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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

Mutation of acetylated lysine residues results in mislocalization of NDPK-D.(A) Inhibition of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D, treated with SIRT1 inhibitor Ex527, and cultured for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and the immunoprecipitates were immunoblotted with anti-acetylated lysine (Ac-Lys) antibody. The Ac-Lys signal intensity was quantified by densitometry and normalized to the signal intensity of precipitated Myc-NDPK-D. *P < 0.05. n = 3. (B) Knockdown of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D and control or SIRT1 shRNA. The acetylation level of NDPK-D was tested as described in (A). **P < 0.01. n = 6. SIRT1 shRNA efficiently reduced Sirt1 mRNA expression in N1E-115 cells (right panel). **P < 0.01. n = 3. (C, D) SIRT1, but not catalytic-dead point mutant (H363Y) deacetylates NDPK-D in N1E-115 cells. Cells were transfected with Myc-NDPK-D and HA-SIRT1 (C) or HA-SIRT1 H363Y (D), and the acetylation levels of Myc-NDPK-D were determined by anti-Ac-Lys antibody. NS: not significant. **P < 0.01. n = 5 (C), n = 3 (D). (E) Schematic representation of lysine residues in NDPK-D. Lys-45, Lys-72, and Lys-91 were candidate acetylation sites. (F) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the K45R, K72R, or K91R mutants. Acetylation levels were determined as described in (A). Replacement of lysine residues with arginine decreased acetylation levels. n = 3. (G) N1E-115 cells were transfected with Myc-tagged wild-type (WT) NDPK-D or the K45/72/91R (3KR) mutant. Acetylation levels were determined as described in (A). **P < 0.01. n = 6. Statistical analyses were performed using Welch’s t-test (A-D, G).
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pone.0139616.g004: Mutation of acetylated lysine residues results in mislocalization of NDPK-D.(A) Inhibition of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D, treated with SIRT1 inhibitor Ex527, and cultured for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and the immunoprecipitates were immunoblotted with anti-acetylated lysine (Ac-Lys) antibody. The Ac-Lys signal intensity was quantified by densitometry and normalized to the signal intensity of precipitated Myc-NDPK-D. *P < 0.05. n = 3. (B) Knockdown of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D and control or SIRT1 shRNA. The acetylation level of NDPK-D was tested as described in (A). **P < 0.01. n = 6. SIRT1 shRNA efficiently reduced Sirt1 mRNA expression in N1E-115 cells (right panel). **P < 0.01. n = 3. (C, D) SIRT1, but not catalytic-dead point mutant (H363Y) deacetylates NDPK-D in N1E-115 cells. Cells were transfected with Myc-NDPK-D and HA-SIRT1 (C) or HA-SIRT1 H363Y (D), and the acetylation levels of Myc-NDPK-D were determined by anti-Ac-Lys antibody. NS: not significant. **P < 0.01. n = 5 (C), n = 3 (D). (E) Schematic representation of lysine residues in NDPK-D. Lys-45, Lys-72, and Lys-91 were candidate acetylation sites. (F) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the K45R, K72R, or K91R mutants. Acetylation levels were determined as described in (A). Replacement of lysine residues with arginine decreased acetylation levels. n = 3. (G) N1E-115 cells were transfected with Myc-tagged wild-type (WT) NDPK-D or the K45/72/91R (3KR) mutant. Acetylation levels were determined as described in (A). **P < 0.01. n = 6. Statistical analyses were performed using Welch’s t-test (A-D, G).

Mentions: SIRT1 mediated NAD+-dependent deacetylation regulates the function of multiple target proteins. Since we employed the SIRT1 deacetylation domain as bait in the yeast two-hybrid screen, it is possible that SIRT1 modulates NDPK-D activity by deacetylation. To test this hypothesis, we performed deacetylation assay. N1E-115 cells were transfected with expression vector encoding Myc-tagged NDPK-D and incubated with or without SIRT1 inhibitor Ex527. NDPK-D was immunoprecipitated with anti-Myc antibody and subjected to western blot analysis using anti-acetylated lysine antibody to examine the acetylation levels of NDPK-D. Elevated levels of acetylated Myc-NDPK-D were detected in the presence of Ex527 relative to the signals in the absence of Ex527, showing that SIRT1 can deacetylate NDPK-D (Fig 4A). We also examined the effect of knockdown of SIRT1 on the acetylation level of NDPK-D. Efficient downregulation of Sirt1 mRNA was found in SIRT1 shRNA-transfected N1E-115 cells (Fig 4B, right graph). The signal for acetylated Myc-NDPK-D increased in SIRT1 shRNA-transfected cells (Fig 4B, left panel and graph). We next examined whether overexpression of SIRT1 deacetylates NDPK-D. N1E-115 cells were transfected with Myc-tagged NDPK-D and HA-tagged SIRT1. The acetylation level of NDPK-D was investigated as described above. The signal for acetylated Myc-NDPK-D was decreased in the presence of HA-tagged SIRT1 (Fig 4C), whereas deacetylase-deficient mutant of SIRT1 (H363Y) did not affect the acetylation level of Myc-NDPK-D (Fig 4D). These results indicate that SIRT1 can modulate the deacetylation level of NDPK-D.


