INTERACTIONS OF MYOGENIC BHLH TRANSCRIPTION FACTORS WITH CALCIUM-BINDING CALMODULIN AND S100A (ALPHA-ALPHA) PROTEINS

MyoD belongs to a family of myogenic basic helix-loop-helix (bHLH) tra nscription factors that activate muscle-specific genes. The basic heli x I sequence of the bHLH motif contains a consensus sequence for prote in kinase C (PKC) substrates. We show here that MyoD is indeed phospho rylated by PKC in vitro on Thr 115 within the basic part of the bHLH m otif. By analogy with calmodulin-target peptide models, we also identi fied within the consensus basic helix I moth of myogenic proteins a co nserved putative calmodulin/S100-binding domain. Calcium-dependent int eraction between MyoD with calmodulin and the abundant muscle S100a(al pha alpha) proteins was demonstrated by affinity chromatography and cr osslinking experiments. The binding of calmodulin and S100a inhibited MyoD phosphorylation by PKC as well as MyoD DNA binding activity. S100 a was found to be more efficient than calmodulin in antagonizing DNA b inding to MyoD. We next developped a rapid purification method for bac terial recombinant MyoD-bHLH domain by affinity chromatography using a calmodulin-Sepharose column and investigated the phosphorylation of t hat 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 K-m of 0.8 mu M. Calmodulin and S100a binding inhi bited MyoD-bHLH phosphorylation by PKC. A strict calcium-dependent int eraction between calcium binding proteins and the MyoD-bHLH was identi fied by native gel electrophoresis and fluorescence spectroscopy with 5-(dimethylamino)naphthalene-1-sulfonylcalmodulin. The MyoD-bHLH bound to fluorescently labeled 5-(dimethylamino)naphthalene-1-sulfonylcalmo dulin with a dissociation constant around 20 nM. S100a inhibited stoic hiometrically 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 interactio n between S100a and MyoD, we report that S100a- and MyoD-like immunore activities 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 t hat myogenic proteins represent a new family of calmodulin/S100-bindin g PKC substrates and that calmodulin/S100a could participate in the re gulation of the bHLH myogenic protein-activities.