Proteomic screen reveals Fbw7 as a modulator of the NF-κB pathway

Abstract

Fbw7 is a ubiquitin-ligase that targets several oncoproteins for proteolysis, but the full range of Fbw7 substrates is not known. Here we show that by performing quantitative proteomics combined with degron motif searches, we effectively screened for a more complete set of Fbw7 targets. We identify 89 putative Fbw7 substrates, including several disease-associated proteins. The transcription factor NF-κB2 (p100/p52) is one of the candidate Fbw7 substrates. We show that Fbw7 interacts with p100 via a conserved degron and that it promotes degradation of p100 in a GSK3β phosphorylation-dependent manner. Fbw7 inactivation increases p100 levels, which in the presence of NF-κB pathway stimuli, leads to increased p52 levels and activity. Accordingly, the apoptotic threshold can be increased by loss of Fbw7 in a p100-dependent manner. In conclusion, Fbw7-mediated destruction of p100 is a regulatory component restricting the response to NF-κB2 pathway stimulation.

Figure 1. Identification of SCF^Fbw7 substrates.

(a) Screen design and proteomic workflow for global identification of candidate Fbw7 targets. Subcellular fractionation of HCT116 FBW7 WT and KO cells into cytosolic (Cyt) and nuclear-/organellar-(Nuc/org) enriched fractions was followed by TMT labelling, sample pooling and peptide fractionation by HiRIEF. Analysis of quantitative data by PQPQ-SAM identified upregulated proteins. (b) Venn diagram showing the total number and overlap of (1) the proteins found by quantitative proteomics to be upregulated in KO cells (2) proteins with ≥3 Fbw7 degron motifs (RKS/TPRKXS/T/E/D) and (3) proteins with motifs predicted to be phosphorylated by GSK3. (c) Validation of quantitative MS-data. WB analysis comparing endogenous protein levels in the different fractions from WT and KO cells. Equal protein amount from each cell type were analysed. (d) FBW7 KO cells were transiently transfected with Fbw7 and GSK-3β as indicated for 24 h. Whole cell extracts were analysed by WB.

Figure 2. NF-κB2 ubiquitylation and levels are regulated by Fbw7.

(a) p100 turnover rate is prolonged in FBW7 KO HCT116 cells. p100 protein half-life was analysed following treatment with cycloheximide. p100 band intensity was normalized to β−actin and then normalized to t=0 controls. p100 half-life in WT=6.7 h (R²=0.87) and in KO=14.7 h (R²=0.78) (b) RelB turnover rate is prolonged in FBW7 KO HCT116 cells. RelB protein half-life was measured as in (a). WT=1.9 h (R²=0.91) and in KO=4.6 h (R²=0.71) (c) Total RNA was prepared from WT and KO cells and NF-κB2 mRNA expression was analysed by qRT–PCR. The results are shown as means. Error bars indicate s.d. (n=3). (d) p100 protein levels increase on Fbw7 silencing. HCT116 FBW7 WT cells were transfected with the scrambled (scr) or FBW7 siRNA for 48 h. Endogenous p100 protein was analysed by WB. ±indicated s.d. (n=3). (e) qRT–PCR of FBW7 and NF-κB2 mRNA levels from siRNA transfected cells in (d), n=3, error bars indicate s.d. (f) Fbw7 knockdown stabilizes p100 protein. HEK293 cells were transfected with the indicated siRNAs for 48 h and NIK for 24 h and treated with cycloheximide. p100 and p52 half-lives were analysed. The graphs to the right show quantification of the protein bands in the blot. p100 half-life control=12 h (R²=0.99) and Fbw7=58 h (R²=0.72). Degradation of p52 was marginal under these conditions and time frames. (g) Fbw7 ubiquitylates endogenous p100. FBW7 WT and KO cells were transfected with HA-ubiquitin. NF-κB2 was immunoprecipitated from whole-cell extracts and NF-κB2 ubiquitylation was analysed by WB using HA antibodies (see also methods). (h) Ubiquitylation of endogenous p100 is restored by ectopic Fbw7 expression. FBW7 KO cells were transiently transfected with FBW7. Ubiquitination assay was performed as described in (g).

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.

Figure 4. Fbw7 modulates p100/p52 levels and activity.

