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Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response.

Choi HK, Choi Y, Park ES, Park SY, Lee SH, Seo J, Jeong MH, Jeong JW, Jeong JH, Lee PC, Choi KC, Yoon HG - Nat Commun (2015)

Bottom Line: Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage.Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients.Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

No MeSH data available.


Related in: MedlinePlus

PDCD5 selectively binds to and mediates caspase-3-dependent cleavage of HDAC3 at Asp-391.(a) PDCD5 is an HDAC3-associating protein among PDCD proteins. Proteins from HCT-116 (p53+/+) whole-cell lysate were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. (b) Overexpression of PDCD5 induces C-terminal cleavage of HDAC3. Cells were transfected with increasing amounts of Flag-PDCD5 plasmid. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (c) Overexpression of PDCD5 selectively triggers the cleavage of HDAC3, but not other class I HDACs. Cells were transfected with increasing amounts of PDCD plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (d,e) In vivo validation of PDCD5-mediated HDAC3 cleavage at Asp-391. HCT-116 cells were transfected with the indicated plasmids. Permeabilized cells were incubated with antibodies against HA and Flag, and then PLA probes were added. Positive signals were analysed using confocal microscopy. Red dots display uncleaved HDAC3. Representative images of three independent experiments are shown. (f) Inhibition of caspase-3 abrogates PDCD5-induced HDAC3 cleavage. HCT-116 cells were transfected with Flag-PDCD5 plasmid and treated with the indicated caspase inhibitors. (g) Depletion of caspase-3 abrogates ET-induced HDAC3 cleavage. Cells were transfected with siRNAs as indicated and treated with ET (100 μM, 12 h). Whole-cell lysates were immunoblotted with the indicated antibodies. (h) PDCD5 is required for caspase-3-dependent HDAC3 cleavage during ET treatment. Either shcontrol or stable shPDCD5-expressing HCT-116 cells was treated with ET. Whole-cell lysates were analysed by western blotting with the indicated antibodies. Scale bar, 10 μm.
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f1: PDCD5 selectively binds to and mediates caspase-3-dependent cleavage of HDAC3 at Asp-391.(a) PDCD5 is an HDAC3-associating protein among PDCD proteins. Proteins from HCT-116 (p53+/+) whole-cell lysate were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. (b) Overexpression of PDCD5 induces C-terminal cleavage of HDAC3. Cells were transfected with increasing amounts of Flag-PDCD5 plasmid. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (c) Overexpression of PDCD5 selectively triggers the cleavage of HDAC3, but not other class I HDACs. Cells were transfected with increasing amounts of PDCD plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (d,e) In vivo validation of PDCD5-mediated HDAC3 cleavage at Asp-391. HCT-116 cells were transfected with the indicated plasmids. Permeabilized cells were incubated with antibodies against HA and Flag, and then PLA probes were added. Positive signals were analysed using confocal microscopy. Red dots display uncleaved HDAC3. Representative images of three independent experiments are shown. (f) Inhibition of caspase-3 abrogates PDCD5-induced HDAC3 cleavage. HCT-116 cells were transfected with Flag-PDCD5 plasmid and treated with the indicated caspase inhibitors. (g) Depletion of caspase-3 abrogates ET-induced HDAC3 cleavage. Cells were transfected with siRNAs as indicated and treated with ET (100 μM, 12 h). Whole-cell lysates were immunoblotted with the indicated antibodies. (h) PDCD5 is required for caspase-3-dependent HDAC3 cleavage during ET treatment. Either shcontrol or stable shPDCD5-expressing HCT-116 cells was treated with ET. Whole-cell lysates were analysed by western blotting with the indicated antibodies. Scale bar, 10 μm.

Mentions: To better understand the functional role of HDAC3 in apoptotic signalling, we performed the yeast two-hybrid screen using human testis, ovary and breast tissue libraries and identified 23 HDAC3-interacting proteins (Supplementary Table 1). Among the identified proteins, PDCD5, a tumour suppressor that was recently found to act as a pro-apoptotic factor, was particularly interesting and thus selected for this study. Moreover, we detected HDAC3 in an immunopurified PDCD5 complex with a high confidence using liquid chromatography–mass spectrometry (MS)/MS analysis, suggesting an in vivo interaction between HDAC3 and PDCD5 (Supplementary Table 2). Endogenous co-immunoprecipitation analysis demonstrated that PDCD5 selectively interacts with HDAC3 among class I HDACs. Reciprocally, HDAC3 specifically interacted with PDCD5 among PDCD proteins, which are known to be involved in apoptotic signalling. These results indicate that HDAC3, among class I HDACs, is a unique PDCD5-associating protein (Fig. 1a). Same results were also obtained with A2780 ovarian cancer cells (Supplementary Fig. 1a,b). Mapping analysis showed that the N-terminal domain of PDCD5 (1–30 amino acids (a.a.)) directly interacts with the N-terminal domain (1–106 a.a.) of HDAC3 (Supplementary Fig. 1c,d).


Programmed cell death 5 mediates HDAC3 decay to promote genotoxic stress response.