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

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

Mutation of acetylated lysine residues results in mislocalization of NDPK-D.(A) Inhibition of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D, treated with SIRT1 inhibitor Ex527, and cultured for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and the immunoprecipitates were immunoblotted with anti-acetylated lysine (Ac-Lys) antibody. The Ac-Lys signal intensity was quantified by densitometry and normalized to the signal intensity of precipitated Myc-NDPK-D. *P < 0.05. n = 3. (B) Knockdown of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D and control or SIRT1 shRNA. The acetylation level of NDPK-D was tested as described in (A). **P < 0.01. n = 6. SIRT1 shRNA efficiently reduced Sirt1 mRNA expression in N1E-115 cells (right panel). **P < 0.01. n = 3. (C, D) SIRT1, but not catalytic-dead point mutant (H363Y) deacetylates NDPK-D in N1E-115 cells. Cells were transfected with Myc-NDPK-D and HA-SIRT1 (C) or HA-SIRT1 H363Y (D), and the acetylation levels of Myc-NDPK-D were determined by anti-Ac-Lys antibody. NS: not significant. **P < 0.01. n = 5 (C), n = 3 (D). (E) Schematic representation of lysine residues in NDPK-D. Lys-45, Lys-72, and Lys-91 were candidate acetylation sites. (F) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the K45R, K72R, or K91R mutants. Acetylation levels were determined as described in (A). Replacement of lysine residues with arginine decreased acetylation levels. n = 3. (G) N1E-115 cells were transfected with Myc-tagged wild-type (WT) NDPK-D or the K45/72/91R (3KR) mutant. Acetylation levels were determined as described in (A). **P < 0.01. n = 6. Statistical analyses were performed using Welch’s t-test (A-D, G).
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pone.0139616.g004: Mutation of acetylated lysine residues results in mislocalization of NDPK-D.(A) Inhibition of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D, treated with SIRT1 inhibitor Ex527, and cultured for 48 h. Cell lysates were immunoprecipitated with anti-Myc antibody and the immunoprecipitates were immunoblotted with anti-acetylated lysine (Ac-Lys) antibody. The Ac-Lys signal intensity was quantified by densitometry and normalized to the signal intensity of precipitated Myc-NDPK-D. *P < 0.05. n = 3. (B) Knockdown of SIRT1 increased acetylation level of NDPK-D. N1E-115 cells were transfected with Myc-NDPK-D and control or SIRT1 shRNA. The acetylation level of NDPK-D was tested as described in (A). **P < 0.01. n = 6. SIRT1 shRNA efficiently reduced Sirt1 mRNA expression in N1E-115 cells (right panel). **P < 0.01. n = 3. (C, D) SIRT1, but not catalytic-dead point mutant (H363Y) deacetylates NDPK-D in N1E-115 cells. Cells were transfected with Myc-NDPK-D and HA-SIRT1 (C) or HA-SIRT1 H363Y (D), and the acetylation levels of Myc-NDPK-D were determined by anti-Ac-Lys antibody. NS: not significant. **P < 0.01. n = 5 (C), n = 3 (D). (E) Schematic representation of lysine residues in NDPK-D. Lys-45, Lys-72, and Lys-91 were candidate acetylation sites. (F) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the K45R, K72R, or K91R mutants. Acetylation levels were determined as described in (A). Replacement of lysine residues with arginine decreased acetylation levels. n = 3. (G) N1E-115 cells were transfected with Myc-tagged wild-type (WT) NDPK-D or the K45/72/91R (3KR) mutant. Acetylation levels were determined as described in (A). **P < 0.01. n = 6. Statistical analyses were performed using Welch’s t-test (A-D, G).
Mentions: SIRT1 mediated NAD+-dependent deacetylation regulates the function of multiple target proteins. Since we employed the SIRT1 deacetylation domain as bait in the yeast two-hybrid screen, it is possible that SIRT1 modulates NDPK-D activity by deacetylation. To test this hypothesis, we performed deacetylation assay. N1E-115 cells were transfected with expression vector encoding Myc-tagged NDPK-D and incubated with or without SIRT1 inhibitor Ex527. NDPK-D was immunoprecipitated with anti-Myc antibody and subjected to western blot analysis using anti-acetylated lysine antibody to examine the acetylation levels of NDPK-D. Elevated levels of acetylated Myc-NDPK-D were detected in the presence of Ex527 relative to the signals in the absence of Ex527, showing that SIRT1 can deacetylate NDPK-D (Fig 4A). We also examined the effect of knockdown of SIRT1 on the acetylation level of NDPK-D. Efficient downregulation of Sirt1 mRNA was found in SIRT1 shRNA-transfected N1E-115 cells (Fig 4B, right graph). The signal for acetylated Myc-NDPK-D increased in SIRT1 shRNA-transfected cells (Fig 4B, left panel and graph). We next examined whether overexpression of SIRT1 deacetylates NDPK-D. N1E-115 cells were transfected with Myc-tagged NDPK-D and HA-tagged SIRT1. The acetylation level of NDPK-D was investigated as described above. The signal for acetylated Myc-NDPK-D was decreased in the presence of HA-tagged SIRT1 (Fig 4C), whereas deacetylase-deficient mutant of SIRT1 (H363Y) did not affect the acetylation level of Myc-NDPK-D (Fig 4D). These results indicate that SIRT1 can modulate the deacetylation level of NDPK-D.

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