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Degradation of MyoD mediated by the SCF (MAFbx)

ubiquitin ligase

L.A. Tintignac, Julie Lagirand, S. Batonnet, Valentina Sirri, M.P. Leibovitch,

S.A. Liebovitch

To cite this version:

L.A. Tintignac, Julie Lagirand, S. Batonnet, Valentina Sirri, M.P. Leibovitch, et al.. Degradation of

MyoD mediated by the SCF (MAFbx) ubiquitin ligase. Journal of Biological Chemistry, American

Society for Biochemistry and Molecular Biology, 2005, 280 (4), pp.2847-2856. �hal-02679960�

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Degradation of MyoD Mediated by the SCF (MAFbx)

Ubiquitin Ligase*

S

Received for publication, October 5, 2004, and in revised form, October 28, 2004 Published, JBC Papers in Press, November 5, 2004, DOI 10.1074/jbc.M411346200

Lionel A. Tintignac‡§, Julie Lagirand‡¶, Sabrina Batonnet‡¶, Valentina Sirri, Marie Pierre Leibovitch‡, and Serge A. Leibovitch‡**

From the ‡Laboratoire de Ge´nomique Fonctionnelle et Myoge´ne`se, UMR866 Diffe´renciation Cellulaire et Croissance, INRA

UM II, Campus INRA/ENSA, 2 Place Pierre Viala, 34060, Montpellier, Cedex 1, France and theInstitut Jacques Monod,

UMR 7592 CNRS, 75251 Paris, France

MyoD controls myoblast identity and differentiation and is required for myogenic stem cell function in adult skeletal muscle. MyoD is degraded by the ubiquitin-pro-teasome pathway mediated by different E3 ubiquitin ligases not identified as yet. Here we report that MyoD interacts with Atrogin-1/MAFbx (MAFbx), a striated muscle-specific E3 ubiquitin ligase dramatically up-reg-ulated in atrophying muscle. A core LXXLL motif se-quence in MyoD is necessary for binding to MAFbx. MAFbx associates with MyoD through an inverted LXXLL motif located in a series of helical leucine-charged residue-rich domains. Mutation in the LXXLL core motif represses ubiquitination and degradation of MyoD induced by MAFbx. Overexpression of MAFbx suppresses MyoD-induced differentiation and inhibits myotube formation. Finally the purified recombinant SCFMAFbx complex (SCF, Skp1, Cdc53/Cullin 1, F-box

protein) mediated MyoD ubiquitination in vitro in a ly-sine-dependent pathway. Mutation of the lysine 133 in MyoD prevented its ubiquitination by the recombinant SCFMAFbx complex. These observations thus

demon-strated that MAFbx functions in ubiquitinating MyoD via a sequence found in transcriptional coactivators. These transcriptional coactivators mediate the binding to liganded nuclear receptors. We also identified a novel protein-protein interaction module not yet identified in F-box proteins. MAFbx may play an important role in the course of muscle differentiation by determining the abundance of MyoD.

Ubiquitin-dependent proteolysis regulates protein abun-dance and serves a central regulatory function in many biolog-ical processes (1). The ubiquitination of the target protein is mediated by the ubiquitin ligases, which represent a diverse family of proteins and protein complexes. The SCF (Skp1, Cdc53/Cullin1, F-box protein) and SCF-like complexes are the largest family of ubiquitin ligases. They ubiquitinate a broad

range of proteins involved in cell cycle progression, signal transduction, transcription, and development (2). The forma-tion of ubiquitin-protein conjugates involves three components that participate in a cascade of ubiquitin transfer reactions, an ubiquitin-activation enzyme (E1),1 an ubiquitin-conjugating

enzyme (E2) and an ubiquitin ligase (E3) that acts at the last step of the cascade (3). The specificity of the SCF complexes derives of the variable components called the F-box proteins that serve as receptor for the target protein (4).

Myogenic differentiation is under the control of the MyoD family of basic helix-loop-helix transcription factors (MRFs) that includes MyoD, myogenin, Myf5, and MRF4/herculin/ Myf-6 (5, 6). Activation of muscle-specific genes by the MRFs occurs through their heterodimerization with the E protein basic helix-loop-helix factors that bind to an E-box DNA consensus sequence (CANNTG) to transactivate muscle spe-cific genes and to efficiently convert non-muscle cells to a myogenic lineage (7). p300/CBP and P/CAF coactivators have acetyltransferase activities and regulate transcription, cell cycle progression, and differentiation. They are both required for MyoD activity and muscle differentiation (8, 9). In con-trast, histone deacetylation inhibits gene activation, and the interaction between histone deacetylase 1 and MyoD pre-vents premature activation of the myogenic program in grow-ing myoblasts (10). The MRFs contain several functionally distinct domains responsible for transcriptional activation, chromatin remodeling, DNA binding, nuclear localization, and heterodimerization (11).

The mechanism by which MyoD induces myogenesis involves both the withdrawal from cell cycle and the activation of mus-cle-specific gene expression. Recent data demonstrate a direct link between MyoD and cell cycle regulation (12, 13) and also its requirement for myogenic stem cell function in adult skel-etal muscle (14) where MyoD protein levels decline from post-natal stage onward (15). Accurate synchronization of dividing myoblasts revealed that MyoD is subject to specific cell cycle-dependent regulation (16 –18). Phosphorylation of MyoD at serine 200 plays a crucial role in modulating its half-life and transcriptional activity during myoblast proliferation (19, 20). Linking this phosphorylation to the cell cycle-dependent drop in MyoD protein before S phase leads to a mechanism impli-cating Cdk2-cyclin E and up-regulation of its inhibitors (p57kip2

and p21cip1) in the tight control of MyoD levels and subsequent

myoblast cell cycle progression or exit into differentiation (17,

* This work was supported in part by grants from Association Fran-c¸aise contre les myophaties (AFM), Ligue Nationale contre le Cancer, l’Association pour le Recherche contre le Cancer (ARC), and INRA. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked

“adver-tisement” in accordance with 18 U.S.C. Section 1734 solely to indicate

this fact.

