Phosphorylation inhibits the DNA-binding activity of MyoD homodimers but not MyoD-E12 heterodimers.
MyoD is a member of the basic helix-loop-helix (bHLH) family of muscle gene regulatory proteins that includes myogenin, myf-5, and MRF4. These proteins have been shown to heterodimerize with E2A bHLH proteins, E12/E47, and to bind to a consensus sequence known as an E-box, CANNTG, the target for transcriptional activation by these myogenic regulators. MyoD is also a phosphorylated nuclear protein that is present in muscle cells prior to the transcriptional activation of the muscle-specific genes, many of which contain E-box elements in their regulatory regions. Here we report that phosphorylated chicken MyoD, called CMD1, produced in sf9 cells using the baculovirus system, is qualitatively similar to CMD1 isolated by immunoaffinity purification from primary cultures of embryonic chick breast muscle. Functional analysis of phosphorylated and dephosphorylated CMD1 produced in sf9 cells indicates that, in the presence of magnesium, DNA binding of phosphorylated CMD1 is inhibited whereas binding in association with E12 is not affected. However, CMD1 binding alone is equally efficient when either EDTA is added in excess or dephosphorylated or bacterially expressed CMD1 is used in the assay. Our results suggest that cellular phosphorylation changes the CMD1 homodimer-heterodimer equilibrium which, in turn, modulates and/or eliminates binding site competition between CMD1 homodimers and CMD1/E-protein heterodimers in the cell.
Mitsui
K
K
Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Shirakata
M
M
Paterson
B M
BM
D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D002478:Cells, Cultured; D002642:Chick Embryo; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009036:Moths; D017570:MyoD Protein / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors
D004268:DNA-Binding Proteins; D017570:MyoD Protein; D051820:TCF Transcription Factors; D058125:Transcription Factor 7-Like 1 Protein; D014157:Transcription Factors; D004247:DNA