cdk1- and cdk2-mediated phosphorylation of MyoD Ser200 in growing C2 myoblasts: role in modulating...

MOLECULAR AND CELLULAR BIOLOGY,0270-7306/99/$04.0010

Apr. 1999, p. 3167–3176 Vol. 19, No. 4

Copyright © 1999, American Society for Microbiology. All Rights Reserved.

cdk1- and cdk2-Mediated Phosphorylation of MyoD Ser200 inGrowing C2 Myoblasts: Role in Modulating MyoD

Half-Life and Myogenic ActivityMAGALI KITZMANN, MARIE VANDROMME,* VALERIE SCHAEFFER, GILLES CARNAC,

JEAN-CLAUDE LABBE, NED LAMB, AND ANNE FERNANDEZ

Institut de Genetique Humaine, Centre National de Recherche Scientifique, UPR 1142,34396 Montpellier cedex 5, France

Received 2 October 1998/Returned for modification 6 November 1998/Accepted 30 December 1998

We have examined the role of protein phosphorylation in the modulation of the key muscle-specific tran-scription factor MyoD. We show that MyoD is highly phosphorylated in growing myoblasts and undergoessubstantial dephosphorylation during differentiation. MyoD can be efficiently phosphorylated in vitro by eitherpurified cdk1-cyclin B or cdk1 and cdk2 immunoprecipitated from proliferative myoblasts. Comparativetwo-dimensional tryptic phosphopeptide mapping combined with site-directed mutagenesis revealed that cdk1and cdk2 phosphorylate MyoD on serine 200 in proliferative myoblasts. In addition, when the seven proline-directed sites in MyoD were individually mutated, only substitution of serine 200 to a nonphosphorylatablealanine (MyoD-Ala200) abolished the slower-migrating hyperphosphorylated form of MyoD, seen either invitro after phosphorylation by cdk1-cyclin B or in vivo following overexpression in 10T1/2 cells. The MyoD-Ala200 mutant displayed activity threefold higher than that of wild-type MyoD in transactivation of anE-box-dependent reporter gene and promoted markedly enhanced myogenic conversion and fusion of 10T1/2fibroblasts into muscle cells. In addition, the half-life of MyoD-Ala200 protein was longer than that of wild-typeMyoD, substantiating a role of Ser200 phosphorylation in regulating MyoD turnover in proliferative myoblasts.Taken together, our data show that direct phosphorylation of MyoD Ser200 by cdk1 and cdk2 plays an integralrole in compromising MyoD activity during myoblast proliferation.

Skeletal muscle differentiation is characterized by with-drawal of myoblasts from the cell cycle, induction of muscle-specific gene expression, and cell fusion into multinucleatedmyotubes. All of these events are coordinated by a family ofmuscle-specific transcription factors including MyoD (8), Myf5(4), myogenin (12, 56), and MRF4 (39). These proteins showhomology within a basic helix-loop-helix (bHLH) domain thatmediates both heterodimerization with ubiquitous activatingbHLH proteins such as E12 and E47 and DNA binding to aspecific sequence, CANNTG, called the E box (9, 25, 30). Oneof the most remarkable properties of myogenic factors is thattheir ectopic expression in nonmuscle cells forces these cellsinto muscle differentiation, a process known as myogenic con-version (6, 8). Although capable of inhibiting cell proliferation(7, 47) and inducing differentiation, MyoD is constitutivelyexpressed in proliferating myoblasts long before differentiationtakes place, implying that its activity is regulated in replicatingcells (26, 49). Indeed, when cultured myoblasts are exposed toserum or growth factors such as basic fibroblast growth factorand transforming growth factor b, both muscle differentiationand MyoD activity are inhibited (34, 48). One of the inhibitorymechanisms that target MyoD in proliferative myoblasts in-volves the Id family of proteins. These HLH proteins, whichare devoid of DNA-binding basic domains, can heterodimerizewith bHLH factors, thus inhibiting their binding to DNA (3).In addition, like most transcription factors (23), MyoD is aphosphoprotein (49), and its phosphorylation could constitute

an important mechanism by which mitogens negatively regu-late its activity. Protein kinase C (PKC), which is activated inresponse to fibroblast growth factor, was first shown to inhibitthe DNA binding activity of myogenin (28) by phosphorylatinga site conserved in the basic region of all myogenic HLHproteins. This same site was shown not to be required for theinhibition of MRF4 by PKC (19). Protein kinase A (PKA) wasalso demonstrated to repress the activity of Myf5 and MyoD,albeit via an indirect mechanism (55).

Because differentiation requires withdrawal from the cellcycle, kinases involved in cell cycle control are likely candidatesfor the inhibition of MyoD in the proliferative state. Cyclin-dependent kinases (CDKs), in association with their regulatorypartners, the cyclins, are key regulators of cell cycle progres-sion. cdk2-cyclin A/E and cdk4-cdk6/cyclin D are involved inthe G1/S transition, whereas cdk1 (also called cdc2)-cyclin A/Bis implicated in the G2/M transition of the cell cycle (32, 40).Several lines of evidence support the involvement of CDKs inthe regulation of muscle differentiation. Overexpression of cy-clin D1 inhibits MyoD muscle-specific gene transactivation (38,42, 43). Cyclins A and E have, to a lesser extent, the same effectalone or in combination with cdk2, whereas the effects ob-served with cyclins B, D2, and D3 remain controversial (17, 38,42, 43). Cyclin-dependent inhibition of muscle gene transacti-vation requires CDK activation and can be reversed by over-expression of p21 (Waf1, Cip1), one of the general CDK in-hibitors. Interestingly, induction of p21 constitutes one of theearliest markers of cell cycle exit associated with myoblastdifferentiation (2) and depends on MyoD (16, 18). AlthoughCDKs appear to be involved in the inhibition of MyoD inproliferating myoblasts, no direct phosphorylation of MyoD byCDKs has been described.

In this report, we show that MyoD phosphorylation is high in

* Corresponding author. Mailing address: Institut de Genetique Hu-maine, Centre National de Recherche Scientifique UPR 1142, 141 Ruede la Cardonille, 34396 Montpellier cedex 5, France. Phone: 33 (0)49961 99 13. Fax: 33 (0)499 61 99 01. E-mail: [email protected].

