reviewed in Ref. 40).Heat-shock proteins interact with many intracellular proteins,explaining why such high quantities of Hsc/Hsp70 wereobtained in smooth conditions.In smooth conditions, we observed that the two isoforms

of cyclin B2 (i.e. the active and the inactive forms) were asso-ciated with an unknownO-GlcNAc partner in immature andmature oocytes. However, in drastic conditions, staining ofenriched-O-GlcNAc proteins with the anti-cyclin B2 anti-body revealed that cyclin B2 was not itself O-GlcNAc-mod-ified, since SDS does not preserve binding of cyclin B2 toWGA-beads. As a control experiment, treatment of oocytelysates with peptide N-glycosidase F was performed in orderto avoid the eventual presence of N-linked oligosaccharides(data not shown).

DISCUSSION

M-phase entry is a particularly delicate step, preparing cellsfor division, which requires highly organized spatio-temporalevents. These events are mainly driven by post-translationalmodifications modulating protein expression or activities orpromoting protein degradation. Among these modifications,phosphorylation and ubiquitination have been extensivelystudied. The highly dynamic and ubiquitous PTM O-GlcNAchas been proposed to play a role in cell cycle protein function,thus presumably regulating cell cycle progression (24) and check-points. Consistent with previous observations inX. laevis oocytes,which reported an overall increase in O-GlcNAc modification

FIGURE 10. Xenopus oocyte UDP-GlcNAc pools are not perturbed by GFATinhibition. Since GFAT inhibition did not block meiotic resumption, contraryto OGT inhibition, UDP-GlcNAc pools of mature, of immature, and of DON- orazaserine-treated oocytes were assayed using high performance anionexchange chromatography. A, profiles obtained for mature and immatureoocytes. The UDP-GlcNAc standard profile is shown at the bottom. B, percent-age of UDP-GlcNAc pools obtained for the different conditions comparedwith the mature oocytes (arbitrarily assigned as 100%). Results are from fourindependent experiments.

FIGURE 11. Cyclin B2 interacts with an O-GlcNAc partner. Batches of twentyimmature (I) and mature (M) oocytes were lysed in homogenization buffer,and enrichments of O-GlcNAc proteins on WGA-agarose beads were per-formed in smooth conditions (A) to permit the recovery of all O-GlcNAc pro-teins and their associated partners or in stringent conditions (B) as describedunder “Experimental Procedures.” Bound proteins were separated by SDS-PAGE and electrotransferred onto a nitrocellulose sheet. Western blot analy-ses were performed using anti-Hsc/Hsp70 and anti-cyclin B2 antibodies. Con-trol of enrichment on WGA-beads was performed using the anti-O-GlcNAcantibody. Protein mass markers (kDa) are indicated to the left. WB, Westernblot.




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during G2/M transition (22), we show here that O-GlcNAc isclearly requested for M-phase entry during meiosis in Xenopusoocytes.O-GlcNAc is the most widespread glycosylation type found

within the cytosolic and nuclear compartments of eukaryoticcells, and it differs from other glycosylation types by its highdynamism. Moreover, O-GlcNAc can compete with phospho-rylation either at the same or at an adjacent site. Despite theintensive study and interest devoted toO-GlcNAc, many func-tions ruled by this glycosylation remain to be elucidated. Takinginto account the most recent observations, which have pointedout the active role played byO-GlcNAc in the regulation of cellcycle (22, 24, 27), we explored the effects of preventing theO-GlcNAc dynamism on cell cycle progression by inhibition ofOGT and O-GlcNAcase, expecting disruption of its progres-sion. We took advantage of the Xenopus oocyte model, whereG2/M transition may be triggered either by hormonal stimula-tion or by cytoplasm injection; in our hands, both were blockedwhen the OGT inhibitor alloxan was added in the medium.Historically, alloxan has been used to induce experimental

