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

MyoD controls myoblast identity and differentiation and is required for myogenic stem cell function in adult skeletal muscle. MyoD is degraded by the ubiquitin-proteasome 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-regulated in atrophying muscle. A core LXXLL motif sequence 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 lysine-dependent pathway. Mutation of the lysine 133 in MyoD prevented its ubiquitination by the recombinant SCFMAFbx complex. These observations thus demonstrated 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. MyoD controls myoblast identity and differentiation and is required for myogenic stem cell function in adult skeletal muscle. MyoD is degraded by the ubiquitin-proteasome 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-regulated in atrophying muscle. A core LXXLL motif sequence 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 lysine-dependent pathway. Mutation of the lysine 133 in MyoD prevented its ubiquitination by the recombinant SCFMAFbx complex. These observations thus demonstrated 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 abundance and serves a central regulatory function in many biological processes (1Hershko A. Ciechanover A. Annu. Rev. Biochem. 1998; 67: 425-479Crossref PubMed Scopus (6959) Google Scholar). 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 (2Elledge S.J. Harper J.W. Biochim. Biophys. Acta. 1998; 1377: 61-70PubMed Google Scholar). The formation of ubiquitin-protein conjugates involves three components that participate in a cascade of ubiquitin transfer reactions, an ubiquitin-activation enzyme (E1), 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, hemagglutinin; LCD, leucine-charged domain; wt, wild type; PGC-1α, peroxisome proliferator-activated receptor-γ coactivator-1α. an ubiquitin-conjugating enzyme (E2) and an ubiquitin ligase (E3) that acts at the last step of the cascade (3Ciechanover A. EMBO J. 1998; 17: 7151-7160Crossref PubMed Scopus (1200) Google Scholar). The specificity of the SCF complexes derives of the variable components called the F-box proteins that serve as receptor for the target protein (4Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 2: 209-219Abstract Full Text Full Text PDF Scopus (1032) Google Scholar). 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 (5Edmondson D.G. Olson E.N. J. Biol. Chem. 1993; 268: 755-768Abstract Full Text PDF PubMed Google Scholar, 6Olson E.N. Klein W.H. Genes Dev. 1994; 1: 1-8Crossref Scopus (605) Google Scholar). 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 specific genes and to efficiently convert non-muscle cells to a myogenic lineage (7Weintraub H. Cell. 1993; 75: 1241-1244Abstract Full Text PDF PubMed Scopus (933) Google Scholar). 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 (8Polesskaya A. Duquet A. Naguibneva I. Weise C. Vervisch A. Bengal E. Hucho F. Robin P. Harel-Bellan A. J. Biol. Chem. 2000; 275: 34359-34364Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar, 9Sartorelli V. Puri P. Hamamori Y. Ogryzko V.G.C. Nakatani Y. Wang J. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). In contrast, histone deacetylation inhibits gene activation, and the interaction between histone deacetylase 1 and MyoD prevents premature activation of the myogenic program in growing myoblasts (10Mal A. Sturniolo M. Schiltz R.L. Ghosh M.K. Harter M.L. EMBO J. 2001; 20: 1739-1753Crossref PubMed Scopus (208) Google Scholar). The MRFs contain several functionally distinct domains responsible for transcriptional activation, chromatin remodeling, DNA binding, nuclear localization, and heterodimerization (11McKinsey T.A. Zhang C.L. Olson E.N. Curr. Opin. Genet. Dev. 2001; 5: 497-504Crossref