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  • Original Paper
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An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo

Oncogene volume 19, pages 2312–2323 (2000)Cite this article

Abstract

The p53 tumour suppressor protein is down-regulated by the action of Mdm2, which targets p53 for rapid degradation by the ubiquitin-proteasome pathway. The p14ARF protein is also a potent tumour suppressor that acts by binding to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing. We have screened a series of overlapping synthetic peptides derived from the p14ARF protein sequence and found that a peptide corresponding to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2. The binding site for Peptide 3 on Mdm2 was determined by deletion mapping and lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which on microinjection into cells can activate p53-dependent transactivation of a reporter plasmid. To determine whether Peptide 3 could similarly activate p53, we expressed a fusion of green fluorescent protein and Peptide 3 in MCF7 and U-2 OS cells and were able to demonstrate induction of p53 protein and p53-dependent transcription. Peptide 3 was able to block in vitro ubiquitination of p53 mediated by Mdm2. Small peptides which are sufficient to block degradation of p53 could provide therapeutic agents able to restore p53-dependent cell death pathways in tumours that retain wild-type p53 expression.

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Acknowledgements

This work was funded by the Cancer Research Campaign. JMP Desterro was supported by the BBSRC and JNICTPraxis XXI (Portugal). DP Lane is a Gibb Fellow of the CRC.

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Authors and Affiliations

  1. Department of Biochemistry, Cancer Research Campaign Cell Transformation Group, University of Dundee, WTB/MSI Building, Dow Street, Dundee, DD1 5EH, Scotland, UK

    Carol A Midgley, Mark K Saville, Stephanie Howard, Alison Sparks & David P Lane

  2. School of Biomedical Science, BMS Building, University of St Andrews, Fife, KY16 9TS, St Andrews, Scotland, UK

    Joana MP Desterro & Ronald T Hay

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  1. Carol A Midgley
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  2. Joana MP Desterro
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Midgley, C., Desterro, J., Saville, M. et al. An N-terminal p14ARF peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo. Oncogene 19, 2312–2323 (2000). https://doi.org/10.1038/sj.onc.1203593

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