An N-terminal p14(ARF) 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 p14(ARF) protein is also a potent tumour suppressor that acts by bind ing to Mdm2 and blocking Mdm2-dependent p53 degradation and transcriptional silencing, We have screened a series of overlapping synthetic peptides der ived from the p14(ARF) protein sequence and found that a peptide correspond ing to the first 20 amino acids of ARF (Peptide 3) could bind human Mdm2, T he binding site for Peptide 3 on Mdm2 was determined by deletion mapping an d lies adjacent to the binding site of the anti-Mdm2 antibody 2A10, which o n 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 MC F7 and U-2 OS cells and were able to demonstrate induction of p53 protein a nd p53-dependent transcription. Peptide 3 was able to block in vitro ubiqui tination of p53 mediated by Mdm2, Small peptides which are sufficient to bl ock degradation of p53 could provide therapeutic agents able to restore p53 -dependent cell death pathways in tumours that retain wild-type p53 express ion.