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Ubiquitination of mammalian AP endonuclease (APE1) regulated by the p53-MDM2 signaling pathway.

Busso CS, Iwakuma T, Izumi T - Oncogene (2009)

Bottom Line: In addition, unlike the wild-type APE1, ubiquitin-APE1 fusion proteins were predominantly present in the cytoplasm.Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells.These results reveal a novel regulation of APE1 through ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology, Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.

ABSTRACT
APE1/Ref-1 is an essential DNA repair/gene regulatory protein in mammals of which intracellular level significantly affects cellular sensitivity to genotoxicants. The apurinic/apyrimidinic endonuclease 1 (APE1) functions are altered by phosphorylation and acetylation. We here report that APE1 is also modified by ubiquitination. APE1 ubiquitination occurred specifically at Lys residues near the N-terminus, and was markedly enhanced by mouse double minute 2 (MDM2), the major intracellular p53 inhibitor. Moreover, DNA-damaging reagents and nutlin-3, an inhibitor of MDM2-p53 interaction, increased APE1 ubiquitination in the presence of p53. Downmodulation of MDM2 increased APE1 level, suggesting that MDM2-mediated ubiquitination can be a signal for APE1 degradation. In addition, unlike the wild-type APE1, ubiquitin-APE1 fusion proteins were predominantly present in the cytoplasm. Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells. These results reveal a novel regulation of APE1 through ubiquitination.

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APE1 ubiquitination in vivo and in vitro(A) AML cell line Kasumi-1 was treated with 1mM H2O2 for 15 (lane 2) or 60 min (lanes 3), and the total extracts were analyzed with APE1 immunoblot as described in Materials and Methods. (Top) HWB APE1 with intensified signals. A bar at the right side indicates the appearance of HWB APE1 (top panel). (Bottom) Intact APE1 and β-actin (re-blot) of the same sample set. (B) Detection of APE1 ubiquitination in cells. (Left) Molecular weight references for ubiquitinated APE1. Total protein extracts from HCT116 cells were blotted with anti-APE1 antibody. Cells were expressing (lane 1) none, (lane 2) wtAPE1, and (lane 3) ubiquitin-APE1 fusion (ubi-APE1). (Right) Protein extracts from HCT116 cells expressing wtAPE1 and (lane 4) the pcDNA3.1 control vector or (lane 5) His-tagged ubiquitin were purified through Ni-NTA magnet beads under the denaturing condition (Materials and Methods). (C) In vitro ubiquitination. Recombinant APE1 was incubated with ubiquitin and HeLa S100 fraction, and then analyzed with anti-APE1 in immunoblot. (A-C) Protein positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), polyubiquitinated APE1 (double filled arrow), a truncated APE1 due to degradation (*), and non-specific bands (>).
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Figure 1: APE1 ubiquitination in vivo and in vitro(A) AML cell line Kasumi-1 was treated with 1mM H2O2 for 15 (lane 2) or 60 min (lanes 3), and the total extracts were analyzed with APE1 immunoblot as described in Materials and Methods. (Top) HWB APE1 with intensified signals. A bar at the right side indicates the appearance of HWB APE1 (top panel). (Bottom) Intact APE1 and β-actin (re-blot) of the same sample set. (B) Detection of APE1 ubiquitination in cells. (Left) Molecular weight references for ubiquitinated APE1. Total protein extracts from HCT116 cells were blotted with anti-APE1 antibody. Cells were expressing (lane 1) none, (lane 2) wtAPE1, and (lane 3) ubiquitin-APE1 fusion (ubi-APE1). (Right) Protein extracts from HCT116 cells expressing wtAPE1 and (lane 4) the pcDNA3.1 control vector or (lane 5) His-tagged ubiquitin were purified through Ni-NTA magnet beads under the denaturing condition (Materials and Methods). (C) In vitro ubiquitination. Recombinant APE1 was incubated with ubiquitin and HeLa S100 fraction, and then analyzed with anti-APE1 in immunoblot. (A-C) Protein positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), polyubiquitinated APE1 (double filled arrow), a truncated APE1 due to degradation (*), and non-specific bands (>).