Scopus (353) Google Scholar). The mechanism by which MyoD induces myogenesis involves both the withdrawal from cell cycle and the activation of muscle-specific gene expression. Recent data demonstrate a direct link between MyoD and cell cycle regulation (12Zhang J-M. Wei Q. Zhao X. Paterson B.M. EMBO J. 1999; 18: 926-933Crossref PubMed Scopus (109) Google Scholar, 13Zhang J-M. Zhao X. Wei Q. Paterson B.M. EMBO J. 1999; 18: 6983-6993Crossref PubMed Scopus (89) Google Scholar) and also its requirement for myogenic stem cell function in adult skeletal muscle (14Megeney L.A. Kablar B. Garrett K. Anderson J.E Rudnicki M.A. Genes Dev. 1996; 10: 1173-1183Crossref PubMed Scopus (561) Google Scholar) where MyoD protein levels decline from postnatal stage onward (15Asakura A. Seale P. Girgis-Gabardo A. Rudnicki M.A. J. Cell Biol. 2002; 1: 123-134Crossref Scopus (585) Google Scholar). Accurate synchronization of dividing myoblasts revealed that MyoD is subject to specific cell cycle-dependent regulation (16Kitzmann M. Carnac G. Vandromme M. Primig M. Lamb N. Fernandez A. J. Cell Biol. 1998; 142: 1447-1459Crossref PubMed Scopus (249) Google Scholar, 17Tintignac L.A. Leibovitch M.P. Kitzmann M. Fernandez A. Ducommun B. Meijer L. Leibovitch S.A. Exp. Cell Res. 2000; 259: 300-307Crossref PubMed Scopus (53) Google Scholar, 18Tintignac L.A. Sirri V. Leibovitch M-P. Lecluse Y. Castedo M. Kroemer Metivier G.D. Leibovitch S.A. Mol. Cell. Biol. 2004; 24: 1809-1821Crossref PubMed Scopus (34) Google Scholar). Phosphorylation of MyoD at serine 200 plays a crucial role in modulating its half-life and transcriptional activity during myoblast proliferation (19Song A. Wang Q. Goebl M.G. Harrington M.A. Mol. Cell. Biol. 1998; 18: 4994-4999Crossref PubMed Scopus (140) Google Scholar, 20Kitzmann M. Vandromme M. Schaeffer V. Carnac G. Labbe J-C. Lamb N. Fernandez A. Mol. Cell. Biol. 1999; 19: 3167-3176Crossref PubMed Scopus (95) Google Scholar). Linking this phosphorylation to the cell cycle-dependent drop in MyoD protein before S phase leads to a mechanism implicating 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 (17Tintignac L.A. Leibovitch M.P. Kitzmann M. Fernandez A. Ducommun B. Meijer L. Leibovitch S.A. Exp. Cell Res. 2000; 259: 300-307Crossref PubMed Scopus (53) Google Scholar, 21Reynaud E.G. Pelpel K Guillier M Leibovitch M-P. Leibovitch S.A. Mol. Cell. Biol. 1999; 19: 7621-7629Crossref PubMed Scopus (88) Google Scholar). In addition MyoD phosphorylated on serine 200 is degraded by the proteasome and CDC34 ubiquitin-conjugating enzyme activity (E2) (19Song A. Wang Q. Goebl M.G. Harrington M.A. Mol. Cell. Biol. 1998; 18: 4994-4999Crossref PubMed Scopus (140) Google Scholar). Altogether these findings indicate that targeted degradation of MyoD following Cdk2-cyclin E phosphorylation of serine 200 during G1 to 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 signal(s) and/or mechanism(s). Up-regulation of the cyclin/Cdk inhibitor p57Kip2 stabilizes MyoD by direct interaction (22Reynaud E.G. Leibovitch M.P. Tintignac L.A.J. Pelpel K. Guillier M. Leibovitch S.A. J. Biol. Chem. 2000; 275: 18767-18776Abstract Full Text Full Text PDF PubMed Scopus (85) Google Scholar), whereas specific DNA binding stabilizes MyoD (23Abu-Hatoum O.A Gross-Mesilaty S. Breitschopf K. Hoffman A. Gonen H. Ciechanover A. Bengal E. Mol. Cell. Biol. 1998; 18: 5670-5677Crossref PubMed Google Scholar) and in vitro MyoD is degraded via the ubiquitin-proteasome pathway in HeLa nucleoplasm (24Floyd Z.E. Trausch-Azar J.S. Reinstein E. Ciechanover A. Schwartz A.L. J. Biol. Chem. 2001; 276: 22468-22475Abstract Full Text Full Text PDF PubMed Scopus (65) Google Scholar), and in differentiated myotubes, MyoD protein turns over rapidly as observed in myoblasts (25Thayer