diabetes in animals (for a review, see Ref. 37). The mechanismbywhich diabetes ismimicked in animals relates to the produc-tion of ROS, such as hydroxyl radicals. Hydroxyl radicals arehighly toxic for cells, especially for pancreatic beta cells, and, inconjunction with an increased flux of calcium, they lead to betacell death and consequently to an inhibition of insulin secre-tion, generating diabetes. We tested the hypothesis that ROSreleased under alloxan treatment could affect G2/M transitioninXenopus oocytes. Indeed, ROS-mediated signal transductionpathways are involved in phosphorylation events by activatingprotein-tyrosine kinases and protein-serine/threonine kinasesthrough a process known as “receptor transactivation.” Forinstance, ROS inhibits Erk1/2, c-Jun N-terminal kinase, andp38 MAPK activations induced by angiotensin II and platelet-derived growth factor in fibroblast (41), but ROS could alterna-tively sustain MAPK activity through the inhibition of MAPKphosphatases, which inactivates MAPK. ROS have beendescribed as potent inhibitors of phosphotyrosine phosphata-ses, like Cdc25C (42). Thus, evidence has accumulated thatROS interfere with cell cycle progression (36, 43). First, andunexpectedly, increasing concentrations ofH2O2 had no effectsonG2/M transition induced by hormonal stimulation;MPF andMAPK were detected in their active forms, whereas �-cateninaccumulated normally in H2O2-treated oocytes (Fig. 6). Sec-ond, we counteracted potential ROS production via the allox-an-dialuric acid redox cycle by the addition of SOD and cata-lase, two detoxifying enzymes (Fig. 5).Whatever the conditionswe used (i.e. increasing the two enzyme amounts ormodulatingthe time period post-progesterone addition), SOD and catalasedid not suppress the alloxan-mediated block of G2/M transi-tion. Because both SOD and catalase failed to impair alloxaneffects and due to the lack of effect of a high concentration ofH2O2 on meiosis progression, it can be assumed that alloxandoes not act on the G2/M transition through ROS generation.To address a potential effect of alloxan on an enzymeother thanOGT, which could use uridine as a substrate, we used a deriva-tive of benzoxazolinone, an OGT inhibitor that was recentlyidentified (28). Since it is not a substrate for OGT, the benzox-

azolinone derivative should exhibit a better selectivity. There-fore, the benzoxazolinone derivative appears to be a useful toolto probe hypo-O-GlcNAc glycosylation. Indeed, we observedthat it decreasedO-GlcNAc to a greater extent than alloxan, atone-tenth the concentration. In the presence of benzoxazolin-one derivative (XI), reduction ofO-GlcNAc levels also impairedmeiotic progression as well as the activation of both MPF andMAPK pathways. This leads us to the conclusion that O-Glc-NAc modification is necessary for M-phase entry in Xenopusoocytes. O-GlcNAcase inhibition also reinforced these obser-vations, since PUGNAc had opposite effects compared withthose of alloxan and the benzoxazolinone derivative, since thischemical compound accelerated GVBD.Inhibition ofXenopus oocyte G2/M transition (stimulated by

either progesterone or cytoplasm injection) by alloxan was cor-related to a drop in theO-GlcNAc level. Although the decreasein the O-GlcNAc content prevented MPF and MAPK activa-tions, as well as �-catenin accumulation, these effects appearedto be reversible and nontoxic for oocytes, since removal ofalloxan from the medium allowed oocytes still to undergo andcomplete G2/M transition in the presence of progesterone. Incontrast to hormonal stimulation, cytoplasm injection droveM-phase entry independently of protein synthesis (12). It mustbe emphasized that the alloxan concentration required washigher to inhibit egg cytoplasm versus progesterone-inducedGVBD (4 versus 3 mM, respectively). One might argue thatmechanisms of protein synthesis induced by hormonal stimu-lation, which play a crucial role in meiotic resumption, arehighly sensitive to O-GlcNAc modifications in Xenopusoocytes. In these conditions, the decrease inO-GlcNAc contentalso led to the absence of GBVD, MPF, and MAPK activations.This last observation demonstrates that O-GlcNAc modifica-tion is required for the activation of theMPF autoamplificationloop.An alternative strategy to decrease O-GlcNAc content is to

target the enzyme responsible for UDP-GlcNAc synthesis,GFAT. Intriguingly, and has previously observed (27), azaserineandDON,which are both inhibitors of GFAT, had no effects onprogesterone-induced GVBD and activation of both MPF andMAPK. Such inefficiency to affect theO-GlcNAc levelmight berelated to an absence of GFAT activity in M-phase-enteredoocytes. This hypothesis has been reinforced by UDP-GlcNAcpool assays; there was no difference in the UDP-GlcNAc con-tent between immature and mature oocytes. Thus, the use ofGFAT inhibitors appears not to be a relevant strategy todecrease O-GlcNAc content and to study its role in Xenopusoocytes. In addition to these observations, it has long beenknown that carbon metabolism in Xenopus oocytes is mainlydirected to glycogen synthesis rather than to glycolysis (44).From these considerations, it can be assumed that in Xenopusoocytes, glucose tends to be stored rather than beingdirected toward the hexosamine biosynthetic pathway.Because early cleavages in Xenopus embryos rely on the useof amino acids as its main source of carbon, glycolysis startsonly at the onset of gastrulation (45). Taken together, theseobservations lead to the conclusion that oocytes glycosylatetheir proteins on an existing UDP-GlcNAc pool in a processthat does not need UDP-GlcNAc production. Thus, Xenopus




