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. 2012 Mar 4;14(4):375-85.
doi: 10.1038/ncb2463.

Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma

Luca Busino  1 , Scott E Millman, Luigi Scotto, Christos A Kyratsous, Venkatesha Basrur, Owen O'Connor, Alexander Hoffmann, Kojo S Elenitoba-Johnson, Michele Pagano
Affiliations

Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma

Luca Busino (VSports app下载) et al. Nat Cell Biol. .

Abstract

Fbxw7α is a member of the F-box family of proteins, which function as the substrate-targeting subunits of SCF (Skp1/Cul1/F-box protein) ubiquitin ligase complexes. Using differential purifications and mass spectrometry, we identified p100, an inhibitor of NF-κB signalling, as an interactor of Fbxw7α. p100 is constitutively targeted in the nucleus for proteasomal degradation by Fbxw7α, which recognizes a conserved motif phosphorylated by GSK3 VSports手机版. Efficient activation of non-canonical NF-κB signalling is dependent on the elimination of nuclear p100 through either degradation by Fbxw7α or exclusion by a newly identified nuclear export signal in the carboxy terminus of p100. Expression of a stable p100 mutant, expression of a constitutively nuclear p100 mutant, Fbxw7α silencing or inhibition of GSK3 in multiple myeloma cells with constitutive non-canonical NF-κB activity results in apoptosis both in cell systems and xenotransplant models. Thus, in multiple myeloma, Fbxw7α and GSK3 function as pro-survival factors through the control of p100 degradation. .

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The authors declare no competing financial interests.

