✦ LIBER ✦

Determining the effect of inducible protein phosphorylation on the DNA-binding activity of transcription factors

✍ Scribed by Athanasios G. Papavassiliou; Kerstin Bohmann; Dirk Bohmann

Publisher: Elsevier Science
Year: 1992
Tongue: English
Weight: 856 KB
Volume: 203
Category: Article
ISSN: 0003-2697
DOI: 10.1016/0003-2697(92)90317-z

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✦ Synopsis

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.

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