Arf is a tumor suppressor that regulates p53 and is a frequent target for loss in human cancers. Through two novel mechanisms, Arf inhibits the oncoprotein Hdm2, which is itself a negative regulator of p53. (1) Arf sequesters Hdm2 within nucleoli and (2) Arf inhibits Hdm2-dependent E3-like ubiquitination of p53. These activities promote p53-mediated cell cycle arrest and apoptosis and are dependent on interactions between Arf and Hdm2. We will identify the domains responsible for Arf/Hdm2 interactions and study them using structural techniques. We will prepare a series of Hdm2 fragments and Arf peptides and monitor their interactions using surface plasmon resonance and circular dichroism spectroscopy. Further, we will establish assays for both Arf functions and determine the functional properties of our domains to correlate their roles in molecular interaction and function. We hypothesize that allosteric interactions between the central acidic domain and the RING domain of Hdm2 regulate both nucleolar localization and E3 activity and that Arf disrupts these interactions by binding the central Hdm2 domain. We will use the above techniques to characterize domain/domain interactions to test this model. Also, we will determine high-resolution solution structures for individual Arf and Hdm2 domains as well as their biologically relevant complexes using NMR spectroscopy. These structures will reveal the molecular basis for Arf/Hdm2 interactions and for Arf-dependent regulation of Hdm2. Since structural information for these domains is not yet available, these studies are poised to contribute novel insights into the molecular mechanisms of this unusual tumor suppressor system. Finally, Arf also functions through a p53-independent mechanism to produce cell cycle arrest. We hypothesize that Arf interacts with other nuclear proteins to produce this effect and will extend our molecular studies to these putative Arf targets; importantly, novel targets have already been identified.
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