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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

Deacetylation of NDPK-D promoted its nuclear localization.(A) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. WT and 3KQ NDPK-D localized in both cytoplasm and nucleus, whereas the majority of 3KR mutants were retained in the nucleus. NDPK-D localization was classified in the transfected N1E cells. “C > N” indicates the cells predominantly exhibiting Myc-NDPK-D in the cytosol. “C = N” indicates the cells exhibiting cytosol and nuclear Myc-NDPK-D. “C < N” indicates the cells showing nuclear-accumulated Myc-NDPK-D. The percentage of transfected cells exhibiting indicated subcellular localization for NDPK-D is shown in the graph. Scale bar: 50 μm. **P < 0.01. n = 5. (B) N1E-115 cells were transfected with Myc-NDPK-D and fractionated into cytosol and nuclear fractions. Each fraction was subjected to deacetylation assay as described in Fig 4. n = 4. (C) The HDAC inhibitor (TSA) seems to increase the acetylation level of NDPK-D in the cytosol fraction. n = 3. (D) Leptomycin B (LMB) treatment did not cause nuclear accumulation of NDPK-D. The cells transfected with Myc-tagged NDPK-D or HA-tagged zyxin (used as positive control) were cultured for 36 h, in the presence or absence of LMB during the last 6 h. The cells were stained with anti-Myc antibody for Myc-NDPK-D or anti-HA antibody for HA-zyxin. Scale bar: 50 μm. (E) SIRT1 causes nuclear accumulation of NDPK-D. N1E-115 cells were transfected with Myc-tagged WT NDPK-D and HA-tagged WT SIRT1 or deacetylase-deficient mutant SIRT1 (H363Y). After 36 h, cells were fixed and immunostained with anti-Myc and HA antibodies. NDPK-D localization was classified in N1E cells expressing both Myc-tagged NDPK-D and HA-tagged SIRT1. Scale bar: 20 μm. *P < 0.05, **P < 0.01. n = 4. (F) Acetylation-mimetic form of NDPK-D induced apoptosis. N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. Cells were immunostained with anti-cleaved caspase-3 and anti-Myc antibodies. The frequencies of cleaved caspase-3-positive cells in the transfected cells were normalized to that of control. **P < 0.01. n = 5. (G) The treatment with SIRT1 inhibitor EX527 tended to increase the number of cleaved caspase-3-positive cells. N1E-115 cells were treated with or without EX527, and cultured for 48 h. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s multiple comparison tests (A, D, E), or Welch’s t-test (B).
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pone.0139616.g005: Deacetylation of NDPK-D promoted its nuclear localization.(A) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. WT and 3KQ NDPK-D localized in both cytoplasm and nucleus, whereas the majority of 3KR mutants were retained in the nucleus. NDPK-D localization was classified in the transfected N1E cells. “C > N” indicates the cells predominantly exhibiting Myc-NDPK-D in the cytosol. “C = N” indicates the cells exhibiting cytosol and nuclear Myc-NDPK-D. “C < N” indicates the cells showing nuclear-accumulated Myc-NDPK-D. The percentage of transfected cells exhibiting indicated subcellular localization for NDPK-D is shown in the graph. Scale bar: 50 μm. **P < 0.01. n = 5. (B) N1E-115 cells were transfected with Myc-NDPK-D and fractionated into cytosol and nuclear fractions. Each fraction was subjected to deacetylation assay as described in Fig 4. n = 4. (C) The HDAC inhibitor (TSA) seems to increase the acetylation level of NDPK-D in the cytosol fraction. n = 3. (D) Leptomycin B (LMB) treatment did not cause nuclear accumulation of NDPK-D. The cells transfected with Myc-tagged NDPK-D or HA-tagged zyxin (used as positive control) were cultured for 36 h, in the presence or absence of LMB during the last 6 h. The cells were stained with anti-Myc antibody for Myc-NDPK-D or anti-HA antibody for HA-zyxin. Scale bar: 50 μm. (E) SIRT1 causes nuclear accumulation of NDPK-D. N1E-115 cells were transfected with Myc-tagged WT NDPK-D and HA-tagged WT SIRT1 or deacetylase-deficient mutant SIRT1 (H363Y). After 36 h, cells were fixed and immunostained with anti-Myc and HA antibodies. NDPK-D localization was classified in N1E cells expressing both Myc-tagged NDPK-D and HA-tagged SIRT1. Scale bar: 20 μm. *P < 0.05, **P < 0.01. n = 4. (F) Acetylation-mimetic form of NDPK-D induced apoptosis. N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. Cells were immunostained with anti-cleaved caspase-3 and anti-Myc antibodies. The frequencies of cleaved caspase-3-positive cells in the transfected cells were normalized to that of control. **P < 0.01. n = 5. (G) The treatment with SIRT1 inhibitor EX527 tended to increase the number of cleaved caspase-3-positive cells. N1E-115 cells were treated with or without EX527, and cultured for 48 h. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s multiple comparison tests (A, D, E), or Welch’s t-test (B).