□S The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1–3.

§ Present address: Growth Control Laboratory, FMI, Maulbeer-strasse 66, 4058, Basel, Switzerland.

¶Fellows of Ministe`re de la Recherche et de la Technologie (MRT). ** To whom correspondence should be addressed. Tel.: 33-04-99-61-29-76; Fax: 33-04-67-54-56-94; E-mail: serge.leibovitch@ensam.inra.fr.

1The abbreviations used are: E1, ubiquitin-activating enzyme; E2,

ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; IRES, internal ribosome entry site; GFP, green fluorescent protein; HA, he-magglutinin; LCD, leucine-charged domain; wt, wild type; PGC-1␣, peroxisome proliferator-activated receptor-␥ coactivator-1␣.

© 2005 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

This paper is available on line at http://www.jbc.org

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21). In addition MyoD phosphorylated on serine 200 is de-graded by the proteasome and CDC34 ubiquitin-conjugating enzyme activity (E2) (19). Altogether these findings indicate that targeted degradation of MyoD following Cdk2-cyclin E phosphorylation of serine 200 during G1to S phase progression

in myoblasts and identify a mechanism that probably requires a SCF complex. On the other hand, recent data have shown that MyoD could be degraded by the ubiquitin-proteasome pathway independently of its phosphorylation state suggesting that attachment of ubiquitin implies other(s) recognition sig-nal(s) and/or mechanism(s). Up-regulation of the cyclin/Cdk inhibitor p57Kip2 stabilizes MyoD by direct interaction (22),

whereas specific DNA binding stabilizes MyoD (23) and in vitro MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm (24), and in differentiated myotubes, MyoD protein turns over rapidly as observed in myoblasts (25). Alto-gether these data indicate that MyoD is degraded by the ubiq-uitin-proteasome pathway mediated by different E3 ubiquitin ligases not identified as yet.

To determine candidate molecular mediators of MyoD ubiq-uitination, we employed a modified version of the yeast two-hybrid screen of the human skeletal muscle library to identify proteins that first interact with human Skp1. This

Skp1-en-riched cDNA library was then screened to isolate F-box pro-teins that bind MyoD and identify positive clones. Here, we report the identification of human Atrogin-1/MAFbx (MAFbx), an E3 ubiquitin ligase up-regulated during skeletal muscle atrophy that interacts with MyoD via a novel leucine-rich in-teraction interface. This interface found in transcriptional co-activators mediates the binding to liganded nuclear receptors and promote its degradation. We demonstrated that the puri-fied recombinant SCFMAFbxcomplex mediated ubiquitination

of MyoD in vitro in a manner that appeared to depend on the presence of the lysine 133. This lysine 133 has been previously shown to play a critical role in the nuclear degradation of MyoD (26). Finally, we show that up-regulation of MAFbx in prolif-erating myoblasts antagonizes differentiation inducing MyoD degradation and preventing muscle specific-gene activation.

EXPERIMENTAL PROCEDURES

Yeast Two-hybrid Screen and DNA Manipulations—We used the

Matchmaker Two-Hybrid Kit with a human skeletal muscle cDNA library kit (Clontech). The assays were performed as recommended by the manufacturer. The full-length cDNA clone of human Skp1 (bait) was subcloned in-frame with the Gal4 DNA-binding domain in the pGBT9 vector, resulting in pGBT9-Skp1. We screened 6⫻ 106clones

(corresponding to two library titers) and selected the positive clones as suggested by the manufacturer. The liquid␤-galactosidase assay was performed according to the manufacturer’s instruction. The Skp1-en-riched cDNA library was then screened against pGBT9-MyoD (bait) and we selected 100 positive clones. Oligonucleotides were synthesized by Proligo. Deletion in the F-box of MAFbx was introduced by PvuII digestion of pACT2-MAFbx and self-annealing of the resulting plasmid. The full-length coding sequences as well as mutants of MAFbx and MyoD were amplified by PCR to introduce BamHI and EcoRI sites on each side of the open reading frame and cloned as BamHI-EcoRI frag-ments into the eukaryotic expression vectors. The bicistronic expression plasmid for MAFbx was carrying out first by using a 1400-bp EcoRI-HindIII fragment containing the internal ribosome entry site (IRES) and GFP from the pMIGR vector and subcloned at the EcoRI and HindIII sites of pcDNA4T0 (Invitrogen). MAFbx and the⌬F-box mutant were then subcloned at the EcoRI site of pcDNA4-IRES-GFP.

Cell Culture, Transfections, and Luciferase Assays—The mouse

skel-etal muscle cell line C2C12 and the fibroblast cell line 10T1/2 were maintained in growth Dulbecco’s modified Eagle’s medium supple-mented with antibiotics and, respectively, 20 and 15% fetal calf serum. Cells were transfected by using the Jet PEI (QBiogene). Luciferase activity was determined using 10-␮l aliquots of cell extracts from har-vested cells in 1⫻ reporter lysis buffer (Promega) with equivalent quantities of proteins in triplicate and repeated at least twice.

