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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

NDPK-D Knockdown induces apoptosis of N1E-115 cells.(A) NDPK-D siRNAs reduced NDPK-D mRNA expression. N1E-115 cells were transfected with the indicated siRNAs. Total RNA isolated at 72 h post-transfection was analyzed by real-time PCR. **P < 0.01. n = 3. (B) siRNA-mediated knockdown of NDPK-D increased LDH release. N1E-115 cells were transfected with indicated siRNA and cultured for 48 h. The relative LDH activities were measured in the culture medium and normalized with control values. **P < 0.01. n = 3. (C) z-VAD-FMK rescues NDPK-D siRNA induced apoptosis. N1E-115 cells were transfected with indicated siRNA and treated with or without 50 μM z-VAD-fmk. Cells were cultured for 48 h and LDH activities were measured as described in (B). LOW: no transfection; TX: 0.1% triton-X 100; *P < 0.05. n = 3. (D, E) siRNA-mediated NDPK-D knockdown increased the number of cleaved caspase-3-positive cells. N1E-115 cells transfected with indicated siRNAs were immunostained with anti-cleaved caspase-3 antibody. The representative images of transfected N1E-115 were shown (D). Percentage of cleaved caspase-3-positive cells were demonstrated in the graph (E). *P < 0.05. Scale bar: 100 μm. n = 3. (F) NDPK-D knockdown produces cleaved caspase-3. N1E-115 cells were transfected with indicated siRNAs. Cell lysates were prepared 72 h after transfection and subjected to western blotting. Cont: control siRNA; si #1, 2, 3, NDPK-D siRNA #1, 2, 3. (G) Knockdown of NDPK-D increased cell death in mouse embryo. Representative images of E17 brain sections from embryos that were co-transfected with GFP and NDPK-D siRNA #1 at E14. The sections were immunostained with anti-GFP and anti-Iba1 antibodies. Scale bar: 600 μm. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s (A, B), or Tukey-Kramer’s (C, E) multiple comparison tests.
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pone.0139616.g002: NDPK-D Knockdown induces apoptosis of N1E-115 cells.(A) NDPK-D siRNAs reduced NDPK-D mRNA expression. N1E-115 cells were transfected with the indicated siRNAs. Total RNA isolated at 72 h post-transfection was analyzed by real-time PCR. **P < 0.01. n = 3. (B) siRNA-mediated knockdown of NDPK-D increased LDH release. N1E-115 cells were transfected with indicated siRNA and cultured for 48 h. The relative LDH activities were measured in the culture medium and normalized with control values. **P < 0.01. n = 3. (C) z-VAD-FMK rescues NDPK-D siRNA induced apoptosis. N1E-115 cells were transfected with indicated siRNA and treated with or without 50 μM z-VAD-fmk. Cells were cultured for 48 h and LDH activities were measured as described in (B). LOW: no transfection; TX: 0.1% triton-X 100; *P < 0.05. n = 3. (D, E) siRNA-mediated NDPK-D knockdown increased the number of cleaved caspase-3-positive cells. N1E-115 cells transfected with indicated siRNAs were immunostained with anti-cleaved caspase-3 antibody. The representative images of transfected N1E-115 were shown (D). Percentage of cleaved caspase-3-positive cells were demonstrated in the graph (E). *P < 0.05. Scale bar: 100 μm. n = 3. (F) NDPK-D knockdown produces cleaved caspase-3. N1E-115 cells were transfected with indicated siRNAs. Cell lysates were prepared 72 h after transfection and subjected to western blotting. Cont: control siRNA; si #1, 2, 3, NDPK-D siRNA #1, 2, 3. (G) Knockdown of NDPK-D increased cell death in mouse embryo. Representative images of E17 brain sections from embryos that were co-transfected with GFP and NDPK-D siRNA #1 at E14. The sections were immunostained with anti-GFP and anti-Iba1 antibodies. Scale bar: 600 μm. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s (A, B), or Tukey-Kramer’s (C, E) multiple comparison tests.