Mentions: Since one of the acetylation target lysine residues are located within the RRK motif (R89–R90–K91), which is important for NDPK-D mitochondrial localization [37] (Fig 3B), we tested whether acetylation might affect the localization of NDPK-D. N1E-115 cells were transfected with plasmids encoding Myc-tagged NDPK-D WT, deacetylated mimetic form (3KR), or acetylated mimetic form (3KQ). While the NDPK-D WT was distributed in both the cytoplasm and the nucleus, 3KR mutants demonstrated a predominantly nuclear localization (Fig 5A). These observations suggest that deacetylation can lead to increased nuclear levels of NDPK-D. We also examined the acetylation levels of NDPK-D in the cytoplasm and the nucleus. The difference of NDPK-D acetylation between cytoplasm and nucleus was not significant (Fig 5B). To address the possibility that other molecules such as histone deacetylases (HDACs) regulate the acetylation level of NDPK-D, we treated the cells with HDAC inhibitor trichostatin A (TSA). The TSA treatment increased the acetylation level of NDPK-D in the cytosol fraction compared with nuclear fraction (Fig 5C). These results suggest that both SIRT1 and HDACs regulate the acetylation 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)

Deacetylation of NDPK-D promoted its nuclear localization.(A) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. WT and 3KQ NDPK-D localized in both cytoplasm and nucleus, whereas the majority of 3KR mutants were retained in the nucleus. NDPK-D localization was classified in the transfected N1E cells. “C > N” indicates the cells predominantly exhibiting Myc-NDPK-D in the cytosol. “C = N” indicates the cells exhibiting cytosol and nuclear Myc-NDPK-D. “C < N” indicates the cells showing nuclear-accumulated Myc-NDPK-D. The percentage of transfected cells exhibiting indicated subcellular localization for NDPK-D is shown in the graph. Scale bar: 50 μm. **P < 0.01. n = 5. (B) N1E-115 cells were transfected with Myc-NDPK-D and fractionated into cytosol and nuclear fractions. Each fraction was subjected to deacetylation assay as described in Fig 4. n = 4. (C) The HDAC inhibitor (TSA) seems to increase the acetylation level of NDPK-D in the cytosol fraction. n = 3. (D) Leptomycin B (LMB) treatment did not cause nuclear accumulation of NDPK-D. The cells transfected with Myc-tagged NDPK-D or HA-tagged zyxin (used as positive control) were cultured for 36 h, in the presence or absence of LMB during the last 6 h. The cells were stained with anti-Myc antibody for Myc-NDPK-D or anti-HA antibody for HA-zyxin. Scale bar: 50 μm. (E) SIRT1 causes nuclear accumulation of NDPK-D. N1E-115 cells were transfected with Myc-tagged WT NDPK-D and HA-tagged WT SIRT1 or deacetylase-deficient mutant SIRT1 (H363Y). After 36 h, cells were fixed and immunostained with anti-Myc and HA antibodies. NDPK-D localization was classified in N1E cells expressing both Myc-tagged NDPK-D and HA-tagged SIRT1. Scale bar: 20 μm. *P < 0.05, **P < 0.01. n = 4. (F) Acetylation-mimetic form of NDPK-D induced apoptosis. N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. Cells were immunostained with anti-cleaved caspase-3 and anti-Myc antibodies. The frequencies of cleaved caspase-3-positive cells in the transfected cells were normalized to that of control. **P < 0.01. n = 5. (G) The treatment with SIRT1 inhibitor EX527 tended to increase the number of cleaved caspase-3-positive cells. N1E-115 cells were treated with or without EX527, and cultured for 48 h. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s multiple comparison tests (A, D, E), or Welch’s t-test (B).