M.J. Tapscott S.J. Davis R.L. Wright W.E. Lassar A.B. Weintraub H. Cell. 1989; 58: Full Text PDF PubMed Scopus Google Scholar). Altogether these data indicate that MyoD is degraded by the ubiquitin-proteasome pathway mediated by different E3 ubiquitin ligases not identified as yet. of MyoD we a of the of the skeletal muscle to identify proteins that with to F-box proteins that bind MyoD and identify we report the of Atrogin-1/MAFbx (MAFbx), an E3 ubiquitin ligase up-regulated during skeletal muscle that interacts with MyoD via a novel interaction found in transcriptional coactivators the binding to liganded nuclear and its We demonstrated that the purified recombinant SCFMAFbx complex mediated ubiquitination of MyoD in vitro in a that to on the of the lysine lysine 133 shown to play a role in the nuclear degradation of MyoD S Leibovitch M-P. Tintignac L.A. Leibovitch S.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). we that up-regulation of MAFbx in myoblasts differentiation MyoD degradation and muscle and DNA used the with a skeletal muscle The as by the The of with the in the in We to and the as by the The to the The and we by in the F-box of MAFbx by of and of the The as as of MAFbx and MyoD by to and on of the and as into the The for MAFbx by a the internal ribosome entry and from the and at the and of MAFbx and the at the of Cell and skeletal muscle cell and the cell in with and by the activity of cell from cells in with of proteins in and at and by with the to and to of the MAFbx cell in 1 1 and and for with and by proteins before transfer a of cells in and into of and 1 at for and the and with by an to the and from from from from from and from proteasome cells with for In and in as (17Tintignac L.A. Leibovitch M.P. Kitzmann M. Fernandez A. Ducommun B. Meijer L. Leibovitch S.A. Exp. Cell Res. 2000; 259: 300-307Crossref PubMed Scopus (53) Google Scholar, S Leibovitch M-P. Tintignac L.A. Leibovitch S.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar) by in vitro The recombinant SCFMAFbx complex in cells by with Skp1, and purified by and in to the In ubiquitination have S Leibovitch M-P. Tintignac L.A. Leibovitch S.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). and from cells 1 of used to transcription with and of as a for for the of the gene MAFbx and on and in for at and with for at The cells with and with The of used to the of MAFbx as an F-box with CDC34 ubiquitin-conjugating enzyme activity (E2) in MyoD ubiquitination (19Song A. Wang Q. Goebl M.G. Harrington M.A. Mol. Cell. Biol. 1998; 18: 4994-4999Crossref PubMed Scopus (140) Google Scholar) suggesting the requirement of an protein complex identify of MyoD ubiquitination and we used a skeletal muscle for genes and MyoD as in the We identified with the for and the a of with the F-box protein Atrogin-1/MAFbx A. A.L. S. A. 2001; PubMed Scopus Google Scholar, E. S. L. N. K. G.D. 2001; PubMed Scopus Google Scholar). The MAFbx the and consensus to with protein C. D. M. M. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar, C. Elledge S.J. Harper J.W. Curr. Biol. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar) In addition to the motif and family binding located in the MAFbx nuclear a and a leucine-charged residue-rich which are between and The of protein-protein domains that MAFbx could different of MAFbx and MyoD during is the of MAFbx in myogenic we MAFbx during myogenic differentiation the cell a for differentiation. These cells as myoblasts in and be induced to by the from to myoblasts into with an of A that the MAFbx up-regulated in the course of differentiation in and revealed that the MAFbx protein is in myoblasts and in this MyoD is up-regulated cells into the differentiation and during a skeletal muscle is observed in