Fig. 7: NIK is neddylated and neddylation promotes its ubiquitination and degradation.
a Twenty-four hours after transfection with the indicated constructs, HepG2 cells were treated with or without 2.0 µM MLN for 24 h. The neddylation of NIK-HA was examined by IB after denaturing IP. Cell lysates were IB as input. N8WT, wild-type NEDD8; N8ΔGG, conjugation-deficient NEDD8 mutant. b The neddylation of NIK-HA was examined by IB after denaturing IP in 293 T cells transfected with the indicated constructs with scrambled DsiRNA or DsiRNA against UBC12. c, d NIK neddylation was assessed after denaturing IP in (c) 293 T cells overexpressing SENP8 WT and C153A mutant, (d) WT and SENP8-KO MEFs, and 293 T cells with SENP8 knocked down. Cells were co-transfected with indicated NIK-HA and/or FLAG-N8WT. e Western blot after IP with an anti-UBC12 antibody or a control IgG antibody in HepG2 and primary hepatocyte (PH) lysates treated with BZM (100 nM, 12 h). f A schematic diagram of the lysine sites on NIK is shown. ·: conserved, x: non-conserved lysines. Yellow lines indicate lysine residues previously identified with K-Ɛ-GG remnants. Catalytic kinase domain (KD): 405-646 amino acids (aa). g Neddylation of various NIK K to R mutants examined by IB after denaturing IP in HepG2 cells. h Proteomic analyses identified the four potential neddylation sites (K385, K387, K650 indicated by yellow lines, and K607 in purple). Forty-eight hours after transfection in HepG2 cells, the neddylation of NIK-HA (WT) and the corresponding 4 R mutant (all of four lysine residues mutated to arginine) was then examined by IB after denaturing IP. i NIK ubiquitination was analyzed after denaturing IP in 293 T cells transfected with the indicated constructs or (D)siRNAs against UBC12 or NEDD8. BZM was added 8 h before harvesting. j, k Twenty-four hours after transfection with the same (j) or adjusted (k) amounts of the indicated plasmids, HepG2 cells were treated with BZM (100 nM, 8 h) or MLN (1.0 µM, 24 h) or 20 µg/ml CHX for indicated periods followed by Western blot. Quantifications were shown as numbers under each image. Three biologically independent experiments except for mass spectroscopic analysis. Source data are provided as a Source Data file.