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proliferative C2 myoblasts and diminishes during the course ofmuscle differentiation. By tryptic phosphopeptide mappingand mutational analysis of MyoD, we show that a CDK con-sensus site comprising Ser200 is phosphorylated in vivo inmyoblasts and in vitro by cdk1 (cdc2) and cdk2. We demon-strate that a nonphosphorylable Ser200 mutant of MyoDshows both higher activity in transactivating muscle-specificgene expression through the E box and greater ability to con-vert 10T1/2 fibroblasts to muscle cells. We also report thatSer200 phosphorylation is involved in specifying the short half-life of MyoD in proliferative myoblasts by showing that MyoD-Ala200 displays a half-life threefold higher than that of wild-type MyoD protein (MyoD-wt). These data show that directCDK-dependent phosphorylation of MyoD on Ser200 is in-volved in negatively regulating MyoD activity.

MATERIALS AND METHODS

Cell culture. C2.7 myoblasts (36) were kept in growth medium (50% Dulbeccomodified Eagle medium [DMEM; ICN, Orsay, France], 50% HaM F12 [GibcoBRL, Life Technologies, Cergy Pontoise, France]) supplemented with 10% fetalcalf serum (FCS; DAP, Neuf-Brisach, France). To induce terminal differentia-tion, myoblasts were placed in differentiation medium (DMEM, 2% FCS). Anearly complete differentiation is obtained in 60 h. Mouse 10T1/2 cells (Amer-ican Type Culture Collection, Biovaley, France) were maintained in growthmedium and moved to differentiation medium following transfection to inducemyogenic conversion.

Purified proteins. Production and purification of full-length murine MyoDhave been described elsewhere (52); MyoD-Ala5 and MyoD-Ala200 were puri-fied by using the same protocol. The active kinase cdk1-cyclin B was purifiedfrom starfish oocytes (24).

2D gel electrophoresis. Proteins extracts from proliferating and differentiatedC2.7 cells were analyzed by two-dimensional (2D) electrophoresis by the methodof O’Farrell (33). First-dimension electrofocusing gels contained 9.5 M urea, 2%(wt/vol) Nonidet P-40 (NP-40), and 5% dithiothreitol (DTT). The ampholinemixture used was composed of 60% (vol/vol) ampholine pH 3 to 10 and 40%(vol/vol) ampholine pH 5 to 7. The second dimension was performed on sodiumdodecyl sulfate (SDS)–12% polyacrylamide gels. Following transfer onto nitro-cellulose membranes, MyoD isoforms were revealed by Western blotting withanti-MyoD monoclonal antibody 5.8A (kindly provided by P. Dias and P. Hough-ton, Memphis, Tenn.). For calf intestinal phosphatase (CIP) treatment, nuclearextracts from C2.7 myoblasts were treated with 20 U of CIP (Promega, Char-bonnieres, France) for 30 min at 37°C.

Western blotting. Nitrocellulose membranes were blocked with phosphate-buffered saline (PBS) containing 10% dry milk and incubated either with anti-CDKs (Santa Cruz Biotechnology, Santa Cruz, Calif.) or anti-MyoD polyclonalantibody C20 (Santa Cruz Biotechnology) diluted 1/300 or with monoclonalanti-a-tubulin (Sigma, St. Quentin Fallavier, France) diluted 1/2,000 in PBScontaining 0.5% bovine serum albumin for 1 h at room temperature. After threewashes in PBS, blots were incubated with secondary antibodies (horseradishperoxidase-conjugated goat anti-rabbit or goat anti-mouse; Amersham, les Ulis,France) and developed by using the Amersham ECL (enhanced chemilumines-cence) reagent.

In vivo labeling and immunoprecipitation. Cells (myoblasts and myotubes)cultured in 60-mm-diameter dishes were labeled with [32P]orthophosphate (1mCi/ml) for 2 h at 37°C. After three washes with PBS, cells were lysed in 100 mlof a mixture composed of (by volume) Laemmli buffer–2% NP-40, 10 mMb-glycerophosphate, and 1 mM phenylmethylsulfonyl fluoride. Lysates wereboiled for 3 min, diluted to 500 ml in radioimmunoprecipitation assay (RIPA)buffer (10 mM Na2HPO4, 100 mM NaCl, 5 mM EDTA, 50 mM NaF, 1 mM DTT,0.1% NP-40, 5% sodium deoxycholate, 0.1% SDS), and homogenized by pas-sages through a 21-gauge needle. Following 10 min of centrifugation at 13,000rpm, supernatants were precleared by incubation with protein G-Sepharosebeads (Pharmacia, Orsay, France) and incubated for 2 h at 4°C with anti-MyoDmonoclonal antibody 5.8A; 20 ml of protein G-Sepharose beads was added for 30min at 4°C, and the beads were washed three times with RIPA buffer and oncewith PBS before loading onto a SDS–12% gel for polyacrylamide gel electro-phoresis (PAGE). The radioactivity was analyzed by autoradiography. Theamount of immunoprecipitated MyoD was estimated by Western blotting usingantibody C20 anti-MyoD polyclonal as described above.

Immunoprecipitation and CDK assays. Cells (myoblasts and myotubes) werewashed twice in 13 PBS and scraped in 1 ml of PBS. After centrifugation at 3,000rpm, pellets were resuspended in lysis buffer (50 mM Tris [pH 7.4], 150 mMNaCl, 0.4% NP-40, 2 mM EDTA, 50 mM NaF, 10 mM b-glycerophosphate, 1mM ATP, 2 mg each of leupeptin and aprotinin per ml, 2 mM sodium vanadate,2 mM DTT). After 10 passages through a 21-gauge needle, cell lysates werecleared by centrifugation at 13,000 rpm. Protein concentrations were determinedby using a Bio-Rad DC kit. Extracts (200 mg) were immunoprecipitated with

either monoclonal anti-cdk1 (C7) or polyclonal anti-cdk2 (M2) or anti-cdk5 (C8)antibodies for 2 h at 4°C. All antibodies (Santa Cruz Biotechnology) were usedat a 1/50 dilution. Depending on antibody species, protein A- or G-Sepharosewas added for 1 h at 4°C. After centrifugation, pellets were washed three timeswith lysis buffer, twice in lysis buffer containing 400 mM NaCl, and twice inkinase buffer (25 mM HEPES [pH 7.4], 25 mM MgCl2, 25 mM b-glycerophos-phate, 2 mM DTT, 0.1 mM NaVO3). Purified cdk1-cyclin B or beads containingCDKs immunoprecipitated from C2.7 cells were incubated in 20 ml of kinasebuffer containing 50 mM ATP and 5 mCi of [g-32P]ATP (Kodak X-ray films) andthen used for Western blot analyses.

