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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 is degraded through the ubiquitin–proteasome pathway. (A) ts20 and ts41 cells were transfected with HA-p73α and either left at 34°C (lanes 1 and 3) or shifted to 40°C (lanes 2 and 4). Cell lysates were analyzed by immunoblot (IB) with anti-HA and anti–β-actin (top) antibodies. Expression of mRNA for p73 and β-actin was assessed by semi-quantitative RT-PCR analysis (bottom). (B) HCT-116(3) cells were either left untreated (lane 1) or incubated with 10 μM or lactacystin (lane 2) for 12 h. Cellular extracts were analyzed with anti-p73 antibody (clone 5B429). (C) Cos-1 cells were either transfected with the empty vector (lane 1) or with HA-Ub (lane 2) and treated with MG132 for 6 h. Cell lysates were immunoprecipitated with a rabbit polyclonal anti-p73 antibody followed by IB with anti-HA antibody. (D) ts20 cells grown at 34°C were transiently transfected with HA-p73α alone or in combination with a constitutively active form of MKK6. 8 h after transfection, some cultures were shifted to 40°C. (E) Phosphorylation of p73 in H1299 cells transfected with HA-p73α alone (lane 1) or in combination with HA-MKK6 (lane 2). Cells were cultured in [32P]orthophosphate-containing medium for 3 h, and phosphorylation was assessed by autoradiography. (F) In vitro phosphorylation of human purified His-p73α by p38 MAPK. 6 μg His-p73α was incubated with 1.2 U of recombinant purified p38 MAPK. ATF-2 was included as a control.
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fig1: p73 is degraded through the ubiquitin–proteasome pathway. (A) ts20 and ts41 cells were transfected with HA-p73α and either left at 34°C (lanes 1 and 3) or shifted to 40°C (lanes 2 and 4). Cell lysates were analyzed by immunoblot (IB) with anti-HA and anti–β-actin (top) antibodies. Expression of mRNA for p73 and β-actin was assessed by semi-quantitative RT-PCR analysis (bottom). (B) HCT-116(3) cells were either left untreated (lane 1) or incubated with 10 μM or lactacystin (lane 2) for 12 h. Cellular extracts were analyzed with anti-p73 antibody (clone 5B429). (C) Cos-1 cells were either transfected with the empty vector (lane 1) or with HA-Ub (lane 2) and treated with MG132 for 6 h. Cell lysates were immunoprecipitated with a rabbit polyclonal anti-p73 antibody followed by IB with anti-HA antibody. (D) ts20 cells grown at 34°C were transiently transfected with HA-p73α alone or in combination with a constitutively active form of MKK6. 8 h after transfection, some cultures were shifted to 40°C. (E) Phosphorylation of p73 in H1299 cells transfected with HA-p73α alone (lane 1) or in combination with HA-MKK6 (lane 2). Cells were cultured in [32P]orthophosphate-containing medium for 3 h, and phosphorylation was assessed by autoradiography. (F) In vitro phosphorylation of human purified His-p73α by p38 MAPK. 6 μg His-p73α was incubated with 1.2 U of recombinant purified p38 MAPK. ATF-2 was included as a control.

Mentions: p73 protein levels have been shown to increase upon proteasome inhibitor treatment (12, 36, 38). However, whether p73 turnover is directly regulated by ubiquitin-mediated proteolysis remains to be proven. The mutant ts20 cell line has a thermolabile ubiquitin-activating E1 enzyme that is inactivated at the nonpermissive temperature of 40°C, which leads to disruption of the ubiquitinylation process. Complete degradation of p73α occurred at 34°C (Fig. 1 A, lane 1), whereas incubation of ts20 cells at 40°C resulted in marked accumulation of p73 protein (Fig. 1 A, lane 2), thus indicating that an intact ubiquitin system is required for p73 degradation. On the contrary, p73 mRNA levels were considerably unaltered upon temperature switch (Fig. 1 A, bottom left). Proteasome inhibitor treatment of ts20 cells resulted in p73 accumulation in a polyubiquitinylated state (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1) and in the appearance of higher molecular weight immunoreactive bands of p73 that contained ubiquitin (Fig. S1 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)

