Figure 3: Fbw7 interacts with NF-κB2 through a conserved phospho-degron.

(a) NF-κB2 binds Fbw7. HEK293 cells were transfected with FLAG-tagged Fbw7 or Fbw7ΔF. Cells were treated with MG132 for 3 h before cell lysis. p100 was immunopurified with anti-NF-κB2 antibodies and Fbw7 binding was analysed by WB using anti-FLAG antibody. * indicates control reaction with beads only. (b) HEK cells were transfected with p100 empty GST vector (EV) or GST-tagged Fbw7. Binding of NF-κB2/p100 protein was analysed following GST-pull-down. (c) Schematic representation of the full-length and different C-terminal NF-κB2 deletions. The arrow indicates the p52 cleavage site. ARD, Ankryn repeat domain. Black boxes indicate putative Fbw7 degron motifs. The motif most similar to known degrons is localized at amino acids 705–711. (d) Comparison of amino acid sequence of the NF-κB2 motif with defined degrons in cyclin E1 and c-Myc. (e) Fbw7 interacts with the NF-κB2 C-terminus. Cells were co-transfected with GST-tagged Fbw7ΔF and the NF-κB2 constructs as indicated. Fbw7 was pulled down with GST-beads followed by WB with anti-NF-κB2 antibody. (f) HEK293 cells were co-transfected with the indicated plasmids and NF-κB2 protein level was analysed by WB. (g) p100 protein with mutated degron motif is stable. Half-life of the WT p100 and the pSA-mutant (serines 707 and 711 replaced by alanines) were compared by CHX-chase analysis in HEK293 cells. The graph shows quantification of the p100 band in the blot on a log2 scale as compared to t=0. (h) GSK-3β induces p100 and RelB degradation by Fbw7. HCT116 FBW7 WT and KO cells were treated with the GSK3β inhibitor (BIO) or transfected with empty vector, GSK3β or GSK3β and Fbw7 as indicated. Lower panel shows quantified relative p100 protein levels. Error bars indicate s.d. One sample t-tests were used to determine statistical significance (n=3 for WT and n=4 for KO). *P<0.05; **P<0.01 and ***P<0.001.