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pone.0139616.g005: Deacetylation of NDPK-D promoted its nuclear localization.(A) N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. WT and 3KQ NDPK-D localized in both cytoplasm and nucleus, whereas the majority of 3KR mutants were retained in the nucleus. NDPK-D localization was classified in the transfected N1E cells. “C > N” indicates the cells predominantly exhibiting Myc-NDPK-D in the cytosol. “C = N” indicates the cells exhibiting cytosol and nuclear Myc-NDPK-D. “C < N” indicates the cells showing nuclear-accumulated Myc-NDPK-D. The percentage of transfected cells exhibiting indicated subcellular localization for NDPK-D is shown in the graph. Scale bar: 50 μm. **P < 0.01. n = 5. (B) N1E-115 cells were transfected with Myc-NDPK-D and fractionated into cytosol and nuclear fractions. Each fraction was subjected to deacetylation assay as described in Fig 4. n = 4. (C) The HDAC inhibitor (TSA) seems to increase the acetylation level of NDPK-D in the cytosol fraction. n = 3. (D) Leptomycin B (LMB) treatment did not cause nuclear accumulation of NDPK-D. The cells transfected with Myc-tagged NDPK-D or HA-tagged zyxin (used as positive control) were cultured for 36 h, in the presence or absence of LMB during the last 6 h. The cells were stained with anti-Myc antibody for Myc-NDPK-D or anti-HA antibody for HA-zyxin. Scale bar: 50 μm. (E) SIRT1 causes nuclear accumulation of NDPK-D. N1E-115 cells were transfected with Myc-tagged WT NDPK-D and HA-tagged WT SIRT1 or deacetylase-deficient mutant SIRT1 (H363Y). After 36 h, cells were fixed and immunostained with anti-Myc and HA antibodies. NDPK-D localization was classified in N1E cells expressing both Myc-tagged NDPK-D and HA-tagged SIRT1. Scale bar: 20 μm. *P < 0.05, **P < 0.01. n = 4. (F) Acetylation-mimetic form of NDPK-D induced apoptosis. N1E-115 cells were transfected with Myc-tagged wild-type NDPK-D, the 3KR or the K45/72/91Q (3KQ) mutant. Cells were immunostained with anti-cleaved caspase-3 and anti-Myc antibodies. The frequencies of cleaved caspase-3-positive cells in the transfected cells were normalized to that of control. **P < 0.01. n = 5. (G) The treatment with SIRT1 inhibitor EX527 tended to increase the number of cleaved caspase-3-positive cells. N1E-115 cells were treated with or without EX527, and cultured for 48 h. Statistical analyses were performed using one-way ANOVA followed by Scheffe’s multiple comparison tests (A, D, E), or Welch’s t-test (B).
Mentions: Since one of the acetylation target lysine residues are located within the RRK motif (R89–R90–K91), which is important for NDPK-D mitochondrial localization [37] (Fig 3B), we tested whether acetylation might affect the localization of NDPK-D. N1E-115 cells were transfected with plasmids encoding Myc-tagged NDPK-D WT, deacetylated mimetic form (3KR), or acetylated mimetic form (3KQ). While the NDPK-D WT was distributed in both the cytoplasm and the nucleus, 3KR mutants demonstrated a predominantly nuclear localization (Fig 5A). These observations suggest that deacetylation can lead to increased nuclear levels of NDPK-D. We also examined the acetylation levels of NDPK-D in the cytoplasm and the nucleus. The difference of NDPK-D acetylation between cytoplasm and nucleus was not significant (Fig 5B). To address the possibility that other molecules such as histone deacetylases (HDACs) regulate the acetylation level of NDPK-D, we treated the cells with HDAC inhibitor trichostatin A (TSA). The TSA treatment increased the acetylation level of NDPK-D in the cytosol fraction compared with nuclear fraction (Fig 5C). These results suggest that both SIRT1 and HDACs regulate the acetylation 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