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Identification of a cryptic nucleolar-localization signal in MDM2

Nature Cell Biology volume 2, pages 179–181 (2000)Cite this article

The tumour suppressor protein p53 is a potent inhibitor of cell growth, activating both cell-cycle arrest and apoptotic pathways in stressed cells1. One of the principal regulators of p53 function and stability is the MDM2 protein, which can target p53 for ubiquitin-dependent degradation by the proteasome, as well as regulating its own stability2. p53 is stabilized in response to stress through several mechanisms, including the expression of ARF (p14ARF in humans and p19ARF in mouse) in response to abnormal proliferative signals3. The ARF protein binds directly to MDM2 and blocks p53 degradation4,5,6,7 by inhibiting the E3 ubiquitin-ligase activity associated with MDM2 (ref. 8) and preventing nuclear export of MDM2 (refs 9, 10). The ability of ARF to inhibit MDM2 function is related to the localization of ARF to the nucleolus11, and nucleolar-localization signals (NoLSs) have been identified in ARF. Here we identify an NoLS in the carboxy-terminal region of human MDM2 that does not function in unstressed cells, but is necessary to cooperate with nucleolar-localization signals in p14ARF to allow relocalization of both proteins.

The nucleolar expression of MDM2 Δ222–437 in these ARF-null cells indicated that MDM2 may itself contain an NoLS, and examination of the sequences retained in the MDM2 Δ222–437 mutant revealed the presence of a stretch of basic residues within the C-terminal region that might function in this way (Fig. 1b). To test the ability of these sequences to function in nucleolar localization, we expressed a peptide corresponding to MDM2 residues 466–473 in the active-site loop of thioredoxin14. The resulting protein contained nuclear-localization signals derived from SV40 and a Myc-epitope tag, to facilitate detection. The thioredoxin protein expressed alone was localized to the nucleus and showed general nucleoplasmic staining, with nucleolar exclusion (Fig. 1c). However, introduction of MDM2 residues 466–473 resulted in the relocalization of this protein to the nucleolus, indicating that this highly conserved region of MDM2 can function as an NoLS.

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Figure 1: Nucleolar-localization signal in MDM2.
Figure 2: Subnuclear localization of p14ARF with MDM2 mutants.
Figure 3: Function of the MDM2 NoLS.

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Acknowledgements

We thank C. Sherr, J. Weber and G. Peters for advice and sharing unpublished results.

Correspondence and requests for materials should be addressed to K.H.V.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://www.nature.com/ncb) or as paper copy from the London editorial office of Nature Cell Biology.

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  1. Michael H. G. Kubbutat

    Present address: Institute of Molecular Medicine, Tumor Biology Center, Breisacher Strasse 117, 79106, Freiburg, Germany

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  1. NCI-FCRDC, Building 560, Room 22-96, West 7th Street, Frederick, 21702-1201, Maryland, USA

    Marion A. E. Lohrum, Margaret Ashcroft, Michael H. G. Kubbutat & Karen H. Vousden

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  1. Marion A. E. Lohrum
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Correspondence to Karen H. Vousden.

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Lohrum, M., Ashcroft, M., Kubbutat, M. et al. Identification of a cryptic nucleolar-localization signal in MDM2 . Nat Cell Biol 2, 179–181 (2000). https://doi.org/10.1038/35004057

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