title: Defining the molecular basis of Arf and Hdm2 interactions.
abstract: Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and 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 limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.
authors.affiliation: Department of Structural Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA.
authors.forename: B
authors.initials: B
authors.lastname: Bothner
authors.forename: W S
authors.initials: WS
authors.lastname: Lewis
authors.forename: E L
authors.initials: EL
authors.lastname: DiGiammarino
authors.forename: J D
authors.initials: JD
authors.lastname: Weber
authors.forename: S J
authors.initials: SJ
authors.lastname: Bothner
authors.forename: R W
authors.initials: RW
authors.lastname: Kriwacki
mesh_terms: D016475:3T3 Cells; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002466:Cell Nucleolus / Q000737:chemistry / Q000378:metabolism; D002942:Circular Dichroism; D019941:Cyclin-Dependent Kinase Inhibitor p16; D017353:Gene Deletion; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011518:Proto-Oncogene Proteins / Q000737:chemistry* / Q000378:metabolism*; D051736:Proto-Oncogene Proteins c-mdm2; D016415:Sequence Alignment; D020349:Surface Plasmon Resonance; D025803:Tumor Suppressor Protein p14ARF / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: C417743:Cdkn2a protein, mouse; D019941:Cyclin-Dependent Kinase Inhibitor p16; D009687:Nuclear Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D025803:Tumor Suppressor Protein p14ARF; D016159:Tumor Suppressor Protein p53; C497206:MDM2 protein, human; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2