NEDD8 controls the localization and mobility of L11. (A) H1299 cells were transfected with 5 μg Flag–L11, 2 μg His₆–NEDD8 and 5 μg of NEDP1 constructs as indicated. Subcellular fractionation was carried out as described in the Methods. Tubulin and lamin were used as cytoplasmic and nuclear markers, respectively. (B) H1299 cells were transfected with 3 μg Flag–L11 (WT) or 12 μg Flag–K0 (K0) mutant and His₆–NEDD8 as described above. NEDDylated proteins and total cell extracts were analysed by western blotting as indicated. (C) The localizations of WT L11 and the K0 mutant were analysed in MCF7 cells as described in the Methods. Fixed cells were stained with anti-Flag. (D) H1299 cells were transfected with 1 μg p53, 1 μg MDM2, 1, 3 or 5 μg of WT L11 and 4, 8 or 12 μg of K0 mutant as indicated. Cells were lysed in 2 × SDS LB and analysed by western blotting. Transfection efficiency and loading was monitored using β-gal (3 μg). (E) U2OS cells were transfected with 0.5 μg WT L11, 2 μg K0 mutant and 2 μg MDM2, and treated with ActD as indicated. IPs were carried out using anti-Flag and blotted with the indicated antibodies. WCLs were used for western blotting. The asterisk indicates a non-specific band. (F) H1299 cells, stably expressing L11–EGFP, were transfected with either control or NEDD8 siRNAs. At 48 h post-transfection, cells were treated as indicated with ActD for 1 h before the FRAP experiment. After the experiment, cells were lysed in 2 × SDS LB and analysed by western blotting (top right panel). The half-life of recovery and the mobile fraction were calculated as described in the Methods. C, cytoplasm; DAPI, 4′,6-diamidino-2-phenylindole; EGFP, enhanced green fluorescent protein; FRAP, fluorescence recovery after photobleaching; β-gal, β-galactosidase; IP, immunoprecipitation; LB, loading buffer; MDM2, mouse double minute 2; N, nucleus; NEDP1, NEDD8-specific protease 1; siRNA, small interfering RNA; T, total extract; WCL, whole cell lysate; WT, wild type.

MDM2 controls the stability of L11 through NEDDylation and is required for p53 stabilization on nucleolar stress. (A) H1299 cells were transfected with control or MDM2 siRNA. The next day, cells were re-seeded and the day after were transfected with 1 μg His₆–NEDD8 and either 0.5 μg p53 (left panel) or 3 μg Flag–L11 (right panel) with Fugene as indicated. NEDDylated proteins and WCL were analysed by Western blotting. The asterisk indicates the presence of unmodified p53. (B) Left panel: wild-type MDM2 was transfected (2, 5, 10 μg) as indicated with 5 μg Flag–L11 and 2 μg His₆–NEDD8 in H1299 cells. Western blot analysis on the NEDDylated proteins and WCL was carried out as before. Right panel: the experiment was carried out as in the left panel, including 5 μg WT MDM2 or the NLS mutant. (C) MCF7 cells were transfected with the indicated siRNAs and treated with ActD as indicated. Cells were lysed and analysed by Western blotting as before. Signals were quantified with Image Gauge. (D) Model for the role of NEDD8 in nucleolar signalling. NEDDylation of L11 is required for the correct localization of L11 in the nucleolus. Stress signals, such as ActD treatment, result in rapid de-NEDDylation of L11 causing either its release from the nucleolus and/or impairment in nucleolar import. This results in L11 nucleoplasmic localization, not only providing the signal for p53 activation, but also making L11 susceptible to degradation. The MDM2–p53 complex acts as a sensor of mislocalized L11, leading to p53 activation. ActD, actinomycin D; MDM2, mouse double minute 2; NEDP1, NEDD8-specific protease 1; NLS, nuclear localization signal; siRNA, small interfering RNA; WCL, whole cell lysate; WT, wild type.

NEDD8 controls L11 signalling to p53 on nucleolar stress. (A) H1299 cells were transfected with 1 μg p53, MDM2, 5 μg Flag–L11 and 2, 5 or 10 μg of NEDP1 constructs as indicated. Cells were lysed in 2 × SDS loading buffer and an equal amount of protein was analysed by western blotting. Transfection efficiency and loading was monitored by co-transfection of β-gal (3 μg). (B) MCF7 cells were transfected with control or NEDD8 siRNA and 48 h later were treated with ActD as indicated. Cells were lysed and analysed by western blotting as in (A). (C) MCF7 cells were transfected with control or individual siRNAs for NEDD8 and analysed as in (A). (D) Transfections were carried out as in (C) and cells were re-seeded the next day; the day after they were transfected with a siRNA-insensitive NEDD8 construct. After 24 h, cells were treated with ActD and used for western blotting. (E) Transfections in MCF7 cells were carried out as in (B) and cell extracts were used for IPs using anti-cullin-1. Eluates and WCL were used for western blotting as indicated. (F) MCF7 cells were transfected with control or NEDD8 siRNAs and treated with ActD. Real-time PCR for the indicated genes was carried out as described in the Methods. Data are presented as mean±s.e.m. from three independent experiments. ActD, actinomycin D; β-gal, β-galactosidase; bax, BCL2-associated X protein; IP, immunoprecipitation; LB, loading buffer; MDM2, mouse double minute 2; NEDP1, NEDD8-specific protease 1; siRNA, small interfering RNA; WCL, whole cell lysate.

NEDDylation of L11 is specifically reduced by ActD. (A) MCF7 cells were transfected with 3 μg Flag–L11 and 1 μg of His₆–NEDD8 constructs, and ActD was applied as indicated. Western blot analysis on the NEDDylated eluates and WCL was carried out using the indicated antibodies. (B) H1299 cells were transfected with 2 μg His₆–NEDD8 and treated with ActD as indicated. NEDDylated proteins or WCL were analysed by western blotting. (C) Experiment carried out as in (B), including 5 μg UBC12C111S mutant. (D) MCF7 cells were treated with ActD as indicated and IPs were carried out using anti-cullin-1. Eluates and WCL were analysed by western blotting. ActD, actinomycin D; HA, haemagglutinin; IP, immunoprecipitation; UBC12, ubiquitin conjugating enzyme 12; WB, western blot; WCL, whole cell lysate.