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Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus

Nature Cell Biology volume 3, pages 445–452 (2001)Cite this article

Abstract

The alternative product of the human INK4a/ARF locus, p14ARF, has the potential to act as a tumour suppressor by binding to and inhibiting the p53 antagonist MDM2. Current models propose that ARF function depends on its ability to sequester MDM2 in the nucleolus. Here we describe situations in which stabilization of MDM2 and p53 occur without relocalization of endogenous MDM2 from the nucleoplasm. Conversely, forms of ARF that do not accumulate in the nucleolus retain the capacity to stabilize MDM2 and p53. We therefore propose that nucleolar localization is not essential for ARF function but may enhance the availability of ARF to inhibit MDM2.

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Figure 1: Time dependence of p14ARF expression and function.
Figure 2: Localization of endogenous MDM2 and p53 after induction of p14ARF.
Figure 3: Induction of endogenous ARF in primary human fibroblasts.
Figure 4: Association of ARF, MDM2 and p53 in NARF cells.
Figure 5: Distribution of p14ARF and MDM2 in different subnuclear fractions.
Figure 6: Localization and function of the N- and C-terminal halves of p14ARF.
Figure 7: Context-dependence of nucleolar-localization signals in p14ARF.
Figure 8: Stabilization of MDM2 and p53 by non-nucleolar ARF.

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References

  1. Ruas, M. & Peters, G. The p16INK4a/CDKN2A tumor suppressor and its relatives. Biochim. Biophys. Acta 1378, 115–177 (1998).

    Google Scholar 

  2. Sherr, C. J. Tumor surveillance via the ARF-p53 pathway. Genes Dev. 12, 2984–2991 (1998).

    Article  CAS  Google Scholar 

  3. Sherr, C. J. & Roberts, J. M. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev. 13, 1501–1512 (1999).

    Article  CAS  Google Scholar 

  4. James, M. C. & Peters, G. Alternative product of the p16/CDKN2A locus connects the Rb and p53 tumor suppressors. Prog. Cell Cycle Res. 4, 71–81 (2000).

    Article  CAS  Google Scholar 

  5. Sharpless, N. E. & DePinho, R. A. The INK4A/ARF locus and its two gene products. Curr. Opin. Genet. Dev. 9, 22–30 (1999).

    Article  CAS  Google Scholar 

  6. Kamijo, T. et al. Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2. Proc. Natl Acad. Sci. USA 95, 8292–8297 (1998).

    Article  CAS  Google Scholar 

  7. Pomerantz, J. et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53. Cell 92, 713–723 (1998).

    Article  CAS  Google Scholar 

  8. Stott, F. J. et al. The alternative product from the human CDKN2A locus, p14ARF, participates in a regulatory feedback loop with p53 and MDM2. EMBO J. 17, 5001–5014 (1998).

    Article  CAS  Google Scholar 

  9. Zhang, Y., Xiong, Y. & Yarbrough, W. G. ARF promotes MDM2 degradation and stabilizes p53:ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways. Cell 92, 725–734 (1998).

    Article  CAS  Google Scholar 

  10. Honda, R. & Yasuda, H. Association of p19ARF with Mdm2 inhibits ubiquitin ligase activity of Mdm2 for tumour suppressor p53. EMBO J. 18, 22–27 (1999).

    Article  CAS  Google Scholar 

  11. Midgley, C. A. et al. An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo. Oncogene 19, 2312–2323 (2000).

    Article  CAS  Google Scholar 

  12. DeGregori, J., Leone, G., Miron, A., Jakoi, L. & Nevins, J. R. Distinct roles for E2F proteins in cell growth control. Proc. Natl Acad. Sci. USA 94, 7245–7250 (1997).

    Article  CAS  Google Scholar 

  13. Bates, S. et al. p14ARF links the tumour suppressors RB and p53. Nature 395, 124–125 (1998).

    Article  CAS  Google Scholar 

  14. Lohrum, M. A. E., Ashcroft, M., Kubbutat, M. H. G. & Vousden, K. H. Contribution of two independent MDM2-binding domains in p14ARF to p53 stabilization. Curr. Biol. 10, 539–542 (2000).

    Article  CAS  Google Scholar 

  15. Weber, J. D. et al. Cooperative signals governing ARF-MDM2 interaction and nucleolar localization of the complex. Mol. Cell. Biol. 20, 2517–2528 (2000).

    Article  CAS  Google Scholar 

  16. Quelle, D. E., Zindy, F., Ashmun, R. A. & Sherr, C. J. Alternative reading frames of the INK4a tumor suppressor gene encode two unrelated proteins capable of inducing cell cycle arrest. Cell 83, 993–1000 (1995).

    Article  CAS  Google Scholar 

  17. Weber, J. D., Taylor, L. J., Roussel, M. F., Sherr, C. J. & Bar-Sagi, D. Nucleolar Arf sequesters Mdm2 and activates p53. Nature Cell Biol. 1, 20–26 (1999).

    Article  CAS  Google Scholar 

  18. Zhang, Y. & Xiong, Y. Mutations in human ARF exon 2 disrupt its nucleolar localization and impair its ability to block nuclear export of MDM2 and p53. Mol. Cell 3, 579–591 (1999).

    Article  CAS  Google Scholar 

  19. Lindström, M. S. et al. Immunolocalization of human p14ARF to the granular component of the interphase nucleus. Exp. Cell Res. 256, 400–410 (2000).

    Article  Google Scholar 

  20. Rizos, H., Darmanian, A. P., Mann, G. J. & Kefford, R. F. Two arginine rich domains in the p14ARF tumour suppressor mediate nucleolar localization. Oncogene 19, 2978–2985 (2000).

