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Reconstitution of Mdm2-dependent post-translational modifications of p53 in yeast.

Di Ventura B, Funaya C, Antony C, Knop M, Serrano L - PLoS ONE (2008)

Bottom Line: Its activity is subject to a tight regulation involving a multitude of post-translational modifications.The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network.These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany. barbara.diventura@bzh.uni-heidelberg.de

ABSTRACT
p53 mediates cell cycle arrest or apoptosis in response to DNA damage. Its activity is subject to a tight regulation involving a multitude of post-translational modifications. The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network. To circumvent this complexity, we studied here the minimal requirements for functionally relevant p53 post-translational modifications by expressing human p53 together with its best characterized modifier Mdm2 in budding yeast. We find that expression of the human p53-Mdm2 module in yeast is sufficient to faithfully recapitulate key aspects of p53 regulation in higher eukaryotes, such as Mdm2-dependent targeting of p53 for degradation, sumoylation at lysine 386 and further regulation of this process by p14(ARF). Interestingly, sumoylation is necessary for the recruitment of p53-Mdm2 complexes to yeast nuclear bodies morphologically akin to human PML bodies. These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies. The reductionist yeast model that was established and validated in this study will now allow to incrementally study simplified parts of the intricate p53 network, thus helping elucidate the core mechanisms of p53 regulation as well as test novel strategies to counteract p53 malfunctions.

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Mdm2 ubiquitylates p53 in yeast leading to p53 degradation.(A) Total yeast cell extracts from exponentially growing cells were immunoblotted for the indicated proteins over time. 3-phosphoglycerate kinase (PGK) was used as loading control. (B) Quantifications of protein levels are shown. Data represent the mean ± standard error (SE), for n = 3 independent experiments. (C,D) as in (A,B) but for p53W23S. (E,F) as in (A,B) but for p53Lys2Arg.
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pone-0001507-g002: Mdm2 ubiquitylates p53 in yeast leading to p53 degradation.(A) Total yeast cell extracts from exponentially growing cells were immunoblotted for the indicated proteins over time. 3-phosphoglycerate kinase (PGK) was used as loading control. (B) Quantifications of protein levels are shown. Data represent the mean ± standard error (SE), for n = 3 independent experiments. (C,D) as in (A,B) but for p53W23S. (E,F) as in (A,B) but for p53Lys2Arg.

Mentions: We then co-expressed p53 and Mdm2. While the mdm2 cDNA was under the control of a constitutive promoter (TEF), the p53 cDNA was under the control of the inducible GAL1 promoter. In this way, Mdm2 proteins were constantly produced, while only a pool of p53 proteins was produced which we followed over time. p53 was degraded in the presence of Mdm2 (Figures 2A and 2B). When a truncated Mdm2 protein (Mdm2NORING) missing the RING finger domain–shown to be responsible for Mdm2 E3 ubiquitin ligase activity in human cells (Fang et al. 2000)–was used instead of wild type Mdm2, p53 showed little degradation (Figures 2A and 2B), as in the absence of Mdm2 (Figures 1A and 1B). Similarly, degradation was severely reduced when we co-expressed Mdm2 with either a mutant p53 with impaired binding to it (p53W23S) (17) (Figures 2C and 2D) or with a mutant p53 in which the C-terminal lysines at positions 372, 373, 382 and 383 were replaced by arginines (p53Lys2Arg), to prevent ubiquitylation keeping the overall charge of the protein unchanged [18] (Figures 2E and 2F). Taken together, these data suggest that Mdm2 ubiquitylates p53 targeting it for degradation in yeast.


Reconstitution of Mdm2-dependent post-translational modifications of p53 in yeast.

Di Ventura B, Funaya C, Antony C, Knop M, Serrano L - PLoS ONE (2008)

Mdm2 ubiquitylates p53 in yeast leading to p53 degradation.(A) Total yeast cell extracts from exponentially growing cells were immunoblotted for the indicated proteins over time. 3-phosphoglycerate kinase (PGK) was used as loading control. (B) Quantifications of protein levels are shown. Data represent the mean ± standard error (SE), for n = 3 independent experiments. (C,D) as in (A,B) but for p53W23S. (E,F) as in (A,B) but for p53Lys2Arg.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2200829&req=5

pone-0001507-g002: Mdm2 ubiquitylates p53 in yeast leading to p53 degradation.(A) Total yeast cell extracts from exponentially growing cells were immunoblotted for the indicated proteins over time. 3-phosphoglycerate kinase (PGK) was used as loading control. (B) Quantifications of protein levels are shown. Data represent the mean ± standard error (SE), for n = 3 independent experiments. (C,D) as in (A,B) but for p53W23S. (E,F) as in (A,B) but for p53Lys2Arg.
Mentions: We then co-expressed p53 and Mdm2. While the mdm2 cDNA was under the control of a constitutive promoter (TEF), the p53 cDNA was under the control of the inducible GAL1 promoter. In this way, Mdm2 proteins were constantly produced, while only a pool of p53 proteins was produced which we followed over time. p53 was degraded in the presence of Mdm2 (Figures 2A and 2B). When a truncated Mdm2 protein (Mdm2NORING) missing the RING finger domain–shown to be responsible for Mdm2 E3 ubiquitin ligase activity in human cells (Fang et al. 2000)–was used instead of wild type Mdm2, p53 showed little degradation (Figures 2A and 2B), as in the absence of Mdm2 (Figures 1A and 1B). Similarly, degradation was severely reduced when we co-expressed Mdm2 with either a mutant p53 with impaired binding to it (p53W23S) (17) (Figures 2C and 2D) or with a mutant p53 in which the C-terminal lysines at positions 372, 373, 382 and 383 were replaced by arginines (p53Lys2Arg), to prevent ubiquitylation keeping the overall charge of the protein unchanged [18] (Figures 2E and 2F). Taken together, these data suggest that Mdm2 ubiquitylates p53 targeting it for degradation in yeast.

Bottom Line: Its activity is subject to a tight regulation involving a multitude of post-translational modifications.The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network.These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies.

View Article: PubMed Central - PubMed

Affiliation: European Molecular Biology Laboratory, Heidelberg, Germany. barbara.diventura@bzh.uni-heidelberg.de

ABSTRACT
p53 mediates cell cycle arrest or apoptosis in response to DNA damage. Its activity is subject to a tight regulation involving a multitude of post-translational modifications. The plethora of functional protein interactions of p53 at present precludes a clear understanding of regulatory principles in the p53 signaling network. To circumvent this complexity, we studied here the minimal requirements for functionally relevant p53 post-translational modifications by expressing human p53 together with its best characterized modifier Mdm2 in budding yeast. We find that expression of the human p53-Mdm2 module in yeast is sufficient to faithfully recapitulate key aspects of p53 regulation in higher eukaryotes, such as Mdm2-dependent targeting of p53 for degradation, sumoylation at lysine 386 and further regulation of this process by p14(ARF). Interestingly, sumoylation is necessary for the recruitment of p53-Mdm2 complexes to yeast nuclear bodies morphologically akin to human PML bodies. These results suggest a novel role for Mdm2 as well as for p53 sumoylation in the recruitment of p53 to nuclear bodies. The reductionist yeast model that was established and validated in this study will now allow to incrementally study simplified parts of the intricate p53 network, thus helping elucidate the core mechanisms of p53 regulation as well as test novel strategies to counteract p53 malfunctions.

Show MeSH
Related in: MedlinePlus