The MDM2 RING Domain and Central Acidic Domain Play Distinct Roles in MDM2 Protein Homodimerization and MDM2-MDMX Protein Heterodimerization

Patrick L Leslie,Hengming Ke,Yanping Zhang

doi:10.1074/jbc.m115.644435

Abstract

The oncoprotein murine double minute 2 (MDM2) is an E3 ligase that plays a prominent role in p53 suppression by promoting its polyubiquitination and proteasomal degradation. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. Intriguingly, deletion of a portion of the MDM2 central acidic domain selectively inhibits interaction with MDM2 while leaving intact the ability of MDM2 to interact with MDMX and to ubiquitinate p53. Further analysis of an MDM2 C-terminal deletion mutant reveals that the C-terminal residues of MDM2 are required for both MDM2 and MDMX interaction. Collectively, our results suggest a model in which MDM2-MDMX heterodimerization requires the extreme C terminus and proper RING domain structure of MDM2, whereas MDM2 homodimerization requires the extreme C terminus and the central acidic domain of MDM2, suggesting that MDM2 homo- and heterodimers utilize distinct MDM2 domains. Our study is the first to report mutations capable of separating MDM2 homo- and heterodimerization.

Highlights

murine double minute 2 (MDM2) dimerization is required for p53 ubiquitination and degradation
MDM2 Residue Asn-447 Is Important for p53 Degradation— Kostic et al [20] performed a detailed investigation of MDM2MDMX really interesting new gene (RING) domain binding by NMR [20]
In this study, radiolabeled purified MDM2 RING domains were titrated with unlabeled purified MDMX RING domains, and differences in the heteronuclear single quantum coherence spectra were obtained for each MDM2 residue within the RING domain upon MDMX binding

Summary

Background

MDM2 dimerization is required for p53 ubiquitination and degradation. Results: MDM2-MDM2 and MDM2-MDMX interactions occur through different mechanisms. In its active form, MDM2 forms homodimers as well as heterodimers with the homologous protein murine double minute 4 (MDMX), both of which are thought to occur through their respective C-terminal RING (really interesting new gene) domains. In this study, using multiple MDM2 mutants, we show evidence suggesting that MDM2 homo- and heterodimerization occur through distinct mechanisms because MDM2 RING domain mutations that inhibit MDM2 interaction with MDMX do not affect MDM2 interaction with WT MDM2. We use several known and novel MDM2 mutant constructs in the context of the full-length protein to show that MDM2-MDM2 interaction requires the central acidic domain and the extreme C-terminal residues of MDM2, whereas MDM2-MDMX interaction requires the proper RING domain structure and the extreme C-terminal residues of MDM2

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION