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An intact HDM2 RING-finger domain is required for nuclear exclusion of p53

Nature Cell Biology volume 2, pages 563–568 (2000)Cite this article

Abstract

The p53 tumour-suppressor protein is negatively regulated by HDM2. Recent reports indicate that the leucine-rich nuclear-export sequence (NES) of HDM2 enables it to shuttle to the cytoplasm, and that this activity is required for degradation of p53. However, it is unclear whether HDM2 is involved in nuclear export of p53, partly because p53 has itself been shown to contain a functional NES within its tetramerization domain. Here we show that co-expression of HDM2 with green fluorescent protein (GFP)-tagged p53 causes redistribution of p53 from the nucleus to the cytoplasm of the cell. This activity is dependent on binding of p53 to HDM2, and requires an intact p53 NES, but is independent of the HDM2 NES. A mutant of the HDM2 RING-finger domain that is unable to ubiquitinate p53 does not cause relocalization of p53, indicating that ubiquitin ligation or other activities of this region of HDM2 may be necessary for its regulation of p53 localization.

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Figure 1: Co-transfection of p53–GFP and HDM2 in NIH/3T3 fibroblasts and U2OS osteosarcoma cells.
Figure 2: Co-transfection of GFP–p53(L22Q,W23S) and GFP–p53(NES–) with HDM2.
Figure 3: Co-expression of HDM2(NES–) with p53–GFP.
Figure 4: Co-expression of HDM2(C464A) with wild-type p53–GFP.
Figure 5: HDM2 cannot relocalize p53–GFP in ts20 cells with an inactivated E1 ubiquitin-charging enzyme.

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Acknowledgements

We thank B. Vogelstein, J. Lin, J. Xiao and A. Schwartz for plasmids, and H. Ozer for providing ts20 cells. We also thank H. Schwartz for reading the manuscript and S. Joshi for helpful comments. T.J. is an Associate Investigator of the Howard Hughes Medical Institute. K.Y.T. is supported by the Medical Scientist Training Program. S.D.B. is a Predoctoral Fellow of the Howard Hughes Medical Institute.

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  1. Department of Biology Massachusetts Institute of Technology, MIT Center for Cancer Research, Massachusetts Avenue , Cambridge, 02139, USA

    Scott D. Boyd, Kenneth Y. Tsai & Tyler Jacks

  2. Harvard-MIT Division of Health Sciences Technology, Cambridge, 02139, Massachusetts, USA

    Scott D. Boyd & Kenneth Y. Tsai

  3. Howard Hughes Medical Institute,

    Scott D. Boyd & Tyler Jacks

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Correspondence to Tyler Jacks.

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Boyd, S., Tsai, K. & Jacks, T. An intact HDM2 RING-finger domain is required for nuclear exclusion of p53. Nat Cell Biol 2, 563–568 (2000). https://doi.org/10.1038/35023500

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