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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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PML is required for p73 transcriptional and proapoptotic activity. (A) A bax promoter-driven luciferase reporter plasmid (bax-Luc) alone or in combination with p73α was transfected into wild type and Pml−/− MEFs, and luciferase activity was assayed 48 h after transfection. pRL-TK vector was included to normalize transfection efficiency, and reporter basal luciferase activity was normalized as 1. Values are mean ± SEM of two separate experiments each performed in duplicate. (B) Wild type and Pml−/− MEFs were retrovirally transduced with a pBabe-p73α construct for 48 h, selected in puromycin-containing medium for 3 d, and scored for apoptosis by FACS® analysis using propidium iodide staining. Values are mean ± SEM of two separate experiments each performed in duplicate. (C) A model for PML-mediated protection against p73 degradation. PML inhibits p73 ubiquitin-dependent degradation (left). PML controls p73 stability by regulating p73 acetylation levels in the PML-NB, thereby preventing its ubiquitinylation (right).
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fig7: PML is required for p73 transcriptional and proapoptotic activity. (A) A bax promoter-driven luciferase reporter plasmid (bax-Luc) alone or in combination with p73α was transfected into wild type and Pml−/− MEFs, and luciferase activity was assayed 48 h after transfection. pRL-TK vector was included to normalize transfection efficiency, and reporter basal luciferase activity was normalized as 1. Values are mean ± SEM of two separate experiments each performed in duplicate. (B) Wild type and Pml−/− MEFs were retrovirally transduced with a pBabe-p73α construct for 48 h, selected in puromycin-containing medium for 3 d, and scored for apoptosis by FACS® analysis using propidium iodide staining. Values are mean ± SEM of two separate experiments each performed in duplicate. (C) A model for PML-mediated protection against p73 degradation. PML inhibits p73 ubiquitin-dependent degradation (left). PML controls p73 stability by regulating p73 acetylation levels in the PML-NB, thereby preventing its ubiquitinylation (right).

Mentions: Overexpression of PML resulted in enhancement of the transactivation potential of various p73 isoforms on both the bax-Luc and the p21-Luc promoters (Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1, and not depicted). Accordingly, Bax and p21 protein levels were enhanced by coexpression of p73 and PML as compared with cells expressing p73 alone (Fig. S5 C). Pml inactivation resulted in a marked impairment of p73-dependent transcription. Indeed, p73α transcriptional activity was diminished up to 65% in Pml−/− cells (Fig. 7 A and not depicted). In agreement with the inability of the PML-ΔRING mutant to efficiently stabilize p73, the transcriptional defect observed in the Pml−/− MEFs could be rescued by PML, but not by PML-ΔRING add-back (Fig. S5 D). Furthermore, SB 202190 treatment reduced the ability of p73 to transactivate the bax-Luc promoter (up to 44% inhibition; Fig. S5 E). Remarkably, PML coactivation of p73-dependent transcription was additionally impaired upon SB 202190 treatment (up to 56% inhibition; Fig. S5 E).


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)

PML is required for p73 transcriptional and proapoptotic activity. (A) A bax promoter-driven luciferase reporter plasmid (bax-Luc) alone or in combination with p73α was transfected into wild type and Pml−/− MEFs, and luciferase activity was assayed 48 h after transfection. pRL-TK vector was included to normalize transfection efficiency, and reporter basal luciferase activity was normalized as 1. Values are mean ± SEM of two separate experiments each performed in duplicate. (B) Wild type and Pml−/− MEFs were retrovirally transduced with a pBabe-p73α construct for 48 h, selected in puromycin-containing medium for 3 d, and scored for apoptosis by FACS® analysis using propidium iodide staining. Values are mean ± SEM of two separate experiments each performed in duplicate. (C) A model for PML-mediated protection against p73 degradation. PML inhibits p73 ubiquitin-dependent degradation (left). PML controls p73 stability by regulating p73 acetylation levels in the PML-NB, thereby preventing its ubiquitinylation (right).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2211783&req=5

fig7: PML is required for p73 transcriptional and proapoptotic activity. (A) A bax promoter-driven luciferase reporter plasmid (bax-Luc) alone or in combination with p73α was transfected into wild type and Pml−/− MEFs, and luciferase activity was assayed 48 h after transfection. pRL-TK vector was included to normalize transfection efficiency, and reporter basal luciferase activity was normalized as 1. Values are mean ± SEM of two separate experiments each performed in duplicate. (B) Wild type and Pml−/− MEFs were retrovirally transduced with a pBabe-p73α construct for 48 h, selected in puromycin-containing medium for 3 d, and scored for apoptosis by FACS® analysis using propidium iodide staining. Values are mean ± SEM of two separate experiments each performed in duplicate. (C) A model for PML-mediated protection against p73 degradation. PML inhibits p73 ubiquitin-dependent degradation (left). PML controls p73 stability by regulating p73 acetylation levels in the PML-NB, thereby preventing its ubiquitinylation (right).
Mentions: Overexpression of PML resulted in enhancement of the transactivation potential of various p73 isoforms on both the bax-Luc and the p21-Luc promoters (Fig. S5, A and B, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1, and not depicted). Accordingly, Bax and p21 protein levels were enhanced by coexpression of p73 and PML as compared with cells expressing p73 alone (Fig. S5 C). Pml inactivation resulted in a marked impairment of p73-dependent transcription. Indeed, p73α transcriptional activity was diminished up to 65% in Pml−/− cells (Fig. 7 A and not depicted). In agreement with the inability of the PML-ΔRING mutant to efficiently stabilize p73, the transcriptional defect observed in the Pml−/− MEFs could be rescued by PML, but not by PML-ΔRING add-back (Fig. S5 D). Furthermore, SB 202190 treatment reduced the ability of p73 to transactivate the bax-Luc promoter (up to 44% inhibition; Fig. S5 E). Remarkably, PML coactivation of p73-dependent transcription was additionally impaired upon SB 202190 treatment (up to 56% inhibition; Fig. S5 E).

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