Post-translational phosphorylation plays an important role in the regulation of transcription factor activity. Certain transcription factors reside in the cell cytoplasm and are translocated into the nucleus in a phosphorylation-dependent manner. This type of regulation is exemplified by the NFKB family of transcription factors which are complexed in the cytosol by members of the I-кB family. Phosphorylation of the I-кB protein by protein kinase A or protein kinase C releases NF-кB, which is then translocated to the nucleus in an active, DNA-binding state. Within the nucleus, post-translational phosphorylation dramatically affects both DNA-binding activity and transcriptional activity. This chapter provides both in vivo and in vitro methods to (1) determine whether the transcription factor under study is phosphorylated in vivo, (2) analyze the sites of phosphorylation, and (3) examine the nature of the protein kinase(s) that mediates this phosphorylation. These methods require either transcription factor-specific antisera or the cloned cDNA for the protein of interest. Examples of these protocols using the c-Jun transcription factor are described in the chapter.
ASJC Scopus subject areas
- Molecular Biology