differentiated cells during the course of differentiation MyoD and MAFbx a of expression. Phosphorylation or of MyoD for with the binding of MyoD with MAFbx in cells by a In myogenic MyoD with and an in observed proteasome activity In MyoD and MAFbx have a nuclear localization, and MyoD with MAFbx not with other F-box proteins as or A and the that is also in not with MAFbx in of target proteins are in the recognition by E3 ubiquitin ligases and subsequent degradation by the S proteasome (4Skowyra D. Craig K.L. Tyers M. Elledge S.J. Harper J.W. Cell. 1997; 2: 209-219Abstract Full Text Full Text PDF Scopus (1032) Google Scholar, M. K. H. J. M. A. 2001; PubMed Scopus Google Scholar, Y. F. H. K. Y. K. M. K. 2002; PubMed Scopus Google Scholar). On the other hand, ubiquitination of requires direct binding of specific domains of the F-box protein and the M. M. Hoffman M.A. N. V. Cell Biol. 2000; 2: PubMed Scopus Google Scholar). The of MyoD that phosphorylation of and L.A. Sirri V. Leibovitch M-P. Lecluse Y. Castedo M. Kroemer Metivier G.D. Leibovitch S.A. Mol. Cell. Biol. 2004; 24: 1809-1821Crossref PubMed Scopus (34) Google Scholar, 20Kitzmann M. Vandromme M. Schaeffer V. Carnac G. Labbe J-C. Lamb N. Fernandez A. Mol. Cell. Biol. 1999; 19: 3167-3176Crossref PubMed Scopus (95) Google Scholar) and/or by (8Polesskaya A. Duquet A. Naguibneva I. Weise C. Vervisch A. Bengal E. Hucho F. Robin P. Harel-Bellan A. J. Biol. Chem. 2000; 275: 34359-34364Abstract Full Text Full Text PDF PubMed Scopus (110) Google Scholar) and V. Puri P. Hamamori Y. Ogryzko V.G.C. Nakatani Y. Wang J. Kedes L. Mol. Cell. 1999; 4: 725-734Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar) for their interaction with MAFbx. cells with MAFbx or with MyoD which serine 200 MyoD and in which to and the MyoD with MAFbx suggesting that phosphorylation and are not in interaction these that of the phosphorylation and/or MyoD and MAFbx complexes in of MAFbx with a LXXLL in that MAFbx to MyoD, the of protein binding found in other F-box proteins to for of MAFbx important for The domains of both proteins required for interaction by in protein binding cells with and/or MyoD or with and the for the of MAFbx by an between MyoD and MAFbx mediated by the of MyoD of revealed a LXXLL consensus sequence that protein-protein and found in a of coactivators D. Mol. 2000; PubMed Scopus Google Scholar). the of this motif to the we the to and its interaction with MAFbx as Mutation in the LXXLL motif to MyoD interaction with MAFbx In binding to the sequence in MAFbx that to complex formation with MyoD. between the and the MAFbx we used a that the central of MAFbx in the MyoD binding a that a consensus inverted LXXLL motif by and in We found that in the inverted LXXLL motif dramatically the binding to MyoD These demonstrated that complex formation the of the inverted LXXLL motif in MAFbx. of MyoD by of MAFbx on the MyoD half-life protein with and/or in the not MyoD to Overexpression of MAFbx in degradation of MyoD by its half-life to and/or on MyoD In cells with SCFMAFbx MyoD degradation the MAFbx of the F-box required for an MyoD half-life In cells with LXXLL motif interaction between MyoD and the degradation of MyoD to that of and that of MyoD is in cells with SCFMAFbx activity and the LXXLL motif that Overexpression of MAFbx in in E. S. L. N. K. G.D. 2001; PubMed Scopus Google Scholar), and on the other hand, during the course of differentiation MyoD and MAFbx a of these data that MyoD We observed that of MAFbx on myogenic of cells a of activity and MyoD protein abundance and the of MAFbx on MyoD myoblasts with or the and myoblasts with and/or induced to in In myoblasts with MAFbx MyoD whereas in myoblasts with the and/or the MyoD observed in the of observed during myotube and the