Characterization of the ubiquitin-protein ligase activity of the Mdm2/MdmX complex
Final Report Abstract
The RING domain proteins Mdm2 and MdmX are critical regulators of the tumor suppressor protein p53. Besides the RING domain, Mdm2 and MdmX share significant structural similarity, with the most conserved regions being the p53-binding domain within the N terminus of the respective protein and a central Zn binding domain of unknown function. In many cases, RING domains represent interaction sites for ubiquitin conjugating enzymes (E2) and, thus, the presence of a RING domain is commonly assumed to be indicative for proteins with the function of an E3 ubiquitin-protein ligase. Indeed, Mdm2 has E3 ligase activity and in concert with members of the UbcH5 subfamily of E2s targets p53 for ubiquitination and degradation within cells. Unlike Mdm2, MdmX has no or only little E3 activity but by forming heterodimeric complexes with Mdm2 via their RING domains, MdmX affects Mdm2 activity. At the beginning of this project, however, the actual consequence of the interaction of Mdm2 with MdmX with respect to the E3 activity of Mdm2 was controversially discussed. In this study, we used a combination of in vitro assays (e.g. ubiquitination of p53, Mdm2 auto-ubiquitination, coprecipitation) and overexpression assays within cells (p53 ubiquitination and degradation) to show that that the ability of Mdm2 to form homodimeric complexes is required for its E3 activity. Furthermore, we showed that the E3 activity of inactive Mdm2 mutants is efficiently rescued by MdmX in vitro and within cells, supporting the notion that the Mdm2-MdmX complex has intrinsic E3 activity and is actively involved in ubiquitination and degradation of p53 in vivo (i.e. MdmX acts as a positive cofactor in Mdm2- mediated ubiquitination of p53). To identify the regions within the RING domain of Mdm2 that render it an active E3 ligase for p53, we performed a mutational analysis of the RING domain of MdmX. This resulted in the identification of two distinct regions that when replaced by the respective regions of Mdm2, turn MdmX into an active E3 ligase for p53. One of the regions localizes to the dimer interface indicating that subtle conformational changes in this region either affect dimer stability and/or the interaction with UbcH5. The second region contains the nucleolar localization signal of Mdm2 but is also assumed to be involved in the interaction with UbcH5. Indeed, we showed that this region has a significant impact on the ability of respective MdmX mutants to functionally interact with UbcH5 in vitro supporting the notion that this region serves two distinct functional purposes, nucleolar localization and E3 ligase activity. Finally, we obtained evidence to suggest that the RING domain of Mdm2 not only binds to UbcH5 but moreover, acts as an allosteric activator of UbcH5. In conclusion, the results obtained provide a more detailed understanding of the E3 activity of Mdm2 and the Mdm2-MdmX complex in particular and contribute to the elucidation of the mechanisms involved in E3-mediated ubiquitination in general, since most of the known E3s are members of the RING domain family of E3s.
Publications
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(2007). Hetero-oligomerization with MdmX rescues the ubiquitin/Nedd8 ligase activity of RING finger mutants of Mdm2. J Biol Chem 282, 10901-10907
Singh RK, Iyappan S, Scheffner M
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(2010). Ubiquitin family members in the regulation of the tumor suppressor p53. In Groettrup M (Ed): Conjugation and deconjugation of ubiquitin family modifiers; Landes Bioscience, Austin, Texas
Xirodimas DP, Scheffner M