Phosphopeptide mapping. 32P-labeled MyoD (immunoprecipitated from myo-blasts) and bacterially expressed MyoD-wt, MyoD-Ala5, and MyoD-Ala200phosphorylated in vitro by cdk1-cyclin B were excised from SDS-gels and di-gested twice with 10 mg of trypsin for 12 h at 37°C in buffer containing 200 mMNH4H2CO3. Digests were desalted by repeated lyophilization and loaded ontothin-layer chromatography plates (Merck-Coger, Paris, France) for 2D peptidemapping. The first dimension was run for 30 min at 1,000 V at pH 1.9 (formicacid-acetic acid-water [50:150:1,800]); second-dimension chromatography wasperformed in phosphochromo buffer (isobutyric acid, 1-butanol–pyridine–aceticacid–water [15:10:3:2]). 32P-labeled peptides were subsequently visualized byautoradiography of the thin-layer chromatography plates.

Mutation of the seven proline-directed sites present on MyoD. The MyoDcDNA was mutagenized in the Moloney sarcoma virus long terminal repeatexpression vector pEMSV-scribe. MyoD mutants were obtained by oligonucle-otide-directed mutagenesis using a QuickChange site-directed mutagenesis kit(Stratagene, Ozyme, Montigny le Bretonneux, France) as instructed by the man-ufacturer. Oligonucleotides were 30 to 32 nucleotides in length, with 14 to 15nucleotides of exact homology with MyoD in the region flanking the substitution.Mutant clones were screened with the oligonucleotide used for mutagenesis,which had been labeled with T4 polynucleotide kinase by using [g-32P]ATP.Selected clones were used for preparative plasmid isolation and then sequencedby using a Sequenase 2.0 kit (U.S. Biochemical) and [35S]dATP (3,000 Ci/mmol;Amersham). The mutants resulting from a change of serine or threonine toalanine were designated MyoD-Ala5, MyoD-Ala37, MyoD-Ala200, MyoD-Ala262, MyoD-Ala277, MyoD-Ala296, and MyoD-Ala298. Substitution of ala-nine for serine at amino acids 5 and 200 was also performed in the T7 procaryoteexpression construct pET3a-MyoD (52).

Phosphorylation of MyoD wild-type and mutant proteins. MyoD-wt andMyoD alanine mutants were obtained by in vitro translation as described by themanufacturer (TnT coupled reticulocyte lysate system; Promega) and were phos-phorylated by cdk1-cyclin B as described above but in the absence of [g-32P]ATP.35S-radiolabeled proteins were visualized by autoradiography.

Transfection and chloramphenicol acetyltransferase (CAT) assays. Plasmidsused for transfection were pEMSV-MyoD wild type and mutants, pCMV-bgal(Stratagene, Paris, France), paAch-CAT1 and paAchmutCAT1 (gifts from J.Piette, Montpellier, France) (35), and p4E-TK-CAT and pTK-CAT (gifts fromH. Weintraub) (54). For Western blot analyses, transfection of 10T1/2 cells werecarried out with a ratio of 5 ml of Lipofectamine to 1 mg of DNA as described bythe manufacturer (Gibco BRL, Life Technologies).

In CAT assays comparing MyoD-wt and MyoD-Ala200 transactivation activ-ities, transfections were done in 60-mm-diameter dishes with 2 mg of total DNAcomposed of pEMSV-MyoD-wt, pEMSV-MyoD-Ala200, pEMSV–pCMV-bgal–paAch-CAT1, paAchmutCAT1, p4E-TK-CAT, or pTK-CAT (at a ratio of1.6/0.2/0.2) and 10 ml of Lipofectamine. Transfected cells were kept in prolifer-ative medium for 36 h and harvested for CAT assay. CAT assays were performedon cell extracts by using 1-deoxy-(dichloroacetyl-1-3H)chloramphenicol (200mCi/mmol; Amersham) by a nonchromatographic method as described byNielsen et al. (31). Promoter activities were expressed as CAT activity units perb-galactosidase unit.

Myogenic conversion. 10T1/2 cells were transfected with 1 mg of plasmidexpressing either MyoD-wt or MyoD-Ala200; 24 h after transfection, cells werecollected for Western blot analyses or moved to differentiation medium for 60 hand either used for Western blot analyses as described above or processed forimmunofluorescence as previously described (51). Anti-MyoD polyclonal anti-body C20 (Santa Cruz Biotechnology) was used to identify transfected cells, andanti-troponin T antibody JLT-12 (Sigma) was used to quantify the level ofdifferentiation. MyoD antibodies were visualized with biotinylated anti-rabbitantibodies and Texas red-streptavidin (Amersham). Fluorescein-conjugated an-ti-mouse antibodies were used to detect troponin T antibodies. DNA was stainedwith Hoechst dye (Sigma).

Cycloheximide treatment. 10T1/2 cells were transfected with either pEMSV-MyoD-wt or pEMSV-MyoD-Ala200 in 35-mm-diameter dishes as describedabove. Transfected cells were treated with cycloheximide (Sigma) at 15 mg/ml forthe indicated times and harvested for Western blot analyses. MyoD was stainedwith anti-MyoD antibody C20 as described above. For each experiment, a-tu-bulin was used as an internal control. Western blots were scanned and quantifiedby using ImgCalc sensitivity software (developed by N. J. C. Lamb; details uponrequest) on a Silicon Graphics Indigo2 Workstation.

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RESULTSHyperphosphorylation of MyoD in proliferative myoblasts.

To examine the posttranslational modifications of MyoD dur-ing myogenesis, we have analyzed MyoD protein expression inthe course of C2.7 differentiation by 2D gel electrophoresisfollowed by western blotting. As shown in Fig. 1A, four majorMyoD isoforms of similar intensities are detected in prolifer-ative myoblasts, with some other minor spots in the moreacidic part of the gel. By contrast, only two major spots arevisible after 60 h of differentiation, a stage when most of thecells have differentiated into myotubes. To confirm that post-translational modifications of MyoD involve mainly phosphor-ylation, we treated nuclear extracts from proliferative myo-blasts with CIP and analyzed the mobility of MyoD by 2D gelelectrophoresis and western blotting as before. As shown inFig. 1A, phosphatase treatment resulted in only one majorMyoD isoform, which resolved at the basic side of the gel. Toconfirm that MyoD is more phosphorylated in myoblasts thanin myotubes, C2 proliferative myoblasts and myotubes werelabeled with [32P]orthophosphate, MyoD was immunoprecipi-tated and separated by SDS-PAGE, and its phosphorylationwas analyzed by autoradiography of the gel. MyoD phosphor-ylation was higher in myoblasts than in myotubes (Fig. 1B,top); Western blot analysis of the immunoprecipitate showsthat the phosphorylated band corresponds to MyoD, and theamounts of immunoprecipitated MyoD were comparable be-tween myoblasts and myotubes (Fig. 1B, bottom).