of the cells The myoblasts the and not MyoD. In contrast, cells with MyoD and into myotubes, with a in the of the Altogether these data that levels of MAFbx are with MyoD protein expression. of MyoD by the MAFbx identified as a that is up-regulated in of skeletal muscle in MAFbx protein in of in and the of MAFbx with the and specific components of an E3 ubiquitin-protein by MyoD as a in the ubiquitination The of MAFbx protein in skeletal muscle during and/or from the for the of and with MAFbx. of and observed in skeletal and found in These complexes not observed with a In an in vitro ubiquitination a MyoD with the three as the the SCFMAFbx from skeletal muscle ubiquitination which in muscle Finally to in vitro the SCFMAFbx complex ubiquitination of MyoD, we the of the complex (Skp1, and in We purified the recombinant complex to and for its to the ubiquitination of MyoD in vitro in the of E1, and A ubiquitination with E1, E2, and SCFMAFbx the of the MyoD dramatically We also the SCFMAFbx complex mediated the ubiquitination of a MyoD that shown to play a role in the nuclear degradation of MyoD S Leibovitch M-P. Tintignac L.A. Leibovitch S.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). observed in the MyoD a dramatically suggesting the of this internal lysine in MyoD ubiquitination by the SCFMAFbx recombinant complex MyoD. the recombinant SCFMAFbx complex purified from cells by and by and/or with the recombinant SCFMAFbx complex for the to mediate the ubiquitination of in the of E1, and ubiquitin of in the LXXLL motif on in vitro ubiquitination of MyoD. of the lysine 133 in represses MyoD ubiquitination by the recombinant SCFMAFbx to MAFbx could MyoD ubiquitination in MAFbx or the with MyoD and into MyoD protein with and with to MyoD proteins. In the of MyoD a of ubiquitination with the The not the ubiquitination of MyoD, with MyoD. In of MAFbx MyoD ubiquitination whereas the MAFbx the also observed in cells by and/or the MAFbx The ubiquitination levels of the to of MyoD in the or of MAFbx 1, and These data that MAFbx via the LXXLL core motif may control the of MyoD. these thus demonstrated that MyoD is a target of the SCFMAFbx complex. a of the to identify F-box proteins that are to with MyoD, we identified a muscle-specific ubiquitin ligase that dramatically during skeletal muscle We have direct biological and that MAFbx interacts with MyoD and induces its MAFbx a in which the central is an inverted LXXLL motif that direct binding to a LXXLL motif found in MyoD The LXXLL is as a which the of coactivators by the nuclear D. Mol. 2000; PubMed Scopus Google Scholar, E. S. M.G. 1997; PubMed Scopus Google Scholar) as as of receptor coactivators to receptor by Genes Dev. 1998; PubMed Scopus Google Scholar). the nuclear receptor shown to be by the transcriptional peroxisome proliferator-activated receptor-γ The binding revealed that the three LXXLL which is an inverted plays a role in binding the nuclear receptor and a novel of nuclear receptor binding motif on J. Biol. Chem. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). The LXXLL motif in MyoD is not observed in the E proteins basic helix-loop-helix factors and not with MAFbx not not with the motif suggesting that in LXXLL core the and of MAFbx for the S. S. M.G. H. J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus (140) Google Scholar). MAFbx other three domains to be for protein-protein the family and the that these domains are for binding of MyoD and could mediate the interaction with MAFbx is the of an E3 ligase that involves a a LXXLL inverted motif for MyoD is degraded in the by the ubiquitin-proteasome implicating at distinct an pathway K. Bengal E. A. Ciechanover A. EMBO J. 1998; 17: PubMed Scopus Google Scholar) and a lysine-dependent pathway