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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 mediates ubiquitin-dependent proteasomal degradation of HDAC3 via C-terminal cleavage of HDAC3.(a) MG132 treatment induces accumulation of cleaved HDAC3. Cells were treated with ET (50 μM) and/or MG132 (10 μM, 3 h). Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3 (left panel). Intensities of protein bands obtained from the immunoblotting assay were quantified with ImageJ (right panel) and normalized with respect to that of β-actin. Relative % intensity was calculated by dividing the normalized intensity by the sum of intensities from both cleaved and full-length HDAC3. Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (b) PDCD5 knockdown diminishes the reduction and cleavage of full-length HDAC3. shcontrol or stable shPDCD5-expressing HCT-116 cells were treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (left panel). Relative % intensity was calculated as described above (right panel). Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (c) HDAC3 ubiquitination increases in a time-dependent manner in response to ET treatment. Cells were transfected with HA-Ub plasmid and treated with MG1432 and/or ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (d) PDCD5 knockdown diminishes HDAC3 ubiquitination in response to ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (e) Mutation of Asp-391 abolishes ET-induced HDAC3 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (f) Mutation of Asp-391 potentiates the action of HDAC3 in the inhibition of PDCD5-mediated p53 acetylation. Cells were transfected with the indicated Flag-tagged plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies.
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f3: PDCD5 mediates ubiquitin-dependent proteasomal degradation of HDAC3 via C-terminal cleavage of HDAC3.(a) MG132 treatment induces accumulation of cleaved HDAC3. Cells were treated with ET (50 μM) and/or MG132 (10 μM, 3 h). Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3 (left panel). Intensities of protein bands obtained from the immunoblotting assay were quantified with ImageJ (right panel) and normalized with respect to that of β-actin. Relative % intensity was calculated by dividing the normalized intensity by the sum of intensities from both cleaved and full-length HDAC3. Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (b) PDCD5 knockdown diminishes the reduction and cleavage of full-length HDAC3. shcontrol or stable shPDCD5-expressing HCT-116 cells were treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (left panel). Relative % intensity was calculated as described above (right panel). Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (c) HDAC3 ubiquitination increases in a time-dependent manner in response to ET treatment. Cells were transfected with HA-Ub plasmid and treated with MG1432 and/or ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (d) PDCD5 knockdown diminishes HDAC3 ubiquitination in response to ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (e) Mutation of Asp-391 abolishes ET-induced HDAC3 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (f) Mutation of Asp-391 potentiates the action of HDAC3 in the inhibition of PDCD5-mediated p53 acetylation. Cells were transfected with the indicated Flag-tagged plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies.

Mentions: HDAC3 cleavage by PDCD5 correlates with increased p53 acetylation; however, HDAC3 cleavage is not observed during ET treatment at the time (4 h) when p53 is activated. We next examined whether the cleaved form of HDAC3 was rapidly degraded during ET treatment. At 2 h after the start of ET treatment, p53 acetylation at multiple lysine residues increased when the cleavage of HDAC3 was observed in the presence of MG132 (Supplementary Fig. 9a). At 4 h after the start of ET treatment, ∼20% of HDAC3 was observed as the cleaved form in the presence of MG132. At 12 h after the start of ET treatment, we found that ∼60% of full-length HDAC3 in the cell lysate was cleaved and dissociated from p53, which is consistent with maximum p53 activation (Fig. 3a). However, PDCD5 knockdown strongly reduced the percentage of HDAC3 cleavage and the dissociation of HDAC3 from p53 in response to ET treatment (Fig. 3b; Supplementary Fig. 9a). Similar results were observed by PDCD5 overexpression in the presence of MG132 (Supplementary Fig. 9b). We found that ET treatment induced the time-dependent ubiquitination and cleavage of HDAC3 (Fig. 3c); however, depletion of PDCD5 substantially reduced HDAC3 ubiquitination (Fig. 3d). By contrast, uncleaved mutant HDAC3D391A was not ubiquitinated in response to ET treatment (Fig. 3e). Confocal microscopy analysis showed the nuclear retention of uncleaved HDAC3D391A even after ET treatment, suggesting that cytoplasmic translocation and cleavage of HDAC3 is required for HDAC3 ubiquitination (Supplementary Fig. 9c). Moreover, HDAC3D391A overexpression conferred stronger inhibition of PDCD5-induced p53 acetylation and activation that was conferred by HDAC3WT (Fig. 3f). These results indicate that PDCD5 induces caspase-3-dependent HDAC3 cleavage, which leads to ubiquitin-dependent proteasomal degradation of HDAC3.


