Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Original Article
  • Published:

Phosphorylation-dependent regulation of SCFFbx4 dimerization and activity involves a novel component, 14-3-3ɛ

Oncogene volume 30, pages 1995–2002 (2011)Cite this article

Subjects

Abstract

Fbx4 is an F-box constituent of Skp-Cullin-F-box (SCF) ubiquitin ligases that directs ubiquitylation of cyclin D1. Ubiquitylation of cyclin D1 requires phosphorylation of both cyclin D1 and Fbx4 by GSK3β. GSK3β-mediated phosphorylation of Fbx4 Ser12 during the G1/S transition regulates Fbx4 dimerization, which in turn governs Fbx4-driven E3 ligase activity. In esophageal carcinomas that overexpress cyclin D1, Fbx4 is subject to inactivating mutations that specifically disrupt dimerization, highlighting the biological significance of this regulatory mechanism. In an effort to elucidate the mechanisms that regulate dimerization, we sought to identify proteins that differentially bind to wild-type Fbx4 versus a cancer-derived dimerization-deficient mutant. We provide evidence that phosphorylation of Ser12 generates a docking site for 14-3-3ɛ. 14-3-3ɛ binds to endogenous Fbx4 and this association is impaired by mutations that target either Ser8 or Ser12 in Fbx4, suggesting that this N-terminal motif in Fbx4 directs its interaction with 14-3-3ɛ. Knockdown of 14-3-3ɛ inhibited Fbx4 dimerization, reduced SCFFbx4 E3 ligase activity and stabilized cyclin D1. Collectively, the current results suggest a model wherein 14-3-3ɛ binds to Ser12-phosphorylated Fbx4 to mediate dimerization and function.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1
Figure 2
Figure 3
Figure 4
Figure 5

Similar content being viewed by others

References

  • Aggarwal P, Lessie MD, Lin DI, Pontano L, Gladden AB, Nuskey B et al. (2007). Nuclear accumulation of cyclin D1 during S phase inhibits Cul4-dependent Cdt1 proteolysis and triggers p53-dependent DNA rereplication. Genes Dev 21: 2908–2922.

    Article  CAS  Google Scholar 

  • Aggarwal P, Vaites LP, Kim JK, Mellert H, Gurung B, Nakagawa H et al. (2010). Nuclear cyclin D1/CDK4 kinase regulates CUL4 expression and triggers neoplastic growth via activation of the PRMT5 methyltransferase. Cancer Cell 18: 329–340.

    Article  CAS  Google Scholar 

  • Alt JR, Cleveland JL, Hannink M, Diehl JA . (2000). Phosphorylation-dependent regulation of cyclin D1 nuclear export and cyclin D1-dependent cellular transformation. Genes Dev 14: 3102–3114.

    Article  CAS  Google Scholar 

  • Barbash O, Egan E, Pontano LL, Kosak J, Diehl JA . (2009). Lysine 269 is essential for cyclin D1 ubiquitylation by the SCF(Fbx4/alphaB-crystallin) ligase and subsequent proteasome-dependent degradation. Oncogene 28: 4317–4325.

    Article  CAS  Google Scholar 

  • Barbash O, Zamfirova P, Lin DI, Chen X, Yang K, Nakagawa H et al. (2008). Mutations in Fbx4 inhibit dimerization of the SCF(Fbx4) ligase and contribute to cyclin D1 overexpression in human cancer. Cancer Cell 14: 68–78.

    Article  CAS  Google Scholar 

  • Benzeno S, Lu F, Guo M, Barbash O, Zhang F, Herman JG et al. (2006). Identification of mutations that disrupt phosphorylation-dependent nuclear export of cyclin D1. Oncogene 25: 6291–6303.

    Article  CAS  Google Scholar 

  • Che XH, Chen H, Xu ZM, Shang C, Sun KL, Fu WN . (2010). 14–3–3epsilon contributes to tumour suppression in laryngeal carcinoma by affecting apoptosis and invasion. BMC Cancer 10: 306.