p73 is degraded through the ubiquitin–proteasome pathway. (A) ts20 and ts41 cells were transfected with HA-p73α and either left at 34°C (lanes 1 and 3) or shifted to 40°C (lanes 2 and 4). Cell lysates were analyzed by immunoblot (IB) with anti-HA and anti–β-actin (top) antibodies. Expression of mRNA for p73 and β-actin was assessed by semi-quantitative RT-PCR analysis (bottom). (B) HCT-116(3) cells were either left untreated (lane 1) or incubated with 10 μM or lactacystin (lane 2) for 12 h. Cellular extracts were analyzed with anti-p73 antibody (clone 5B429). (C) Cos-1 cells were either transfected with the empty vector (lane 1) or with HA-Ub (lane 2) and treated with MG132 for 6 h. Cell lysates were immunoprecipitated with a rabbit polyclonal anti-p73 antibody followed by IB with anti-HA antibody. (D) ts20 cells grown at 34°C were transiently transfected with HA-p73α alone or in combination with a constitutively active form of MKK6. 8 h after transfection, some cultures were shifted to 40°C. (E) Phosphorylation of p73 in H1299 cells transfected with HA-p73α alone (lane 1) or in combination with HA-MKK6 (lane 2). Cells were cultured in [32P]orthophosphate-containing medium for 3 h, and phosphorylation was assessed by autoradiography. (F) In vitro phosphorylation of human purified His-p73α by p38 MAPK. 6 μg His-p73α was incubated with 1.2 U of recombinant purified p38 MAPK. ATF-2 was included as a control.
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Related In: Results  -  Collection

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fig1: p73 is degraded through the ubiquitin–proteasome pathway. (A) ts20 and ts41 cells were transfected with HA-p73α and either left at 34°C (lanes 1 and 3) or shifted to 40°C (lanes 2 and 4). Cell lysates were analyzed by immunoblot (IB) with anti-HA and anti–β-actin (top) antibodies. Expression of mRNA for p73 and β-actin was assessed by semi-quantitative RT-PCR analysis (bottom). (B) HCT-116(3) cells were either left untreated (lane 1) or incubated with 10 μM or lactacystin (lane 2) for 12 h. Cellular extracts were analyzed with anti-p73 antibody (clone 5B429). (C) Cos-1 cells were either transfected with the empty vector (lane 1) or with HA-Ub (lane 2) and treated with MG132 for 6 h. Cell lysates were immunoprecipitated with a rabbit polyclonal anti-p73 antibody followed by IB with anti-HA antibody. (D) ts20 cells grown at 34°C were transiently transfected with HA-p73α alone or in combination with a constitutively active form of MKK6. 8 h after transfection, some cultures were shifted to 40°C. (E) Phosphorylation of p73 in H1299 cells transfected with HA-p73α alone (lane 1) or in combination with HA-MKK6 (lane 2). Cells were cultured in [32P]orthophosphate-containing medium for 3 h, and phosphorylation was assessed by autoradiography. (F) In vitro phosphorylation of human purified His-p73α by p38 MAPK. 6 μg His-p73α was incubated with 1.2 U of recombinant purified p38 MAPK. ATF-2 was included as a control.
Mentions: p73 protein levels have been shown to increase upon proteasome inhibitor treatment (12, 36, 38). However, whether p73 turnover is directly regulated by ubiquitin-mediated proteolysis remains to be proven. The mutant ts20 cell line has a thermolabile ubiquitin-activating E1 enzyme that is inactivated at the nonpermissive temperature of 40°C, which leads to disruption of the ubiquitinylation process. Complete degradation of p73α occurred at 34°C (Fig. 1 A, lane 1), whereas incubation of ts20 cells at 40°C resulted in marked accumulation of p73 protein (Fig. 1 A, lane 2), thus indicating that an intact ubiquitin system is required for p73 degradation. On the contrary, p73 mRNA levels were considerably unaltered upon temperature switch (Fig. 1 A, bottom left). Proteasome inhibitor treatment of ts20 cells resulted in p73 accumulation in a polyubiquitinylated state (Fig. S1 A, available at http://www.jem.org/cgi/content/full/jem.20031943/DC1) and in the appearance of higher molecular weight immunoreactive bands of p73 that contained ubiquitin (Fig. S1 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