Immunoprecipitation and Immunoblotting—Two MAFbx

anti-bodies were generated by injecting rabbits with the amino acid peptide KKRKKDMLNSKTKT(C) corresponding to amino acids 61–74 and amino acid peptide KGTDHPCTANNPE(C) corresponding to amino acids 326 –339 of the human MAFbx protein. Antibodies were affinity-purified against antigenic peptides. For immunoprecipitation, cell ly-sates in IP buffer (50 mMTris, pH 7.4, 150 mMNaCl, 10% glycerol, 0.5% Nonidet P-40, 0.5 mMsodium-orthovanadate, 50 mMNaF, 80␮M ␤-glyc-erophosphate, 10 mMsodium-pyrophosphate, 1 mMdithiothreitol, 1 mM EGTA, and 10 ␮g/ml leupeptin, 10 ␮g/ml pepstatin, and 10 ␮g/ml aprotinin) were precleaned for 30 min with protein-G beads and immu-noprecipitated by using standard procedures. Immuimmu-noprecipitated pro-teins were loaded onto 10% SDS-polyacrylamide gels before electro-phoretic transfer onto a nitrocellulose membrane. For detection of MyoD-ubiquitin conjugates, cells were rinsed in phosphate-buffered saline and scraped into 800 ␮l of radioimmune precipitation assay buffer (20 mMTris, pH7.5, 5 mMEDTA, 150 mMNaCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1% SDS, and 1 mMphenylmethylsulfonyl fluo-ride). Cells were then spun at 15,000⫻ g for 15 min, and the superna-tant was denatured and then precipitated with anti-MyoD antibody. Western blotting was performed by using an ECL kit (Amersham Bio-sciences) according to the manufacturer’s instructions. Anti-MyoD poly-clonal (C20) and anti-HA polypoly-clonal (Y-11) were purchased from Santa Cruz Biotechnology. Anti-ubiquitin (P4D1) antibodies were purchased from Calbiochem, anti-Troponin T monoclonal (JLT-12) was purchased from Sigma, anti-HA epitope (12CA5) was purchased from Roche Diag-nostics, MyoD monoclonal (5.A8) was from Pharmingen, and anti-FLAG (M2) was from Kodak. To inhibit proteasome activity, cells were FIG. 1. Characterization of human MAFbx protein. A, schematic

representation of the putative domains present in human skeletal mus-cle cDNA clones R5 and R8 encoding MAFbx. B, reverse transcription-PCR analysis of MAFbx mRNA expression in proliferating myoblasts and during the course of differentiation of C2C12 cells (24, 48, and 72 h after switching into differentiation medium). C, Western blot analysis of MAFbx and MyoD proteins during C2C12 differentiation. Asterisk marks a nonspecific band. LZ, leucine zipper; GAPDH, glyceraldehyde-3-phosphate dehydrogenase, NLS, nuclear localization signal.

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treated with 50␮MMG132 (Sigma) for 2 h.

In Vitro and in Vivo Ubiquitination Assay—Ubiquitination assays

were determined essentially as described (17, 26) by using [35

S]-methionine-labeled in vitro translated HA-MyoD. The recombinant SCFMAFbxcomplex was produced in insect cells by infection with

baculovirus encoding HA-MAFbx-His6, Skp1, Cul1, and Rbx1,

puri-fied by nickel-agarose chromatography, and added in roughly similar amount to the reaction. In vivo ubiquitination assays have been described (26).

RNA Extraction and Reverse Transcription-PCR Analyses—Total

RNA was isolated from C2C12 cells using TRI Reagent (Sigma). For semiquantitative reverse transcription-PCR analysis, 1␮g of RNA was used to perform reverse transcription with Superscript II RnaseH Re-verse Transcriptase (Invitrogen) and hexanucleotides (Roche Diagnos-tics). 2␮l of each reaction served as a template for PCR analysis. Primer pairs for the amplification of the gene products were MAFbx 5⬘-GGG-GGAAGCTTTCAACAG-3⬘ forward, 5⬘-TGAGGCCTTTGAAGGCAG-3⬘ reverse and glyceraldehyde-3-phosphate dehydrogenase 5⬘-GAGCTGA-ACGGGAAGCTCACT-3⬘ forward, 5⬘-TTGTCATACCAGGAAATGAG-C-3⬘ reverse.

Immunofluorescence Staining—Cells were cultured on coverslips and

fixed in 3% paraformaldehyde for 10 min at 4 °C, and permeabilized with 0.25% Triton X-100 for 30 min at room temperature. The cells were treated with 5% normal goat serum and immunostained with anti-MyoD mAb (5.A8) The Texas Red-conjugated F(ab⬘)2fragment of donkey

anti-mouse IgG was used to visualize the anti-mouse monoclonal antibodies.

RESULTS

Identification of MAFbx as an F-box Protein Interacting with MyoD—The CDC34 ubiquitin-conjugating enzyme activity (E2)

has been implicated in MyoD ubiquitination (19) suggesting the requirement of an Skp1/Cul1/F-box protein complex (E3). To identify candidate molecular mediators of MyoD ubiquitination and degradation, we used a human skeletal muscle library en-riched for F-box-expressing genes and MyoD as bait in the yeast two-hybrid screen. We identified two positive clones with the same open reading frame, one clone (clone R8, 1.6-kb insert) coded for 350 amino acids and the second clone (clone R5, 0.95-kb insert) encoded a polypeptide of 259 amino acids (Fig. 1A) highly homologous with the mouse F-box protein Atrogin-1/MAFbx (MAFbx) (27, 28). The full-length human MAFbx lacks the LRR and WD40 consensus motifs known to interact with protein sub-strates (29, 30) In addition to the PDZ motif and cytochrome c family heme binding site located in the C terminus domain, MAFbx contains two potential nuclear localization signals, a leucine zipper domain and a leucine-charged residue-rich domain

FIG. 2. MAFbx and MyoD proteins interact via a LXXLL motif.

