Mapping of functional domains in Fos and Jun proteins using epitope-specific antibodies.
A panel of epitope-specific antibodies, directed against c-Fos, c-Jun, and FosB derived oligopeptide sequences, was generated and used to study the interaction of Fos and Jun proteins and the binding of the Fos/Jun complex to the AP1-binding site (TRE). Our results strongly support results previously obtained by site-directed mutagenesis experiments. The leucine zipper is the major site of interaction between Fos and Jun. Antibodies directed against this domain of Fos bound free Fos protein efficiently, but were unable to recognize Fos within the Fos/Jun complex. In contrast, all other Fos epitope-specific antibodies showed similar reactivity with both free and complexed Fos. Antibodies directed against sequences adjacent to the leucine zipper inhibited formation of the complex. This may suggest that amino acids in the vicinity of the leucine zipper may also play some role in the formation of the protein complex. Binding of Fos/Jun to the TRE was inhibited only by antibodies directed against the basic regions in Fos or Jun previously suggested to represent the DNA binding sites. The fact that very similar results were obtained by two totally different strategies, i.e., mutagenesis experiments and domain mapping using epitope-specific antibodies, lends strong support to the proposed domain structure of Fos and Jun family members.
Institut für Molekularbiologie und Tumorforschung (IMT), Philipps-Universität Marburg, Federal Republic of Germany.
J
J
Adamkiewicz
H J
HJ
Brüller
H
H
Gausepohl
R
R
Frank
R
R
Müller
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D000911:Antibodies, Monoclonal; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000276:immunology; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes / Q000032:analysis*; D005455:Fluorescent Antibody Technique; D051379:Mice; D008807:Mice, Inbred BALB C / Q000276:immunology; D008969:Molecular Sequence Data; D009842:Oligopeptides / Q000138:chemical synthesis / Q000276:immunology; D014176:Protein Biosynthesis; D011505:Protein-Tyrosine Kinases / Q000235:genetics*; D011518:Proto-Oncogene Proteins / Q000235:genetics* / Q000276:immunology; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors / Q000235:genetics* / Q000276:immunology
D000906:Antibodies; D000911:Antibodies, Monoclonal; D004268:DNA-Binding Proteins; D000939:Epitopes; D009842:Oligopeptides; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D011505:Protein-Tyrosine Kinases