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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

Relative NDPK-D expression patterns in mouse brain at different developmental stages.(A) Expression profiles of mouse cDNA from cerebrum at various developmental stages. The expression levels of NDPK-D relative to those of GAPDH were measured by ΔΔCt method. The results were the mean ± SE from three independent experiments. The relative NDPK-D expression levels are presented as fold changes relative to the level in P1. (B) In situ hybridization of NDPK-D mRNA in sagittal section of E15 mouse brain. Expression of NDPK-D mRNA was detected in both cortical plate and ventricular zone. Scale bar: 500 μm (low magnification image), 200 μm (high magnification image).
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pone.0139616.g001: Relative NDPK-D expression patterns in mouse brain at different developmental stages.(A) Expression profiles of mouse cDNA from cerebrum at various developmental stages. The expression levels of NDPK-D relative to those of GAPDH were measured by ΔΔCt method. The results were the mean ± SE from three independent experiments. The relative NDPK-D expression levels are presented as fold changes relative to the level in P1. (B) In situ hybridization of NDPK-D mRNA in sagittal section of E15 mouse brain. Expression of NDPK-D mRNA was detected in both cortical plate and ventricular zone. Scale bar: 500 μm (low magnification image), 200 μm (high magnification image).

Mentions: We first examined the NDPK-D expression profile by real-time PCR, using total RNA extracted from mouse brains at various developmental stages. NDPK-D expression was relatively higher in the embryonic stage than in the adult stage (Fig 1A). Next, we investigated the distribution of NDPK-D in the brain by in situ hybridization. Sagittal sections of mouse brain at E15 were prepared and hybridized with NDPK-D antisense or sense probe. Hybridization with NDPK-D antisense RNA probe displayed widespread expression, with relatively higher expression of NDPK-D mRNA in several regions including ventricular zone and cortical plate (Fig 1B). The absence of hybridization signals with sense control probe eliminated any background noise and confirmed specific NDPK-D mRNA expression in the brain tissues (Fig 1B).


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

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

Relative NDPK-D expression patterns in mouse brain at different developmental stages.(A) Expression profiles of mouse cDNA from cerebrum at various developmental stages. The expression levels of NDPK-D relative to those of GAPDH were measured by ΔΔCt method. The results were the mean ± SE from three independent experiments. The relative NDPK-D expression levels are presented as fold changes relative to the level in P1. (B) In situ hybridization of NDPK-D mRNA in sagittal section of E15 mouse brain. Expression of NDPK-D mRNA was detected in both cortical plate and ventricular zone. Scale bar: 500 μm (low magnification image), 200 μm (high magnification image).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4591271&req=5

pone.0139616.g001: Relative NDPK-D expression patterns in mouse brain at different developmental stages.(A) Expression profiles of mouse cDNA from cerebrum at various developmental stages. The expression levels of NDPK-D relative to those of GAPDH were measured by ΔΔCt method. The results were the mean ± SE from three independent experiments. The relative NDPK-D expression levels are presented as fold changes relative to the level in P1. (B) In situ hybridization of NDPK-D mRNA in sagittal section of E15 mouse brain. Expression of NDPK-D mRNA was detected in both cortical plate and ventricular zone. Scale bar: 500 μm (low magnification image), 200 μm (high magnification image).
Mentions: We first examined the NDPK-D expression profile by real-time PCR, using total RNA extracted from mouse brains at various developmental stages. NDPK-D expression was relatively higher in the embryonic stage than in the adult stage (Fig 1A). Next, we investigated the distribution of NDPK-D in the brain by in situ hybridization. Sagittal sections of mouse brain at E15 were prepared and hybridized with NDPK-D antisense or sense probe. Hybridization with NDPK-D antisense RNA probe displayed widespread expression, with relatively higher expression of NDPK-D mRNA in several regions including ventricular zone and cortical plate (Fig 1B). The absence of hybridization signals with sense control probe eliminated any background noise and confirmed specific NDPK-D mRNA expression in the brain tissues (Fig 1B).

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