Identification and characterization of JunD missense mutants that lack menin binding

Menin, the product of the MEN1 tumor suppressor gene, binds to the AP1 transcription factor JunD and represses JunD transcriptional activity. The effects of human or mouse JunD missense mutations upon menin interaction were studied by random and alanine scanning mutagenesis of the menin binding region of JunD (amino acids 1-70). JunD mutant proteins were tested for menin binding in a reverse yeast two-hybrid assay, and for transcriptional regulation by menin in AP1-reporter assays. Random mutagenesis identified two different mutations that disrupted menin interaction at mouse JunD amino acid 42 (G42E and G42R). Mutation G42A generated by alanine scanning did not affect menin binding, likely reflecting the conserved nature of this amino acid substitution. Furthermore, by size exclusion chromatography menin co-migrated with wild type JunD but not with the JunD mutant tested (G42E). Alanine scanning mutagenesis of residues 30-55 revealed two different amino acids, P41 and P44, of mouse JunD that were critical for interaction with menin. Mouse JunD missense mutants P41A, G42R, G42E and P44A failed to bind menin and also escaped menin's control over their transcriptional activity. At lower amounts of transfected menin, the transcriptional effect of menin on the mutants P41A, G42R and G42E was changed from repression to activation, similar to that with c-jun. In conclusion, a small N-terminal region of JunD mediates a key difference between JunD and c-jun, and a component of this difference is dependent on JunD binding to menin.