Figures

Figure 1
Figure 1
p100 interacts with Fbxw7α through a conserved degron phosphorylated by GSK3. (a) p100 binds Fbxw7α. HEK293 cells were transfected with cDNAs encoding the indicated FLAG-tagged F-box proteins (FBPs), Cdh1 or Cdc20 and treated with the proteasome inhibitor MG132 for 6 h. FLAG-tagged immunoprecipitates (IPs) from cell extracts with anti-FLAG resin were immunoblotted as indicated. Lane 1 shows whole-cell extract (WCE) from empty vector (EV)-transfected cells. (b) Ser 707 and Ser 711 in p100 are required for the interaction with Fbxw7α. HEK293 cells were transfected with HA-tagged Fbxw7α and constructs encoding FLAG-tagged p100 or p100(Ser707/711Ala). FLAG-tagged p100 was immunoprecipitated from cell extracts, followed by immunoblotting as indicated. The right panel shows whole-cell extract. (c) The p100 degron requires phosphorylation to bind Fbxw7α. In vitro-translated Fbxw7α and βTrCP1 were incubated with beads coupled to the indicated p100 peptides or BimEL peptide. Beads were washed and eluted proteins were immunoblotted as indicated. The first lane shows 10% of in vitro-translated protein inputs. (d) GSK3 phosphorylates p100 in vitro. In vitro-translated p100 or p100(Ser707/711Ala) was incubated at 30 °C for 1 h in the presence or absence of GSK3β. Reaction products were immunoblotted as indicated. (e) GSK3-mediated phosphorylation of p100 is required for p100 binding to Fbxw7α in vitro. In vitro-translated, FLAG-tagged p100 was incubated with or without GSK3β before incubation with in vitro-translated Fbxw7α. FLAG-tagged immunoprecipitates were immunoblotted as indicated. 5% of inputs are shown. (f) GSK3 phosphorylates p100 in vivo. MEFs were treated with dimethylsulphoxide (DMSO) or the GSK3 inhibitor GSK3i IX (5 μM). Cell extracts were immunoblotted as indicated. (g) In vivo binding between p100 and Fbxw7α depends on GSK3 activity. Nfκb2−/− MEFs were infected with retroviruses expressing FLAG-tagged p100 or p100(Ser707/711Ala). Cells were treated with the indicated concentrations of GSK3i IX for 12 h. FLAG-tagged immunoprecipitates were immunoblotted as indicated. (h) p100 is ubiquitylated in vitro in a degron- and SCFFbxw7α-dependent manner. [35S]-in vitro-translated (IVT) p100 or p100(Ser707/711Ala) was incubated at 30 °C with a ubiquitylation mix. SCFFbxw7α was added to the reaction for the indicated times. Reactions were subjected to SDS–PAGE and analysed by autoradiography. Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 2
Figure 2
Fbxw7α controls p100 stability in the nucleus. (a) A constitutively nuclear mutant of p100 (p100(ΔNES)) is stabilized by proteasome inhibitor treatment or silencing of Fbxw7. HeLa cells were infected with a retrovirus expressing HA-tagged p100(ΔNES) and treated with either siRNAs or MG132. Cells were treated with cycloheximide (CHX) for the indicated times, and total cell lysates were analysed by immunoblotting as indicated. (b) A constitutively nuclear mutant of p100 (p100(ΔNES)) is stabilized by mutation of Ser 707 and Ser 711 to alanine. HeLa cells were infected with retroviruses expressing either HA-tagged p100(ΔNES) or HA-tagged p100(ΔNES; Ser707/711Ala) and treated with cycloheximide for the indicated times. Total cell lysates were analysed by immunoblotting as indicated (left). mRNA levels of retrovirally expressed p100 were analysed by quantitative PCR (right). (c) Identification of an NES in p100. MEFs were infected with retroviruses expressing either HA-tagged p100(4×NES) or HA-tagged p100(4×NES; Ser707/711Ala) and stained with an antibody against HA. Scale bar, 20 μm. The identified consensus NES is shown on the bottom. aa, amino acids. (d) A constitutively nuclear mutant of p100 (p100(4×NES)) is stabilized by mutation of Ser 707 and Ser 711 to alanine. Nfκb2−/− MEFs were infected with a retrovirus expressing HA-tagged p100(4×NES), HA-tagged p100(4×NES; Ser707/711Ala), HA-tagged p100(NLS) or HA-tagged-p100(NLS; Ser707/711Ala), treated with cycloheximide for the indicated times and total cell lysates were analysed by immunoblotting as indicated (left). IκBα is shown as a positive control for cycloheximide activity. mRNA levels of retrovirally expressed p100 were analysed by quantitative PCR (right). Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 3
Figure 3