A, association between MAFbx and MyoD in C2C12 myoblasts and

myotubes. Cells were grown in proliferation medium (Mb) and/or in differentiation medium (Mt) with 50␮MMG132 (⫹) for 2h, after which cell lysates were immunoprecipitated with anti-MAFbx 61–74

antibod-ies. The resulting precipitates were subjected to immunoblot (IB) anal-ysis with antibodies to MyoD or MAFbx 326 –339, as indicated. B, interaction between MAFbx and various mutants of MyoD. The ability of each MyoD mutant to bind MAFbx is summarized (⫹ or ⫺). Amino acid regions corresponding to the putative MAFbx-MyoD interacting sequence and schematic representation of mutant MyoD L164Q gener-ated by site-directed mutagenesis. C, empty plasmid (lane 1) or expres-sion plasmids encoding HA-MyoD L164Q (lane 2) or HA-MyoD-wt (lane

3) were cotransfected with an expression plasmid encoding

FLAG-MAFbx into 10T1/2 cells. Cell lysates were processed for immunopre-cipitation with anti-FLAG antibodies (M2). Immunoprecipitates were then analyzed by Western blot with anti-Flag and anti-HA, respectively (bottom panel). Aliquots of lysates were processed for Western blotting with anti-Flag and anti-HA (top panel). Note that anti-FLAG antibodies failed to immunoprecipitate HA-MyoD. D, identification of the sequence in MAFbx required for MyoD interaction. 10T1/2 cells were cotrans-fected with MyoD and wild type (full) and/or deletion mutants of MAFbx. (⫹) and (⫺) indicate whether or not the various mutants interact with MyoD. Representation of the putative MyoD-binding do-main in MAFbx. Highly conserved LCD in mammals is underlined, and leucine residues are in bold. E, empty vector (lane 1) or expression plas-mids encoding FLAG-MAFbx-wt (lane 2) or FLAG-MAFbx L169Q (lane 3) were transfected into 10T1/2 cells together with an expression plasmid encoding HA-MyoD. Whole cell lysates were processed for immunopre-cipitation with anti-FLAG antibodies. Immunoprecipitates were then an-alyzed by Western blot with anti-FLAG and anti-HA, respectively (bottom

panel). Aliquots of lysates were processed for Western blotting with

anti-FLAG and anti-HA (top panel). WCE, whole cell extract.

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FIG. 3. MAFbx increases MyoD turnover. A, 10T1/2 cells were cotransfected using the expression vector encoding HA-MyoD and various FLAG-tagged F-box proteins as indicated. The cells were treated with cycloheximide (CHX) 36 h after transfection to block protein synthesis. Cell lysates were prepared at the indicated time and analyzed by Western blotting with anti-HA and anti-FLAG, respectively (left panel). Quantitation of MyoD turnover following cycloheximide treatment based on densitometric scanning of experiments is shown (right panel). Note that the⌬F-box mutant stabilized MyoD. B, effects of mutations of LXXLL motif in MAFbx on MyoD degradation. MyoD turnover was analyzed in 10T/1/2 cells transfected with the indicated expression vectors followed by pulse chase to measure MyoD stability in the presence of MAFbx wild type and/or the mutant L169Q. Quantitation of MyoD turnover following pulse chase based on densitometric scanning is shown (right panel). C, pulse-chase analysis was used to measure MyoD stability in wild type and mutant MyoD L164Q in the absence or in the presence of MAFbx as in B.

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(LCD), which are conserved between human, rat, and mouse species (supplemental Fig. 1). The presence of various protein-protein interacting domains suggests that MAFbx could poten-tially recognize different substrates.

Expression of MAFbx and MyoD during C2C12 Muscle Dif-ferentiation—Because little is known about the expression of

MAFbx in myogenic differentiation, we initially characterized MAFbx expression during early myogenic differentiation using

FIG. 4. Overexpression of MAFbx degrades MyoD and suppresses myogenic differentiation. A, effect of MAFbx on MyoD-dependent transcriptional transactivation of muscle-specific genes. 10T1/2 cells were cotransfected with 0.5␮g of MCK-Luc reporter plasmid together with 2␮g of pEMSV-MyoD in combination with 0.5 and 2 ␮g of expression vector encoding FLAG-tagged MAFbx. Expression vector without insert was included to normalize DNA in all transfections. Luciferase levels were determined 48 h after transfections in proliferating medium and represent the average of three independent experiments (errors bars, S.D.). Protein concentrations were equalized by Bradford assay, and aliquots were analyzed by Western blotting for MyoD and MAFbx expression using specific antibodies (upper panel). B, overexpression of MAFbx suppressed MyoD in C2C12 myoblasts (a– o). Myoblasts were transfected with empty pcDNA4-IRES-GFP vector (a– e) and either MAFbx-IRES-GFP (f–j) or the mutant MAFbx⌬F-IRES-GFP (k–o). Forty-eight hours later, cells were fixed and stained with anti-MyoD and revealed with goat anti-mouse antibody conjugated to Texas Red (d, i, and n). Hoechst 33258 dye shows nuclei (b, g, and i). Merged images are shown in e, j, and o. Images of a representative field were obtained by indirect immunofluorescence microscopy (400⫻). C, overexpression of MAFbx suppresses myogenic differ-entiation. C2C12 myoblasts were transfected with pcDNA4-IRES-GFP expression plasmid (a– e), the mutant MAFbx⌬F-IRES-GFP (f–j), or MAFbx-IRES-GFP (k– o). Twenty-four hours later, cells were switched to differentiation medium (DM) for 72 h, fixed, and stained with anti-MyoD as in (B) and with Hoechst 33258 dye (b, g, and l). Merged images are shown in e, j, and o. Images of a representative field were obtained by indirect immunofluorescence microscopy (400⫻). DAPI, 4,6-diamidino-2-phenylindole.

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the C2C12 cell line, a well defined model for ex vivo differenti-ation. These cells proliferate as myoblasts in high serum con-centrations and can be induced to differentiate by reducing the serum concentration from 20 to 2%. Under our conditions, C2C12 myoblasts fuse into myotubes within 48 – 60 h with an efficiency of 75– 80%. A semiquantitative reverse transcription-PCR analysis showed that the MAFbx mRNA was up-regulated

(2–3-fold) in the course of C2C12 differentiation (Fig. 1B). Anti-MAFbx antibodies were developed in our laboratory (see “Experimental Procedures”), and Western blot analysis re-vealed that the MAFbx protein is barely detectable in myo-blasts and accumulates in C2C12 myotubes. By this time, MyoD is up-regulated after switching cells into the differenti-ation medium (DM) and then decreases during differentidifferenti-ation; troponin T, a skeletal muscle marker, is observed only in dif-ferentiated cells (Fig. 1C). Thus, during the course of C2C12 differentiation MyoD and MAFbx showed a contrasting pattern of expression.