Mentions: To investigate the physiological function of NDPK-D, we performed loss-of-function experiments using small interfering RNA (siRNA). We first examined the knockdown efficacy of NDPK-D siRNA in neuroblastoma N1E-115 cells. Efficient NDPK-D mRNA downregulation specifically occurred in NDPK-D siRNA-transfected, but not in the control, non-target siRNA-transfected cells (Fig 2A). We then examined whether NDPK-D knockdown regulated cellular viability by the lactate dehydrogenase (LDH) activity assay. N1E-115 cells were transfected with control or NDPK-D siRNA and cultured for 48 h. LDH release was greater in NDPK-D siRNA-transfected cells than in the control siRNA-transfected cells (Fig 2B). Moreover, pan-caspase inhibitor, z-VAD-FMK suppressed the release of LDH from the cells (Fig 2C). In addition, immunostaining demonstrated a significant increase (P < 0.05) in cleaved caspase-3-positive cells in the presence of NDPK-D siRNA (Fig 2D). z-VAD-FMK significantly inhibited (P < 0.05) the NDPK-D siRNA-induced caspase activation (Fig 2E). Western blot analysis further confirms that NDPK-D downregulation induces caspase-3 activation (Fig 2F). Taken together, these results indicate that knockdown of NDPK-D increases apoptosis through caspase-3 activation in N1E-115 cells. Then, to assess the effect of NDPK-D inhibition in vivo, we performed in utero electroporation. Embryonic day (E) 14 mouse embryos were co-transfected with GFP and NDPK-D siRNA #1. Embryos were fixed and analyzed 3 days after the in utero electroporation. Knockdown of NDPK-D increased the accumulation of Iba1-positive microglia (Fig 2G), suggesting phagocytosis of dead cells by microglia. These results support the notion that NDPK-D is required for neuronal survival both in vitro and in vivo.


