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

Oncogene. 2000 May 4;19(19):2312-23. doi: 10.1038/sj.onc.1203593.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Antibodies, Monoclonal / metabolism
  • Antibodies, Monoclonal / pharmacology
  • Base Sequence
  • Binding Sites
  • Gene Expression Regulation, Neoplastic
  • Green Fluorescent Proteins
  • Humans
  • Ligases / metabolism
  • Luminescent Proteins / genetics
  • Luminescent Proteins / metabolism
  • Microinjections
  • Molecular Sequence Data
  • Nuclear Proteins*
  • Peptide Fragments / genetics
  • Peptide Fragments / immunology
  • Peptide Fragments / metabolism*
  • Peptide Mapping
  • Proteins / genetics
  • Proteins / metabolism*
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / immunology
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins c-mdm2
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Tumor Cells, Cultured
  • Tumor Suppressor Protein p14ARF
  • Tumor Suppressor Protein p53 / drug effects
  • Tumor Suppressor Protein p53 / genetics
  • Tumor Suppressor Protein p53 / metabolism*
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases
  • Ubiquitins / metabolism*


  • Antibodies, Monoclonal
  • Luminescent Proteins
  • Nuclear Proteins
  • Peptide Fragments
  • Proteins
  • Proto-Oncogene Proteins
  • Recombinant Fusion Proteins
  • Tumor Suppressor Protein p14ARF
  • Tumor Suppressor Protein p53
  • Ubiquitins
  • Green Fluorescent Proteins
  • Ubiquitin-Conjugating Enzymes
  • MDM2 protein, human
  • Proto-Oncogene Proteins c-mdm2
  • Ubiquitin-Protein Ligases
  • Ligases