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oocytes offer a unique opportunity to uncouple inhibition ofO-GlcNAc and GFAT in vivo.Further targets and function of O-GlcNAc modification

remain to be determined during the cell cycle. Although theinvolvement of O-GlcNAc at the G2/M transition (27) and atthe metaphase/anaphase transition may be suspected from theobservations of Slawson et al. (24, 27), we report for the firsttime here a crucial role for OGT, theO-GlcNAc key enzyme, inM-phase entry, and association of cyclin B2 with an unidenti-fiedO-GlcNAc partnermight account for unsuspected levels ofregulation. If post-translational modifications, such as phos-phorylation, have been described for cyclin B isoforms duringG2/M progression, their functions remain unclear except forcyclin B1. The latter can be phosphorylated by MAPK andpolokinase, regulating MPF nuclear export but not enzymaticactivity of the heterodimer complex (46). In our hands, cyclinB2 appeared to be in association with an O-GlcNAc-modifiedpartner throughoutG2- andM-phases, because interactionwasnot associated within the active or inactive states of the cyclinB2-Cdk1 complex. Nevertheless, it may be hypothesized thatO-GlcNAc is involved in the association between cyclin B2 andCdc2 to form the heterodimer MPF or to play a role in theinteractions between MPF and its downstream effectors. Itshould be also noted that O-GlcNAc level immediatelyincreases after hormonal stimulation (2 h post-progesterone;data not shown), indicating thatO-GlcNAc is necessary for theearly steps of the G2/M transition. Because MAPK activity orcyclin B synthesis is not essential forM-phase entry inXenopusoocytes (17, 18, 21, 47), mechanisms sensitive to O-GlcNAcvariation, which are essential for G2/M transition, remain to beelucidated.As a valuable tool for studyingO-GlcNAc function in the cell

cycle, OGT inhibitors have opened a narrow field of investiga-tion for deciphering the role of O-GlcNAc modification at theG2/M transition. Further efforts are required to seek modifica-tion ofO-GlcNAc content on the key regulators of the cell cycle.

Acknowledgments—We thank Arlette Lescuyer for technical assist-ance during this work. We are indebted to Dr. Gannon for the gener-ous gift of anti-cyclin B2 antibodies (JG103) and Prof. Jerome Lemoinefor providing PUGNAc. We thank Dr. Anne Harduin-Lepers for Eng-lish grammatical corrections.

REFERENCES1. Masui, Y. (1967) J. Exp. Zool. 166, 365–3752. Smith, L. D., Ecker, R. E., and Subtelny, S. (1968) Dev. Biol. 17, 627–6433. Schmitt, A., and Nebreda, A. R. (2002) J. Cell Sci. 115, 2457–24594. Nurse, P., Masui, Y., and Hartwell, L. (1998) Nat. Med. 4, 1103–11065. Masui, Y. (2001) Differentiation 69, 1–176. Kumagai, A., and Dunphy, W. G. (1991) Cell 64, 903–9147. Lorca, T., Labbe, J. C., Devault, A., Fesquet, D., Capony, J. P., Cavadore,

J. C., Le Bouffant, F., and Doree, M. (1992) EMBO J. 11, 2381–23908. Gautier, J., and Maller, J. L. (1991) EMBO J. 10, 177–1829. Rime,H., Huchon,D., De Smedt, V., Thibier, C.Galaktionov, K., Jessus, C.,

and Ozon, R. (1994) Biol. Cell 82, 11–2210. Hoffmann, I., Clarke, P. R., Marcote, M. J., Karsenti, E., and Draetta, G.

(1993) EMBO J. 12, 53–63

11. Strausfeld, U., Fernandez, A., Capony, J. P., Girard, F., Lautredou, N.,Derancourt, J., Labbe, J. C., and Lamb, N. J. (1994) J. Biol. Chem. 269,5989–6000

12. Wasserman, W. J., and Masui, Y. (1975) Exp. Cell Res. 91, 381–38813. Ferrell, J. E., Jr., Wu, M., Gerhart, J. C., and Martin, G. S. (1991)Mol. Cell.

Biol. 11, 1965–197114. Sagata, M., Oskarsson, M., Copeland, T., Brumbaugh, J., and Vande

Woude, G. F. (1988) Nature 335, 519–52515. Palmer, A., Gavin, A. C., and Nebreda, A. R. (1998) EMBO J. 17,

5037–504716. Fischer, D. L., Brassac, T., Galas, S., and Doree, M. (1999) Development

126, 4537–454617. Dupre, A., Suziedelis, K., Valuckaite, R., de Gunzburg, J., Ozon, R., Jessus,

C., and Haccart, O. (2002) Oncogene 21, 6425–643318. Baert, F., Bodart, J. F., Bocquet-Muchembled, B., Lescuyer-Rousseau, A.,

and Vilain, J. P. (2003) J. Biol. Chem. 278, 49714–4972019. Abrieu,A.,Magnaghi-Jaulin, L., Kahana, J. A., Peter,M., Castro,A., Vigneron,

