In this study, we show that Fbxw7α promotes degradation of the NF-κB inhibitor protein p100. Phosphorylation of p100 on Ser 707 and Ser 711 by GSK3 is a prerequisite for the p100–Fbxw7α interaction. Although predominantly cytoplasmic, p100 shuttles continuously between the cytoplasm and the nucleus. We show that p100 degradation by Fbxw7α and GSK3 occurs in the nucleus, independently from NF-κB activation. However, although independent from NF-κB signalling, p100 clearance from the nucleus (through Fbxw7α-mediated degradation or nuclear exclusion by a newly identified nuclear export signal in the C-terminus of p100) is a prerequisite for binding of RelB at the promoters of target genes and efficient activation of NF-κB-dependent gene transcription. In fact, deletion of the Fbxw7
gene, inhibition of GSK3, expression of a stable p100 mutant or expression of a constitutively nuclear p100 mutant attenuates the NF-κB response. The function of Fbxw7α in the NF-κB response is consistent with mouse genetic data showing that conditional knockout of Fbxw7
in haematopoietic stem cells induces a significant decrease in the number of both immature and mature B cells41
, which rely on BAFF-mediated NF-κB signalling for homeostasis42
. Moreover, in agreement with our findings, fibroblasts from GSK3
-deficient embryos exhibit reduced activation of NF-κB signalling43
Whereas phosphorylation of p100 on Ser 707 is constitutive, phosphorylation of p100 on Ser 866/870 is induced by NF-κB stimuli, leading to p100 cleavage into p52 (refs 23,24
). Accordingly, p52 can be generated from p100(Ser707/711Ala), but not from a p100(Ser866/870) mutant. Interestingly, cytoplasmic p100 is phosphorylated on both Ser 707 and Ser 866/870, whereas nuclear p100 is phosphorylated only on Ser 707. Furthermore, p100 phosphorylated on Ser 707 binds only nuclear Fbxw7α, whereas p100 phosphorylated on Ser 866/870 binds only βTrCP. Thus, differentially localized and modified fractions of p100 undergo distinct regulation by the ubiquitin proteasome system.
From yeast to mammals, inducible phosphorylation is the dominant mechanism controlling the recruitment of substrates to F-box proteins2,5
. As phosphorylation of the p100 degron is constitutive, we investigated how Fbxw7α-mediated proteolysis of p100 is regulated. We found that NF-κB proteins are able to induce p100 stabilization through Fbxw7α displacement. These observations indicate that the nuclear accumulation of NF-κB proteins (p52 and RelB) induced by NF-κB signalling may lead to p100 stabilization through competition with Fbxw7α. Thus, we propose a control mechanism, in which recognition of a substrate by an F-box protein is negatively regulated by accumulation of the substrate’s interacting partners, physically disrupting the ligase–substrate interaction.
The mechanism of p100 degradation has implications for B-cell malignancies. Constitutive activation of the NF-κB pathway has been found in many lymphoid diseases and contributes to the malignant transformation of B-cell lineages, including plasma cells27–30
. Pharmacologic inhibition of the proteasome28
or Cullin–RING ligases44
induces apoptosis in multiple myeloma and B-cell lymphoma cell lines, partially by inhibiting NF-κB. Here, we have shown that inhibition of GSK3-mediated signalling (for example, by expressing a phospho-defective mutant of p100 or through pharmacologic inhibition of GSK3) impairs the survival of HMMCLs both in cell systems and xenotransplant models. Interestingly, the toxicity of GSK3 inhibition can be substantially reduced by knockdown of p100. Thus, the intersection of Fbxw7–GSK3–p100 may serve as a potential intervention point for the treatment of multiple myeloma. Moreover, it is likely that the effects observed in multiple myeloma may be generalized to other B-cell neoplasms, especially those addicted to NF-κB.
Together, our studies have revealed an unexpected pro-survival role for Fbxw7. A number of cancers, including T-ALL, breast cancers, cholangiocarcinoma, gastric adenocarcinoma and head and neck squamous carcinoma7,8,11,45
, often carry hemizygous or homozygous mutations in the FBXW7
gene. Most cancer-associated FBXW7
point mutations result in the accumulation of substrates (for example, Notch, c-Myc, cyclin E and Mcl1) due to reduced binding. Interestingly, mutations of Fbxw7 have not been detected in 38 multiple myeloma samples13
, 24 HMMCLs (L.B., W. M. Kuehl and M.P., unpublished results), 70 primary B-ALLs (I. Aifantis, personal communication, and ref. 12
), 20 B-CLLs (ref. 12
) and 13 DLBCLs (ref. 46
). Our findings indicate that, in contrast to T cells, B cells are uniquely dependent on Fbxw7 function for survival. Although characterized as a tumour suppressor, Fbxw7 therefore functions as a pro-survival gene in multiple myeloma through its control of NF-κB activity.