Taken together, these data clearly show that MyoD phos-phorylation changes during the course of differentiation of C2cells, being hyperphosphorylated in myoblasts compared tomyotubes.

CDK-dependent phosphorylation of MyoD. CDKs, a familyof kinases implicated throughout the cell cycle, are potentiallyinvolved in both phosphorylation of MyoD and inhibition of itsactivity in myoblasts (16–18, 38, 42, 43). Analysis of the aminoacid sequence of MyoD revealed seven putative CDK phos-phorylation sites distributed in the NH2- and COOH-terminalregions of the protein outside the bHLH domain (see Fig. 5Aand below). As such, MyoD represents a potential target fordirect phosphorylation by CDKs.

To investigate if MyoD could be phosphorylated in a CDK-dependent manner, cdk1 (also called cdc2) and cdk2 wereimmunoprecipitated from myoblasts or myotubes and assayedfor their activities against MyoD, with histone H1 used as an

internal control. Since we have previously shown that cdk5 is apositive regulator of myogenesis, its involvement in MyoDinhibition is unlikely (27); therefore, cdk5 activity was alsoexamined as a control. As shown in Fig. 2A (top), both cdk1and cdk2 isolated from myoblasts phosphorylate H1, whereasthey show little or no H1 kinase activity when immunoprecipi-tated from myotubes. In contrast, cdk5 H1 kinase activity isdetected in both myoblasts and myotubes, in agreement withour previous study (27). With respect to MyoD phosphoryla-tion (Fig. 2A, bottom), both cdk1 and cdk2 display a highkinase activity toward MyoD in myoblasts which is stronglyreduced in myotubes, whereas cdk5 shows no MyoD phosphor-ylation activity in either myoblasts or myotubes. Immunopre-cipitation efficiency was controlled by Western blot analysis ofthe immunoprecipitated CDKs. As shown in Fig. 2B, the lossof kinase activity observed for cdk2 in myotubes correlates withthe presence of a single slower-migrating inactive form of cdk2(15). The level of immunoprecipitated cdk5 is the same inmyoblasts and myotubes, and as previously described (27), thecdk1 protein level is significantly decreased in differentiatedcells (1). Compared to their H1 kinase activities, cdk1 ap-peared to phosphorylate MyoD more efficiently than cdk2. Tofurther demonstrate that MyoD could be directly phosphory-lated by cdk1, we analyzed recombinant MyoD in an in vitrokinase assay using purified cdk1-cyclin B (purified as dimerfrom starfish oocytes [24]). A study of the phosphorylation ofMyoD revealed that cdk1-cyclin B-purified kinase efficientlyphosphorylates MyoD, causing a decrease in its electro-phoretic mobility on SDS-PAGE (Fig. 3A). Interestingly,MyoD from proliferative myoblasts migrates as two bands of

FIG. 1. Hyperphosphorylation of MyoD in C2.7 myoblasts. (A) Total cellularproteins were extracted from C2.7 cells and separated by 2D gel electrophoresis.The different isoforms of MyoD (arrows) were detected by Western blot analysisusing extracts made from proliferative myoblasts (P) or cells placed for 60 h indifferentiation medium or nuclear from extracts from proliferative myoblaststreated with CIP (P1cip). Migrations of the first dimension electrofocusing(IEF) gel (with the acid side indicated) and the second SDS-PAGE dimensionare illustrated by arrows. (B) MyoD was immunoprecipitated from [32P]orthophos-phate-labeled proliferative (Prol) or differentiated (Diff) C2.7 cells as describedin Materials and Methods. MyoD was resolved by SDS-PAGE, its phosphoryla-tion analyzed by autoradiography (32p), and the amount of immunoprecipitatedMyoD protein was controlled by Western blot (WB) analysis.

FIG. 2. cdk1 and cdk2 from C2 myoblasts phosphorylate efficiently MyoD.(A) cdk1, cdk2, and cdk5 were immunoprecipitated from proliferative (P) anddifferentiated (72) C2.7 cells and assayed for kinase activity against H1 histone(top panel) and MyoD (bottom panel). Shown are autoradiograms of the differ-ent kinase reactions following SDS-PAGE. (B) Western blot (WB) analysis ofcdk1, cdk2, and cdk5 after immunoprecipitation from proliferative (P) and dif-ferentiated (72) C2.7 cells.

FIG. 3. Electrophoretic shift of MyoD after phosphorylation by cdk1-cyclinB. (A) Bacterially produced MyoD protein was phosphorylated by purified cdk1-cyclin B kinase for the time indicated. Shown is the autoradiogram from the 32Pphosphorylation reaction. Unphosphorylated MyoD migrates as 45-kDa bandthat shifts to 47 kDa in the course of phosphorylation by cdk1-cyclin B. (B)Western blot showing MyoD migration after SDS-PAGE of mitotic cells extracts(M) and proliferative C2.7 nuclear extracts before (C2P) and after (C2P cip)treatment with CIP.

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approximately 45 and 47 kDa (Fig. 3B). The 47-kDa band canbe converted to 45 kDa following CIP treatment of C2 nuclearextracts, showing that the slower-migrating form correspondsto hyperphosphorylated MyoD, in agreement with a previousreport by Tapscott et al. (49). Because cdk1 is known to beactive at the G2/M transition and during mitosis, we also ana-lyzed MyoD phosphorylation in vivo, in mitotic C2 cells col-lected by mitotic shake from asynchronous myoblasts. Asshown in Fig. 3B, only the slower-migrating hyperphosphory-lated MyoD is present in mitotic C2 cells.

Together, these results demonstrate that cdk1 (cdc2) andcdk2 isolated from proliferating myoblasts efficiently phos-phorylate MyoD, whereas cdk5 does not. Phosphorylation ofMyoD by purified cdk1-cyclin B causes a decrease of its elec-trophoretic mobility similarly to the hyperphosphorylated formof MyoD present in both growing or mitotic C2 myoblasts,further supporting an involvement of this kinase in the phos-phorylation of MyoD in proliferative myoblasts.