p100 is degraded by Fbxw7α and GSK3 independently of NF-κB signalling, but NF-κB proteins compete with Fbxw7α for binding to p100. (a) p100 accumulates in the nucleus on LTβR activation. MEFs were treated with agonistic anti-LTβR antibodies, fixed and stained with antibodies against the N- or C-terminus of p100 (green). A similar pattern was observed with an antibody against either the N-terminus (recognizing both p100 and p52) or the C-terminus (recognizing only p100). Scale bar, 100 μm. (b) Levels of p100 are higher in Fbxw7 −/− MEFs than in Fbxw7flox/flox MEFs. MEFs were stimulated with agonistic anti-LTβR antibodies and collected at the indicated times. Nuclear and cytoplasmic fractions were analysed by immunoblotting as indicated. (c) Levels of p100 are increased on GSK3 inhibition. MEFs were incubated with GSK3i IX and stimulated with agonistic anti-LTβR antibodies. Nuclear and cytoplasmic fractions were analysed by immunoblotting as indicated. (d) p52 induces the accumulation of nuclear p100. Nfκb2−/− MEFs were infected with retroviruses expressing p100 and HA-tagged p52, fixed and stained with antibodies against the C-terminus of p100 (green) and the HA tag (red). DNA was stained with DAPI. Scale bar, 100 μm. (e) p52 induces p100 accumulation in control cells, but not in cells depleted of Fbxw7. HeLa Tet-ON HA-tagged p52 cells were treated with siRNA against LacZ or FBXW7. Doxycycline (DOX) was added to cells at the indicated times. Total cell extracts were immunoblotted as indicated. (f) p52 competes with Fbxw7α for binding to p100 in vivo. HEK293 cells were transfected with cDNAs encoding either FLAG-tagged p100 or FLAG-tagged p100(Ser707/711Ala). Increasing amounts of HA-tagged p52 plasmid were transfected. Proteins were immunoprecipitated from cell extracts with anti-FLAG resin (anti-FLAG), and the immunoprecipitates (IPs) were immunoblotted as indicated. The first lane shows cells transfected with an empty vector (EV). (g) The interaction of Fbxw7 with p100 is disrupted after treatment of cells with anti-LTβR. MEFs were treated with anti-LTβR for 18 h, collected, lysed and endogenous Fbxw7α was immunoprecipitated. Immunocomplexes were analysed by western blotting for the indicated proteins. Fbxw7−/− cells were used as a negative control. Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 4
Figure 4
Clearance of p100 from the nucleus is crucial for non-canonical NF-κB signalling. (a) LTβR-dependent gene transcription is impaired in cells lacking Fbxw7. Fbxw7flox/flox and Fbxw7 −/− MEFs were treated with agonistic anti-LTβR antibodies and collected at the indicated times. Levels of the indicated mRNAs were determined by quantitative real-time PCR (±s.d., n = 3). The value for the amount of PCR product present in Fbxw7flox/flox MEFs was set as 1. (b) LTβR-dependent gene transcription is impaired in cells expressing stable p100(Ser707/711Ala). MEFs were infected with retroviruses expressing empty vector (EV), p100 or p100(Ser707/711Ala), stimulated with agonistic anti-LTβR antibodies and processed as in a. Levels of the indicated mRNAs were determined by quantitative PCR (±s.d., n = 3). The value for the amount of PCR product present in empty-vector-infected MEFs was set to 1. (c) LTβR-dependent gene transcription is impaired in cells expressing constitutively nuclear p100(4×NES). MEFs were infected with retroviruses expressing empty vector, p100, p100(Ser707/711Ala), p100(4×NES) or p100(4×NES; Ser707/711Ala) and stimulated with agonistic anti-LTβR antibodies. After 12 h of stimulation, levels of the indicated mRNAs were determined by quantitative PCR (±s.d., n = 3) and normalized to the transcriptional activation measured in cells infected with empty vector. (d) LTβR-dependent binding of RelB to NF-κB elements is impaired in cells expressing p100(Ser707/711Ala) or p100(4×NES). MEFs were infected with retroviruses expressing p100, p100(Ser707/711Ala) or p100(4×NES) and stimulated with agonistic anti-LTβR antibodies. DNA precipitated with an anti-RelB antibody was amplified by real-time PCR using primers flanking the indicated gene promoters (±s.d., n = 3). The LTβR-unresponsive promoter of IL-6 was used as a negative control. The value for the amount of PCR product present in MEFs infected with wild-type p100 was set as 1.
Figure 5
Figure 5
p100 degradation promotes multiple myeloma cell survival. (a) In B-cell lines, p100 is cytoplasmic, whereas Fbxw7α is largely nuclear. Subcellular fractionation was carried out on HMMCLs (ARP-1, KMS11, MM1.S and U266B1), DLBCL (Ly10, SudHL6 and Farage) and Burkitt’s lymphoma (Raji) cell lines. Lysates were immunoblotted as indicated. IκBα and α-tubulin, cytoplasmic controls. c-Myc, nuclear control. (b) Stable p100(Ser707/711Ala) and p100(4×NES) are correctly processed in HMMCLs. HMMCLs were infected with retroviruses encoding HA-tagged p100, p100(Ser707/711Ala), p100(4×NES) or empty vector (EV). Cell extracts were immunoblotted as indicated. Short exposure (s.e.) and long exposure (l.e.) are shown. (c) Expression of stable p100 mutant impairs HMMCL growth. HMMCLs were infected with retroviruses expressing p100, p100(Ser707/711Ala) or empty vector. Cell proliferation was monitored by MTS assay and normalized on empty vector at day 1, arbitrarily set as 100%. Error bars represent s.d., n = 4. (d) Stable p100 expression impairs NFκB-dependent transcription in HMMCLs. HMMCLs were infected as in c. Steady-state levels of the indicated mRNAs were analysed by quantitative PCR (±s.d., n = 3). PCR product amount in empty vector was set as 1. (e) Expression of stable p100 promotes apoptosis of HMMCLs. HMMCLs were infected as in c. At 