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 element, 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 beads as revealed by immunoblot analyses. We also show that the leucine zipper domain of ATFa is essential for this interaction.
Chatton
B
B
LGME/CNRS, Strasbourg, France.
Bahr
A
A
Acker
J
J
Kedinger
C
C
D051696:Activating Transcription Factors; D000595:Amino Acid Sequence; D001483:Base Sequence; D001798:Blood Proteins / Q000378:metabolism*; D005822:Genetic Vectors; D005982:Glutathione Transferase / Q000235:genetics*; D008969:Molecular Sequence Data; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*; D014162:Transfection
D051696:Activating Transcription Factors; D001798:Blood Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005982:Glutathione Transferase