Fig. 4

Akt phosphorylates MSY3 and reduces its binding to myogHCE in vitro. a Phosphorylation of MSY3 by Akt. GST-MSY3 (*) was phosphorylated by Akt, (lanes 7 and 8) but not by GSK3β (11 12). Auto-phosphorylated Akt protein (°) and GSK3β (§) are present. The Comassie gel is shown (lanes 1–4). For each sample, there are two experimental replicates. b Schematic of the myogenic locus. This diagram describes the myogenin promoter (the 130 bp upstream region from the TSS) and the genomic alignments of six vertebrates. The schematic also shows the relative locations of myogHCE element—the region to which MSY3 binds and other well-known motifs (myogHCE-brown, MEF3box-light blue, MEF2box-green, TATA box-purple, and E-box-yellow. MyogHCE is MSY3 binding site [16]; MEF3box is Dach2 binding site [13]; MEF2box is MEF2/HDAC9 binding site [12]. c MSY3 phosphorylation by Akt impairs its binding at the myogenin promoter. Sequences of MyogHCE (MYOwt) and mutant myogHCE (MYOmutL) oligonucleotides used in EMSA are shown. Mutant MYOmutL disrupts the myogHCE sequence, which incudes the MSY3 site. Mobility shift assay (EMSA) of GST-MSY3 binding to EMSA assay of GST and GSTMSY3 fusion protein with myogHCE (MYOwt) and mutant myogHCE (MYOmutL) ³²P labeled oligonucleotide in double-strand form without (first three lanes) and with prior treatment by Akt (lanes 4 and 5) and by GSK3β (lanes 6 and 7)