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Acetylation of NDPK-D Regulates Its Subcellular Localization and Cell Survival.

Fujita Y, Fujiwara K, Zenitani S, Yamashita T - PLoS ONE (2015)

Bottom Line: NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation.Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells.Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo, Japan.

ABSTRACT
Nucleoside diphosphate kinases (NDPK) are ubiquitous enzymes that catalyze the reversible phosphotransfer of γ-phosphates between di- and triphosphonucleosides. NDPK-D (Nm23-H4) is the only member of the NDPK family with a mitochondrial targeting sequence. Despite the high expression of NDPK-D in the developing central nervous system, its function remains to be determined. In this study, we show that NDPK-D knockdown induces apoptosis in neuroblastoma cells as well as in mouse cortex, suggesting that NDPK-D is required for neuronal survival. We identified NDPK-D as a binding partner of NAD+-dependent histone deacetylase, SIRT1, by yeast two-hybrid screening. NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation. Inhibition of SIRT1 increases the acetylation of NDPK-D. Overexpression of NDPK-D along with SIRT1, or mutation in the acetylated lysine residues in NDPK-D, increases its nuclear accumulation. Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells. Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis.

No MeSH data available.


Related in: MedlinePlus

SIRT1 interacts with NDPK-D.(A) Localization of SIRT1 and NDPK-D. COS-7 cells transfected with plasmids encoding HA-SIRT1 and Myc-NDPK-D were cultured for 36 h, and immunostained with anti-HA and anti-Myc antibodies. Scale bar: 100 μm. (B) Cytosolic NDPK-D partially co-localized with mitochondria-targeted GFP. Cells transfected with plasmids encoding Myc-NDPK-D and mitochondria-targeted GFP were immunostained with anti-Myc and anti-GFP antibodies. Scale bar: 20 μm. (C) NDPK-D localized to both cytoplasmic and nuclear fractions. COS-7 cells were transfected with Myc-NDPK-D, and cultured for 36 h. Cytoplasmic and nuclear fractions were isolated and subjected to western blotting using indicated antibodies. (D) Co-immunoprecipitation of SIRT1 and NDPK-D. Cells were transiently transfected with the indicated plasmids and lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-HA antibody. (E) Co-immunoprecipitation of Endogenous SIRT1 and Myc-tagged NDPK-D. Cells were immunoprecipitated with anti-SIRT1 or control IgG antibody. The immunoprecipitates were immunoblotted with anti-Myc antibody.
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pone.0139616.g003: SIRT1 interacts with NDPK-D.(A) Localization of SIRT1 and NDPK-D. COS-7 cells transfected with plasmids encoding HA-SIRT1 and Myc-NDPK-D were cultured for 36 h, and immunostained with anti-HA and anti-Myc antibodies. Scale bar: 100 μm. (B) Cytosolic NDPK-D partially co-localized with mitochondria-targeted GFP. Cells transfected with plasmids encoding Myc-NDPK-D and mitochondria-targeted GFP were immunostained with anti-Myc and anti-GFP antibodies. Scale bar: 20 μm. (C) NDPK-D localized to both cytoplasmic and nuclear fractions. COS-7 cells were transfected with Myc-NDPK-D, and cultured for 36 h. Cytoplasmic and nuclear fractions were isolated and subjected to western blotting using indicated antibodies. (D) Co-immunoprecipitation of SIRT1 and NDPK-D. Cells were transiently transfected with the indicated plasmids and lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-HA antibody. (E) Co-immunoprecipitation of Endogenous SIRT1 and Myc-tagged NDPK-D. Cells were immunoprecipitated with anti-SIRT1 or control IgG antibody. The immunoprecipitates were immunoblotted with anti-Myc antibody.

Mentions: We previously conducted yeast two-hybrid screen with human fetal brain cDNA library using the conserved sirtuin catalytic domain as the bait [32]. Among the possible SIRT1 binding partners, we focused on human NDPK-D because of its role in mitochondrial biogenesis. To validate the association of SIRT1 with NDPK-D in mammalian cells, we first examined the subcellular localization of SIRT1 and NDPK-D in COS-7 cells. The cells were co-transfected with expression vector carrying Myc-tagged NDPK-D and HA-tagged SIRT1, and immunostained with antibodies against Myc and HA. While SIRT1 predominantly localized to nucleus (Fig 3A), NDPK-D showed mostly cytosol localization with certain variations. NDPK-D was mainly localized in the cytosol with puncta in some cells, and in other cells, was partially localized in the nucleus with diffuse staining in the cytosol (Fig 3A). The punctate NDPK-D signals partially merged with mitochondria-targeted GFP, suggesting that NDPK-D localized to mitochondria (Fig 3B). Western blot analysis on the cytoplasmic and the nuclear lysates were prepared from Myc-NDPK-D transfected COS-7 cells detected NDPK-D protein in both cytoplasmic and nuclear fractions (Fig 3C). These results suggest that NDPK-D localizes to both cytosol and nucleus and that it shares the same subcellular compartments when co-expressed with SIRT1.


Acetylation of NDPK-D Regulates Its Subcellular Localization and Cell Survival.

