EUKARYOTIC GST FUSION VECTOR FOR THE STUDY OF PROTEIN-PROTEIN ASSOCIATIONS IN-VIVO - APPLICATION TO INTERACTION OF ATFA WITH JUN AND FOS

We describe a multipurpose eukaryotic expression vector that incorporates the following features: restriction sites for in-frame insertion of cDNAs of interest between sequences encoding the glutathione-S-transferase (GST) and an oligohistidine elements, allowing expression of the corresponding fusion proteins; a phosphorylation site for protein kinase A for in vitro labeling of the fusion protein; a T7 promoter for in vitro transcription and subsequent translation; and signals for single-stranded DNA production in bacteria. We have used this vector to demonstrate the formation in vivo of complexes between the transcription factor ATFa, a member of the family of ATF/CRE binding proteins, and the c-Jun or c-Fos proteins. Such interactions could be detected in crude extracts from cells transfected with vectors expressing the GST-ATFa fusion protein, as well as the c-Jun or c-Fos proteins. Complexes containing both ATFa and either c-Jun or c-Fos were specifically retained on glutathione (GSH)-agarose bends as revealed by immunoblot analyses. We also show that the leucine zipper do,nain of ATFa is essential for this interaction.