Multiple interacting domains contribute to p14ARF mediated inhibition of MDM2

The small basic protein p14(Arf), encoded by one of the alternative transcripts from the human INK4A/ARF locus, interferes with MDM2-mediated ubiquitination of the p53 tumour suppressor protein. The resultant stabilization of p53 leads to increased expression of p53-regulated genes, such as MDM2 itself and the cyclin-dependent kinase inhibitor p21(CIP1). Here we relate physical interactions between p][4111 and MDM2, as determined using synthetic peptides and systematic deletions of p14(Arf), with consequential effects on p53 stabilization and transcriptional activity. The data imply that the amino terminal half of p14(Arf), encoded by the alternative first exon (exon 1beta) contacts MDM2 through multiple domains that can independently impede MDM2-mediated degradation of p53, provided that they are localized in the cell nucleus. As well as identifying previously unrecognized functional domains, our findings offer an explanation for the relative paucity of missense mutations in exon 1beta in human tumours.