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

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

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

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

Targeted Inactivation of Mdm2 RING Finger E3 Ubiquitin Ligase Activity in the Mouse Reveals Mechanistic Insights into p53 Regulation

It is believed that Mdm2 suppresses p53 in two ways: transcriptional inhibition by direct binding, and degradation via its E3 ligase activity. To study these functions physiologically, we generated mice bearing a single-residue substitution (C462A) abolishing the E3 function without affecting p53 binding. Unexpectedly, homozygous mutant mice died before E7.5, and deletion of p53 rescued the lethality. Furthermore, reintroducing a switchable p53 by crossing with mice surprisingly demonstrated that the mutant Mdm2 was rapidly degraded in a manner indistinguishable from that of the wild-type Mdm2. Hence, our data indicate that (1) the Mdm2-p53 physical interaction, without Mdm2-mediated p53 ubiquitination, cannot control p53 activity sufficiently to allow early mouse embryonic development, and (2) Mdm2's E3 function is not required for Mdm2 degradation

A) Quantitative real-time PCR analysis of p53 target genes in MEF cells pre-treated with 4-OHT for 24 hours to activate p53^ER.

<p>Values represent an average of three samples measured relative to GAPDH, and error bars indicated standard deviation. All samples are of the genotype <i>p53^ER/−</i> with Mdm2 status as indicated below graph. B) Western blot analysis of p21 expression in MEF cells of indicated genotypes at 0, 12, and 24 hours following treatment with 4-OHT to activate p53^ER. Actin is shown as loading control.</p

A potential mechanism for differential regulation of p53 transcriptional activity by wild-type Mdm2 and Mdm2^C462A.

<p>A) Heterodimerization between wild-type Mdm2 and Mdmx is necessary for inhibiting the p53-p300 interaction and suppressing p300-mediated acetylation of p53, reducing p53 activity. B) Absence of Mdm2 permits p300-p53 interaction, allowing p300-mediated acetylation of p53, and thereby enhancing p53 transcriptional activity compared to that in Mdm2-positive cells. C) Mdm2^C462A cannot heterodimerize with Mdmx and, therefore, fails to inhibit the p53-p300 interaction, allowing enhanced p300-mediated acetylation and activation of p53. In addition, monomeric Mdm2 (such as RING mutant Mdm2^C462A) promotes p300-p53 binding to further enhance p300-mediated p53 acetylation beyond that which occurs in Mdm2-null cells.</p

Interaction between Mdm2 and Mdmx is impaired in MEFs with Mdm2^C462A compared to those with wild-type Mdm2.

<p>Immunoprecipitation and western blotting were carried out 24 hours after administering 4-OHT to activate p53^ER. Actin is shown as a loading control. Note that the interaction between Mdm2 and its known binding partner L5 is not disrupted by the C462A mutation.</p