Fujita Y, Fujiwara K, Zenitani S, Yamashita T - PLoS ONE (2015)

SIRT1 interacts with NDPK-D.(A) Localization of SIRT1 and NDPK-D. COS-7 cells transfected with plasmids encoding HA-SIRT1 and Myc-NDPK-D were cultured for 36 h, and immunostained with anti-HA and anti-Myc antibodies. Scale bar: 100 μm. (B) Cytosolic NDPK-D partially co-localized with mitochondria-targeted GFP. Cells transfected with plasmids encoding Myc-NDPK-D and mitochondria-targeted GFP were immunostained with anti-Myc and anti-GFP antibodies. Scale bar: 20 μm. (C) NDPK-D localized to both cytoplasmic and nuclear fractions. COS-7 cells were transfected with Myc-NDPK-D, and cultured for 36 h. Cytoplasmic and nuclear fractions were isolated and subjected to western blotting using indicated antibodies. (D) Co-immunoprecipitation of SIRT1 and NDPK-D. Cells were transiently transfected with the indicated plasmids and lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-HA antibody. (E) Co-immunoprecipitation of Endogenous SIRT1 and Myc-tagged NDPK-D. Cells were immunoprecipitated with anti-SIRT1 or control IgG antibody. The immunoprecipitates were immunoblotted with anti-Myc antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591271&req=5

pone.0139616.g003: SIRT1 interacts with NDPK-D.(A) Localization of SIRT1 and NDPK-D. COS-7 cells transfected with plasmids encoding HA-SIRT1 and Myc-NDPK-D were cultured for 36 h, and immunostained with anti-HA and anti-Myc antibodies. Scale bar: 100 μm. (B) Cytosolic NDPK-D partially co-localized with mitochondria-targeted GFP. Cells transfected with plasmids encoding Myc-NDPK-D and mitochondria-targeted GFP were immunostained with anti-Myc and anti-GFP antibodies. Scale bar: 20 μm. (C) NDPK-D localized to both cytoplasmic and nuclear fractions. COS-7 cells were transfected with Myc-NDPK-D, and cultured for 36 h. Cytoplasmic and nuclear fractions were isolated and subjected to western blotting using indicated antibodies. (D) Co-immunoprecipitation of SIRT1 and NDPK-D. Cells were transiently transfected with the indicated plasmids and lysates were immunoprecipitated with anti-Myc antibody. The immunoprecipitates were immunoblotted with anti-HA antibody. (E) Co-immunoprecipitation of Endogenous SIRT1 and Myc-tagged NDPK-D. Cells were immunoprecipitated with anti-SIRT1 or control IgG antibody. The immunoprecipitates were immunoblotted with anti-Myc antibody.
Mentions: We previously conducted yeast two-hybrid screen with human fetal brain cDNA library using the conserved sirtuin catalytic domain as the bait [32]. Among the possible SIRT1 binding partners, we focused on human NDPK-D because of its role in mitochondrial biogenesis. To validate the association of SIRT1 with NDPK-D in mammalian cells, we first examined the subcellular localization of SIRT1 and NDPK-D in COS-7 cells. The cells were co-transfected with expression vector carrying Myc-tagged NDPK-D and HA-tagged SIRT1, and immunostained with antibodies against Myc and HA. While SIRT1 predominantly localized to nucleus (Fig 3A), NDPK-D showed mostly cytosol localization with certain variations. NDPK-D was mainly localized in the cytosol with puncta in some cells, and in other cells, was partially localized in the nucleus with diffuse staining in the cytosol (Fig 3A). The punctate NDPK-D signals partially merged with mitochondria-targeted GFP, suggesting that NDPK-D localized to mitochondria (Fig 3B). Western blot analysis on the cytoplasmic and the nuclear lysates were prepared from Myc-NDPK-D transfected COS-7 cells detected NDPK-D protein in both cytoplasmic and nuclear fractions (Fig 3C). These results suggest that NDPK-D localizes to both cytosol and nucleus and that it shares the same subcellular compartments when co-expressed with SIRT1.

Bottom Line: NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation.Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells.Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Neuroscience, Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita, Osaka, Japan; Core Research for Evolutional Science and Technology, Japan Science and Technology Agency, 5, Sanbancho, Chiyoda-ku, Tokyo, Japan.

ABSTRACT
Nucleoside diphosphate kinases (NDPK) are ubiquitous enzymes that catalyze the reversible phosphotransfer of γ-phosphates between di- and triphosphonucleosides. NDPK-D (Nm23-H4) is the only member of the NDPK family with a mitochondrial targeting sequence. Despite the high expression of NDPK-D in the developing central nervous system, its function remains to be determined. In this study, we show that NDPK-D knockdown induces apoptosis in neuroblastoma cells as well as in mouse cortex, suggesting that NDPK-D is required for neuronal survival. We identified NDPK-D as a binding partner of NAD+-dependent histone deacetylase, SIRT1, by yeast two-hybrid screening. NDPK-D co-localized with SIRT1, and the association of these molecules was confirmed by co-immunoprecipitation. Inhibition of SIRT1 increases the acetylation of NDPK-D. Overexpression of NDPK-D along with SIRT1, or mutation in the acetylated lysine residues in NDPK-D, increases its nuclear accumulation. Furthermore, the NDPK-D acetylation-mimic mutant increased apoptosis in N1E-115 cells. Our data demonstrate that acetylation regulates the shuttling of NDPK-D between nucleus and cytoplasm, and increased acetylation of NDPK-D causes apoptosis.

No MeSH data available.


Related in: MedlinePlus