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

Clegg HV, Itahana Y, Itahana K, Ramalingam S, Zhang Y - PLoS ONE (2012)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

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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Related in: MedlinePlus

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- cells.
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pone-0038212-g005: 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- cells.

Mentions: How might disruption of Mdm2's RING domain enhance the p53-p300 interaction? Is it due to lack of E3 ubiquitin ligase activity, or is it caused by another essential function of the RING domain? E3 ubiquitin ligase activity is not likely to be essential for inhibiting p53-p300 binding, as Mdm2's sister protein, Mdmx, lacks E3 ligase activity yet has been shown to inhibit p300-mediated acetylation of p53. It is possible that another function of the RING domain influences the interaction. One hypothesis is that formation of an Mdm2-Mdmx heterodimer may be necessary for Mdm2 to inhibit p300, as the RING domain was shown to mediate this heterodimerization [22], and Mdmx inhibits p300-mediated acetylation of p53 [23]. That is, the heterodimer may be more efficient at inhibiting p300-p53 binding than Mdm2 or Mdmx alone. To determine whether the RING point mutation affects Mdm2-Mdmx binding in vivo, we carried out a co-immunoprecipitation (co-IP) for Mdm2 in Mdm2+/+;p53ER/− and Mdm2m/m;p53ER/− MEF cells. We found that the C462A RING mutation disrupts the interaction between Mdm2 and Mdmx (Fig. 4), indicating that an intact RING domain is necessary for Mdm2-Mdmx heterodimerization in vivo. Thus, it is possible that the enhanced p53 activity and p300-p53 interaction produced by Mdm2C462A may stem from its inability to heterodimerize with Mdmx. We present a hypothesized model in which the Mdm2-Mdmx heterodimer inhibits p300-p53 binding in vivo, while monomeric Mdm2 promotes this interaction. In this model, heterodimerization between Mdm2 and Mdmx blocks p300-p53 binding and p300-mediated acetylation of p53. In Mdm2- cells, this inhibition is released, permitting p300 to interact with and acetylate p53. When Mdm2 exists as a monomer rather than a heterodimer, as is the case, presumably, with Mdm2C462A, (if not able to dimerize with Mdmx, it is also not likely to dimerize with itself), not only is it unable to inhibit p300-mediated acetylation of p53, but the monomeric Mdm2 further enhances this acetylation beyond the basal level found in Mdm2- cells. This may be a result of monomeric Mdm2 bridging together p300 and p53, as Mdm2 is known to interact with both of these proteins [20], [21]. We speculate that the Mdm2-Mdmx heterodimer is not able to promote p300-p53 binding, perhaps due to bulkiness or another inherent difference between the monomer and the heterodimer (Fig. 5). It should be noted that this study does not specifically differentiate between Mdm2-Mdmx heterodimerization and Mdm2-Mdm2 homodimerization. Ultimately, further research will be needed to determine whether the effect of Mdm2C462A on the p300-p53 interaction is mediated by its ability to heterodimerize with Mdmx, or by another mechanism.


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

Clegg HV, Itahana Y, Itahana K, Ramalingam S, Zhang Y - PLoS ONE (2012)

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- cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3362553&req=5

pone-0038212-g005: 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- cells.
Mentions: How might disruption of Mdm2's RING domain enhance the p53-p300 interaction? Is it due to lack of E3 ubiquitin ligase activity, or is it caused by another essential function of the RING domain? E3 ubiquitin ligase activity is not likely to be essential for inhibiting p53-p300 binding, as Mdm2's sister protein, Mdmx, lacks E3 ligase activity yet has been shown to inhibit p300-mediated acetylation of p53. It is possible that another function of the RING domain influences the interaction. One hypothesis is that formation of an Mdm2-Mdmx heterodimer may be necessary for Mdm2 to inhibit p300, as the RING domain was shown to mediate this heterodimerization [22], and Mdmx inhibits p300-mediated acetylation of p53 [23]. That is, the heterodimer may be more efficient at inhibiting p300-p53 binding than Mdm2 or Mdmx alone. To determine whether the RING point mutation affects Mdm2-Mdmx binding in vivo, we carried out a co-immunoprecipitation (co-IP) for Mdm2 in Mdm2+/+;p53ER/− and Mdm2m/m;p53ER/− MEF cells. We found that the C462A RING mutation disrupts the interaction between Mdm2 and Mdmx (Fig. 4), indicating that an intact RING domain is necessary for Mdm2-Mdmx heterodimerization in vivo. Thus, it is possible that the enhanced p53 activity and p300-p53 interaction produced by Mdm2C462A may stem from its inability to heterodimerize with Mdmx. We present a hypothesized model in which the Mdm2-Mdmx heterodimer inhibits p300-p53 binding in vivo, while monomeric Mdm2 promotes this interaction. In this model, heterodimerization between Mdm2 and Mdmx blocks p300-p53 binding and p300-mediated acetylation of p53. In Mdm2- cells, this inhibition is released, permitting p300 to interact with and acetylate p53. When Mdm2 exists as a monomer rather than a heterodimer, as is the case, presumably, with Mdm2C462A, (if not able to dimerize with Mdmx, it is also not likely to dimerize with itself), not only is it unable to inhibit p300-mediated acetylation of p53, but the monomeric Mdm2 further enhances this acetylation beyond the basal level found in Mdm2- cells. This may be a result of monomeric Mdm2 bridging together p300 and p53, as Mdm2 is known to interact with both of these proteins [20], [21]. We speculate that the Mdm2-Mdmx heterodimer is not able to promote p300-p53 binding, perhaps due to bulkiness or another inherent difference between the monomer and the heterodimer (Fig. 5). It should be noted that this study does not specifically differentiate between Mdm2-Mdmx heterodimerization and Mdm2-Mdm2 homodimerization. Ultimately, further research will be needed to determine whether the effect of Mdm2C462A on the p300-p53 interaction is mediated by its ability to heterodimerize with Mdmx, or by another mechanism.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Lineberger Comprehensive Cancer Center, School of Medicine, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, United States of America.

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.

Show MeSH
Related in: MedlinePlus