p14ARF interacts with DAXX: effects on HDM2 and p53.

The p14(ARF) (ARF) tumour suppressor plays an important role in the cellular response to oncogene activation. In this report, we demonstrate an interaction between ARF and DAXX, a highly conserved protein with identified roles in the regulation of gene expression. HDM2 was shown to interact with each of ARF and DAXX upon upregulation of expression as well as at lower expression levels following transfection of ARF and DAXX. Through immunofluorescence analysis, we observed that endogenous ARF and DAXX colocalize both to nucleoli and to nuclear bodies in cell lines that co-express both proteins. Similar results were obtained upon co-transfection of ARF and DAXX. Co-expression of ARF and DAXX was further found to inhibit ARF-mediated HDM2 sumoylation and to induce sumoylation and ubiquitination of DAXX itself, implicating DAXX as a substrate of ARF-mediated post-translational events. We also observed induction of p53 sumoylation in the presence of ARF and DAXX, an effect that was inhibited by upregulation of HDM2 expression. In summary, we have identified DAXX as a novel ARF binding partner and substrate of ARF-mediated sumoylation and suggest that DAXX acts as a modifier of both p53-dependent and p53-independent ARF function.