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 mediates ubiquitin-dependent proteasomal degradation of HDAC3 via C-terminal cleavage of HDAC3.(a) MG132 treatment induces accumulation of cleaved HDAC3. Cells were treated with ET (50 μM) and/or MG132 (10 μM, 3 h). Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3 (left panel). Intensities of protein bands obtained from the immunoblotting assay were quantified with ImageJ (right panel) and normalized with respect to that of β-actin. Relative % intensity was calculated by dividing the normalized intensity by the sum of intensities from both cleaved and full-length HDAC3. Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (b) PDCD5 knockdown diminishes the reduction and cleavage of full-length HDAC3. shcontrol or stable shPDCD5-expressing HCT-116 cells were treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (left panel). Relative % intensity was calculated as described above (right panel). Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (c) HDAC3 ubiquitination increases in a time-dependent manner in response to ET treatment. Cells were transfected with HA-Ub plasmid and treated with MG1432 and/or ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (d) PDCD5 knockdown diminishes HDAC3 ubiquitination in response to ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (e) Mutation of Asp-391 abolishes ET-induced HDAC3 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (f) Mutation of Asp-391 potentiates the action of HDAC3 in the inhibition of PDCD5-mediated p53 acetylation. Cells were transfected with the indicated Flag-tagged plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies.
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f3: PDCD5 mediates ubiquitin-dependent proteasomal degradation of HDAC3 via C-terminal cleavage of HDAC3.(a) MG132 treatment induces accumulation of cleaved HDAC3. Cells were treated with ET (50 μM) and/or MG132 (10 μM, 3 h). Whole-cell lysates were immunoblotted with the indicated antibodies. Arrow indicates cleaved HDAC3 (left panel). Intensities of protein bands obtained from the immunoblotting assay were quantified with ImageJ (right panel) and normalized with respect to that of β-actin. Relative % intensity was calculated by dividing the normalized intensity by the sum of intensities from both cleaved and full-length HDAC3. Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (b) PDCD5 knockdown diminishes the reduction and cleavage of full-length HDAC3. shcontrol or stable shPDCD5-expressing HCT-116 cells were treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies (left panel). Relative % intensity was calculated as described above (right panel). Error bars, s.d. (n=3). (*P<0.01 versus without ET.) (c) HDAC3 ubiquitination increases in a time-dependent manner in response to ET treatment. Cells were transfected with HA-Ub plasmid and treated with MG1432 and/or ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (d) PDCD5 knockdown diminishes HDAC3 ubiquitination in response to ET. Whole-cell lysates were immunoprecipitated with anti-HDAC3 (C) antibody and immunoblotted with the indicated antibodies. (e) Mutation of Asp-391 abolishes ET-induced HDAC3 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with ET and/or MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (f) Mutation of Asp-391 potentiates the action of HDAC3 in the inhibition of PDCD5-mediated p53 acetylation. Cells were transfected with the indicated Flag-tagged plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies.
Mentions: HDAC3 cleavage by PDCD5 correlates with increased p53 acetylation; however, HDAC3 cleavage is not observed during ET treatment at the time (4 h) when p53 is activated. We next examined whether the cleaved form of HDAC3 was rapidly degraded during ET treatment. At 2 h after the start of ET treatment, p53 acetylation at multiple lysine residues increased when the cleavage of HDAC3 was observed in the presence of MG132 (Supplementary Fig. 9a). At 4 h after the start of ET treatment, ∼20% of HDAC3 was observed as the cleaved form in the presence of MG132. At 12 h after the start of ET treatment, we found that ∼60% of full-length HDAC3 in the cell lysate was cleaved and dissociated from p53, which is consistent with maximum p53 activation (Fig. 3a). However, PDCD5 knockdown strongly reduced the percentage of HDAC3 cleavage and the dissociation of HDAC3 from p53 in response to ET treatment (Fig. 3b; Supplementary Fig. 9a). Similar results were observed by PDCD5 overexpression in the presence of MG132 (Supplementary Fig. 9b). We found that ET treatment induced the time-dependent ubiquitination and cleavage of HDAC3 (Fig. 3c); however, depletion of PDCD5 substantially reduced HDAC3 ubiquitination (Fig. 3d). By contrast, uncleaved mutant HDAC3D391A was not ubiquitinated in response to ET treatment (Fig. 3e). Confocal microscopy analysis showed the nuclear retention of uncleaved HDAC3D391A even after ET treatment, suggesting that cytoplasmic translocation and cleavage of HDAC3 is required for HDAC3 ubiquitination (Supplementary Fig. 9c). Moreover, HDAC3D391A overexpression conferred stronger inhibition of PDCD5-induced p53 acetylation and activation that was conferred by HDAC3WT (Fig. 3f). These results indicate that PDCD5 induces caspase-3-dependent HDAC3 cleavage, which leads to ubiquitin-dependent proteasomal degradation of HDAC3.

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