Phosphorylation or Acetylation of MyoD Is Not Necessary for Interaction with MAFbx—We first determined the binding of

MyoD with MAFbx in mammalian cells by using a coimmuno-precipitation assay. In C2C12 myogenic cells, endogenous MyoD coimmunoprecipitated with endogenous MAFbx, and an increase in MyoD/MAFbx association was observed when pro-teasome activity was inhibited (Fig. 2A). In transiently trans-fected 10T1/2 cells, MyoD and MAFbx have a nuclear localiza-tion, and MyoD coimmunoprecipitated with MAFbx but did not associate with other F-box proteins such as Skp2 or FBX03 (supplemental Fig. 2, A and B). Furthermore, Myf-5, the second determination factor that is also expressed in myoblasts, did not coimmunoprecipitate with MAFbx in cotransfection exper-iments (supplemental Fig. 2C).

Posttranslational modifications of target proteins are impcated in the recognition by certain SCF-type E3 ubiquitin li-gases and subsequent degradation by the 26 S proteasome (4, 31, 32). On the other hand, ubiquitination of substrates re-quires direct binding of specific domains of the F-box protein and the substrate (33). The main posttranslational modifica-tions of MyoD that implicate phosphorylation of Ser-5 and Ser-200 (18, 20) and/or acetylation by CBP/p300 (8) and p/CAF (9) were tested for their interaction with MAFbx. 10T1/2 cells were transiently transfected with plasmids encoding MAFbx alone or with MyoD-wt, MyoD S200D, which mimics constitu-tive serine 200 phosphorylation, MyoD S5A/S200A, and MyoD/K 3 R in which all lysines were mutated to arginines. Surprisingly MyoD-wt and the MyoD mutants all interacted with MAFbx suggesting that major phosphorylation and acety-lation are not implicated in MyoD/MAFbx interaction (supple-mental Fig. 2D). Altogether, these results show that independ-ent of the phosphorylation and/or acetylation, MyoD and MAFbx form complexes in vivo

LCD of MAFbx Interacts with a Highly Conserved LXXLL Motif in MyoD—The results outlined above show that MAFbx

binds to MyoD, but the lack of known protein binding motifs found in other F-box proteins prompted us to search for regions of MAFbx important for substrate recognition. The domains of both proteins required for interaction were mapped by in vivo protein binding experiments. 10T1/2 cells were cotransfected with expression vectors encoding MyoD-wt and/or MyoD dele-tion or substitudele-tion mutants together with MAFbx, and the immunoprecipitates were then examined for the presence of MAFbx by an immunoblot analysis (supplemental Fig. 3A). Stable association between MyoD and MAFbx was mediated by the helix 2 domain of MyoD (Fig. 2B). Sequence analysis of amino acids 143–172 revealed a LXXLL consensus sequence that mediates protein-protein interactions and was originally found in a variety of coactivators (34). To assess the contribu-tion of this motif to the interaccontribu-tion, we mutated the leucine 164 to glutamine (L164Q) and tested its interaction with MAFbx as described above. Mutation in the LXXLL motif was sufficient to reduce MyoD interaction with MAFbx (Fig. 2C).

In vivo reciprocal binding experiments were performed to

define the sequence in MAFbx that contributes to complex

FIG. 5. Accumulation of MAFbx during atrophy induced by

aging and fasting promotes ubiquitin conjugation of MyoD in

vitro. A, total muscle lysates were prepared from hind limb skeletal

muscle of control (N, lanes 1 and 3) and 3 days food-deprived mice (Atro, lanes 2 and 4) and immunoprecipitated (IP) with anti-MAFbx 61–74 antibodies. Immunoprecipitates were then analyzed by immu-noblot (IB) with anti-MAFbx 326 –339 antibodies. B, total muscle lysates from hind limb skeletal muscle of control (N, lanes 1 and 2) and 3 days food-deprived mice (Atro, lane 3) were immunoprecipi-tated with anti-MAFbx 61–74 antibodies. Immunoprecipitates were then analyzed by Western blot with MAFbx 326 –339, anti-Skp1, and anti-Cul1 antibodies, respectively. C, 35S-labeled

HA-tagged MyoD-wt subjected to the in vitro ubiquitination reaction in the presence of normal and/or atrophic mouse skeletal muscle lysates.

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formation with MyoD. To avoid confusion between the IgG band and the MAFbx mutants, we used a mammalian expres-sion plasmid encoding GST-MyoD (supplemental Fig. 3B). Overlapping deletion mutant analysis showed that the central part of MAFbx (amino acids 140 –222) was implicated in the MyoD binding (Fig. 2D). This region contains a LCD that shares a consensus inverted LXXLL motif flanked by LXXL and LXXXL sequences highly conserved in mammals. We found that mutation (L169Q) in the inverted LXXLL motif dramatically reduced the binding to MyoD (Fig. 2E). These results demonstrated that complex formation depends upon the integrity of the inverted LXXLL motif in MAFbx.

Accelerated Degradation of MyoD by MAFbx—The effect of

MAFbx expression on the MyoD half-life was investigated after blocking protein synthesis with cycloheximide and/or in pulse-chase experiments. As expected the empty vector did not affect MyoD stability (half-life was to ⬃45 min). Overexpression of MAFbx resulted in enhanced degradation of MyoD by decreas-ing its half-life to⬃30 min. By contrast Skp2 and/or FBL5 was ineffective on MyoD degradation. In cells with compromised SCFMAFbxactivity, MyoD degradation was affected; the MAFbx

mutant⌬F-box (deleted of the F-box domain required for Skp1 interaction) showed an increased MyoD half-life (⬃150 min) (Fig. 3A). In cells with compromised LXXLL motif interaction between MyoD and MAFbx, the degradation rate of MyoD remained similar to that of MyoD-wt alone (Fig. 3, B and C). It appears that destabilization of MyoD is increased in cells with SCFMAFbxE3-ligase activity and highlights the LXXLL motif

that mediates MyoD/MAFbx interaction.

