Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors.
Inducible phosphorylation or dephosphorylation of transcription factors is an important mechanism of signal-dependent gene regulation in eukaryotic cells. This paper describes a combination of techniques that can be used to study the effect of this covalent protein modification on the DNA-binding activity of transcription factors. The protein of interest is genetically tagged with oligohistidine to allow rapid purification on nickel-chelate columns after being transiently overexpressed in eukaryotic tissue culture cells. Phosphorylation-dependent DNA-binding activity is determined in a blotting assay including an in situ dephosphorylation step. Studies on the protooncogene-encoded transcription factor c-Jun employing this assay revealed a TPA-inducible protein dephosphorylation event that strongly increases the DNA-binding potential of the protein. Our data confirm the results published recently by others and provide a rapid, efficient, sensitive, and well-controlled experimental system to analyze the phosphorylation-regulated modulations in the DNA-binding activity of transcription factors.
Papavassiliou
A G
AG
European Molecular Biology Laboratory, Differentiation Programme, Heidelberg, Germany.
Bohmann
K
K
Bohmann
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010766:Phosphorylation; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D004247:DNA