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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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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- cells, and not following immunoprecipitation with IgG.
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pone-0038212-g002: 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- cells, and not following immunoprecipitation with IgG.

Mentions: We explored potential mechanisms for the increased p53 activity observed in cells with the Mdm2 C462A mutation. Mdm2 has been thought to inhibit p53's transcriptional activity by interacting with p53 on its target gene promoters and masking the transactivation domain of p53. As shown above, Mdm2C462A retains its ability to interact with p53, yet does not suppress p53 activity [9]. However, it is possible that the mutant Mdm2 may not interact with p53 while located on p53's target gene promoters and is unable to control p53 activity due to this defect. To rule out this possibility, it is essential to determine whether the Mdm2C462A-p53 interaction can take place on the promoter of a p53 target gene. To address this directly, chromatin immunoprecipitation (ChIP) analysis was carried out to assess p53-Mdm2 binding on the p21 promoter in Mdm2m/m;p53ER/− MEFs cells. Mdm2m/m;p53−/− cells were included as a negative control. The cells were incubated with 4-OHT for 24 hours to activate p53, and formaldehyde was applied to crosslink proteins to DNA. The cells were lysed, sonicated to shear DNA, and immunoprecipitated with p53 antibody or IgG (negative control). A subset of each sample was resolved by SDS PAGE and western blotting, while another portion was subject to reverse crosslinking and PCR targeting the p21 (CDKN1A) promoter (Fig. 2A). PCR product indicating presence of the p21 promoter was detected equally in all three input samples, but following immunoprecipitation with p53 antibody, was present only in the sample from Mdm2m/m;p53ER/− MEFs. DNA from the p21 promoter was not detected in Mdm2m/m;p53ER/− MEFs immunoprecipitated with IgG or in p53- MEFs immunoprecipitated with p53 antibody (negative controls) (Fig. 2B). Western blotting of the samples showed that both Mdm2 and p53 were present in Mdm2m/m;p53ER/− MEFs immunoprecipitated with p53 antibody. No Mdm2 or p53 was detected in control samples immunoprecipitated with IgG alone or from p53- MEFs (Fig. 2C). These data indicate that Mdm2C462A interacts with p53 on the p21 gene promoter.


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) 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- cells, and not following immunoprecipitation with IgG.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3362553&req=5

pone-0038212-g002: 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- cells, and not following immunoprecipitation with IgG.
Mentions: We explored potential mechanisms for the increased p53 activity observed in cells with the Mdm2 C462A mutation. Mdm2 has been thought to inhibit p53's transcriptional activity by interacting with p53 on its target gene promoters and masking the transactivation domain of p53. As shown above, Mdm2C462A retains its ability to interact with p53, yet does not suppress p53 activity [9]. However, it is possible that the mutant Mdm2 may not interact with p53 while located on p53's target gene promoters and is unable to control p53 activity due to this defect. To rule out this possibility, it is essential to determine whether the Mdm2C462A-p53 interaction can take place on the promoter of a p53 target gene. To address this directly, chromatin immunoprecipitation (ChIP) analysis was carried out to assess p53-Mdm2 binding on the p21 promoter in Mdm2m/m;p53ER/− MEFs cells. Mdm2m/m;p53−/− cells were included as a negative control. The cells were incubated with 4-OHT for 24 hours to activate p53, and formaldehyde was applied to crosslink proteins to DNA. The cells were lysed, sonicated to shear DNA, and immunoprecipitated with p53 antibody or IgG (negative control). A subset of each sample was resolved by SDS PAGE and western blotting, while another portion was subject to reverse crosslinking and PCR targeting the p21 (CDKN1A) promoter (Fig. 2A). PCR product indicating presence of the p21 promoter was detected equally in all three input samples, but following immunoprecipitation with p53 antibody, was present only in the sample from Mdm2m/m;p53ER/− MEFs. DNA from the p21 promoter was not detected in Mdm2m/m;p53ER/− MEFs immunoprecipitated with IgG or in p53- MEFs immunoprecipitated with p53 antibody (negative controls) (Fig. 2B). Western blotting of the samples showed that both Mdm2 and p53 were present in Mdm2m/m;p53ER/− MEFs immunoprecipitated with p53 antibody. No Mdm2 or p53 was detected in control samples immunoprecipitated with IgG alone or from p53- MEFs (Fig. 2C). These data indicate that Mdm2C462A interacts with p53 on the p21 gene promoter.

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