An efficient screening assay for the rapid and precise determination of affinities between leucine zipper domains.
The protein products of the jun and fos oncogenes require a functional protein-protein interaction domain, called the "leucine zipper domain", to exert their transcriptional regulatory activity. A scintillation proximity assay was developed in which the biotinylated leucine zipper domain of the Jun protein (275-315) was immobilized on streptavidin-coated microfluorospheres and in which the leucine zipper domain of the Fos protein (160-200) was used as free, labeled ligand. The Fos leucine zipper peptide specifically bound to the Jun leucine zipper peptide, and for the first time, a dissociation constant (Kd = 110 +/- 12 nM in PBS/0.1% Tween) could be determined. Optimal heterodimer formation was reached at neutral pH. Both acidic and alkaline pH decreased the association of the peptides which was, furthermore, completely abolished by 500 mM NaCl, confirming that charged residues are critical for heterodimerization. A commercially obtained recombinant Jun protein competed as efficiently as the Jun leucine zipper peptide for binding to the Fos peptide, confirming the feasibility of using the two leucine zipper peptides to study the interactions between the two transcription factors. We also injected leucine zipper peptides individually into Xenopus oocytes to study whether they would interfere with the activity of the Fos/Jun heterodimer in vivo. Both peptides blocked selectively insulin-mediated oocyte maturation with an IC50 in the range of 15 ng per oocyte. In conclusion, the scintillation proximity assay described here may be used to investigate protein-protein interactions mediated by leucine zipper structures and to identify compounds that inhibit leucine zipper association.
Pernelle
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Laboratoire Oncologie Moléculaire, Rhone Poulenic Rorer SA, Vitry sur Seine, France.
Clerc
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Dureuil
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Bracco
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Tocque
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D000595:Amino Acid Sequence; D000818:Animals; D001710:Biotin; D005260:Female; D066298:In Vitro Techniques; D007457:Iodine Radioisotopes; D016350:Leucine Zippers / Q000502:physiology*; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects; D010446:Peptide Fragments / Q000138:chemical synthesis / Q000378:metabolism / Q000494:pharmacology; D011485:Protein Binding; D016760:Proto-Oncogene Proteins c-fos / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D016755:Proto-Oncogene Proteins c-jun / Q000737:chemistry* / Q000378:metabolism / Q000502:physiology; D015398:Signal Transduction / Q000187:drug effects; D014982:Xenopus laevis
D007457:Iodine Radioisotopes; D010446:Peptide Fragments; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D001710:Biotin