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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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Related in: MedlinePlus

PML protects p73 from ubiquitinylation and proteasome-mediated degradation. (A and B) PML increases the half-life of both exogenous and endogenous p73. p73 half-life was measured upon CHX (20 μg/ml) addition in H1299 cells transfected with either HA-p73α alone or with PML (A) and in wild type and Pml−/− MEFs (B). Cells lysates were examined by IB using anti-HA (A) or anti-p73 (B, clone 5B429) antibody. (C) H1299 cells were cotransfected with the indicated plasmids for 24 h. Some cultures were incubated with MG132 (lanes 5 and 6). p73-Ub immunocomplexes were analyzed by IB analysis with anti-HA (top) and anti-GFP (bottom) antibodies. (D) Cos-1 cells were either transfected with the empty vector (lane 1) or with Flag-PML (lane 2) for 24 h. Cell lysates were analyzed with rabbit polyclonal anti-p73, anti-Flag, and anti–β-actin antibody. Semi-quantitative RT-PCR analysis on p73 and β-actin mRNAs in Cos-1 cells was treated as aforementioned (bottom). (E) Cell lysates from wild type and Pml−/− MEFs were examined by IB using anti-p73 (clone 5B429) antibody. (F) Cos-1 cells were either left untreated (lane 1) or transfected with 200 pmol PML (lane 2) and Lamin A/C (lane 3) siRNA. After 48 h, cell lysates were prepared and analyzed for p73, PML, Lamin A/C, and β-actin expression by IB.
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fig2: PML protects p73 from ubiquitinylation and proteasome-mediated degradation. (A and B) PML increases the half-life of both exogenous and endogenous p73. p73 half-life was measured upon CHX (20 μg/ml) addition in H1299 cells transfected with either HA-p73α alone or with PML (A) and in wild type and Pml−/− MEFs (B). Cells lysates were examined by IB using anti-HA (A) or anti-p73 (B, clone 5B429) antibody. (C) H1299 cells were cotransfected with the indicated plasmids for 24 h. Some cultures were incubated with MG132 (lanes 5 and 6). p73-Ub immunocomplexes were analyzed by IB analysis with anti-HA (top) and anti-GFP (bottom) antibodies. (D) Cos-1 cells were either transfected with the empty vector (lane 1) or with Flag-PML (lane 2) for 24 h. Cell lysates were analyzed with rabbit polyclonal anti-p73, anti-Flag, and anti–β-actin antibody. Semi-quantitative RT-PCR analysis on p73 and β-actin mRNAs in Cos-1 cells was treated as aforementioned (bottom). (E) Cell lysates from wild type and Pml−/− MEFs were examined by IB using anti-p73 (clone 5B429) antibody. (F) Cos-1 cells were either left untreated (lane 1) or transfected with 200 pmol PML (lane 2) and Lamin A/C (lane 3) siRNA. After 48 h, cell lysates were prepared and analyzed for p73, PML, Lamin A/C, and β-actin expression by IB.

Mentions: PML Protects p73 from Proteasome-mediated Degradation. PML isoform IV regulates p53 induction and transcriptional activation (32–34, 41) and protects p53 from MDM2-mediated degradation (42, 43). Therefore, we set out to test whether PML isoform IV might regulate p73 stability. PML overexpression indeed led to a dose-dependent p73 accumulation (Fig. S2, A and B, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1). To test whether PML regulates p73 stability, we followed the half-life of ectopically expressed p73 and found that it was greatly increased by PML overexpression (Fig. 2 A), thus demonstrating that PML stabilizes p73 in vivo. Importantly, and in agreement with these results, we observed that the half-life of endogenous p73α was prolonged in cells overexpressing PML (not depicted) and resulted markedly shortened in Pml−/− compared with wild-type MEFs (Fig. 2 B).


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 protects p73 from ubiquitinylation and proteasome-mediated degradation. (A and B) PML increases the half-life of both exogenous and endogenous p73. p73 half-life was measured upon CHX (20 μg/ml) addition in H1299 cells transfected with either HA-p73α alone or with PML (A) and in wild type and Pml−/− MEFs (B). Cells lysates were examined by IB using anti-HA (A) or anti-p73 (B, clone 5B429) antibody. (C) H1299 cells were cotransfected with the indicated plasmids for 24 h. Some cultures were incubated with MG132 (lanes 5 and 6). p73-Ub immunocomplexes were analyzed by IB analysis with anti-HA (top) and anti-GFP (bottom) antibodies. (D) Cos-1 cells were either transfected with the empty vector (lane 1) or with Flag-PML (lane 2) for 24 h. Cell lysates were analyzed with rabbit polyclonal anti-p73, anti-Flag, and anti–β-actin antibody. Semi-quantitative RT-PCR analysis on p73 and β-actin mRNAs in Cos-1 cells was treated as aforementioned (bottom). (E) Cell lysates from wild type and Pml−/− MEFs were examined by IB using anti-p73 (clone 5B429) antibody. (F) Cos-1 cells were either left untreated (lane 1) or transfected with 200 pmol PML (lane 2) and Lamin A/C (lane 3) siRNA. After 48 h, cell lysates were prepared and analyzed for p73, PML, Lamin A/C, and β-actin expression by IB.
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Related In: Results  -  Collection

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fig2: PML protects p73 from ubiquitinylation and proteasome-mediated degradation. (A and B) PML increases the half-life of both exogenous and endogenous p73. p73 half-life was measured upon CHX (20 μg/ml) addition in H1299 cells transfected with either HA-p73α alone or with PML (A) and in wild type and Pml−/− MEFs (B). Cells lysates were examined by IB using anti-HA (A) or anti-p73 (B, clone 5B429) antibody. (C) H1299 cells were cotransfected with the indicated plasmids for 24 h. Some cultures were incubated with MG132 (lanes 5 and 6). p73-Ub immunocomplexes were analyzed by IB analysis with anti-HA (top) and anti-GFP (bottom) antibodies. (D) Cos-1 cells were either transfected with the empty vector (lane 1) or with Flag-PML (lane 2) for 24 h. Cell lysates were analyzed with rabbit polyclonal anti-p73, anti-Flag, and anti–β-actin antibody. Semi-quantitative RT-PCR analysis on p73 and β-actin mRNAs in Cos-1 cells was treated as aforementioned (bottom). (E) Cell lysates from wild type and Pml−/− MEFs were examined by IB using anti-p73 (clone 5B429) antibody. (F) Cos-1 cells were either left untreated (lane 1) or transfected with 200 pmol PML (lane 2) and Lamin A/C (lane 3) siRNA. After 48 h, cell lysates were prepared and analyzed for p73, PML, Lamin A/C, and β-actin expression by IB.
Mentions: PML Protects p73 from Proteasome-mediated Degradation. PML isoform IV regulates p53 induction and transcriptional activation (32–34, 41) and protects p53 from MDM2-mediated degradation (42, 43). Therefore, we set out to test whether PML isoform IV might regulate p73 stability. PML overexpression indeed led to a dose-dependent p73 accumulation (Fig. S2, A and B, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1). To test whether PML regulates p73 stability, we followed the half-life of ectopically expressed p73 and found that it was greatly increased by PML overexpression (Fig. 2 A), thus demonstrating that PML stabilizes p73 in vivo. Importantly, and in agreement with these results, we observed that the half-life of endogenous p73α was prolonged in cells overexpressing PML (not depicted) and resulted markedly shortened in Pml−/− compared with wild-type MEFs (Fig. 2 B).

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