Mdm2 RING Mutation Enhances p53 Transcriptional Activity and p53-p300 Interaction

Abstract

The p53 transcription factor and tumor suppressor is regulated primarily by the E3 ubiquitin ligase Mdm2, which ubiquitinates p53 to target it for proteasomal degradation. Aside from its ubiquitin ligase function, Mdm2 has been believed to be capable of suppressing p53's transcriptional activity by binding with and masking the transactivation domain of p53. The ability of Mdm2 to restrain p53 activity by binding alone, without ubiquitination, was challenged by a 2007 study using a knockin mouse harboring a single cysteine-to-alanine point mutation (C462A) in Mdm2's RING domain. Mouse embryonic fibroblasts with this mutation, which abrogates Mdm2's E3 ubiquitin ligase activity without affecting its ability to bind with p53, were unable to suppress p53 activity. In this study, we utilized the Mdm2C462A mouse model to characterize in further detail the role of Mdm2's RING domain in the control of p53. Here, we show in vivo that the Mdm2C462A protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2C462A actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2C462A facilitates the interaction between p53 and the acetyltransferase CBP/p300, and it fails to heterodimerize with its homolog and sister regulator of p53, Mdmx, suggesting that a fully intact RING domain is required for Mdm2's inhibition of the p300-p53 interaction and for its interaction with Mdmx. These findings help us to better understand the complex regulation of the Mdm2-p53 pathway and have important implications for chemotherapeutic agents targeting Mdm2, as they suggest that inhibition of Mdm2's E3 ubiquitin ligase activity may be sufficient for increasing p53 activity in vivo, without the need to block Mdm2-p53 binding.

Modulation of p53 C-Terminal Acetylation by mdm2, p14ARF, and Cytoplasmic SirT2

“…These findings provide an insight into why inhibition of both SirT1 and SirT2 seems to be required to achieve effective p53 activation with small-molecule sirtuin inhibitors (25). Finally, our observations are in line with previously suggested tumor suppressor roles for mdm2 (26), and the recently described positive effect of mdm2 on p53-dependent transcription when the E3 ubiquitin ligase function of mdm2 is impaired (27).…”

“…We speculate that additional mechanisms are likely to be required to enable a positive role for mdm2 in p53-dependent transcription in our system. In a recent groundbreaking publication, Clegg and colleagues (27) obtained enhanced expression of p53 target genes in mouse embryonic fibroblasts from an mdm2-C462A knock-in mouse model. This extremely interesting observation shows that in this model system, there are mechanisms that make it possible for mdm2 to enhance p53 transcriptional activity when mdm2 E3 ubiquitin ligase function is impaired.…”

Targeting p53-MDM2-MDMX Loop for Cancer Therapy

“…A study using an MDM2 RING finger (C462A) mutant knockin mouse model by the Zhang group has shown that the stability of MDM2 is in fact not regulated by autoubiquitination in vivo [140], nor it is capable of blocking p53 activity by binding alone. This finding suggests that the MDM2 RING finger E3 ubiquitin ligase function plays an important role in suppressing p53, although this mutant is also defective in binding to MDMX [141, 142].…”

Rhythmic expression of DEC2 protein in vitro and in vivo

“…The MCF-7 human breast cancer and U2OS human osteosarcoma cell lines were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). Primary MEF were prepared as previously described (37). The cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (Sigma Chemical Co., St. Louis, MO, USA) or in DMEM without glucose (Gibco-Life Technologies, Grand Island, NY, USA) supplemented with 10% fetal bovine serum at 37˚C in a humidified atmosphere of 95% air and 5% CO 2 .…”