Regulation of the E3 ubiquitin ligase activity of MDM2 by an N-terminal pseudo-substrate motif

Journal of Chemical Biology

Bartosz Wawrzynow²  Liam Worrall³  Erin G. Worrall²  Kathryn L. Ball²  Ted R. Hupp²  Malcolm Walkinshaw¹ 

[1] Institute for Translational and Chemical Biology, University of Edinburgh, Edinburgh, Scotland UK EH9 3JR;Institute of Genetics and Molecular Medicine, University of Edinburgh, Edinburgh, Scotland UK EH4 2XR;Biochemistry and Molecular Biology and the Center for Blood Research, University of British Columbia, Vancouver, British Columbia Canada V6T 1Z3

关键词: MDM2;    p53;    Allostery;    Kinase;    Ubiquitination;   

DOI  :  10.1007/s12154-009-0019-5

学科分类：分子生物学，细胞生物学和基因

来源: Springer

【 摘 要 】

The tumor suppressor p53 has evolved a MDM2-dependent 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 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 phospho-mimetic 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.

【 授权许可】

Unknown