Defining the molecular basis of Arf and Hdm2 interactions

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Ar f) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and su bsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that eith er p53 or p14(Arf) are lost or modified in virtually all human cancers. Iso lated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon bindi ng, domains of both Arf and Hdm2 undergo ad structures dramatic transition from disordered conformations to extended comprised of beta -strands. The p resence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sit es within Arf and Hdm2 that interact at a resolution of five amino acid res idues using surface plasmon resonance. Surface plasmon resonance and circul ar dichroism spectropolarimetry confirm the presence of multiple interactio n domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) i nteract with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences loca ted in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 1 5 amino acids in length, that contain the binding motifs. Micro-injection a nd live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the l imited size of the protein domains involved provide opportunities for the d evelopment of anticancer therapeutics. (C) 2001 Academic Press.