1. Volume 1, Issue 5, Pages 434-443 (May 2012)
SCFFbw7 Modulates the NFκB Signaling Pathway by Targeting NFκB2 for Ubiquitination and Destruction 
Hidefumi Fukushima, Akinobu Matsumoto, Hiroyuki Inuzuka, Bo Zhai, Alan W. Lau, Lixin Wan, Daming Gao, Shavali Shaik, Min Yuan, Steven P. Gygi, Eijiro Jimi, John M. Asara, Keiko Nakayama, Keiichi I. Nakayama, Wenyi Wei 
Cell Reports 
Volume 1, Issue 5, Pages (May 2012)
DOI: /j.celrep
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2. Cell Reports 2012 1, 434-443DOI: (10.1016/j.celrep.2012.04.002)
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3. Figure 1
Identification of NFκB2 as a Specific Fbw7-Interacting Protein
(A) Immunoblot (IB) analysis of whole-cell lysates (WCLs) derived from Fbw7−/− DLD1 cells infected with Fbw7-expressing lentiviral constructs.
(B) IB analysis of WCLs and HA-immunoprecipitates (IPs) derived from Fbw7−/− DLD1 cells infected with the HA-R465H-Fbw7-expressing lentiviral construct.
(C) The list of top candidates identified in the two mass spectrometry screenings searching for putative SCFFbw7 ubiquitin substrates.
(D) IB analysis of WCLs and IPs derived from 293T cells transfected with HA-β-TRCP1 together with Flag-NFκB1 or Flag-NFκB2 construct.
(E) IB analysis of WCLs and IPs derived from 293T cells transfected with HA-Fbw7 together with Flag-NFκB1 or Flag-NFκB2 construct.
(F) IB analysis of WCLs and anti-NFκB2 IPs derived from HeLa cells. Mouse IgG was used as a negative control.
(G) IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-NFκB2 together with a panel of GST-tagged F-box proteins.
(H) IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-NFκB2 together with the indicated Myc-tagged Cullin family of proteins.
(I and J) IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-NFκB2 together with Myc-Skp1 (I) or Myc-Rbx1 (J).
See also Figure S1.
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4. Figure 2 Fbw7 Negatively Regulates the Stability of NFκB2
(A) IB analysis of WT or Fbw7−/− DLD1 and HCT116 cells.
(B–E) Real-time RT-PCR analysis examining the relative mRNA expression levels of NFκB1 (B), NFκB2 (C), IκBα (D), and RelB (E) in WT and Fbw7−/− DLD1 cells. The error bars represent 1 SD, and the p value was generated with the use of the Student's t test. ∗: p < 0.01, n = 3.
(F) WT or Fbw7−/− DLD1 cell lines were treated with 20 μg/ml cycloheximide. At the indicated time points, WCLs were prepared and IBs were probed with the indicated antibodies.
(G) IB analysis of WT or Fbw7−/− DLD1 cells.
(H) IB analysis of HeLa cells transfected with the indicated siRNA oligonucleotides.
(I) IB analysis of T98G cells infected with the indicated shRNA lentiviral vectors.
(J) IB analysis of Fbw7−/− DLD1 cells infected with the indicated Fbw7-expressing lentiviral vectors.
(K) IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-NFκB2 together with the indicated Fbw7 encoding constructs.
(L) IB analysis of HeLa cells infected with the indicated lentiviral shRNA constructs.
(M and N) IB analysis of WCLs derived from WT or Fbw7−/− DLD1 cells stimulated with TWEAK (M) or TNFα (N) and harvested after stimulation at the indicated time periods.
See also Figure S2.
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5. Figure 3
Fbw7 Promotes NFκB2 Ubiquitination and Destruction in a GSK3 Phosphorylation-Dependent Manner
(A) Sequence alignment of NFκB2 with the consensus Fbw7 phosphodegrons. The putative Fbw7 phosphodegron sequence present in NFκB2 is conserved across different species.