MyoD is phosphorylated on a CDK site in vivo. To comparethe sites phosphorylated on MyoD in vivo in proliferative myo-blasts with those targeted in vitro by purified cdk1-cyclin B andcdk1 or cdk2 immunoprecipitated from myoblasts, we usedtryptic digestion of MyoD followed by 2D phosphotryptic pep-tide mapping. As illustrated in Fig. 4A, two major phospho-tryptic peptides (spots 1 and 2 in the left panel) are obtainedafter digestion of 32P-labeled MyoD immunoprecipitated fromproliferating myoblasts. In the case of MyoD phosphorylatedby cdk1-cyclin B in vitro, two major phosphotryptic peptidesare also resolved (arrowed in the middle panel). We haveobserved the same pattern when analyzing MyoD phosphory-lated in vitro by cdk1 or cdk2 immunoprecipitated from pro-liferating C2.7 cells (unpublished observations). When in vivo-and in vitro-phosphorylated MyoD tryptic peptides are mixed(right panel), only one of the two peptides resolved in vivo(Spot 2) comigrated with one of the phosphopeptides fromcdk1-phosphorylated MyoD (the other major site phosphory-lated in vitro was never observed in vivo). To estimate which

sites were phosphorylated, we used the PhosPepSort programto obtain a prediction of the mobility map for the trypticphosphopeptides expected after phosphorylation of the sevenproline-directed sites on MyoD (Fig. 4B). As shown in Fig. 4B,the seven sites should lie in four phosphopeptides spanningamino acids (aa) 1 to 9 (Ser5), aa 10 to 41 (Ser37), aa 188 to202 (Ser200), and aa 258 to 319 (Ser262, Ser277, Ser298, andThr296). According to the mobility prediction, the two majorphosphopeptides obtained after in vitro phosphorylation ofMyoD by cdk1 and cdk2 would correspond to phosphorylationof Ser5 and Ser200. Of these two peptides, only one, which ispredicted to contain Ser200, is common between in vitro and invivo maps.

This result shows that at least one site phosphorylated invivo corresponds to a site phosphorylated by both cdk1 andcdk2 in vitro that most likely contains serine 200.

Ser200 is a major site of CDK-dependent phosphorylation.To determine precisely the site for in vivo CDK-dependentphosphorylation of MyoD, we mutated each putative CDK sitein MyoD. As shown in Fig. 5A, seven putative sites are distrib-uted in the NH2 and COOH ends of MyoD, at positions Ser5,Ser37, Ser200, Ser262, Ser277, Thr296, and Ser298. Seven mu-tants were generated by site-directed mutagenesis replacingthe amino acid serine or threonine by a nonphosphorylatablealanine residue and named MyoD-Ala5 to MyoD-Ala298.

Wild-type MyoD and its seven mutants were translated inthe presence of [35S]methionine in rabbit reticulocyte lysateand subjected to phosphorylation by purified cdk1-cyclin B.Phosphorylated proteins were separated by SDS-PAGE andvisualized by autoradiography. As shown in Fig. 5B, phosphor-ylation of MyoD-wt by cdk1-cyclin B resulted in a decrease ofits electrophoretic mobility. This slower-migrating form wasalso observed after phosphorylation of all but one (MyoD-Ala200) of the MyoD mutants, indicating that phosphorylationof Ser200 is responsible for the shift in mobility seen after thephosphorylation of MyoD by cdk1-cyclin B in vitro. To inves-tigate if Ser200 is also responsible for the shift observed in vivo,

FIG. 4. Phosphotryptic map analysis of MyoD phosphorylation in C2 myoblasts and following in vitro phosphorylation by cdk1-cyclin B. (A) Phosphorylation siteson MyoD were analyzed by tryptic digestion followed by 2D peptide mapping. The phosphopeptide map for in vivo MyoD phosphorylation (left panel) was obtainedafter immunoprecipitation of 32P-radiolabeled MyoD from dividing myoblasts. The phosphopeptide map for in vitro-phosphorylated MyoD (middle panel) wasperformed on bacterially produced MyoD phosphorylated in vitro by cdk1-cyclin B. In vivo and in vitro-phosphorylated MyoD were mixed following tryptic digestion,before 2D analysis (right panel). Numbers 1 and 2 indicate the two major phosphopeptides obtained from MyoD in proliferative myoblasts. The two majorphosphopeptides found with MyoD phosphorylated in vitro are pointed to by arrows. (B) Mobility prediction of MyoD tryptic phosphopeptides by using thePhosPepSort program shows the pattern of migration for the four MyoD phosphopeptides that would be generated if all of the seven proline-directed sites present onMyoD were phosphorylated.


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expression vectors coding for MyoD-wt and MyoD mutantswere transfected into 10T1/2 cells. Transfected cells weregrown for 36 h in proliferative medium, and MyoD expressionwas monitored by Western blotting of whole-cell extracts. Asshown in Fig. 5C, MyoD is detected as two bands followingtransfection of MyoD-wt and all but one of the mutants.Among the seven mutants, only MyoD-Ala200 is resolved as asingle band which migrates as the fast-migrating hypophos-phorylated form of MyoD found in both MyoD-wt-transfected10T1/2 and C2 myoblasts.

By 2D tryptic mapping (Fig. 4), we previously predicted thatSer200 was most likely the target of cdk1 and cdk2 phosphor-ylation in vitro and in vivo and that Ser5 could be phosphor-ylated in vitro but not in vivo. To confirm this prediction,mutant forms of MyoD (MyoD-Ala200 and MyoD-Ala5) wereproduced in bacteria, purified, and analyzed by 2D trypticmapping following in vitro phosphorylation by cdk1-cyclin B,as previously described for the wild-type protein. For compar-ison, the same experiment was carried out on MyoD-wt. Asshown in Fig. 6, two major phosphopeptides, S5 and S200(predicted to contain Ser5 and Ser200, respectively), are ob-

tained after in vitro phosphorylation of MyoD-wt by cdk1-cyclin B. MyoD-Ala200 is not phosphorylated on the S200peptide, whereas MyoD-Ala5 is no longer phosphorylated onthe Ser5 peptide. These results confirm that MyoD-wt can bephosphorylated by cdk1-cyclin B in vitro on two sites, Ser5 andSer200. As only the S200 peptide is common between in vitroand in vivo maps (Fig. 4), Ser200 is the only CDK sitephosphorylated on MyoD in vivo.

Together, these results identify Ser200 as a major site ofcdk1- and cdk2-dependent phosphorylation of MyoD both invitro and in vivo.