    Article  CAS  Google Scholar 

  21. Roth, J., Dobbelstein, M., Freedman, D. A., Shenk, T. & Levine, A. J. Nucleo–cytoplasmic shuttling of the hdm2 oncoprotein regulates the levels of the p53 protein via a pathway used by the human immunodeficiency virus rev protein. EMBO J. 17, 554–564 (1998).

    Article  CAS  Google Scholar 

  22. Freedman, D. A. & Levine, A. J. Nuclear export is required for degradation of endogenous p53 by MDM2 and human papillomavirus E6. Mol. Cell. Biol. 18, 7288–7293 (1998).

    Article  CAS  Google Scholar 

  23. Tao, W. & Levine, A. J. Nucleocytoplasmic shuttling of oncoprotein Hdm2 is required for Hdm2-mediated degradation of p53. Proc. Natl Acad. Sci. USA 96, 3077–3080 (1999).

    Article  CAS  Google Scholar 

  24. Tao, W. & Levine, A. J. p19ARF stabilizes p53 by blocking nucleo-cytoplasmic shuttling of Mdm2. Proc. Natl Acad. Sci. USA 96, 6937–6941 (1999).

    Article  CAS  Google Scholar 

  25. Diller, L. et al. p53 functions as a cell cycle control point in osteosarcoma. Mol. Cell. Biol. 10, 5772–5781 (1990).

    Article  CAS  Google Scholar 

  26. Dimri, G. P., Itahana, K., Acosta, M. & Campisi, J. Regulation of a senescence checkpoint response by the E2F1 transcription factor and p14ARF tumor suppressor. Mol. Cell. Biol. 20, 273–285 (2000).

    Article  CAS  Google Scholar 

  27. Kurokawa, K., Tanaka, T. & Kato, J. p19ARF prevents G1 cyclin-dependent kinase activation by interacting with MDM2 and activating p53 in mouse fibroblasts. Oncogene 18, 2718–2727 (1999).

    Article  CAS  Google Scholar 

  28. Ochs, R. L. Methods used to study structure and function of the nucleolus. Methods Cell Biol. 53, 303–321 (1998).

    Article  CAS  Google Scholar 

  29. Quelle, D. E., Cheng, M., Ashmun, R. A. & Sherr, C. J. Cancer-associated mutations at the INK4a locus cancel cell cycle arrest by p16INK4a but not by the alternative reading frame protein p19ARF. Proc. Natl Acad. Sci. USA 94, 669–673 (1997).

    Article  CAS  Google Scholar 

  30. Johnston, J. A., Ward, C. L. & Kopito, R. R. Aggresomes: a cellular response to misfolded proteins. J. Cell Biol. 143, 1883–1898 (1998).

    Article  CAS  Google Scholar 

  31. García-Mata, R., Bebök, Z., Sorscher, E. J. & Sztul, E. S. Characterization and dynamics of aggresome formation by a cytosolic GFP-chimera. J. Cell Biol. 146, 1239–1254 (1999).

    Article  Google Scholar 

  32. Stommel, J. M. et al. A leucine-rich nuclear export signal in the p53 tetramerization domain: regulation of subcellular localization and p53 activity by NES masking. EMBO J. 18, 1660–1672 (1999).

    Article  CAS  Google Scholar 

  33. Lohrum, M. A. E., Ashcroft, M., Kubbutat, M. H. G. & Vousden, K. H. Identification of a cryptic nucleolar-localization signal in MDM2. Nature Cell Biol. 2, 179–181 (2000).

    Article  CAS  Google Scholar 

  34. Marechal, V., Elenbaas, B., Piette, J., Nicolas, J-C. & Levine, A. J. The ribosomal L5 protein is associated with mdm-2 and mdm-2–p53 complexes. Mol. Cell. Biol. 14, 7414–7420 (1994).

    Article  CAS  Google Scholar 

  35. McConnell, B. B., Starborg, M., Brookes, S. & Peters, G. Inhibitors of cyclin-dependent kinases induce features of replicative senescence in early passage human diploid fibroblasts. Curr. Biol. 8, 351–354 (1998).

    Article  CAS  Google Scholar 

  36. Dalton, S. & Treisman, R. Characterization of SAP-1, a protein recruited by serum response factor to the c-fos serum response element. Cell 68, 597–612 (1992).

    Article  CAS  Google Scholar 

  37. Bates, S. et al. CDK6 (PLSTIRE) and CDK4 (PSK-J3) are a distinct subset of the cyclin-dependent kinases that associate with cyclin D1. Oncogene 9, 71–79 (1994).

    CAS  Google Scholar 

  38. Kubbutat, M. H. G., Jones, S. N. & Vousden, K. H. Regulation of p53 stability by Mdm2. Nature 287, 299–303 (1997).

    Article  Google Scholar 

Download references

Acknowledgements

We thank K. Vousden for materials and for communicating data before publication, and M. Fried, M. Owen and D. Ish-Horowicz for discussions and comments on the manuscript. S.L. was the recipient of a long-term EMBO fellowship.

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  1. Imperial Cancer Research Fund, 44 Lincoln's Inn Fields, London, WC2A 3PX, UK

    Susana Llanos, Paula A. Clark, Janice Rowe & Gordon Peters

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Correspondence to Gordon Peters.

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Llanos, S., Clark, P., Rowe, J. et al. Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus. Nat Cell Biol 3, 445–452 (2001). https://doi.org/10.1038/35074506

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