Choi HK, Choi Y, Park ES, Park SY, Lee SH, Seo J, Jeong MH, Jeong JW, Jeong JH, Lee PC, Choi KC, Yoon HG - Nat Commun (2015)

PDCD5 selectively binds to and mediates caspase-3-dependent cleavage of HDAC3 at Asp-391.(a) PDCD5 is an HDAC3-associating protein among PDCD proteins. Proteins from HCT-116 (p53+/+) whole-cell lysate were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. (b) Overexpression of PDCD5 induces C-terminal cleavage of HDAC3. Cells were transfected with increasing amounts of Flag-PDCD5 plasmid. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (c) Overexpression of PDCD5 selectively triggers the cleavage of HDAC3, but not other class I HDACs. Cells were transfected with increasing amounts of PDCD plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (d,e) In vivo validation of PDCD5-mediated HDAC3 cleavage at Asp-391. HCT-116 cells were transfected with the indicated plasmids. Permeabilized cells were incubated with antibodies against HA and Flag, and then PLA probes were added. Positive signals were analysed using confocal microscopy. Red dots display uncleaved HDAC3. Representative images of three independent experiments are shown. (f) Inhibition of caspase-3 abrogates PDCD5-induced HDAC3 cleavage. HCT-116 cells were transfected with Flag-PDCD5 plasmid and treated with the indicated caspase inhibitors. (g) Depletion of caspase-3 abrogates ET-induced HDAC3 cleavage. Cells were transfected with siRNAs as indicated and treated with ET (100 μM, 12 h). Whole-cell lysates were immunoblotted with the indicated antibodies. (h) PDCD5 is required for caspase-3-dependent HDAC3 cleavage during ET treatment. Either shcontrol or stable shPDCD5-expressing HCT-116 cells was treated with ET. Whole-cell lysates were analysed by western blotting with the indicated antibodies. Scale bar, 10 μm.
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Related In: Results  -  Collection

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f1: PDCD5 selectively binds to and mediates caspase-3-dependent cleavage of HDAC3 at Asp-391.(a) PDCD5 is an HDAC3-associating protein among PDCD proteins. Proteins from HCT-116 (p53+/+) whole-cell lysate were immunoprecipitated and subsequently immunoblotted with the indicated antibodies. (b) Overexpression of PDCD5 induces C-terminal cleavage of HDAC3. Cells were transfected with increasing amounts of Flag-PDCD5 plasmid. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (c) Overexpression of PDCD5 selectively triggers the cleavage of HDAC3, but not other class I HDACs. Cells were transfected with increasing amounts of PDCD plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3. (d,e) In vivo validation of PDCD5-mediated HDAC3 cleavage at Asp-391. HCT-116 cells were transfected with the indicated plasmids. Permeabilized cells were incubated with antibodies against HA and Flag, and then PLA probes were added. Positive signals were analysed using confocal microscopy. Red dots display uncleaved HDAC3. Representative images of three independent experiments are shown. (f) Inhibition of caspase-3 abrogates PDCD5-induced HDAC3 cleavage. HCT-116 cells were transfected with Flag-PDCD5 plasmid and treated with the indicated caspase inhibitors. (g) Depletion of caspase-3 abrogates ET-induced HDAC3 cleavage. Cells were transfected with siRNAs as indicated and treated with ET (100 μM, 12 h). Whole-cell lysates were immunoblotted with the indicated antibodies. (h) PDCD5 is required for caspase-3-dependent HDAC3 cleavage during ET treatment. Either shcontrol or stable shPDCD5-expressing HCT-116 cells was treated with ET. Whole-cell lysates were analysed by western blotting with the indicated antibodies. Scale bar, 10 μm.
Mentions: To better understand the functional role of HDAC3 in apoptotic signalling, we performed the yeast two-hybrid screen using human testis, ovary and breast tissue libraries and identified 23 HDAC3-interacting proteins (Supplementary Table 1). Among the identified proteins, PDCD5, a tumour suppressor that was recently found to act as a pro-apoptotic factor, was particularly interesting and thus selected for this study. Moreover, we detected HDAC3 in an immunopurified PDCD5 complex with a high confidence using liquid chromatography–mass spectrometry (MS)/MS analysis, suggesting an in vivo interaction between HDAC3 and PDCD5 (Supplementary Table 2). Endogenous co-immunoprecipitation analysis demonstrated that PDCD5 selectively interacts with HDAC3 among class I HDACs. Reciprocally, HDAC3 specifically interacted with PDCD5 among PDCD proteins, which are known to be involved in apoptotic signalling. These results indicate that HDAC3, among class I HDACs, is a unique PDCD5-associating protein (Fig. 1a). Same results were also obtained with A2780 ovarian cancer cells (Supplementary Fig. 1a,b). Mapping analysis showed that the N-terminal domain of PDCD5 (1–30 amino acids (a.a.)) directly interacts with the N-terminal domain (1–106 a.a.) of HDAC3 (Supplementary Fig. 1c,d).

Bottom Line: Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage.Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients.Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Brain Korea 21 PLUS Project for Medical Sciences, Yonsei University College of Medicine, Seoul 120-752, Korea.

ABSTRACT
The inhibition of p53 activity by histone deacetylase 3 (HDAC3) has been reported, but the precise molecular mechanism is unknown. Here we show that programmed cell death 5 (PDCD5) selectively mediates HDAC3 dissociation from p53, which induces HDAC3 cleavage and ubiquitin-dependent proteasomal degradation. Casein kinase 2 alpha phosphorylates PDCD5 at Ser-119 to enhance its stability and importin 13-mediated nuclear translocation of PDCD5. Genetic deletion of PDCD5 abrogates etoposide (ET)-induced p53 stabilization and HDAC3 cleavage, indicating an essential role of PDCD5 in p53 activation. Restoration of PDCD5(WT) in PDCD5(-/-) MEFs restores ET-induced HDAC3 cleavage. Reduction of both PDCD5 and p53, but not reduction of either protein alone, significantly enhances in vivo tumorigenicity of AGS gastric cancer cells and correlates with poor prognosis in gastric cancer patients. Our results define a mechanism for p53 activation via PDCD5-dependent HDAC3 decay under genotoxic stress conditions.

No MeSH data available.


Related in: MedlinePlus