An N-terminal p14(ARF) peptide blocks Mdm2-dependent ubiquitination in vitro and can activate p53 in vivo

CA Midgley, JM Desterro, S Howard, A Sparks, Ronald Thomas Hay, DP Lane

Research output: Contribution to journalArticlepeer-review

224 Citations (Scopus)

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 p14(ARF) 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 p14(ARF) 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.

Original languageEnglish
Pages (from-to)2312-2323
Number of pages12
JournalOncogene
Volume19
Issue number19
DOIs
Publication statusPublished - 4 May 2000

Keywords

  • p53 tumour suppressor
  • Mdm2
  • p14(ARF)
  • ubiquitination
  • protein stability
  • transcriptional activation
  • TUMOR-SUPPRESSOR P53
  • CELL-CYCLE ARREST
  • NUCLEAR EXPORT
  • DNA-BINDING
  • EMBRYONIC LETHALITY
  • MDM2-DEFICIENT MICE
  • ONCOPROTEIN MDM2
  • MAMMALIAN-CELLS
  • STABILIZES P53
  • HUMAN SARCOMAS

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