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

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.
Mesh Terms:
Amino Acid Sequence, Antibodies, Monoclonal, Base Sequence, Binding Sites, Gene Expression Regulation, Neoplastic, Green Fluorescent Proteins, Humans, Ligases, Luminescent Proteins, Microinjections, Molecular Sequence Data, Nuclear Proteins, Peptide Fragments, Peptide Mapping, Proteins, Proto-Oncogene Proteins, Proto-Oncogene Proteins c-mdm2, Recombinant Fusion Proteins, Tumor Cells, Cultured, Tumor Suppressor Protein p14ARF, Tumor Suppressor Protein p53, Ubiquitin-Conjugating Enzymes, Ubiquitin-Protein Ligases, Ubiquitins
Oncogene
Date: May. 04, 2000
Download Curated Data For This Publication
116600
Switch View:
  • Interactions 1