Abstract A29: Mdm2 E3 ubiquitin ligase function is dispensable for the degradation of Mdm2 itself

Abstract

Abstract Mdm2 has long been considered the major negative regulator of both p53 stability and activity. The RING finger domain of Mdm2 confers its E3 ubiquitin ligase activity, and through this function Mdm2 controls p53 stability, additionally, auto-ubiquitination of Mdm2 is believed to be the primary mechanism of Mdm2 degradation. In vitro, mutation or truncation of the RING domain in Mdm2 resulted in loss of ubiquitin ligase activity towards both Mdm2 and its substrate p53. In contrast, recent knock-in mouse models indicate that under physiological conditions the Mdm2 RING-mediated E3 ubiquitin ligase function is dispensable for Mdm2 degradation. To investigate the in vivo significance of Mdm2 auto-ubiquitination, we compared two Mdm2 knock-in mouse strains each harboring a single amino acid substitution in RING domain (C462A) or C-terminus (Y487A). Both knock-in mutants disrupt Mdm2 E3 ligase activity for p53. We observed that (1) mutant Mdm2 protein (C462A or Y487A) fails to degrade p53 protein. (2) Wild type and mutant Mdm2 are degraded similarly. Despite efficient disruption of p53 degradation, half-life assays demonstrated that neither of the Mdm2 mutants affected the rate of degradation of Mdm2 itself indicating not only that Mdm2 auto-ubiquitination is not the primary mechanism for Mdm2 degradation in vivo, but also that an additional mechanism is in action to regulate Mdm2. (3) Inhibition of proteasomal degradation by MG-132 resulted in similar levels of accumulation of both wild type and mutant Mdm2s suggesting Mdm2 degradation is mediated via the proteasome. In vivo ubiquitination assay reconfirmed that Mdm2 RING domain mutation does not affect degradation and accumulation of Mdm2 itself, further implicating the possibility of an outside E3 ligase activity. In conclusion, auto-ubiquitination of Mdm2 is not necessary for Mdm2 degradation in vivo. The identification of E3 ubiquitin ligases with specificity for Mdm2 is an important next step to gain mechanistic insight into the regulation of Mdm2 regulation, and with that, p53. Citation Format: Tae-Hyung Kim, Yanping Zhang. Mdm2 E3 ubiquitin ligase function is dispensable for the degradation of Mdm2 itself. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr A29.