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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: 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.

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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p53 and Mdm2 co-localize in yeast cells.(A,B) Maximum projections from fluorescence image stacks of live yeast cells expressing the indicated fusion proteins were obtained with a DeltaVision workstation. Bar, 2 µm. (C) Indirect immunofluorescence of fixed yeast cells stained for p53-Mdm2 bodies (primary antibody: goat polyclonal N-19 anti-p53; secondary antibody: Cy2-labeled donkey anti-goat; showed in green), Nop1 (primary antibody: mouse monoclonal; secondary antibody: Cy5-labeled donkey anti-mouse; showed in red) and chromatin (detected with Höechst, showed in blue). (D) Time-lapse microscopy on yeast cells expressing p53 and Mdm2 obtained recording image stacks and then performing maximum projections of live yeast cells at the indicated times. Galactose was added directly into the Petri dish to trigger p53 expression and time-lapse started shortly after. Mdm2 is shown in red and the phase-contrast image of the cells in blue.
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pone-0001507-g003: p53 and Mdm2 co-localize in yeast cells.(A,B) Maximum projections from fluorescence image stacks of live yeast cells expressing the indicated fusion proteins were obtained with a DeltaVision workstation. Bar, 2 µm. (C) Indirect immunofluorescence of fixed yeast cells stained for p53-Mdm2 bodies (primary antibody: goat polyclonal N-19 anti-p53; secondary antibody: Cy2-labeled donkey anti-goat; showed in green), Nop1 (primary antibody: mouse monoclonal; secondary antibody: Cy5-labeled donkey anti-mouse; showed in red) and chromatin (detected with Höechst, showed in blue). (D) Time-lapse microscopy on yeast cells expressing p53 and Mdm2 obtained recording image stacks and then performing maximum projections of live yeast cells at the indicated times. Galactose was added directly into the Petri dish to trigger p53 expression and time-lapse started shortly after. Mdm2 is shown in red and the phase-contrast image of the cells in blue.

Mentions: Notably, co-expressing p53-ECFP and Mdm2-EYFP revealed that the two proteins co-localized to one or more nuclear dots, to which we will refer as p53-Mdm2 bodies (Figure 3A). This co-localization was lost when the mutant p53W23S was used in place of wild type p53 (Figure 3B), indicating that direct binding of the proteins was required for this localization. These structures were visible also when performing indirect immunofluorescence (Figure 3C), therefore they were not triggered by fusing the proteins to the fluorescent tags. Using an antibody against the yeast nucleolar protein Nop1, we analyzed the localization of the p53-Mdm2 bodies in respect to the nucleolus. We did not find substantial overlap between the signals corresponding respectively to the anti-Nop1 and the anti-p53/Mdm2 antibodies, therefore we concluded that p53-Mdm2 bodies did not reside in the nucleolus (Figure 3C). The p53-Mdm2 bodies were most often found in regions of the nucleus adjacent to the nucleolus (Figures 3C and 4A,C,D). Time-lapse microscopy revealed that p53-Mdm2 co-localization is stable over time (Figure 3D). Soon after its expression, p53 is found in the discrete loci where Mdm2 is localized (data not shown), but the p53-Mdm2 complexes then move towards a bigger structure (Figure 3D). We next investigated the morphology and localization within the cell of the p53-Mdm2 bodies using immunoelectron microscopy (Figure 4). The immunostaining detected areas containing electron dense fibrillar spheroids with a doughnut-like shape (13 out of 16 cells analyzed, Figures 4A–D), reminiscent of human PML bodies [19]. Cells expressing only p53 or only Mdm2 showed a diffuse pattern of the gold particles (Figures 4E and 4F), therefore confirming that the formation of the bodies required both p53 and Mdm2.


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)

p53 and Mdm2 co-localize in yeast cells.(A,B) Maximum projections from fluorescence image stacks of live yeast cells expressing the indicated fusion proteins were obtained with a DeltaVision workstation. Bar, 2 µm. (C) Indirect immunofluorescence of fixed yeast cells stained for p53-Mdm2 bodies (primary antibody: goat polyclonal N-19 anti-p53; secondary antibody: Cy2-labeled donkey anti-goat; showed in green), Nop1 (primary antibody: mouse monoclonal; secondary antibody: Cy5-labeled donkey anti-mouse; showed in red) and chromatin (detected with Höechst, showed in blue). (D) Time-lapse microscopy on yeast cells expressing p53 and Mdm2 obtained recording image stacks and then performing maximum projections of live yeast cells at the indicated times. Galactose was added directly into the Petri dish to trigger p53 expression and time-lapse started shortly after. Mdm2 is shown in red and the phase-contrast image of the cells in blue.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2200829&req=5

pone-0001507-g003: p53 and Mdm2 co-localize in yeast cells.(A,B) Maximum projections from fluorescence image stacks of live yeast cells expressing the indicated fusion proteins were obtained with a DeltaVision workstation. Bar, 2 µm. (C) Indirect immunofluorescence of fixed yeast cells stained for p53-Mdm2 bodies (primary antibody: goat polyclonal N-19 anti-p53; secondary antibody: Cy2-labeled donkey anti-goat; showed in green), Nop1 (primary antibody: mouse monoclonal; secondary antibody: Cy5-labeled donkey anti-mouse; showed in red) and chromatin (detected with Höechst, showed in blue). (D) Time-lapse microscopy on yeast cells expressing p53 and Mdm2 obtained recording image stacks and then performing maximum projections of live yeast cells at the indicated times. Galactose was added directly into the Petri dish to trigger p53 expression and time-lapse started shortly after. Mdm2 is shown in red and the phase-contrast image of the cells in blue.
Mentions: Notably, co-expressing p53-ECFP and Mdm2-EYFP revealed that the two proteins co-localized to one or more nuclear dots, to which we will refer as p53-Mdm2 bodies (Figure 3A). This co-localization was lost when the mutant p53W23S was used in place of wild type p53 (Figure 3B), indicating that direct binding of the proteins was required for this localization. These structures were visible also when performing indirect immunofluorescence (Figure 3C), therefore they were not triggered by fusing the proteins to the fluorescent tags. Using an antibody against the yeast nucleolar protein Nop1, we analyzed the localization of the p53-Mdm2 bodies in respect to the nucleolus. We did not find substantial overlap between the signals corresponding respectively to the anti-Nop1 and the anti-p53/Mdm2 antibodies, therefore we concluded that p53-Mdm2 bodies did not reside in the nucleolus (Figure 3C). The p53-Mdm2 bodies were most often found in regions of the nucleus adjacent to the nucleolus (Figures 3C and 4A,C,D). Time-lapse microscopy revealed that p53-Mdm2 co-localization is stable over time (Figure 3D). Soon after its expression, p53 is found in the discrete loci where Mdm2 is localized (data not shown), but the p53-Mdm2 complexes then move towards a bigger structure (Figure 3D). We next investigated the morphology and localization within the cell of the p53-Mdm2 bodies using immunoelectron microscopy (Figure 4). The immunostaining detected areas containing electron dense fibrillar spheroids with a doughnut-like shape (13 out of 16 cells analyzed, Figures 4A–D), reminiscent of human PML bodies [19]. Cells expressing only p53 or only Mdm2 showed a diffuse pattern of the gold particles (Figures 4E and 4F), therefore confirming that the formation of the bodies required both p53 and Mdm2.

Bottom Line: 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.

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