title: Interactions of myogenic bHLH transcription factors with calcium-binding calmodulin and S100a (alpha alpha) proteins.
abstract: MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) transcription factors that activate muscle-specific genes. The basic helix I sequence of the bHLH motif contains a consensus sequence for protein kinase C (PKC) substrates. We show here that MyoD is indeed phosphorylated by PKC in vitro on Thr 115 within the basic part of the bHLH motif. By analogy with calmodulin-target peptide models, we also identified within the consensus basic helix I motif of myogenic proteins a conserved putative calmodulin/S100-binding domain. Calcium-dependent interaction between MyoD with calmodulin and the abundant muscle S100a(alpha alpha) proteins was demonstrated by affinity chromatography and cross-linking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100a was found to be more efficient than calmodulin in antagonizing DNA binding to MyoD. We next developed a rapid purification method for bacterial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of that peptide by PKC and its interactions with calmodulin and S100a. We confirmed the phosphorylation of the threonine residue 115 in the MyoD-bHLH by PKC with a Km of 0.8 microM. Calmodulin and S100a binding inhibited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent interaction between calcium binding proteins and the MyoD-bHLH was identified by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin with a dissociation constant around 20 nM. S100a inhibited stoichiometrically the binding of the bHLH peptide for labeled calmodulin, suggesting an affinity of S100a for the bHLH peptide at least 1 order of magnitude higher than calmodulin. In favor of an in vivo interaction between S100a and MyoD, we report that S100a- and MyoD-like immunoreactivities colocalize in H9c2 cells, and that a significant amount of MyoD-like immunoreactivity is recovered in the S100a immunoprecipitate from crude H9c2 cell extract in the presence of calcium. We propose that myogenic proteins represent a new family of calmodulin/S100-binding PKC substrates and that calmodulin/S100a could participate in the regulation of the bHLH myogenic protein activities.
authors.affiliation: Département de Biologie Moléculaire et Structurale, CFA, DBMS-BMCC, INSERM Unité 309, Grenoble, France.
authors.forename: J
authors.initials: J
authors.lastname: Baudier
authors.forename: E
authors.initials: E
authors.lastname: Bergeret
authors.forename: N
authors.initials: N
authors.lastname: Bertacchi
authors.forename: H
authors.initials: H
authors.lastname: Weintraub
authors.forename: J
authors.initials: J
authors.lastname: Gagnon
authors.forename: J
authors.initials: J
authors.lastname: Garin
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002417:Cattle; D016384:Consensus Sequence; D018257:Helix-Loop-Helix Motifs; D006801:Humans; D008969:Molecular Sequence Data; D017570:MyoD Protein / Q000378:metabolism*; D018006:Myogenic Regulatory Factors / Q000378:metabolism; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011493:Protein Kinase C / Q000378:metabolism; D009418:S100 Proteins; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002135:Calcium-Binding Proteins; D002147:Calmodulin; D017570:MyoD Protein; D018006:Myogenic Regulatory Factors; D009418:S100 Proteins; C110294:S100A1 protein; D014157:Transcription Factors; D011493:Protein Kinase C