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

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

NDPK-D Knockdown induces apoptosis of N1E-115 cells.(A) NDPK-D siRNAs reduced NDPK-D mRNA expression. N1E-115 cells were transfected with the indicated siRNAs. Total RNA isolated at 72 h post-transfection was analyzed by real-time PCR. **P < 0.01. n = 3. (B) siRNA-mediated knockdown of NDPK-D increased LDH release. N1E-115 cells were transfected with indicated siRNA and cultured for 48 h. The relative LDH activities were measured in the culture medium and normalized with control values. **P < 0.01. n = 3. (C) z-VAD-FMK rescues NDPK-D siRNA induced apoptosis. N1E-115 cells were transfected with indicated siRNA and treated with or without 50 μM z-VAD-fmk. Cells were cultured for 48 h and LDH activities were measured as described in (B). LOW: no transfection; TX: 0.1% triton-X 100; *P < 0.05. n = 3. (D, E) siRNA-mediated NDPK-D knockdown increased the number of cleaved caspase-3-positive cells. N1E-115 cells transfected with indicated siRNAs were immunostained with anti-cleaved caspase-3 antibody. The representative images of transfected N1E-115 were shown (D). Percentage of cleaved caspase-3-positive cells were demonstrated in the graph (E). *P < 0.05. Scale bar: 100 μm. n = 3. (F) NDPK-D knockdown produces cleaved caspase-3. N1E-115 cells were transfected with indicated siRNAs. Cell lysates were prepared 72 h after transfection and subjected to western blotting. Cont: control siRNA; si #1, 2, 3, NDPK-D siRNA #1, 2, 3. (G) Knockdown of NDPK-D increased cell death in mouse embryo. Representative images of E17 brain sections from embryos that were co-transfected with GFP and NDPK-D siRNA #1 at E14. The sections were immunostained with anti-GFP and anti-Iba1 antibodies. Scale bar: 600 μm. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s (A, B), or Tukey-Kramer’s (C, E) multiple comparison tests.
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pone.0139616.g002: NDPK-D Knockdown induces apoptosis of N1E-115 cells.(A) NDPK-D siRNAs reduced NDPK-D mRNA expression. N1E-115 cells were transfected with the indicated siRNAs. Total RNA isolated at 72 h post-transfection was analyzed by real-time PCR. **P < 0.01. n = 3. (B) siRNA-mediated knockdown of NDPK-D increased LDH release. N1E-115 cells were transfected with indicated siRNA and cultured for 48 h. The relative LDH activities were measured in the culture medium and normalized with control values. **P < 0.01. n = 3. (C) z-VAD-FMK rescues NDPK-D siRNA induced apoptosis. N1E-115 cells were transfected with indicated siRNA and treated with or without 50 μM z-VAD-fmk. Cells were cultured for 48 h and LDH activities were measured as described in (B). LOW: no transfection; TX: 0.1% triton-X 100; *P < 0.05. n = 3. (D, E) siRNA-mediated NDPK-D knockdown increased the number of cleaved caspase-3-positive cells. N1E-115 cells transfected with indicated siRNAs were immunostained with anti-cleaved caspase-3 antibody. The representative images of transfected N1E-115 were shown (D). Percentage of cleaved caspase-3-positive cells were demonstrated in the graph (E). *P < 0.05. Scale bar: 100 μm. n = 3. (F) NDPK-D knockdown produces cleaved caspase-3. N1E-115 cells were transfected with indicated siRNAs. Cell lysates were prepared 72 h after transfection and subjected to western blotting. Cont: control siRNA; si #1, 2, 3, NDPK-D siRNA #1, 2, 3. (G) Knockdown of NDPK-D increased cell death in mouse embryo. Representative images of E17 brain sections from embryos that were co-transfected with GFP and NDPK-D siRNA #1 at E14. The sections were immunostained with anti-GFP and anti-Iba1 antibodies. Scale bar: 600 μm. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s (A, B), or Tukey-Kramer’s (C, E) multiple comparison tests.
Mentions: To investigate the physiological function of NDPK-D, we performed loss-of-function experiments using small interfering RNA (siRNA). We first examined the knockdown efficacy of NDPK-D siRNA in neuroblastoma N1E-115 cells. Efficient NDPK-D mRNA downregulation specifically occurred in NDPK-D siRNA-transfected, but not in the control, non-target siRNA-transfected cells (Fig 2A). We then examined whether NDPK-D knockdown regulated cellular viability by the lactate dehydrogenase (LDH) activity assay. N1E-115 cells were transfected with control or NDPK-D siRNA and cultured for 48 h. LDH release was greater in NDPK-D siRNA-transfected cells than in the control siRNA-transfected cells (Fig 2B). Moreover, pan-caspase inhibitor, z-VAD-FMK suppressed the release of LDH from the cells (Fig 2C). In addition, immunostaining demonstrated a significant increase (P < 0.05) in cleaved caspase-3-positive cells in the presence of NDPK-D siRNA (Fig 2D). z-VAD-FMK significantly inhibited (P < 0.05) the NDPK-D siRNA-induced caspase activation (Fig 2E). Western blot analysis further confirms that NDPK-D downregulation induces caspase-3 activation (Fig 2F). Taken together, these results indicate that knockdown of NDPK-D increases apoptosis through caspase-3 activation in N1E-115 cells. Then, to assess the effect of NDPK-D inhibition in vivo, we performed in utero electroporation. Embryonic day (E) 14 mouse embryos were co-transfected with GFP and NDPK-D siRNA #1. Embryos were fixed and analyzed 3 days after the in utero electroporation. Knockdown of NDPK-D increased the accumulation of Iba1-positive microglia (Fig 2G), suggesting phagocytosis of dead cells by microglia. These results support the notion that NDPK-D is required for neuronal survival both in vitro and in vivo.

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