72 h post-infection, cells were stained with Annexin V/7-AAD and analysed by flow cytometry. Apoptosis and cell viability were calculated as the percentage of Annexin V/7-AAD double-positive and double-negative cells, respectively. (f) Forced nuclear localization of p100 inhibits HMMCLs proliferation. HMMCLs were infected with retroviruses encoding p100, p100(4×NES) mutant or empty vector. Cell viability was monitored as in c. Values were normalized on the empty vector at time 0. Error bars represent s.d., n = 4. (g) Fbxw7 depletion impairs HMMCL growth. HMMCLs were infected with the indicated shRNA-encoding lentiviruses. Cell viability was monitored as in c (left panels). Values were normalized on Luc shRNA at time 0. Error bars represent s.d., n = 4. Fbxw7 protein levels were analysed by immunoblotting (right panel). (h) Fbxw7 depletion stabilizes nuclear p100. HMMCLs were infected as in g. Total (T), cytoplasmic (C) and nuclear (N) fractions were immunoblotted as indicated. IκBα and Lamin A/C are cytoplasmic and nuclear markers, respectively. Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 6
Figure 6
Expression of a stable p100 mutant impairs the growth of HMMCLs xenotransplanted into immunodeficient mice. (a) KMS11 cells were infected with retroviruses expressing either p100 or p100(Ser707/711Ala) and a firefly luciferase reporter, before intraperitoneal injection of SCID Beige mice. Cell growth was monitored by in vivo imaging. (b) Bioluminescence quantification of a with Living Image software (Xenogen). Error bars represent s.d., n = 3. (c) KMS11 cells were infected with retroviruses expressing a firefly luciferase reporter and an empty vector (EV), p100 or p100(Ser707/711Ala). Three days after infection, cells were injected subcutaneously into SCID Beige mice. Cell growth was monitored by in vivo imaging at 7, 14 and 28 days following injection. Bioluminescence was quantified with Living Image software (Xenogen).
Figure 7
Figure 7
Pharmacologic inhibition of GSK3, Cullin–RING ligases or the proteasome impairs the growth of multiple myeloma cells. (a) GSK3 inhibition induces nuclear accumulation of p100 in HMMCLs. KMS11 and ARP-1 cells were treated with GSK3i IX (2 μM for 8 h). Total (T), cytoplasmic (C) and nuclear (N) fractions were isolated and analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and c-Myc, respectively, are also shown. (b) Proteasome or Cullin–RING ligase inhibitors induce nuclear accumulation of p100 in HMMCLs. KMS11 cells were treated with either bortezomib or MLN4924 for 4 h. Total, cytoplasmic and nuclear fractions were analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and c-Myc, respectively, are also shown. (c) NF-κB-dependent transcription is impaired in HMMCLs treated with a GSK3 inhibitor. KMS11 cells were treated with GSK3i IX and the steady-state levels of the indicated mRNAs were analysed by real-time PCR (±s.d., n = 3). The value for the amount of PCR product present in DMSO-treated cells was set as 1. (d) Inhibition of GSK3, Cullin–RING ligases or the proteasome decreases the viability of HMMCLs. KMS11, MM1.R and ARP-1 cells were treated with increasing concentrations of GSK3i IX, MLN4924 or bortezomib. Cell viability was measured by MTS assay at 72 h after treatment and individually normalized to untreated cells (0 nM), arbitrarily set as 100%. Error bars represent s.d., n = 4. (e) Next-generation GSK3 inhibitors induce accumulation of nuclear p100 in HMMCLs. KMS11 cells were treated with GSK3i XVI or GSK3i XXII (5 μM and 1 μM for 8 h, respectively). Total, cytoplasmic and nuclear fractions were isolated and analysed by immunoblotting as indicated. Cytoplasmic and nuclear markers, IκBα and Lamin A/C, respectively, are also shown. (f) Next-generation GSK3 inhibitors decrease the viability of HMMCLs. KMS11, MM1.R and ARP-1 cells were treated with increasing concentrations of GSK3i XVI or GSK3i XXII. Cell viability was measured by MTS assay at 72 h after treatment. Each value was individually normalized on the DMSO-treated cells, arbitrarily set as 100%. Error bars represent s.d., n = 4. Uncropped images of blots are shown in Supplementary Fig. S8.
Figure 8
Figure 8
p100 contributes to the sensitivity of multiple myeloma cells to GSK3 inhibition. (a) Efficient knockdown of p100 in HMMCLs. ARP-1 cells were infected with lentiviruses encoding shRNAs targeting p100 or a control shRNA against LacZ. Cell extracts were then subjected to immunoblotting as indicated. (b) p100 knockdown desensitized ARP-1 cells to GSK3i IX. ARP-1 cells were infected with the indicated lentiviruses and treated with increasing concentrations of GSK3i IX, MLN4924, bortezomib or dexamethasone. Cell viability was measured by MTS assay at 72 h after treatment and normalized to the untreated LacZ shRNA (0 nM), arbitrarily set as 100%. Error bars represent s.d., n = 4. Uncropped images of blots are shown in Supplementary Fig. S8.
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