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

Nature Cell Biology volume 14, pages 375–385 (2012)Cite this article

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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. 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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Figure 1: p100 interacts with Fbxw7α through a conserved degron phosphorylated by GSK3.
Figure 2: Fbxw7α controls p100 stability in the nucleus.
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.
Figure 4: Clearance of p100 from the nucleus is crucial for non-canonical NF-κB signalling.
Figure 5: p100 degradation promotes multiple myeloma cell survival.
Figure 6: Expression of a stable p100 mutant impairs the growth of HMMCLs xenotransplanted into immunodeficient mice.
Figure 7: Pharmacologic inhibition of GSK3, Cullin–RING ligases or the proteasome impairs the growth of multiple myeloma cells.
Figure 8: p100 contributes to the sensitivity of multiple myeloma cells to GSK3 inhibition.

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Acknowledgements

The authors thank I. Aifantis, H. J. Cho, G. Franzoso, W. M. Kuehl and A. Ventura for reagents; B. Aranda-Orgilles for her contribution; and I. Aifantis, G. Franzoso, K. Nakayama, S. Reed and J. R. Skaar for critically reading the manuscript. M.P. is grateful to T. M. Thor for continuous support. This work was supported in part by grant 5P30CA016087-33 from the National Cancer Institute, a fellowship from the American Italian Cancer Foundation and NIH 5T32HL007151-33 to L.B., NIH T32 CA009161 grant to S.M. and grants from the National Institutes of Health (R01-GM057587, R37-CA076584 and R21-CA161108) and the Multiple Myeloma Research Foundation to M.P. M.P. is an Investigator with the Howard Hughes Medical Institute.

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  1. Christos A. Kyratsous

    Present address: Present address: Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, New York 10591, USA,

  2. Luca Busino and Scott E. Millman: These authors contributed equally to this work

Authors and Affiliations

  1. NYU Cancer Institute, New York University School of Medicine, 522 First Avenue, New York, New York 10016, SRB 1107, USA

    Luca Busino, Scott E. Millman, Luigi Scotto, Christos A. Kyratsous, Owen O’Connor & Michele Pagano

  2. Howard Hughes Medical Institute, USA

    Christos A. Kyratsous & Michele Pagano

  3. Department of Pathology, University of Michigan, Ann Arbor, Michigan 48109, USA

    Venkatesha Basrur & Kojo S. Elenitoba-Johnson

  4. Department of Chemistry and Biochemistry, Signaling Systems Laboratory, University of California, San Diego, La Jolla, California 92093, USA

    Alexander Hoffmann

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Contributions

L.B. conceived and directed the project. L.B. and S.E.M. designed and carried out most experiments. L.S. and O.O. helped with the mouse experiments. C.K. carried out the in vitro experiments shown in Fig. 1d,e and Supplementary Figs S2d and S5b. V.B. and K.S.E-J. carried out the mass spectrometry analysis of the Fbxw7α complex purified by L.B. A.H. provided constructs, cell lines and advice. M.P. coordinated the work and oversaw the results. L.B., S.M. and M.P. wrote the manuscript.

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Correspondence to Christos A. Kyratsous or Michele Pagano.

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Busino, L., Millman, S., Scotto, L. et al. Fbxw7α- and GSK3-mediated degradation of p100 is a pro-survival mechanism in multiple myeloma. Nat Cell Biol 14, 375–385 (2012). https://doi.org/10.1038/ncb2463

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