A dominant negative to activation protein-1 (AP1) that abolishes DNA binding and inhibits oncogenesis.
We describe a dominant negative (DN) to activation protein-1 (AP1) that inhibits DNA binding in an equimolar competition. AP1 is a heterodimer of the oncogenes Fos and Jun, members of the bZIP family of transcription factors. The DN, termed A-Fos, consists of a newly designed acidic amphipathic protein sequence appended onto the N-terminus of the Fos leucine zipper, replacing the normal basic region critical for DNA binding. The acidic extension and the Jun basic region form a heterodimeric coiled coil structure that stabilizes the complex over 3000-fold and prevents the basic region of Jun from binding to DNA. Gel shift assays indicate that A-Fos can inactivate the DNA binding of a Fos:Jun heterodimer in an equimolar competition. Transient transfection assays indicate that A-Fos inhibits Jun-dependent transactivation. Both the acidic extension and the Fos leucine zipper are critical for this inhibition. Expression of A-Fos in mouse fibroblasts inhibits focus formation more than colony formation, reflecting the ability of A-Fos to interfere with the AP1 biological functions in mammalian cells. This reagent is more potent than a deletion of either the Fos or Jun transactivation domain, which has been used previously as a dominant negative to AP1 activity.
Olive
M
M
Laboratory of Biochemistry, NCI, National Institutes of Health, Bethesda, Maryland 20892, USA.
Krylov
D
D
Echlin
D R
DR
Gardner
K
K
Taparowsky
E
E
Vinson
C
C
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002471:Cell Transformation, Neoplastic; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D019084:Fluorescent Antibody Technique, Indirect; D006801:Humans; D016350:Leucine Zippers; D046911:Macromolecular Substances; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D017433:Protein Structure, Secondary; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism*; D016755:Proto-Oncogene Proteins c-jun / Q000378:metabolism; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors* / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D018808:Transcription Factor AP-1; D004247:DNA