Figure 4. Menin binds to JunD and blocks JNK-mediated JunD phosphrylation.

a, Sequence alignment of the MBM sequences of JunD, MLL1, and MLL2. Conserved residues are highlighted in yellow. b, Crystal structure of the menin-JunD_MBM complex. Menin is in surface representation and colored as in Fig. 1c. The JunD_MBM peptide is in stick model and colored in magenta. c, Overlay of JunD_MBM (magenta) and MLL1_MBM (yellow). d, In vitro ITC data of wild-type and mutant menin-JunD_MBM interactions (refer to Supplementary Fig. 10). e, Co-IP of the mutant menin-JunD interactions. Lanes marked “IN” represent 2.5% of input cell lysate used for the immunoprecipitation. f, Sequence alignment of JunD and c-Jun. The MBM sequence of JunD is highlighted in magenta. Key residues in the JNK-docking motif are denoted by blue dots and three phosphorylation sites are labeled above the sequence. The consensus sequence of the JNK-docking motif is shown at the upper-right corner. g, Lys46, Lys47, Leu52, and L54 in the JNK-docking motif are required for JunD phosphorylation by JNK. h, GST-pull down assay of FLAG-JNK3 by GST-JunD in the presence or absence of full-length menin. i, The phosphorylation of wild-type or mutant JunD by JNK1 was examined with in vitro kinase assay. The phosphorylated JunD was detected with anti-JunD phosphor-Ser100 antibody. j, The phosphorylation of JunD by JNK3 was examined as described in (i). k, HEK-293T cells were transfected with wild type or mutant c-Myc-JunD plasmids, either alone or together with FLAG-menin plasmids. Immunoblots were performed with either anti-phosphor-JunD (upper panel), anti c-Myc (middle panel) or anti FLAG (lower panel) antibodies to examine the phosphorylation level of JunD in response to anisomycin stimulation.