(B) IB analysis showing the recovery of HA-Fbw7 bound to the indicated GST-NFκB2 proteins (with GST as a negative control) after incubation with GSK3. Ponceau S staining was performed to indicate equal loading of the indicated GST-fusion proteins.
(C) IB analysis of WCLs and IPs derived from 293T cells transfected with HA-Fbw7 together with the indicated Flag-NFκB2-encoding constructs.
(D) IB analysis of WCLs and IPs derived from 293T cells transfected with HA-Fbw7 and Flag-NFκB2. Where indicated, 25 μM of the GSK3β inhibitor VIII (with DMSO as a negative control) was added for 8 hr before harvesting.
(E) IB analysis of HeLa cells transfected with the indicated siRNA oligos.
(F and G) IB analysis of 293T cells transfected with Flag-NFκB1 or the indicated NFκB2 constructs together with HA-Fbw7 plasmids in the presence or absence of HA-GSK3.
(H) 293T cells were transfected with the indicated Flag-NFκB2 constructs together with the HA-Fbw7 and HA-GSK3 plasmids. Twenty hours after transfection, cells were split into 60 mm dishes, and after another 20 hr, they were treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, WCLs were prepared and IBs were probed with the indicated antibodies.
(I) IB analysis of WCLs and IPs derived from 293T cells transfected with Flag-NFκB2 together with the indicated Fbw7 encoding constructs.
(J) Fbw7−/− DLD1 cells were infected with Fbw7-expressing lentiviral constructs and selected with 1 μg/ml puromycin for 72 hr to eliminate the noninfected cells. Afterwards, cells were split into 60 mm dishes, and after another 20 hr, they were treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, WCLs were prepared and IBs were probed with the indicated antibodies.
(K) Fbw7 promotes the in vivo ubiquitination of NFκB2. IB analysis of WCLs and IPs derived from 293T cells transfected with indicated Flag-NFκB2 constructs together with the HA-Fbw7 and HA-GSK3 plasmids, and Myc-Ub.
See also Figures S3 and S4.
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6. Figure 4
Loss of Fbw7 Leads to Elevated Expression of NFκB2, which Subsequently Inhibits NFκB Activity
(A and B) Normalized luciferase activities of WT and Fbw7−/− DLD1 cells transfected with artificial (A) or naturally occurring (B) NFκB-responsive reporter. IL-6, interleukin 6 promoter. In both cases, the same reporter construct with the NFκB site mutated (Mut-Luc) was used as a negative control for the luciferase assay. Where indicated, cells were treated with the NFκB agonist TNFα before harvesting for luciferase assay.
(C and D) Normalized luciferase activities of WT and NFκB2−/− MEFs transfected with indicated NFκB2-expressing constructs together with the artificial 3XκB NFκB-responsive reporter. Where indicated, cells were treated with the NFκB agonist TNFα (C) or IL-1β (D) before harvesting for luciferase assay.
(E and F) Normalized luciferase activities of WT and Fbw7−/− DLD1 cells transfected with the indicated NFκB2-expressing constructs together with the artificial 3XκB NFκB-responsive reporter. Where indicated, cells were treated with the NFκB agonist TNFα (E) or IL-1β (F) before harvesting for luciferase assay.
(G and H) Normalized luciferase activities of WT and various engineered Fbw7−/− DLD1 cells transfected with the artificial 3XκB NFκB-responsive reporter. Where indicated, cells were treated with the NFκB agonist TNFα (G) or IL-1β (H) before harvesting for luciferase assay.
(I–K) RT-PCR was performed to monitor the changes in IκBα (I), ICAM1 (J), and RelB (K) mRNA levels, normalized to GAPDH mRNA, in indicated cell lines stimulated with 10 ng/ml TNFα for the indicated time period.