Trausch-Azar Ciechanover A. Schwartz A.L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). In also a link between the phosphorylation of MyoD and the ubiquitin-proteasome degradation pathway in myoblasts (17Tintignac L.A. Leibovitch M.P. Kitzmann M. Fernandez A. Ducommun B. Meijer L. Leibovitch S.A. Exp. Cell Res. 2000; 259: 300-307Crossref PubMed Scopus (53) Google Scholar, A. Wang Q. Goebl M.G. Harrington M.A. Mol. Cell. Biol. 1998; 18: 4994-4999Crossref PubMed Scopus (140) Google Scholar, 20Kitzmann M. Vandromme M. Schaeffer V. Carnac G. Labbe J-C. Lamb N. Fernandez A. Mol. Cell. Biol. 1999; 19: 3167-3176Crossref PubMed Scopus (95) Google Scholar). Altogether these data that MyoD may be targeted by different E3 ligases. between the pathway and the lysine-dependent we to the of MyoD protein three have by three and we have shown that in MyoD, lysine 133 is targeted for ubiquitination and degradation in the lysine-dependent pathway and plays an role in MyoD activity in the S Leibovitch M-P. Tintignac L.A. Leibovitch S.A. J. Biol. Chem. 2004; Full Text Full Text PDF PubMed Scopus Google Scholar). We that the SCFMAFbx recombinant complex and the SCFMAFbx from the skeletal induced proteolysis of MyoD, to the that MAFbx is probably not in the ubiquitination of MyoD. we observed that the in MyoD that dramatically its ubiquitination by the of this internal lysine in MyoD Altogether data that MyoD is by the SCFMAFbx via the internal in which lysine 133 may be the ubiquitination On the other hand, the to and represses the ubiquitination and degradation of MyoD probably by and/or the binding for other that the ubiquitin-proteasome pathway via and regulates the of MyoD suggesting that MyoD ubiquitination on the and/or on lysine 133 could be mediated by different ubiquitin ligases during The of MAFbx in skeletal muscle A. A.L. S. A. 2001; PubMed Scopus Google Scholar, E. S. L. N. K. G.D. 2001; PubMed Scopus Google Scholar) and its up-regulation during the course of differentiation that MAFbx is not involved in cell cycle MAFbx could in a state and/or regulate proteins in that signal to cell cycle control in MyoD is to control the exit from the cell cycle as as muscle-specific gene M. Fernandez A. Cell. Mol. 2001; 58: PubMed Scopus Google Scholar). be for that MyoD and that control proliferation in myoblasts to have to the cell cycle N. Mol. Cell. Biol. 2002; 17: Scopus Google Scholar). In is a of muscle protein and muscle not differentiated cells also cells that differentiation and not to the also to the E. Dev. Biol. 1996; PubMed Scopus Google Scholar). In the cells are not for differentiation also for MyoD, and in the state with levels of MyoD protein in their MyoD is induced from cells and is required for these cells to and to skeletal muscle differentiation necessary for the (14Megeney L.A. Kablar B. Garrett K. Anderson J.E Rudnicki M.A. Genes Dev. 1996; 10: 1173-1183Crossref PubMed Scopus (561) Google Scholar). cells are the of MyoD in the muscle Rudnicki M.A. Dev. Biol. 2000; PubMed Scopus Google Scholar). proteolysis via the ubiquitin-proteasome pathway is a of muscle induced by and A.L. Curr. Opin. 2001; 4: PubMed Scopus Google Scholar). In the muscle-specific MAFbx dramatically and protein occurs rapidly protein to of muscle The subsequent on MyoD in myogenesis and that up-regulation of MAFbx is to skeletal muscle regulation and MAFbx is at an stage of muscle before muscle is and its is during muscle a role of these proteins in both and of the proteolysis A. A.L. S. A. 2001; PubMed Scopus Google Scholar, E. S. L. N. K. G.D. 2001; PubMed Scopus Google Scholar). data that of MAFbx be required to MyoD functions the formation of direct of MAFbx may in the muscle with in as as with other We and for and L. for of the with