Role of Mdm2 acid domain interactions in recognition and ubiquitination of the transcription factor IRF-2.
Mdm2 (murine double minute 2)-mediated ubiquitination of the p53 tumour suppressor requires interaction of the ligase at two distinct binding sites that form general multiprotein-docking sites for the p53 protein. The first Mdm2-binding site resides in the transactivation domain of p53 and is an allosteric effector site for Mdm2-mediated p53 ubiquitination; the second site requires the acid domain of Mdm2 to recognize a 'ubiquitination signal' within p53's DNA-binding core. In order to expand on fundamental requirements for a protein to function as an Mdm2 substrate and the role of the acid domain in recognition, we have carried out a bioinformatics search for open reading frames that have homology with the Mdm2-docking sites in p53. IRF-2 [IFN (interferon) regulatory factor-2], an IFN-regulated transcription factor, has been identified as an Mdm2-binding protein and substrate requiring interactions with both the hydrophobic pocket and the acid domain of Mdm2. Mutation of either of the two Mdm2-binding sites on IRF-2 can attenuate substrate ubiquitination, confirming the requirement of a dual-site substrate interaction mechanism. Ligands that bind to the hydrophobic pocket are not sufficient to inhibit Mdm2 E3-ligase activity. Rather, acid domain-binding ligands act as E3-ligase inhibitors, lending additional support to the idea that the acid domain of Mdm2 is key to understanding its mechanism of action. The ability of Mdm2 and IRF-2 to form a complex in cells complements the biochemical assays and together establishes a novel substrate with which to develop insights into E3-ubiquitin ligase-substrate interactions in vitro and in cells.
Pettersson
Susanne
S
CRUK Interferon and Cell Signalling Group, Cell Signalling Unit, IGMM, University of Edinburgh, Edinburgh, UK.
Kelleher
Michael
M
Pion
Emmanuelle
E
Wallace
Maura
M
Ball
Kathryn L
KL
D000818:Animals; D019295:Computational Biology; D050836:Interferon Regulatory Factor-2 / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D017434:Protein Structure, Tertiary; D051736:Proto-Oncogene Proteins c-mdm2 / Q000737:chemistry / Q000502:physiology*; D044767:Ubiquitin-Protein Ligases / Q000037:antagonists & inhibitors; D054875:Ubiquitination / Q000502:physiology*
D050836:Interferon Regulatory Factor-2; C497207:Mdm2 protein, mouse; D051736:Proto-Oncogene Proteins c-mdm2; D044767:Ubiquitin-Protein Ligases