(L and M) RT-PCR was performed to analyze the induction of NFκB signaling pathway by monitoring the changes in IκBα (L) and RelB (M) mRNA levels, normalized to GAPDH mRNA, in the indicated WT and various engineered Fbw7−/− DLD1 cell lines stimulated with 10 ng/ml TNFα for the indicated time period.
(N and O) Viability of the indicated cell lines after exposure to TNFα.
Data are presented as mean ± SD, n = 3. See also Figures S5, S6, and S7.
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7. Figure S1
Development of the Mass-Spectrometry-Based Screen to Identify Novel Fbw7 Ubiquitin Substrates, Related to Figure 1
(A) Schematic illustration of the various T-ALL-derived hotspot Fbw7 mutations.
(B) Fbw7−/− DLD1 cells were infected with the HA-R505C-Fbw7-expressing lentiviral constructs and selected with 1 μg/ml puromycin to eliminate the non-infected cells before HA-immunoprecipitation. The immunoprecipitates were eluted with HA-peptides before mass spectrometry analysis and immunoblot analysis with the indicated antibodies.
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8. Figure S2
Loss of Fbw7 Leads to Elevated NFκB2 Abundance, Related to Figure 2
(A) Immunoblot analysis of wild-type (WT) or Fbw7−/− DLD1 cells after synchronization with nocodazole and release at the indicated time points.
(B) Real-time RT-PCR analysis to examine the relative mRNA expression levels of RelA in wild-type (WT) and Fbw7−/− DLD1 cells. Three independent sets of experiments were performed to generate the error bars. The error bars represent one standard deviation.
(C) Immunoblot analysis of NIH 3T3 cells infected with the indicated shRNA lentiviral vectors. Cells were treated with 1 μg/ml puromycin for 48 hr post-infection to eliminate the non-infected cells before harvesting for immunoblot analysis with the indicated antibodies.
(D) Immunoblot analysis of T98G cells infected with the indicated shRNA lentiviral vectors. Cells were treated with 1 μg/ml puromycin for 48 hr post-infection to eliminate the non-infected cells before harvesting for immunoblot analysis with the indicated antibodies.
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9. Figure S3
Fbw7 Negatively Regulates NFκB2 Stability, Related to Figure 3
(A) GSK3 phosphorylates NFκB2 in vitro at multiple sites. Purified recombinant GSK3 protein (from New England Biolabs) was incubated with 5 μg of the indicated GST-NFκB2 proteins in the presence of γ-32P-ATP. The kinase reaction products were resolved by SDS-PAGE and phosphorylation was detected by autoradiography.
(B) Immunoblot analysis of 293T cells transfected with the Flag-NFκB2 and HA-Fbw7 plasmids in the presence of the indicated kinase-encoding constructs. A plasmid encoding GFP was used as a negative control for transfection efficiency.
(C) Immunoblot analysis of wild-type (WT) DLD1 cells. Where indicated, cells were treated with 15 μM MG132 for 10 hr to block the proteasome pathway, or 25 μM of the GSK3β inhibitor VIII for 8 hr before harvesting.
(D) Immunoblot analysis of 293T cells transfected with Flag-NFκB2 together with HA-Fbw7 plasmids in the presence or absence of HA-GSK3. A plasmid encoding GFP was used as a negative control for transfection efficiency. Where indicated, 15 μM MG132 was added for 10 hr to block the 26S proteasome before harvesting.
(E) Immunoblot analysis of 293T cells transfected with the indicated amount of Flag-RelB together with HA-Fbw7 in the presence or absence of HA-GSK3. A plasmid encoding GFP was used as a negative control for transfection efficiency.
(F) 293T cells were transfected with the indicated Flag-NFκB2 constructs together with the HA-Fbw7 and HA-GSK3 plasmids. Twenty hours post-transfection, cells were split into 60 mm dishes, and after another 20 hr, treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies.