(a) Fbw7 knockdown increases p52 protein levels. HEK293 cells were transfected with control (scr), Fbw7 or βTrCP siRNA oligos followed by transient transfection of p100 with or without NIK for 24 h. Whole-cell extracts were analysed. (b) Fbw7 alters p100 and p52 levels. HEK293 cells were co-transfected with p100 and Fbw7 or Fbw7ΔF, with or without NIK and analysed by WB. Lower panel shows quantified protein levels, for p100 (dark bars) and p52 (light bars). Error bars indicate s.d. (n=3, Fbw7+NIK n=5). (c) Fbw7 reduces p52 levels on CD40 stimulation. MDA-231 cells were transfected with control (EV) or Fbw7 plasmid. 24 h post-transfections cells were stimulated by soluble recombinant CD40 overnight and analysed by WB. (d) HEK293 cells were transfected with control (scr), Fbw7 or βTrCP siRNAs followed by transient transfection of p100 and NIK for 24 h. p100 levels in the cytosolic and nuclear extracts were analysed. (e) HEK293 cells were transfected with βTrCP siRNA oligos followed by transient transfection of control (EV) or Fbw7 for 24 h. p100 levels in the cytosolic and nuclear extracts were analysed. (f) p52:RelB complexes are more abundant in FBW7 KO cells. HCT116 FBW7 WT and KO cells were treated with 10 ng ml⁻¹ TNF-α for 24 h before lysis. 200 μg of protein was used to immunoprecipitate RelB. (g) Increased mRNA expression of NF-κB2 targets in FBW7-deficient cells. FBW7 WT and KO cells were treated or not with 10 ng ml⁻¹ TNF-α for 24 h before total RNA extraction. qPCR was performed for the indicated genes. Actin was used as a normalizing gene and all levels were compared with untreated WT. Graph depicts the mean and s.d. of a minimum of three independent experiments. Statistical significance was tested using ANOVA. *P<0.05; **P<0.01 and ***P<0.001.

Figure 5. Regulation of NF-κB activity by Fbw7 with or without stimuli.

(a) Basal NF-κB activity is lower in HCT116 FBW7 KO compared with WT cells. WT and KO cells were transfected with the kB-luciferase reporter. Relative luciferase activity was measured. Results are shown as means. Error bars indicate s.d. (n=3). (b) p100 overexpression represses kB-luciferase activity in the absence of stimulation. HEK293 or HCT FBW7 WT cells were transfected with the kB-luciferase reporter and empty vector (EV), p100, pSA and NIK as indicated. Results are shown as means. Error bars indicate s.d. (n=3). The p100/EV ratios of fold induction were 29 (s.d.=5) in HEK and 2.9 (s.d.=0.75) in HCT cells. (c) FBW7 WT and KO cells were transfected with the κB-Luciferase reporter with NIK or treated with 5 ng ml⁻¹ TNF-α (24 h). Luciferase activity was analysed and normalized to untreated cells for each cell type. Error bars show s.d. (n=3). (d) U2OS cells were transfected with EV, Fbw7 or Fbw7ΔF as indicated. All cells were also transfected with the cyclin D1 promoter-specific luciferase reporter. Results are shown as means. Error bars indicate s.d. (n=3). (e) Response to the NF-κB pathway stimulation is enhanced in Fbw7ΔF expressing cells. HEK293 cells were transfected with the κB-Luciferase reporter and NIK, Fbw7 and Fbw7ΔF as indicated for 48 h. Luciferase activity was measured. The results were shown as means. Error bars are s.d. (n=3). (f) Protein levels of NF-κB2 targets are higher in FBW7 KO cells. WB analysis of NF-κB2 targets in HCT FBW7 WT and KO cells. Cells were treated with 10 ng ml⁻¹ TNF-α for the indicated times. Statistical significance was tested using ANOVA in (d) and unpaired, two-tailed t-tests elsewhere. *P<0.05; **P<0.01 and ***P<0.001.

Figure 6. Elevated p100 as a result of Fbw7 loss increases survival when processed.

(a) U2OS cells were transfected with the control (scrambled), Fbw7 or/and p100 siRNA as indicated. 48-h post-silencing cells were treated with 1 μM Daunorubicin (DN) for 48 h and cell survival was assayed by crystal violet staining. (b) Cells were treated with 1 μM DN for 48 h and apoptosis was monitored by Annexin V staining. (c) Cells were transfected as in a and treated with 10 μM Etoposide (Etop) for 24 h. Apoptosis was monitored by Annexin V staining. (d) HEK293 cells were transiently transfected with the mutant p100 (p100SA) and NIK and treated with 1 μM Doxorubicin (DX) for 29 h and subjected to Annexin V staining. Welch's t-test was used to determine statistical significance in all panels (n=3). Error bars indicate s.d. *P<0.05; **P<0.01 and ***P<0.001.

Figure 7. Fbw7 modulates the response to NF-κB pathway stimulation through controlling p100 protein levels.

In response to specific stimuli p100 is processed to p52 through NIK-mediated phosphorylation. Increase in p100 levels as a result of Fbw7 inactivation leads to elevated p52 production, alterations in p52/NF-κB complex formation and activity on stimulation. In the absence of stimuli, p100 binds to and inhibits various NF-κB complexes. Consequently, Fbw7-mediated regulation of p100 levels controls downstream transcriptional targets of both the canonical (RelA:p50) and the non-canonical (RelB:p52) NF-κB pathways.