Overexpression of MAFbx in C2C12 myotubes results in atrophy (28), and on the other hand, during the course of C2C12 differentiation MyoD and MAFbx showed a contrasting pattern of expression (Fig. 1C). Together these data suggested that MAFbx-inducing C2C12 atrophy implicated MyoD degra-dation. We observed that increasing amounts of MAFbx on MyoD-mediated myogenic conversion of 10T1/2 cells caused a gradual decrease of MCK-Luc activity and MyoD protein abun-dance (Fig. 4A). To locate and assess the effect of MAFbx overexpression on MyoD proteins, C2C12 myoblasts were transfected with pcDNA4-MAFbx-IRES-GFP or the mutant MAFbx-⌬F-box-IRES-GFP, and 48 h later C2C12 myoblasts (Fig. 4B) were stained with anti-MyoD antibodies and/or in-duced to differentiate in low serum medium (Fig. 4C). In C2C12 myoblasts transfected with MAFbx (green), MyoD staining was selectively lost, whereas in C2C12 myoblasts transfected with the empty vector and/or the ⌬F-box mutant, MyoD staining was observed in the nucleus (Fig. 4B). Furthermore overex-pression of MAFbx-IRES-GFP was never observed during myo-tube formation, and the great majority of the transfected cells were lost. The few MAFbx-expressing C2C12 myoblasts (green) maintained the mononucleated, non-differentiated phenotype and did not express MyoD. In contrast, C2C12 cells transfected with empty vector expressed MyoD and fused into multinucle-ated myotubes, with a higher level in the presence of the mutant ⌬F-box (Fig. 4C). Altogether these data suggest that high levels of MAFbx are incompatible with MyoD protein expression.

Mediation of MyoD Ubiquitination by the SCFMAFbx

—Be-cause MAFbx mRNA was identified as a marker that is up-regulated in multiple models of skeletal muscle atrophy in mice, MAFbx protein expression was first tested in two models of in vivo atrophy, aging and food deprivation. Then the asso-ciation of MAFbx with the essential Skp1 and Cul1 proteins, specific components of an E3 ubiquitin-protein ligase, was tested by using MyoD as a substrate in the ubiquitination assays. The level of MAFbx protein (42 kDa) in skeletal muscle

FIG. 6. The SCFMAFbx recombinant complex ubiquitinates

MyoD. A, the recombinant SCFMAFbxcomplex purified from Sf9 insect

cells lysates was analyzed by SDS-PAGE and then by immunoblotting (IB) and/or staining with Coomassie Brilliant Blue (CBB). B, the re-combinant SCFMAFbxcomplex was assayed for the ability to mediate the

ubiquitination of HA-MyoD in the presence of E1, Cdc34, and ubiquitin (Ub). C, effect of mutation in the LXXLL motif on in vitro ubiquitination of MyoD. D, mutation of the lysine 133 in arginine represses MyoD ubiquitination by the recombinant SCFMAFbxcomplex.

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increased during aging and/or food deprivation (Fig. 5A). Total extracts from the hind leg muscles were then analyzed for the association of Cul1 and Skp1 with MAFbx. Coimmunoprecipi-tation of MAFbx, Cul1, and Skp1 were observed in normal mouse skeletal muscle, and increased amounts were found in atrophic mice. These complexes were not observed after immu-noprecipitation with a preimmune serum (Fig. 5B). In an in

vitro ubiquitination assay using a MyoD mutant with the

ami-no-terminal three amino acids deleted (HA-tagged MyoD) as the physiological substrate, the SCFMAFbxfrom normal skeletal

muscle exhibited ubiquitination activity, which was higher in atrophic muscle (Fig. 5C). Finally to determine in vitro whether the SCFMAFbx complex mediates ubiquitination of MyoD, we

expressed the four subunits of the complex (Skp1, Cul1, Rbx1, and MAFbx) in Sf9 cells. We then purified the recombinant complex to near homogeneity (Fig. 6A) and assayed it for its ability to catalyze the ubiquitination of HA-tagged MyoD in

vitro in the presence of E1, Cdc34 (E2), and ubiquitin. A

sig-nificant ubiquitination was detected with E1, E2, ubiquitin, and SCFMAFbx(Fig. 6B) but the polyubiquitination of the

HA-tagged MyoD mutant L164Q was dramatically reduced (Fig. 6C). We also examined whether the SCFMAFbxcomplex

medi-ated the ubiquitination of a MyoD mutant K133R that has been

recently shown to play a critical role in the nuclear degradation of MyoD (26). As observed in Fig. 6D, the MyoD mutant K133R showed a dramatically reduced polyubiquitination strongly suggesting the implication of this internal lysine in MyoD ubiquitination by the SCFMAFbx.

