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Ubiquitin-dependent degradation of p73 is inhibited by PML.

Bernassola F, Salomoni P, Oberst A, Di Como CJ, Pagano M, Melino G, Pandolfi PP - J. Exp. Med. (2004)

Bottom Line: Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway.We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels.As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021, USA.

ABSTRACT
p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression.

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p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml−/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml−/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml−/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.
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fig4: p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml−/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml−/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml−/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.

Mentions: Because PML interacts with p73, next we investigated whether PML could affect p73 sub-cellular localization. Endogenous p73 showed a diffuse as well as a nuclear punctuate pattern partially colocalizing with PML (Fig. 4 A). Similar results were obtained in HCT116(3) cells (unpublished data). Ectopically expressed GFP-p73α partly accumulated in speckles in wild-type MEFs (in ∼70% of cells), which colocalized with PML (∼40% of PML-NBs per cell were found to contain p73) (Fig. 4 B and see Fig. 5 D). In sharp contrast, GFP-p73α did not accumulate in speckles, and its staining pattern remained entirely diffuse in Pml−/− MEFs (Fig. 4 C).


Ubiquitin-dependent degradation of p73 is inhibited by PML.

Bernassola F, Salomoni P, Oberst A, Di Como CJ, Pagano M, Melino G, Pandolfi PP - J. Exp. Med. (2004)

p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml−/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml−/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml−/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211783&req=5

fig4: p73 and PML colocalization within the PML-NBs is required for p73 stabilization (A) Representative image of Cos-1 cells costained with anti-p73 (green, clone 5B429) and anti-PML (red) antibodies and analyzed by confocal microscopy (left). Nuclei were visualized by 4′,6-diamidino-2-phenylindole (DAPI) staining. The arrows indicate p73-containing speckles. (right) A quantitative analysis of the green and red fluorescence intensity at distinct nuclear speckles as indicated by the yellow arrow. (B–D) GFP-p73α was overexpressed into wild type (B) and Pml−/− (C) MEFs. Full-length PML was cotransfected with GFP-p73α into Pml−/− MEFs (D). Nuclei were visualized by DAPI staining. The arrows indicate p73-containing PML-NBs. (E) Pml−/− MEFs were transiently transfected with HA-p73α alone (lane 1) or in combination with full-length Flag-PML (lane 2), Flag-PML ΔRING (lane 3), or PML-RARα (lane 4). Cell extracts were subjected to IB with anti-HA, anti-Flag, anti-RARα, and anti–β-actin antibodies. (bottom) Normalization of transfection efficiency by quantitation of GFP expression.
Mentions: Because PML interacts with p73, next we investigated whether PML could affect p73 sub-cellular localization. Endogenous p73 showed a diffuse as well as a nuclear punctuate pattern partially colocalizing with PML (Fig. 4 A). Similar results were obtained in HCT116(3) cells (unpublished data). Ectopically expressed GFP-p73α partly accumulated in speckles in wild-type MEFs (in ∼70% of cells), which colocalized with PML (∼40% of PML-NBs per cell were found to contain p73) (Fig. 4 B and see Fig. 5 D). In sharp contrast, GFP-p73α did not accumulate in speckles, and its staining pattern remained entirely diffuse in Pml−/− MEFs (Fig. 4 C).

Bottom Line: Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway.We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels.As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells.

View Article: PubMed Central - PubMed

Affiliation: Molecular Biology Program, Memorial Sloan-Kettering Cancer Center, 1275 York Ave, New York, NY 10021, USA.

ABSTRACT
p73 has been identified recently as a structural and functional homologue of the tumor suppressor p53. Here, we report that p73 stability is directly regulated by the ubiquitin-proteasome pathway. Furthermore, we show that the promyelocytic leukemia (PML) protein modulates p73 half-life by inhibiting its degradation in a PML-nuclear body (NB)-dependent manner. p38 mitogen-activated protein kinase-mediated phosphorylation of p73 is required for p73 recruitment into the PML-NB and subsequent PML-dependent p73 stabilization. We find that p300-mediated acetylation of p73 protects it against ubiquitinylation and that PML regulates p73 stability by positively modulating its acetylation levels. As a result, PML potentiates p73 transcriptional and proapoptotic activities that are markedly impaired in Pml-/- primary cells. Our findings demonstrate that PML plays a crucial role in modulating p73 function, thus providing further insights on the molecular network for tumor suppression.

Show MeSH
Related in: MedlinePlus