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. 2012;7(5):e38212.
doi: 10.1371/journal.pone.0038212. Epub 2012 May 29.

Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction

Hilary V Clegg  1 , Yoko Itahana, Koji Itahana, Sundhar Ramalingam, Yanping Zhang
Affiliations

Mdm2 RING mutation enhances p53 transcriptional activity and p53-p300 interaction

Hilary V Clegg et al. PLoS One. 2012.

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 Mdm2(C462A) 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 Mdm2(C462A) protein not only fails to suppress p53, but compared to the complete absence of Mdm2, Mdm2(C462A) actually enhances p53 transcriptional activity toward p53 target genes p21/CDKN1A, MDM2, BAX, NOXA, and 14-3-3σ. In addition, we found that Mdm2(C462A) 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.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. A) Quantitative real-time PCR analysis of p53 target genes in MEF cells pre-treated with 4-OHT for 24 hours to activate p53ER.
Values represent an average of three samples measured relative to GAPDH, and error bars indicated standard deviation. All samples are of the genotype p53ER/− 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 p53ER. Actin is shown as loading control.
Figure 2
Figure 2. A) Schematic depicting chromatin immunoprecipitation (ChIP) analysis carried out to assess Mdm2-p53 binding on the promoter of the p53 target, p21.
MEF cells were pre-treated with 4-OHT for 24 hours to induce activation of p53ER. Cells were crosslinked using formaldehyde, sonicated to shear chromatin, and immunoprecipitated with p53 antibody. A portion of each sample was subject to reverse crosslinking followed by PCR amplification targeting a region of the p21 promoter, while another portion was used for western blotting to assess the p53-Mdm2 interaction. B) p21/CDKN1A promoter was PCR amplified and resolved in 1% agarose gel following immunoprecipitation with p53 antibody and reverse crosslinking as shown in (A). C) Western blot following immunoprecipitation with p53 antibody as shown in (A). Membrane was blotted for Mdm2, stripped, and re-blotted for p53. Note that a band representing Mdm2 is present in the sample immunoprecipitated with p53 antibody but not in p53-null cells, and not following immunoprecipitation with IgG.
Figure 3
Figure 3. Immunoprecipitation and western blotting of MEF cell lysates 24 hours after administration of 4-OHT.
Note that the p53-p300 interaction is enhanced in the Mdm2m/m MEFs compared to Mdm2-null MEFs despite similar immunoprecipitation of p53 and equivalent loading for p300.
Figure 4
Figure 4. Interaction between Mdm2 and Mdmx is impaired in MEFs with Mdm2C462A compared to those with wild-type Mdm2.
Immunoprecipitation and western blotting were carried out 24 hours after administering 4-OHT to activate p53ER. 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.
Figure 5
Figure 5. A potential mechanism for differential regulation of p53 transcriptional activity by wild-type Mdm2 and Mdm2C462A.
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) Mdm2C462A 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 Mdm2C462A) promotes p300-p53 binding to further enhance p300-mediated p53 acetylation beyond that which occurs in Mdm2-null cells.
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