Finally, to test whether MAFbx could promote MyoD ubiq-uitination in vivo, expression vectors encoding MAFbx or the mutant ⌬F-box were cotransfected with MyoD and HA-ubiq-uitin into 10T1/2 cells. MyoD protein was immunoprecipitated with anti-MyoD antibodies and was probed with anti-HA to detect ubiquitinated MyoD proteins. In the presence of MAFbx, MyoD showed a higher degree of ubiquitination compared with the mock transfectant (Fig. 7A). The mutant ⌬F-box did not promote the ubiquitination of MyoD, although it remained associated with MyoD. In C2C12 myoblasts, overexpression of MAFbx increased MyoD ubiquitination whereas the MAFbx mutant L169Q like the empty vector were ineffective (Fig. 7B). This was also observed in 10T1/2 cells transiently cotransfected by expression plasmids encoding HA-tagged MyoD-wt, FLAG-tagged MAFbx-wt, and/or the MAFbx mutant L169Q (Fig. 7C,

lanes 1–3). The ubiquitination levels of the mutant Myo

DL164Q were roughly similar to those of MyoD in the absence or presence of MAFbx (Fig. 7C, lanes 1, 4, and 5). These data

FIG. 7. MAFbx increases MyoD ubiquitination in vivo. A, 10T1/2 cells were transiently transfected with expression plasmids encoding HA-ubiquitin and MyoD in the absence (lane 2) or in the presence of the mutant⌬F-box (lane 3) or MAFbx (lane 4). Transfected cells were treated for 2 h with 50␮MMG132 before harvesting. Cells were lysed in denaturation buffer containing 1% SDS. Aliquots (10%) were analyzed by Western blotting with anti-HA antibodies (upper panel). Cell lysates were then diluted in immunoprecipitation buffer, subjected to immunoprecipitation with anti-MyoD, and analyzed by Western blotting with anti-HA antibodies (lower panel). After exposure blots were stripped and reprobed with anti-FLAG and anti-MyoD. B, MAFbx but not the mutant MAFbx L169Q increased polyubiquitination of endogenous MyoD in C2C12 myoblasts.

C, mutation of the LXXLL interaction motif impaired MyoD ubiquitination.

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suggest that MAFbx via the LXXLL core motif may control the destabilization of MyoD. Altogether, these experiments thus demonstrated that MyoD is a target of the SCFMAFbxcomplex.

DISCUSSION

By using a modified version of the yeast two-hybrid screen to identify F-box proteins that are able to interact with MyoD, we identified MAFbx, a muscle-specific ubiquitin ligase that dra-matically increases during skeletal muscle atrophy. We have provided direct biological and biochemical evidence that MAFbx interacts with MyoD and thereby induces its ubiquitin-dependent proteolysis. MAFbx contains a LCD in which the central part is an inverted LXXLL motif that mediates direct binding to a LXXLL motif found in all MyoD species. The ␣-helical LXXLL is described as a signature motif, which me-diates the recruitment of coactivators by the nuclear hormone receptors (34, 35) as well as ligand-independent recruitment of steroid receptor coactivators to estrogen receptor by cyclin D1 (36). Recently the cardiac-enriched nuclear receptor ERR␣ has been shown to be coactivated by the transcriptional coactivator peroxisome proliferator-activated receptor-␥ coactivator-1␣ (PGC-1␣). The PGC-1␣/ERR␣ binding interface revealed that among the three potential LXXLL motifs (L1–L3), only L3, which is an inverted motif, plays a major role in binding the nuclear receptor and represents a novel LXXLL-type of nuclear receptor binding motif on PGC-1␣ (37). The LXXLL motif in MyoD (EGLQALLR) is not observed in the E proteins basic helix-loop-helix factors and E12 did not coimmunoprecipitate with MAFbx (data not shown). Myf-5 did not coimmunoprecipi-tate with MAFbx, although it contains the motif (ESLQELLR) suggesting that variant residues in LXXLL core motifs influ-ence the affinity and selectivity of MAFbx for the substrates (38). MAFbx contains other three domains known to be impli-cated for protein-protein interaction, the PDZ-binding motif, cytochrome c family heme-binding site, and the leucine zipper domain (Fig. 1A). It seems likely that these domains are dis-pensable for binding of MyoD and could mediate the interaction with additional substrates. MAFbx is the first example of an E3 ligase that involves a conserved LCD domain containing a LXXLL inverted motif for substrate recognition.

MyoD is degraded in the nucleus by the ubiquitin-protea-some system implicating at least two distinct pathways, an N terminus-dependent pathway (39) and a lysine-dependent pathway (40). In vivo studies also implicate a link between the phosphorylation of MyoD Ser-200 and the ubiquitin-protea-some degradation pathway in myoblasts (17, 19, 20). Alto-gether these data suggest that MyoD may be targeted by dif-ferent E3 ligases. To avoid confusion between the N terminus-dependent pathway and the lysine-terminus-dependent pathway, we introduced modifications to the N terminus of MyoD protein (the first three amino acids have been replaced by three HA epitopes), and we have recently shown that in MyoD, lysine 133 is targeted for ubiquitination and rapid degradation in the lysine-dependent pathway and plays an integral role in com-promising MyoD activity in the nucleus (26). We showed that the SCFMAFbx recombinant complex and the SCFMAFbx from

the skeletal muscles induced ubiquitin-dependent proteolysis of HA-tagged MyoD, leading to the conclusion that MAFbx is probably not implicated in the N terminus-dependent ubiquiti-nation of MyoD. Indeed we observed that the mutation K133R in MyoD that dramatically reduced its ubiquitination by

SCF-MAFbx strengthened the implication of this internal lysine in

MyoD ubiquitination. Altogether our data show that MyoD is ubiquitinated by the SCFMAFbx via the internal lysines in

which lysine 133 may be the major ubiquitination site. On the other hand, the ⌬F-box mutant binds to and represses the ubiquitination and degradation of MyoD probably by blocking

and/or masking the binding sites for other ligase(s). It appears that the ubiquitin-proteasome pathway via phosphorylation-dependent and -inphosphorylation-dependent mechanisms regulates the turn-over of MyoD suggesting that MyoD ubiquitination on the N terminus and/or on lysine 133 could be mediated by different ubiquitin ligases during myogenesis.