(G) The SCFFbw7 complex promotes NFκB2 ubiquitination in vitro. Affinity-purified SCFFbw7 complexes were incubated with purified recombinant GST-NFκB2 proteins, purified E1, E2 and ubiquitin as indicated at 30°C for 60 min. The ubiquitination reaction products were resolved by SDS-PAGE and probed with the anti-NFκB2 antibody.
(H) Schematic illustration of the identified phosphorylation sites in NFκB2 by mass spectrometry analysis as described in the Extended Experimental Procedures. Among them, phosphorylation of Ser707 and Ser711 creates an Fbw7-recognizable phospho-degron.
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10. Figure S4
Fbw7 Negatively Regulates the Stability, but Not the Processing, of NFκB2, Related to Figure 3
(A) Immunoblot analysis of WT or Fbw7−/− DLD1 cells transfected with WT Flag-NFκB2 or S707A/S711A-Flag-NFκB2 together with HA-β-TRCP1 and/or IKKα plasmids. A plasmid encoding GFP was used as a negative control for transfection efficiency.
(B) Schematic representation of the NFκB1/2 and NFκB2/1 chimera protein-encoding constructs used in (C).
(C) Immunoblot analysis of DLD1 cells transfected with the various indicated NFκB chimera protein-encoding constructs together with HA-Fbw7 plasmids in the presence or absence of HA-GSK3. A plasmid encoding GFP was used as a negative control for transfection efficiency.
(D) A schematic illustration of the various tumor derived NFκB2 mutants, which lack the Ser707/Ser711 phospho-degron that can be recognized by Fbw7.
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11. Figure S5
Loss of Fbw7 Leads to Reduced NFκB Signaling Activity, Related to Figure 4
(A and B) Normalized luciferase activities of WT and Fbw7−/− HCT116 cells transfected with artificial (A) or naturally occurring (B) NFκB-responsive reporter. IL-6, Interleukin 6 promoter. In both cases, the same reporter construct with NFκB sites mutated (Mut-Luc) was used as a negative control for the luciferase assay. Where indicated, cells were treated with the NFκB agonist TNFα before harvesting for luciferase assay. Data are presented as mean ± S.D., n = 3.
(C) Normalized luciferase activities of WT and Fbw7−/− DLD1 cells transfected with the artificial 3XIκB NFκB-responsive reporter. NFκB site mutated (Mut-Luc) reporter was used as a negative control for the luciferase assay. Where indicated, cells were treated with the NFκB agonist PMA before harvesting for luciferase assay. Data are presented as mean ± S.D., n = 3.
(D and E) RT-PCR analysis to monitor the changes in cyclin D2 (D) and NFκB2 (E) mRNA levels, normalized to GAPDH mRNA, in indicated cell lines stimulated with 10 ng/ml TNFα for the indicated time period. Data are presented as mean ± S.D., n = 3.
(F) RT-PCR analysis to analyze the induction of NFκB signaling pathway by monitoring the changes in ICAM1 mRNA levels, normalized to GAPDH mRNA, in the indicated WT and various engineered Fbw7−/− DLD1 cell lines stimulated with 10 ng/ml TNFα for the indicated time period. Data are presented as mean ± S.D., n = 3.
(G) Immunoblot analysis of WT and NFκB2−/− MEFs infected with the indicated shRNA lentiviral vectors. Cells were treated with 1 μg/ml puromycin for 48 hr post-infection to eliminate the non-infected cells before harvesting for immunoblot analysis with the indicated antibodies.
(H) The various generated cell lines in (G) were then subjected to the 3xIκB NFκB-responsive luciferase reporter assays. Where indicated, cells were treated with the NFκB agonist TNFα before harvesting for luciferase assays. Data are presented as mean ± S.D., n = 3.
(I) Immunoblot analysis of WT and Fbw7−/− DLD1 cell lines infected with multiple independent shRNA lentiviral vectors to deplete the endogenous c-Myc (with shGFP as a negative control). Cells were treated with 1 μg/ml puromycin for 48 hr post-infection to eliminate the non-infected cells before harvesting for immunoblot analysis with the indicated antibodies.
