Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif
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ORIGINAL ARTICLE
Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif Erin G. Worrall & Bartosz Wawrzynow & Liam Worrall & Malcolm Walkinshaw & Kathryn L. Ball & Ted R. Hupp
Received: 10 February 2009 / Accepted: 25 March 2009 / Published online: 16 May 2009 # Springer-Verlag 2009
Abstract The tumor suppressor p53 has evolved a MDM2dependent feedback loop that promotes p53 protein degradation through the ubiquitin–proteasome system. MDM2 is an E3-RING containing ubiquitin ligase that catalyzes p53 ubiquitination by a dual-site mechanism requiring ligand occupation of its N-terminal hydrophobic pocket, which then stabilizes MDM2 binding to the ubiquitination signal in the DNA-binding domain of p53. A unique pseudo-substrate motif or “lid” in MDM2 is adjacent to its E. G. Worrall : T. R. Hupp CRUK p53 Signal Transduction Group, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR B. Wawrzynow : K. L. Ball CRUK Interferon and Cell Signaling Group, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR E. G. Worrall : B. Wawrzynow : K. L. Ball : T. R. Hupp (*) Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR e-mail: [email protected] L. Worrall Institute for Translational and Chemical Biology, University of Edinburgh, Edinburgh, Scotland, UK, EH4 2XR
N-terminal hydrophobic pocket, and we have evaluated the effects of the flexible lid on the dual-site ubiquitination reaction mechanism catalyzed by MDM2. Deletion of this pseudo-substrate motif promotes MDM2 protein thermoinstability, indicating that the site can function as a positive regulatory element. Phospho-mimetic mutation in the pseudo-substrate motif at codon 17 (MDM2S17D) stabilizes the binding of MDM2 towards two distinct peptide docking sites within the p53 tetramer and enhances p53 ubiquitination. Molecular modeling orientates the phospho-mimetic pseudo-substrate motif in equilibrium over a charged surface patch on the MDM2 at Arg97/ Lys98, and mutation of these residues to the MDM4 equivalent reverses the activating effect of the phosphomimetic mutation on MDM2 function. These data highlight the ability of the pseudo-substrate motif to regulate the allosteric interaction between the N-terminal hydrophobic pocket of MDM2 and its central acidic domain, which stimulates the E3 ubiquitin ligase function of MDM2. This model of MDM2 regulation implicates an as yet undefined lid-kinase as a component of pro-oncogenic pathways that stimulate the E3 ubiquitin ligase function of MDM2 in cells. Keywords MDM2 . p53 . Allostery . Kinase . Ubiquitination
Introduction M. Walkinshaw Institute for Translational and Chemical Biology, University of Edinburgh, Edinburgh, Scotland, UK, EH9 3JR Present Address: L. Worrall Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1Z3
Reconstitution of a multicomponent ubiquitin–enzyme reaction mechanism, including the role of E1, E2, and E3
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