    Article  Google Scholar 

  • Chew EH, Poobalasingam T, Hawkey CJ, Hagen T . (2007). Characterization of cullin-based E3 ubiquitin ligases in intact mammalian cells--evidence for cullin dimerization. Cell Signal 19: 1071–1080.

    Article  CAS  Google Scholar 

  • Dhillon AS, Yip YY, Grindlay GJ, Pakay JL, Dangers M, Hillmann M et al. (2009). The C-terminus of Raf-1 acts as a 14–3–3-dependent activation switch. Cell Signal 21: 1645–1651.

    Article  CAS  Google Scholar 

  • Diehl JA . (2002). Cycling to cancer with cyclin D1. Cancer Biol Ther 1: 226–231.

    Article  CAS  Google Scholar 

  • Diehl JA, Cheng M, Roussel MF, Sherr CJ . (1998). Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization. Genes Dev 12: 3499–3511.

    Article  CAS  Google Scholar 

  • Diehl JA, Zindy F, Sherr CJ . (1997). Inhibition of cyclin D1 phosphorylation on threonine-286 prevents its rapid degradation via the ubiquitin-proteasome pathway. Genes Dev 11: 957–972.

    Article  CAS  Google Scholar 

  • Freed E, Symons M, Macdonald SG, McCormick F, Ruggieri R . (1994). Binding of 14–3–3 proteins to the protein kinase Raf and effects on its activation. Science 265: 1713–1716.

    Article  CAS  Google Scholar 

  • Ichimura T, Isobe T, Okuyama T, Takahashi N, Araki K, Kuwano R et al. (1988). Molecular cloning of cDNA coding for brain-specific 14–3–3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci USA 85: 7084–7088.

    Article  CAS  Google Scholar 

  • Konishi H, Nakagawa T, Harano T, Mizuno K, Saito H, Masuda A et al. (2002). Identification of frequent G(2) checkpoint impairment and a homozygous deletion of 14–3–3epsilon at 17p13.3 in small cell lung cancers. Cancer Res 62: 271–276.

    CAS  Google Scholar 

  • Korcheva VB, Levine J, Beadling C, Warrick A, Countryman G, Olson NR et al. (2010). Immunohistochemical and molecular markers in breast phyllodes tumors. Appl Immunohistochem Mol Morphol (e-pub ahead of print 10 October 2010).

  • Li Y, Hao B . (2010). Structural basis of dimerization-dependent ubiquitination by the SCF(Fbx4) ubiquitin ligase. J Biol Chem 285: 13896–13906.

    Article  CAS  Google Scholar 

  • Lin DI, Barbash O, Kumar KG, Weber JD, Harper JW, Klein-Szanto AJ et al. (2006). Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex. Mol Cell 24: 355–366.

    Article  CAS  Google Scholar 

  • Lin HK, Wang G, Chen Z, Teruya-Feldstein J, Liu Y, Chan CH et al. (2009). Phosphorylation-dependent regulation of cytosolic localization and oncogenic function of Skp2 by Akt/PKB. Nat Cell Biol 11: 420–432.

    Article  CAS  Google Scholar 

  • Liu D, Bienkowska J, Petosa C, Collier RJ, Fu H, Liddington R . (1995). Crystal structure of the zeta isoform of the 14–3–3 protein. Nature 376: 191–194.

    Article  CAS  Google Scholar 

  • Pignataro L, Sambataro G, Pagani D, Pruneri G . (2005). Clinico-prognostic value of D-type cyclins and p27 in laryngeal cancer patients: a review. Acta Otorhinolaryngol Ital 25: 75–85.

    CAS  Google Scholar 

  • Skaar JR, Pagano M . (2009). Control of cell growth by the SCF and APC/C ubiquitin ligases. Curr Opin Cell Biol 21: 816–824.

    Article  CAS  Google Scholar 

  • Suzuki H, Chiba T, Suzuki T, Fujita T, Ikenoue T, Omata M et al. (2000). Homodimer of two F-box proteins betaTrCP1 or betaTrCP2 binds to IkappaBalpha for signal-dependent ubiquitination. J Biol Chem 275: 2877–2884.