MyoD-Ala200 shows enhanced muscle gene-specific transacti-vating activity. To assess the consequence of Ser200 phosphory-lation on MyoD activity, we initially compared the abilities ofMyoD-wt and MyoD-Ala200 to transactivate muscle-specificgene expression. Plasmids expressing either MyoD-wt orMyoD-Ala200 were cotransfected with a CAT reporter genecontaining the acetylcholine receptor a-subunit promoter(paAch-CAT1) in 10T1/2 cells. Transfected cells were kept inproliferative medium for 36 h, and transactivation of the re-porter gene estimated by CAT assay. In each case, plasmidpCMV-bgal was cotransfected as an internal control for trans-fection efficiency. As expected (Fig. 7A), the low basal activityof the wild-type reporter gene was highly enhanced by MyoD-wt; moreover, MyoD-Ala200 further increased the level ofCAT reporter activity threefold over that obtained with MyoD-wt. Such an increase was not observed with any of the otherMyoD mutants (unpublished observations). To demonstratethat this increased transactivation activity of MyoD-Ala200required the E boxes, we performed the same experimentwith a mutant form of the reporter, paAchmutCAT1, wherethe E boxes had been mutated (35). Neither MyoD-wt norMyoD-Ala200 could transactivate the reporter plasmidpaAchmutCAT1 (unpublished observations). We also usedplasmid p4E-TK-CAT, which contains a simplified enhancercomprising four tandem copies of the E-box sequence (fromthe muscle creatine kinase gene enhancer) upstream of theminimal thymidine kinase promoter and, as a control, plasmidpTK-CAT, devoid of E boxes. As shown in Fig. 7A, MyoD-Ala200 was again threefold more efficient than MyoD-wt intransactivating CAT expression from the p4E-TK-CAT con-struct, which confirms the effect observed with paAch-CAT.

Taken together, these results show that mutation of Ser200results in an increased ability of MyoD to transactivate muscle-specific gene expression through the E box. Although a three-

FIG. 5. Among the seven potential CDK-dependent phosphorylation sites,mutation of only Ser200 prevents the phosphorylation shift of MyoD. (A) MyoDprotein exhibits seven proline-directed sites on its amino acid sequence: Ser5,Ser37, Ser200, Ser262, Ser277, Thr296, and Ser298. Each of the Ser and Thrresidues was mutated to Ala as described in Materials and Methods. (B) Invitro-translated 35S-labeled MyoD-wt (WT) and individual Ala mutants of MyoD(Ala5, Ala37, Ala200, Ala262, Ala277, Ala296, and Ala298) were incubated with(circled “p”) or without cdk1-cyclin B. Phosphorylation shifts were visualizedafter SDS-PAGE. Shown is the autoradiogram of the SDS-PAGE analysis. (C)Western blot analysis of MyoD overexpression in 10T1/2 cells transiently trans-fected with either MyoD-wt or each of the seven Ala mutants. Shown is the ECLdetection of MyoD immunoreactivity.

FIG. 6. Phosphotryptic map analysis of MyoD-Ala5 and MyoD-Ala200 fol-lowing in vitro phosphorylation by cdk1-cyclin B. Phosphorylation sites on MyoDwere analyzed by tryptic digestion followed by 2D peptide mapping. Phos-phopeptide maps were obtained for bacterially produced MyoD-wt (left panel),MyoD-Ala200 (middle panel), and MyoD-Ala5 (right panel) phosphorylated invitro by cdk1-cyclin B. The two major phosphopeptides found with MyoD-wtphosphorylated in vitro are pointed to by arrows labeled Ser5 and Ser200.


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fold difference in activity may seem unsufficient to ascribe apredominant role of MyoD Ser200 phosphorylation in control-ling its activity, it should be noted that this value is an under-estimate since only 50% of MyoD-wt is phosphorylated whenoverexpressed, as clearly shown in Fig. 5C.

MyoD-Ala200 promotes complete myogenic conversion of10T1/2 fibroblasts to muscle cells. Since mutation of MyoDserine 200 to alanine increased its capacity to transactivatemuscle-specific gene expression, we next compared the abilitiesof MyoD-wt and MyoD-Ala200 proteins to trigger myogenicconversion. 10T1/2 cells were transfected with expression vec-tors coding for either MyoD-wt or MyoD-Ala200. Transfectedcells were placed in differentiation medium for 60 h and ana-lyzed by immunofluorescence for expression of MyoD andtroponin T as a differentiation marker. The efficiency of myo-genic conversion was estimated as the percentage of cells ex-pressing MyoD that also expressed troponin T. The immuno-fluorescence presented in Fig. 7B reveal that MyoD-Ala200was significantly more efficient than MyoD-wt in converting10T1/2 cells to myotubes. After 60 h of differentiation (Fig.7B), nearly all MyoD-Ala200-expressing cells had differentiatedinto troponin T-positive myotubes whereas 35% of MyoD-wt-expressing cells remained negative for troponin T. A cleardifference in activity between the two MyoD proteins was alsoobserved at the phenotypic level. As shown in Fig. 7B, MyoD-Ala200-expressing cells formed many giant interconnectedmyotubes. We never observed this extent of differentiationwith the wild-type protein even if conversion was allowed forup to 5 days. To accurately quantify the increase in myogenicconversion ability of MyoD-Ala200 versus MyoD-wt, conver-sions were done as before but MyoD and troponin T expres-sion levels were analyzed by western blotting. As shown in Fig.7C, after 24 h (wt P and Ala200 P), similar levels of MyoD-wtand MyoD-Ala200 are expressed (upper panel), with no de-tactable troponin T expression (lower panel). After 60 h indifferentiation medium (wt 60h and Ala200 60h), troponin T isexpressed (lower panel) and is present at levels fivefold higherin MyoD-Ala200- than MyoD-wt-overexpressing cells. It isworth noting that in these culture conditions, MyoD-wt proteinlevel appears to be about twofold lower than the MyoD-Ala200level (upper panel, wt 60h and Ala200 60h), probably as aresult of differences in protein half-life (see below).

Taken together, these data show that the muscle-specifictranscription factor MyoD is phosphorylated in vivo on Ser200by a CDK. This phosphorylation event appears to restrictMyoD activity since mutation of serine 200 to a nonphosphor-ylatable alanine residue significantly enhances both the tran-scriptional activity of MyoD and the ability of MyoD to inducemyogenic conversion of nonmuscle cells.

Ser200 phosphorylation regulates MyoD protein turnover.Because phosphorylation by CDKs has been involved in thetargeted degradation of several factors such as p27 (53), wenext investigated a potential link between CDK-dependentphosphorylation of MyoD and its specific degradation. If phos-phorylation of Ser200 is implicated in MyoD degradation, mu-tation of Ser200 would be expected to increase the half-lifeof MyoD. To test this hypothesis, we transfected MyoD-wtand MyoD-Ala200 in 10T1/2 cells and determined the half-lifeof MyoD following cycloheximide treatment (Fig. 8A). Thehalf-life of MyoD-wt was found to be about 40 min (average ofvalues obtained from two different experiments [Fig. 8B]), inagreement with a previous report from Thayer et al. (50).Expression of a-tubulin, a stable protein, was not modified 2 hafter cycloheximide addition. By contrast, MyoD-Ala200 wasfound to be more stable than MyoD-wt, with an half-life ex-tended to 140 min.