(J) The generated various cell lines in (I) were then subjected to the 3xIκB NFκB-responsive luciferase reporter assays. Where indicated, cells were treated with the NFκB agonist TNFα before harvesting for luciferase assays. Data are presented as mean ± S.D., n = 3.
(K) WT and Fbw7−/− DLD1 cell lines were infected with multiple independent shRNA lentiviral vectors to deplete the endogenous Mcl-1 (with shGFP as a negative control). Cells were treated with 1 μg/ml puromycin for 48 hr post-infection to eliminate the non-infected cells before subjected to the 3xIκB NFκB-responsive luciferase reporter assays. Where indicated, cells were treated with the NFκB agonist TNFα before harvesting for luciferase assays. Data are presented as mean ± S.D., n = 3.
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12. Figure S6
Loss of Fbw7 Perturbs T Cell Development In Part through Stabilizing the NFκB2 Protein, Related to Figure 4
(A) The weight of the thymus derived from the control (Fbw7F/F) or Lck-Cre/Fbw7F/F mice. Three independent sets of experiments were performed to generate the error bars. The error bars represent one standard deviation and the p value was generated with the Student's t test. ∗: p < 0.05.
(B) Schematic illustration of the cell number for the whole thymus derived from the control (Fbw7F/F) or Lck-Cre/Fbw7F/F mice. Three independent sets of experiments were performed to generate the error bars. The error bars represent one standard deviation and the p value was generated with the Student's t test. ∗: p < 0.05.
(C) Cells derived from the thymocytes of the control (Fbw7F/F) or Lck-Cre/Fbw7F/F mice were stained with CD4 and CD8 antibodies before subjective to FACS analysis.
(D) Schematic illustration of the cell numbers for each FACS fraction obtained in (C). Three independent sets of experiments were performed to generate the error bars. The error bars represent one standard deviation and the p value was generated with the Student's t test. ∗: p < 0.05.
(E) Immunoblot analysis of whole cell lysates (WCL) derived from each sorted FACS fraction obtained in (C).
(F) Real-time RT-PCR analysis to examine the relative mRNA expression levels of Bcl-2, a well-characterized NFκB transcriptional target, in thymocytes derived from each sorted FACS fraction obtained in (C). Three independent sets of experiments were performed to generate the error bars. The error bars represent one standard deviation and the p value was generated with the Student's t test. ∗: p < 0.05.
(G and H) The thymocytes from Fbw7F/F or Lck-Cre/Fbw7F/F mice were treated with 20 μg/ml cycloheximide (CHX). At the indicated time points, whole cell lysates were prepared and immunoblots were probed with the indicated antibodies (G). Band intensity was measured, normalized by that of β-actin, and expressed as a percentage of the corresponding normalized value for time zero (H).
(I–L) RT-PCR analysis to monitor the relative changes of mRNA levels in the various well-characterized NFκB transcriptional targets in RelB (I), Bcl-2 (J), IL-2 (K) and IκBα (L), normalized to GAPDH mRNA, in DN thymocyte fraction derived from the Fbw7F/F versus Lck-Cre/Fbw7F/F thymus. Cells were stimulated with 2 μg/ml anti-CD3 antibody for the indicated time period to activate the NFκB signaling pathway before harvesting for the RT-PCR analysis. Data are presented as mean ± S.D., n = 3. p value was generated with the Student's t test. ∗: p < 0.05.
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13. Figure S7
Schematic Overviews of the Role of Fbw7 in Modulating the Activities of the NFkB Signaling Pathways, Related to Figure 4
(A) A schematic illustration of the differences between the processing and the degradation of NFκB2 in regulating the activities of the canonical and/or the non-canonical NFκB signaling pathways.
(B) A schematic illustration of the proposed molecular circuits in governing the NFκB signaling pathways.
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