S., Lorca, T., Cleveland, D.W., and Labbe, J. C. (2001)Cell 106, 83–9320. Bodart, J. F., Gutierrez, D. V., Nebreda, A. R., Buckner, B. D., Reseau, J. R.,

and Duesbery, N. S. (2002) Dev. Biol. 245, 348–36121. Bodart, J. F., Baert, F. Y., Sellier, C., Duesbery, N. S., Flament, S., andVilain,

J. P. (2005) Dev. Biol. 283, 373–38322. Lefebvre, T., Baert, F., Bodart, J. F., Flament, S., Michalski, J. C., and Vilain,

J. P. (2004) J. Cell. Biochem. 93, 999–101023. Slawson,C.,Housley,M.P., andHart,G.W. (2006) J.Cell.Biochem.97,71–8324. Slawson, C., Zachara, N. E., Vosseler, K., Cheung, W. D., Lane, M. D., and

Hart, G. W. (2005) J. Biol. Chem. 280, 32944–3295625. Iyer, S. P., and Hart, G. W. (2003) Biochemistry 42, 2493–249926. Fang, B., and Miller, M. W. (2001) Exp. Cell Res. 263, 243–25327. Slawson, C., Shafii, S., Amburgey, J., and Potter, R. (2002) Biochim. Bio-

phys. Acta 1573, 121–12928. Gross, B. J., Kraybill, B. C., and Walker, S. (2005) J. Am. Chem. Soc. 127,

14588–1458929. Zhu, W., Leber, B., and Andrews, D. W. (2001) EMBO J. 20, 5999–600730. Chesnel, F., Bonnec,G., Tardivel, A., andBoujard, D. (1997)Dev. Biol. 188,

122–13331. Konrad, R. J., Zhang, F., Hale, J. E., Knierman, M. D., Becker, G. W., and

Kudlow, J. E. (2002) Biochem. Biophys. Res. Commun. 293, 207–21232. Kim, Y. H., Song, M., O. H., Y. S., Heo, K., Choi, J. W., Park, J. M., Kim,

S. H., Lim, S., Kwon, H.M., Ryu, S. H., and Suh, P. G. (2006) J. Cell. Physiol.207, 689–696

33. Champattanachai, V., Marchase, R. B., and Chatham, J. C. (2007) Am. J.Physiol. 292, C178–C187

34. Liu, J., Pang., Y., Chang, T., Bounelis, P., Chatham, J. C., and Marchase,R. B. (2006) J. Mol. Cell. Cardiol. 40, 303–312

35. Wu, G., and He, X. (2006) Biochemistry 45, 5319–532336. Rudolph, J. (2005) Antioxid. Redox. Signal. 7, 761–76737. Szkudelski, T. (2001) Physiol. Res. 50, 536–54638. Haltiwanger, R. S., Grove, K., and Philipsberg, J. A. (1998) J. Biol. Chem.

273, 3611–361739. Wells, L., Vosseler, K., and Hart, G. W. (2003) Cell. Mol. Life Sci. 60,

222–22840. Zachara,N. E., andHart, G.W. (2004)Biochim. Biophys. Acta1673, 13–2841. Lander, H. M., Ogiste, J. S., Pearce, S. F., Levi, R., and Novogrodsky, A.

(1995) J. Biol. Chem. 270, 7017–702042. Savitsky, P. A., and Finkel, T. (2002) J. Biol. Chem. 277, 20535–2054043. Cakir, Y., and Ballinger, S. W. (2005) Antioxid. Redox. Signal. 7, 726–74044. Dworkin, M. B., and Dworkin-Rastl, E. (1989) Dev. Biol. 132, 512–52345. Dworkin, M. B., and Dworkin-Rastl, E. (1992) Mol. Reprod. Dev. 32,

354–36246. Walsh, S., Margolis, S. S., and Kornbluth, S. (2003) Mol. Cancer Res. 1,

280–28947. Hochegger, H., Klotzbutcher, A., Kirk, J., Howell, M., le Guellec, K.,

Fletcher, K., Duncan, T., Sohail,M., andHunt, T. (2001)Development128,3795–3807




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Jean-François BodartSuzanne Walker, René Cacan, Jean-Claude Michalski, Jean-Pierre Vilain and

Vanessa Dehennaut, Tony Lefebvre, Chantal Sellier, Yves Leroy, Benjamin Gross, OocytesXenopus laevisin

/M Transition2-Acetylglucosaminyltransferase Inhibition Prevents GN-Linked O

doi: 10.1074/jbc.M700444200 originally published online February 28, 20072007, 282:12527-12536.J. Biol. Chem.

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