DISCUSSION

An essential step during myogenesis is the reorientation ofthe proliferative cell cycle toward differentiation processes inwhich the transcription factor MyoD plays clearly a criticalrole. Although overexpression of MyoD can drive nontrans-formed fibroblasts into differentiation (8), myoblasts prolifer-ate efficiently while expressing MyoD. A mechanism other thanregulation of MyoD expression is therefore required to explainwhy myoblasts do not enter differentiation. In this report, wedemonstrate that phosphorylation plays an active role in pre-venting differentiation through a negative effect on MyoD ac-tivity. We observe that MyoD phosphorylation is high in myo-blasts and reduced during differentiation and show for the firsttime that MyoD is a direct substrate for phosphorylation byCDKs. Comparative peptide mapping combined with site-directed mutagenesis show that MyoD is phosphorylated bycdk1 (cdc2) and cdk2 on Ser200 both in vitro and in prolifer-ating myoblasts. Indeed, substitution of Ser200 by an alanine

FIG. 7. MyoD-Ala200 displays enhanced transactivating and myogenic activ-ities. (A) paAch-CAT1 and p4E-TK-CAT reporter constructs were cotrans-fected in 10T1/2 cells with either pEMSV or encoding plasmid pEMSV-MyoD-wt or pEMSV-MyoD-Ala200 and pCMV-bgal. Transfected cells weregrown for 36 h in DMEM containing 10% FCS, and CAT activity was measuredand corrected with respect to b-galactosidase activity. CAT activities are ex-pressed relative to that of each reporter plasmid transfected with pEMSV-MyoD, set as 100%. (B) 10T1/2 were transfected with either pEMSV-MyoD-wtor pEMSV-MyoD-Ala200 and placed in differentiation medium (DMEM con-taining 2% FCS) for 60 h. Cells were fixed and stained for both MyoD andtroponin T. Shown are the extents of myogenic conversion of 10T1/2 by MyoD-wt(left panels) and MyoD Ala200 (right panels) with staining for MyoD expression(a and b), troponin T expression (c and d), and DNA staining (e and f). Bar, 10mm. The average percentages of cells expressing MyoD-wt or MyoD-Ala200 thatcoexpressed troponin T were calculated from two different experiments and areindicated in the bottom. The total numbers of MyoD-positive cells counted were235 for MyoD-wt and 280 for MyoD-Ala200. (C) 10T1/2 cells were transfectedwith either pEMSV-MyoD-wt or pEMSV-MyoD-Ala200 as described above.Transfected cells were grown in proliferative medium (DMEM containing 10%FCS) for 24 h (P) and placed in differentiation medium for 60 h (60h). Cells werecollected either before (P) or after (60h) myogenic conversion and analyzed bywestern blotting for MyoD and troponin T expression.


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(MyoD-Ala200) prevents the appearance of hyperphosphory-lated MyoD after either its phosphorylation by cdk1-cyclin B invitro or overexpression in 10T1/2 cells. The phosphorylation ofthis site by CDKs is clearly inhibitory to MyoD function, as

demonstrated by the greater myogenic activity of MyoD-Ala200than of MyoD-wt.

cdk1 and cdk2 phosphorylate MyoD on serine 200 in pro-liferative myoblasts. Our data show that the kinases responsi-

d

FIG. 7—Continued.


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ble for the phosphorylation of Ser200 on MyoD in proliferativemyoblasts include the mitotic activator kinase cdk1-cyclin Band cdk2-cyclin A/E kinase, which is present and active frommid-G1 until mitosis. Overexpression of cyclin D1 was shownto promote hyperphosphorylation of MyoD (42, 43), suggest-ing that cdk4-cyclin D1 could directly phosphorylate MyoD.However, in contrast to the efficient phosphorylation of MyoDby cdk2 and cdk1 in vitro, we have been unable to observe aneffective phosphorylation of MyoD in assays using immuno-precipitated cdk4 from C2 myoblasts (unpublished observa-tions). This observation is in agreement with that of Skapek etal. (43), who found that baculovirus-produced cdk4-cyclin D1fails to phosphorylate MyoD. It thus appears that the hyper-phosphorylation of MyoD observed after cyclin D1 overexpres-sion may be the result of an indirect effect rather than a directcdk4-dependent phosphorylation of MyoD. cdk1- and cdk2-dependent phosphorylation requires the Ser/Thr-Pro (S/T-P)cluster to be followed immediately by a basic residue (Lys/Arg[46]), which is the case for the motif containing Ser200 that wehave identified on MyoD. Of the 6 other S/T-P sites present onMyoD, only serine 5 is also a potential site for phosphorylationby cdk1 and cdk2. Although this site is phosphorylated in vitro,as shown by phosphopeptide map analysis (Fig. 4 and 6), it wasnever found phosphorylated in vivo in C2.7 myoblasts (Fig. 4),and mutation of Ser5 to alanine did not cause any significanteffect on MyoD-dependent transcriptional activation of a re-porter gene containing the acetylcholine receptor promoter(unpublished observations). Ser200 is thus the only cdk1- andcdk2-dependent site used in vivo. It is also the only site re-sponsible for the electrophoretic shift in mobility seen whenMyoD is phosphorylated either in vitro or in vivo. It is to benoted that the sequence immediately surrounding and includ-ing Ser200 is highly conserved in MyoD from many differentspecies (unpublished observations). We cannot rule out thepossibility that phosphorylation of Ser200 is a prerequisite for

phosphorylation of MyoD at other sites. In this context, ki-nases other than CDKs may also phosphorylate MyoD andcontribute to its inhibition in proliferative myoblasts. In addi-tion to Ser200, a second phosphopeptide is clearly observed by2D tryptic mapping of MyoD isolated from proliferative myo-blasts. It does not correspond to any of the peptides resolvedafter in vitro phosphorylation of MyoD by cdk1-cyclin B (Fig.4), implying that a kinase other than cdk1 or cdk2 also phos-phorylates MyoD in vivo. PKA and PKC have been implied tonegatively regulate myogenic factors, although this regulationappeared to be indirect in the case of PKA (28, 55). Accordingto the PhosPepSort mobility analysis shown in Fig. 4, it isunlikely that PKC is responsible for this MyoD phosphoryla-tion in myoblasts. Indeed, the predicted map of MyoD-Thr 115phosphopeptide (equivalent to the site phosphorylated by PKCon myogenin [28]) does not correspond to the second trypticphosphopeptide observed in vivo (spot 1 in Fig 4). The kinaseresponsible for this phosphopeptide remains to be identified.

