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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

Casein kinase 2α enhances the stability and IPO13-mediated nuclear translocation of PDCD5 during genotoxic stress responses.(a) CK2α knockdown diminishes the effect of MG132 on PDCD5 stability. HCT-116 (p53+/+) cells were transfected with MG132 or shCK2α and immunoblotted with the indicated antibodies. (b) CK2α knockdown abolishes ET-induced HDAC3 cleavage, PDCD5 induction and PDCD5 phosphorylation. Either shcontrol or stable shCK2α-expressing HCT-116 cells were treated with ET. Whole-cell lysates were immunoblotted with the indicated antibodies. (c) CK2α overexpression inhibits PDCD5 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (d) CK2-mediated phosphorylation is required for ET-induced PDCD5 nuclear translocation. Cells were transfected with the indicated Flag-PDCD5 plasmids and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (e) IPO13 selectively interacts with PDCD5 upon genotoxic stress response. Cells were transfected with HA-PDCD5 and the indicated Flag-tagged plasmids, and treated with ET. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (f) Knockdown of IPO13 abrogated the nuclear translocation of endogenous PDCD5 in response to ET. HCT-116 cells were transfected with the indicated siRNAs and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (g) IPO13 knockdown abolishes the nuclear translocation of phosphor-PDCD5. HCT-116 cells were transfected with the indicated siRNAs and Flag-PDCD5 plasmid, and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (h) Phospho-mimetic PDCD5S119D mutant further promotes p53 acetylation and stabilization. Cells were transfected with the indicated Flag-PDCD5 plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Scale bar, 10 μm.
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f4: Casein kinase 2α enhances the stability and IPO13-mediated nuclear translocation of PDCD5 during genotoxic stress responses.(a) CK2α knockdown diminishes the effect of MG132 on PDCD5 stability. HCT-116 (p53+/+) cells were transfected with MG132 or shCK2α and immunoblotted with the indicated antibodies. (b) CK2α knockdown abolishes ET-induced HDAC3 cleavage, PDCD5 induction and PDCD5 phosphorylation. Either shcontrol or stable shCK2α-expressing HCT-116 cells were treated with ET. Whole-cell lysates were immunoblotted with the indicated antibodies. (c) CK2α overexpression inhibits PDCD5 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (d) CK2-mediated phosphorylation is required for ET-induced PDCD5 nuclear translocation. Cells were transfected with the indicated Flag-PDCD5 plasmids and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (e) IPO13 selectively interacts with PDCD5 upon genotoxic stress response. Cells were transfected with HA-PDCD5 and the indicated Flag-tagged plasmids, and treated with ET. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (f) Knockdown of IPO13 abrogated the nuclear translocation of endogenous PDCD5 in response to ET. HCT-116 cells were transfected with the indicated siRNAs and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (g) IPO13 knockdown abolishes the nuclear translocation of phosphor-PDCD5. HCT-116 cells were transfected with the indicated siRNAs and Flag-PDCD5 plasmid, and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (h) Phospho-mimetic PDCD5S119D mutant further promotes p53 acetylation and stabilization. Cells were transfected with the indicated Flag-PDCD5 plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Scale bar, 10 μm.

Mentions: We next examined how PDCD5 levels are increased and how PDCD5 is translocated into the nucleus in response to ET treatment. CK2α phosphorylated PDCD5 at Ser-119; mutation of this site disrupted the pro-apoptotic function of PDCD5 (ref. 29). Therefore, we tested whether CK2-mediated phosphorylation increased PDCD5 stability. Treatment with MG132 significantly increased PDCD5 levels, whereas CK2α knockdown diminished the effect of MG132 on PDCD5 stabilization (Fig. 4a). A cycloheximide time-course experiment showed that CK2α enhanced PDCD5 stability. CK2α knockdown reduced PDCD5 stability, indicating a crucial role for CK2α in PDCD5 stabilization (Supplementary Fig. 10a). To confirm these results, we generated a PDCD5 antibody that specifically recognized the phosphorylated Ser-119 residue. The phospho-specific antibody detected phosphorylated Ser-119 in PDCD5WT, but not in the PDCD5S119A mutant, verifying its specificity (Supplementary Fig. 10b). We observed that the levels of both PDCD5 and phosphorylated PDCD5 concurrently increase in response to ET treatment. However, CK2 knockdown diminished the levels of PDCD5 and phosphorylated PDCD5, and reduced HDAC3 cleavage and p53 activation in response to ET (Fig. 4b; Supplementary Fig. 10c). CK2 overexpression markedly reduced PDCD5 ubiquitination (Fig. 4c) and increased PDCD5 stability, but not PDCD5 mRNA levels (Supplementary Fig. 10d). These results indicate that CK2-mediated phosphorylation of Ser-119 is required for PDCD5 stabilization.