The expression of MAFbx in normal skeletal muscle tissues (27, 28) and its up-regulation during the course of C2C12 differentiation suggest that MAFbx is not involved in regulat-ing typical cell cycle progress. MAFbx could help maintain myotubes in a postmitotic state and/or regulate proteins in pathways that typically couple signal transduction to cell cycle control in proliferating cells. MyoD is known to control the exit from the cell cycle as well as muscle-specific gene expression (41). It would be critical for myofibers that maintain MyoD and signaling pathways that control proliferation in undifferenti-ated myoblasts to have mechanisms protecting against at-tempts to enter the cell cycle (42). In healthy muscles, there is a continual process of muscle protein production and break-down. Skeletal muscle contains not only differentiated cells but also undifferentiated quiescent cells (satellite cells) that retain differentiation potential and contribute not only to the myo-tubes but also to the satellite pools (43). In the quiescent state, satellite cells are negative not only for differentiation markers but also for MyoD, and in the activated state they proliferate with high levels of MyoD protein in their nuclei. MyoD expres-sion is induced from satellite cells and is required for these cells to proliferate and to reinitiate skeletal muscle differentiation necessary for the repair process (14). MyoD⫺/⫺ satellite cells are differentiation-defective, highlighting the dependence of MyoD expression in the muscle fiber regeneration (44). Accel-erated proteolysis via the ubiquitin-proteasome pathway is a major cause of muscle atrophy induced by denervation, disuse, and various pathological conditions (45). In atrophy, the mus-cle-specific ubiquitin-ligase MAFbx dramatically increases, and protein breakdown occurs more rapidly than protein pro-duction, leading to loss of muscle weight. The subsequent effect on MyoD turnover in myogenesis and regeneration after inju-ries suggests that up-regulation of MAFbx is likely to affect skeletal muscle regulation and regeneration. MAFbx is strongly regulated at an early stage of muscle wasting, even before muscle weight loss is detectable, and its expression is maintained during muscle atrophy. This suggests a role of these proteins in both initiation and maintenance of the accel-erated proteolysis (27, 28). Our data strongly suggested that overexpression of MAFbx would be required to suppress all MyoD functions inhibiting the formation of new myofibers. Thus, direct inhibition of MAFbx expression may prove bene-ficial in reducing the muscle wasting associated with cachexia in cancer patients as well as with other disorders.

Acknowledgments—We thank Dr. Annick Harel-Bellan, Yue Xiong,

Michele Pagano, and Tim Hunt for expression plasmids and Dr. L. Dayan-Grosjean for critical reading of the manuscript.

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24

Supplementary data

Supplementary data Figure 1

Multiple alignments of the amino acid sequences of the mammalian MAFbx proteins

family showing high conservation of the predicted putative domains.

Supplementary data Figure 2

Phosphorylation and acetylation are not implicated in the binding of MyoD with MAFbx.

A, MAFbx colocalizes in nucleus as MyoD. 10T1/2 cells were cotransfected with

mammalian expression plasmids encoding GFP-MAFbx (green) and MyoD. Twenty-four

hours later, the transfected cells were fixed and immunostained with anti-MyoD antibodies

(red) and Hoechst 33258 (blue) and examined by indirect immunofluorescence

microscopy. B/ 10T1/2 cells were cotransfected with expression vectors encoding

Flag-tagged MAFbx (lane 1), Skp2 (lane 2) or FBX03 (lane 3) together with MyoD. Cells were

harvested 24 hours after transfection and total cell lysates were immunoprecipitated with

anti-Flag antibodies, then F-box proteins and MyoD were detected by western blotting

using anti–Flag (M2) and anti-MyoD (C-20) antibodies respectively (right panel). Portions

of the cells lysates corresponding to 10% of the input were subjected to immunoblot

analysis (left panel).

C, MyoD but not Myf-5 binds to MAFbx. 10T1/2 cells were cotransfected with expression

vectors encoding HA-tagged MyoD, HA-tagged Myf-5 or empty vector together with

Flag-tagged MAFbx. Cells were harvested 24 hr after transfection. Whole cell extracts (WCE)

were directly probed with HA and/or Flag antibodies or immunoprecipitated with anti-Flag

antibodies prior to western blotting with HA and Flag antibodies (IP).

D, 10T1/2 cells were cotransfected with expression plasmids: pCMV-Flag-MAFbx alone

(lane 2) or together with pEMSV-MyoD-wt (lane 1), pEMSV-MyoD S200D, (lane 3),

pEMSV-MyoD,S5A/S200A (lane 4) and pEMSV-MyoD K>R (lane 5). Lysates were

directly probed with anti-MyoD and anti-Flag antibodies respectively (WCE) or

immunoprecipitated with anti-Flag antibodies and then MAFbx and MyoD proteins were

detected by western blotting .

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25

Supplementary data Figure 3.

Characterization of MAFbx binding domain in MyoD.

A, 10T1/2 cells were cotransfected with each indicated HA-tagged MyoD mutant together

with expression plasmid encoding Flag-MAFbx. Cells were lysed and aliquots of total cell

lysates (input) processed for western blot analysis with anti-HA and anti-Flag antibodies

(right panel, lanes 1-6). Portions of cell lysates were immunoprecipitated with anti-HA

antibodies (12CA5). Immunoprecipitates were then analyzed by western blot with

anti-Flag antibodies (lower panel). Note that anti-HA antibodies failed to immunoprecipitate

Flag–MAFbx (lane 1).

B/ Identification of sequences in MAFbx required for interaction with MyoD.

10T1/2 cells were transfected with each indicated expression plasmid, then cells were

lysed and aliquots (input) processed for western blot analysis with Flag and

anti-MyoD antibodies (left panels, lanes 1-9) or for GST-pull down with glutathione–agarose

beads). Beads were processed further by immunoblotting with anti-Flag and anti-MyoD

antibodies respectively (right panels, lanes 1-9).

(14)
(15)
(16)
(17)

Leibovitch and Serge A. Leibovitch

Lionel A. Tintignac, Julie Lagirand, Sabrina Batonnet, Valentina Sirri, Marie Pierre

Degradation of MyoD Mediated by the SCF (MAFbx) Ubiquitin Ligase

doi: 10.1074/jbc.M411346200 originally published online November 5, 2004

2005, 280:2847-2856.

J. Biol. Chem.

10.1074/jbc.M411346200

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