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Missense p53 mutants often accumulate in tumors and drive progression through gain of function. MDM2 efficiently degrades wild-type p53 but fails to degrade mutant p53 in tumor cells. Previous studies revealed that mutant p53 inhibits MDM2 autoubiquitination, suggesting that the interaction inhibits MDM2 E3 activity. Recent work showed that MDM2 E3 activity is stimulated by intramolecular interaction between the RING and acidic domains. Here, we show that in the mutant p53-MDM2 complex, the mutant p53 core domain binds to the MDM2 acidic domain with significantly higher avidity than wild-type p53. The mutant p53-MDM2 complex is deficient in catalyzing ubiquitin release from the activated E2 conjugating enzyme. An MDM2 construct with extra copies of the acidic domain is resistant to inhibition by mutant p53 and efficiently promotes mutant p53 ubiquitination and degradation. The results suggest that mutant p53 interferes with the intramolecular autoactivation mechanism of MDM2, contributing to reduced ubiquitination and increased accumulation in tumor cells.

Nucleomethylin (NML), a novel nucleolar protein, is important for mediating the assembly of the energy-dependent nucleolar silencing complex (eNoSC), which also contains SirT1 and SUV39H1. eNoSC represses rRNA transcription during nutrient deprivation, thus reducing energy expenditure and improving cell survival. We found that NML is an RNA binding protein that copurifies with 5S, 5.8S, and 28S rRNA. The SirT1 and RNA binding regions on NML showed partial overlap, and the NML-SirT1 interaction was competitively inhibited by rRNA. Nutrient deprivation triggered downregulation of rRNA transcription, reduced the level of NML-associated rRNA, and stimulated NML-SirT1 binding. Assembly of eNoSC facilitated repression of pre-rRNA transcription. These results suggest that nascent rRNA generates a positive-feedback signal by suppressing the assembly of eNoSC and protecting active ribosomal DNA units from heterochromatin formation. This RNA-mediated mechanism enables the eNoSC to amplify the effects of upstream nutrient-responsive regulators.

The RING domain ubiquitin E3 ligase MDM2 is a key regulator of p53 degradation and a mediator of signals that stabilize p53. The current understanding of the mechanisms by which MDM2 posttranslational modifications and protein binding cause p53 stabilization remains incomplete. Here we present evidence that the MDM2 central acidic region is critical for activating RING domain E3 ligase activity. A 30-amino-acid minimal region of the acidic domain binds to the RING domain through intramolecular interactions and stimulates the catalytic function of the RING domain in promoting ubiquitin release from charged E2. The minimal activation sequence is also the binding site for the ARF tumor suppressor, which inhibits ubiquitination of p53. The acidic domain-RING domain intramolecular interaction is modulated by ATM-mediated phosphorylation near the RING domain or by binding of ARF. These results suggest that MDM2 phosphorylation and association with protein regulators share a mechanism in inhibiting the E3 ligase function and stabilizing p53 and suggest that targeting the MDM2 autoactivation mechanism may be useful for therapeutic modulation of p53 levels.