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)

Casein kinase 2α enhances the stability and IPO13-mediated nuclear translocation of PDCD5 during genotoxic stress responses.(a) CK2α knockdown diminishes the effect of MG132 on PDCD5 stability. HCT-116 (p53+/+) cells were transfected with MG132 or shCK2α and immunoblotted with the indicated antibodies. (b) CK2α knockdown abolishes ET-induced HDAC3 cleavage, PDCD5 induction and PDCD5 phosphorylation. Either shcontrol or stable shCK2α-expressing HCT-116 cells were treated with ET. Whole-cell lysates were immunoblotted with the indicated antibodies. (c) CK2α overexpression inhibits PDCD5 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (d) CK2-mediated phosphorylation is required for ET-induced PDCD5 nuclear translocation. Cells were transfected with the indicated Flag-PDCD5 plasmids and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (e) IPO13 selectively interacts with PDCD5 upon genotoxic stress response. Cells were transfected with HA-PDCD5 and the indicated Flag-tagged plasmids, and treated with ET. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (f) Knockdown of IPO13 abrogated the nuclear translocation of endogenous PDCD5 in response to ET. HCT-116 cells were transfected with the indicated siRNAs and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (g) IPO13 knockdown abolishes the nuclear translocation of phosphor-PDCD5. HCT-116 cells were transfected with the indicated siRNAs and Flag-PDCD5 plasmid, and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (h) Phospho-mimetic PDCD5S119D mutant further promotes p53 acetylation and stabilization. Cells were transfected with the indicated Flag-PDCD5 plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Scale bar, 10 μm.
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f4: Casein kinase 2α enhances the stability and IPO13-mediated nuclear translocation of PDCD5 during genotoxic stress responses.(a) CK2α knockdown diminishes the effect of MG132 on PDCD5 stability. HCT-116 (p53+/+) cells were transfected with MG132 or shCK2α and immunoblotted with the indicated antibodies. (b) CK2α knockdown abolishes ET-induced HDAC3 cleavage, PDCD5 induction and PDCD5 phosphorylation. Either shcontrol or stable shCK2α-expressing HCT-116 cells were treated with ET. Whole-cell lysates were immunoblotted with the indicated antibodies. (c) CK2α overexpression inhibits PDCD5 ubiquitination. Cells were transfected with HA-Ub and the indicated Flag-tagged plasmids, and treated with MG132. Whole-cell lysates were immunoprecipitated with anti-Flag antibody and immunoblotted with the indicated antibodies. (d) CK2-mediated phosphorylation is required for ET-induced PDCD5 nuclear translocation. Cells were transfected with the indicated Flag-PDCD5 plasmids and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (e) IPO13 selectively interacts with PDCD5 upon genotoxic stress response. Cells were transfected with HA-PDCD5 and the indicated Flag-tagged plasmids, and treated with ET. Whole-cell lysates were immunoprecipitated and immunoblotted with the indicated antibodies. (f) Knockdown of IPO13 abrogated the nuclear translocation of endogenous PDCD5 in response to ET. HCT-116 cells were transfected with the indicated siRNAs and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (g) IPO13 knockdown abolishes the nuclear translocation of phosphor-PDCD5. HCT-116 cells were transfected with the indicated siRNAs and Flag-PDCD5 plasmid, and treated with ET. Immunofluorescence analysis was performed as described in the Supplementary Experimental Procedures Section. (h) Phospho-mimetic PDCD5S119D mutant further promotes p53 acetylation and stabilization. Cells were transfected with the indicated Flag-PDCD5 plasmids. Whole-cell lysates were immunoblotted with the indicated antibodies. Scale bar, 10 μm.
Mentions: We next examined how PDCD5 levels are increased and how PDCD5 is translocated into the nucleus in response to ET treatment. CK2α phosphorylated PDCD5 at Ser-119; mutation of this site disrupted the pro-apoptotic function of PDCD5 (ref. 29). Therefore, we tested whether CK2-mediated phosphorylation increased PDCD5 stability. Treatment with MG132 significantly increased PDCD5 levels, whereas CK2α knockdown diminished the effect of MG132 on PDCD5 stabilization (Fig. 4a). A cycloheximide time-course experiment showed that CK2α enhanced PDCD5 stability. CK2α knockdown reduced PDCD5 stability, indicating a crucial role for CK2α in PDCD5 stabilization (Supplementary Fig. 10a). To confirm these results, we generated a PDCD5 antibody that specifically recognized the phosphorylated Ser-119 residue. The phospho-specific antibody detected phosphorylated Ser-119 in PDCD5WT, but not in the PDCD5S119A mutant, verifying its specificity (Supplementary Fig. 10b). We observed that the levels of both PDCD5 and phosphorylated PDCD5 concurrently increase in response to ET treatment. However, CK2 knockdown diminished the levels of PDCD5 and phosphorylated PDCD5, and reduced HDAC3 cleavage and p53 activation in response to ET (Fig. 4b; Supplementary Fig. 10c). CK2 overexpression markedly reduced PDCD5 ubiquitination (Fig. 4c) and increased PDCD5 stability, but not PDCD5 mRNA levels (Supplementary Fig. 10d). These results indicate that CK2-mediated phosphorylation of Ser-119 is required for PDCD5 stabilization.

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