    Article  CAS  Google Scholar 

  • Thorson JA, Yu LW, Hsu AL, Shih NY, Graves PR, Tanner JW et al. (1998). 14–3–3 proteins are required for maintenance of Raf-1 phosphorylation and kinase activity. Mol Cell Biol 18: 5229–5238.

    Article  CAS  Google Scholar 

  • Tzivion G, Luo Z, Avruch J . (1998). A dimeric 14–3–3 protein is an essential cofactor for Raf kinase activity. Nature 394: 88–92.

    Article  CAS  Google Scholar 

  • Tzivion G, Shen YH, Zhu J . (2001). 14–3–3 proteins; bringing new definitions to scaffolding. Oncogene 20: 6331–6338.

    Article  CAS  Google Scholar 

  • van Heusden GP . (2009). 14–3–3 Proteins: insights from genome-wide studies in yeast. Genomics 94: 287–293.

    Article  CAS  Google Scholar 

  • van Heusden GP, Steensma HY . (2006). Yeast 14–3–3 proteins. Yeast 23: 159–171.

    Article  CAS  Google Scholar 

  • Wang J, Lou H, Pedersen CJ, Smith AD, Perez RG . (2009). 14–3–3zeta contributes to tyrosine hydroxylase activity in MN9D cells: localization of dopamine regulatory proteins to mitochondria. J Biol Chem 284: 14011–14019.

    Article  CAS  Google Scholar 

  • Waterman MJ, Stavridi ES, Waterman JL, Halazonetis TD . (1998). ATM-dependent activation of p53 involves dephosphorylation and association with 14–3–3 proteins. Nat Genet 19: 175–178.

    Article  CAS  Google Scholar 

  • Welcker M, Clurman BE . (2007). Fbw7/hCDC4 dimerization regulates its substrate interactions. Cell Div 2: 7.

    Article  Google Scholar 

  • Yaffe MB, Rittinger K, Volinia S, Caron PR, Aitken A, Leffers H et al. (1997). The structural basis for 14–3–3:phosphopeptide binding specificity. Cell 91: 961–971.

    Article  CAS  Google Scholar 

  • Zeng Z, Wang W, Yang Y, Chen Y, Yang X, Diehl JA et al. (2010). Structural basis of selective ubiquitination of TRF1 by SCFFbx4. Dev Cell 18: 214–225.

    Article  CAS  Google Scholar 

  • Zhang L, Wang H, Liu D, Liddington R, Fu H . (1997). Raf-1 kinase and exoenzyme S interact with 14–3–3zeta through a common site involving lysine 49. J Biol Chem 272: 13717–13724.

    Article  CAS  Google Scholar 

  • Zhang W, Koepp DM . (2006). Fbw7 isoform interaction contributes to cyclin E proteolysis. Mol Cancer Res 4: 935–943.

    Article  CAS  Google Scholar 

Download references

Acknowledgements

We wish to thank Margarita Romero for excellent technical assistance. This work was supported by a grant from the NIH (P01-CA098101) and a Leukemia & Lymphoma Scholar award (JAD).

Author information

Author notes

  1. O Barbash and E K Lee: These authors contributed equally to this work.

Authors and Affiliations

  1. The Leonard and Madlyn Abramson Family Cancer Research Institute and Cancer Center, University of Pennsylvania, Philadelphia, PA, USA

    O Barbash, E K Lee & J A Diehl

  2. Department of Cancer Biology, University of Pennsylvania, Philadelphia, PA, USA

    O Barbash, E K Lee & J A Diehl

Authors
  1. O Barbash
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. E K Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J A Diehl
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to J A Diehl.

Ethics declarations

Competing interests

The authors declare no conflict of interest.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Barbash, O., Lee, E. & Diehl, J. Phosphorylation-dependent regulation of SCFFbx4 dimerization and activity involves a novel component, 14-3-3ɛ. Oncogene 30, 1995–2002 (2011). https://doi.org/10.1038/onc.2010.584

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/onc.2010.584

Keywords

This article is cited by

Search

Advanced search

Quick links