Among the members of the MyoD gene family, myogeninhas been shown to be phosphorylated on Ser47 and Ser170,two serine residues which lie in sequences similar to CDK-dependent phosphorylation sites (57). These two sites haveindeed been shown to be phosphorylated by cdk1 in vitro (20).However, the significance of such phosphorylation is unclear.The absence of this myogenic factor in proliferating myoblastsargues against a cell cycle-dependent regulation of myogenin.In addition, by 2D gel analysis, we showed that in contrast toMyoD, the phosphorylation status of myogenin does not un-dergo dramatic changes during differentiation of C2.7 cells(unpublished observations). Thus, the CDK-dependent phos-phorylation of MyoD Ser200 we have shown must play anunique role, one that cannot be extended to myogenin, in theregulation of MyoD activity. During the preparation of thispaper, Song et al. (45) reported that Ser200 is required forMyoD hyperphosphorylation. However, they did not investi-gate the nature of the protein kinase(s) responsible for MyoDphosphorylation or if such phosphorylation of MyoD occurs invivo in myoblasts. In this report, we demonstrated that cdk1and cdk2 are the protein kinases involved in the direct phos-phorylation of MyoD Ser200 in proliferative myoblasts.

Impeding Ser200 phosphorylation enhances MyoD activity.The mutant MyoD-Ala200 was more efficient than MyoD-wt inconverting 10T1/2 cells to muscle cells. This augmentation wascorrelated to an enhanced ability of MyoD-Ala200 (aboutthreefold higher than that of MyoD-wt) to transactivate musclegene expression via the E box (Fig. 7A). However, this in-creased transactivating capability was not linked to significantalteration in MyoD DNA binding affinity. Indeed, by band shiftanalysis, we observed that phosphorylation of MyoD by cdk1-cyclin B did not alter the binding of MyoD homodimer to theE-box and had marginal effects on MyoD-E12 DNA binding(unpublished observations). That DNA-binding and transcrip-tional activities of myogenic factors are not necessarily corre-lated has been reported in previously. For instance, in myo-blasts blocked from differentiating by transforming growthfactor b, myogenic factors appear to retain DNA-binding ac-tivity without activating muscle gene transcription (5). Inter-estingly, MyoD-containing complexes capable of binding to anE box are observable in nuclear extracts from both proliferat-ing myoblasts and differentiated myotubes (reference 41 andour unpublished observations). It thus appears that the tran-scriptional activity of MyoD is not necessarily reflected by itscapacity to bind to DNA. Because DNA-binding activity wasnot the mechanism by which phosphorylation of MyoD Serine200 could control MyoD activity, we have compared the sta-bilities of MyoD-wt and MyoD-Ala200. We found, in agree-

FIG. 8. Impeding MyoD Ser200 phosphorylation stabilizes the protein. (A)10T1/2 cells were transfected with either pEMSV-MyoD-wt or pEMSV-MyoD-Ala200 and grown for 24 h in proliferative medium before addition of cyclohex-imide (15 mg/ml) to the medium for 0, 15, 30, 60, or 120 min. MyoD anda-tubulin protein levels were determined by immunoblot analysis at the indicatedtimes after cycloheximide addition. (B) Immunoblots were quantified by densi-tometric scanning, and MyoD protein levels (corrected with respect to tubulinexpression) were expressed relative to that observed before cycloheximide treat-ment, set as 100%.


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ment with a recent report from Song et al. (45), that MyoD-Ala200 was more stable than MyoD-wt, suggesting thatphosphorylation of Ser200 decreases MyoD activity by reduc-ing its half-life. This phosphorylation seems to be required fortargeting MyoD to the ubiquitin pathway (45). A rapid turn-over of MyoD may allow a fine regulation of its activity inmyoblasts. High-level expression of MyoD obtained by ectopicexpression into nonmuscle cells is known to stop cell cycleprogression before S phase, allowing cells to engage into thedifferentiation process (7, 47). Controlled degradation ofMyoD may be necessary to prevent MyoD from reaching athreshold that can interfere with normal cell cycle events be-fore myoblasts have received the appropriate signal to differ-entiate. By isolating C2 cells that have lost MyoD expression,Horwitz (22) found that the autoactivation loop of MyoD istightly linked to protein stability. In addition, the reducedhalf-life that we observed for phosphorylated MyoD may resultfrom a change in MyoD-associated protein. Ser200 phosphor-ylation may reduce the association of MyoD with partners suchas pRb (14), MEF-2 proteins (29), the coactivator p300 (11,37), or proteins such as Id. cdk2-dependent phosphorylationhas been shown to change the interaction specificity of theHLH protein Id3 (10). CDK-dependent phosphorylation hasalso been shown to change the interaction between the tran-scription factor E2F and pRB (21, 44). In a similar way, freeMyoD could be more sensitive to degradation. Gerber et al.(13) have recently shown that MyoD, in addition to being ableto bind DNA and activate muscle-specific gene expression, canremodel chromatin at binding sites in muscle gene regulatoryregions and activate transcription at previously silent loci. Thisability of MyoD to activate genes within inactive chromatinmapped to a cysteine- and histidine-rich region of the aminoterminus and a region extending from aa 218 to 269 in thecarboxy terminus of MyoD. Interestingly, deletion of a regionbetween aa 170 and 209 (that includes Ser200) increased theability of MyoD to initiate transcription of endogenous genes,implying a repressive role of this region in chromatin remod-eling by MyoD. It is tempting to hypothesize that in addition tomodulating MyoD half-life and transactivating ability, Ser200phosphorylation may cause conformational changes of MyoDand thereby modulate intra- or intermolecular interactions in-volved in remodeling chromatin.

ACKNOWLEDGMENTS

We thank Jacques Demaille for his continued support. We thank P.Dias for the generous gift of monoclonal anti-MyoD antibody, HalWeintraub for coding plasmids p4E-TK-CAT and pTK-CAT, andJacques Piette for plasmids paAch-CAT1 and paAchmutCAT1.

This work was supported by grants from Association Francaise con-tre les Myopathies and Association pour la Recherche contre le Can-cer (contract 1344 and a fellowship to M.K.).

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