Mentions: Decrease of APE1 during apoptosis has been reported in human cytolytic T-cells and myelolytic cells (Fan et al., 2003; Robertson et al., 1997). We examined the stability of APE1 after DNA damage generation by H2O2 using Kasumi-1, a human myeloblastic leukemia cell line (Asou et al., 1991). High-molecular weight-band (HWB) APE1 was detected in an immunoblot assay using an anti-APE1 antibody (top Fig. 1A), while the amount of the intact APE1 was observed to decrease (bottom, Fig. 1A) by about 10% compared to the control, which is consistent with the small ratio of HWB relative to intact APE1. This result implies that APE1 was ubiquitinated.


Ubiquitination of mammalian AP endonuclease (APE1) regulated by the p53-MDM2 signaling pathway.

Busso CS, Iwakuma T, Izumi T - Oncogene (2009)

APE1 ubiquitination in vivo and in vitro(A) AML cell line Kasumi-1 was treated with 1mM H2O2 for 15 (lane 2) or 60 min (lanes 3), and the total extracts were analyzed with APE1 immunoblot as described in Materials and Methods. (Top) HWB APE1 with intensified signals. A bar at the right side indicates the appearance of HWB APE1 (top panel). (Bottom) Intact APE1 and β-actin (re-blot) of the same sample set. (B) Detection of APE1 ubiquitination in cells. (Left) Molecular weight references for ubiquitinated APE1. Total protein extracts from HCT116 cells were blotted with anti-APE1 antibody. Cells were expressing (lane 1) none, (lane 2) wtAPE1, and (lane 3) ubiquitin-APE1 fusion (ubi-APE1). (Right) Protein extracts from HCT116 cells expressing wtAPE1 and (lane 4) the pcDNA3.1 control vector or (lane 5) His-tagged ubiquitin were purified through Ni-NTA magnet beads under the denaturing condition (Materials and Methods). (C) In vitro ubiquitination. Recombinant APE1 was incubated with ubiquitin and HeLa S100 fraction, and then analyzed with anti-APE1 in immunoblot. (A-C) Protein positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), polyubiquitinated APE1 (double filled arrow), a truncated APE1 due to degradation (*), and non-specific bands (>).
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Related In: Results  -  Collection

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Figure 1: APE1 ubiquitination in vivo and in vitro(A) AML cell line Kasumi-1 was treated with 1mM H2O2 for 15 (lane 2) or 60 min (lanes 3), and the total extracts were analyzed with APE1 immunoblot as described in Materials and Methods. (Top) HWB APE1 with intensified signals. A bar at the right side indicates the appearance of HWB APE1 (top panel). (Bottom) Intact APE1 and β-actin (re-blot) of the same sample set. (B) Detection of APE1 ubiquitination in cells. (Left) Molecular weight references for ubiquitinated APE1. Total protein extracts from HCT116 cells were blotted with anti-APE1 antibody. Cells were expressing (lane 1) none, (lane 2) wtAPE1, and (lane 3) ubiquitin-APE1 fusion (ubi-APE1). (Right) Protein extracts from HCT116 cells expressing wtAPE1 and (lane 4) the pcDNA3.1 control vector or (lane 5) His-tagged ubiquitin were purified through Ni-NTA magnet beads under the denaturing condition (Materials and Methods). (C) In vitro ubiquitination. Recombinant APE1 was incubated with ubiquitin and HeLa S100 fraction, and then analyzed with anti-APE1 in immunoblot. (A-C) Protein positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), polyubiquitinated APE1 (double filled arrow), a truncated APE1 due to degradation (*), and non-specific bands (>).
Mentions: Decrease of APE1 during apoptosis has been reported in human cytolytic T-cells and myelolytic cells (Fan et al., 2003; Robertson et al., 1997). We examined the stability of APE1 after DNA damage generation by H2O2 using Kasumi-1, a human myeloblastic leukemia cell line (Asou et al., 1991). High-molecular weight-band (HWB) APE1 was detected in an immunoblot assay using an anti-APE1 antibody (top Fig. 1A), while the amount of the intact APE1 was observed to decrease (bottom, Fig. 1A) by about 10% compared to the control, which is consistent with the small ratio of HWB relative to intact APE1. This result implies that APE1 was ubiquitinated.

Bottom Line: In addition, unlike the wild-type APE1, ubiquitin-APE1 fusion proteins were predominantly present in the cytoplasm.Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells.These results reveal a novel regulation of APE1 through ubiquitination.

View Article: PubMed Central - PubMed

Affiliation: Department of Otolaryngology, Stanley S Scott Cancer Center, Louisiana State University Health Sciences Center, New Orleans, LA 70112, USA.

ABSTRACT
APE1/Ref-1 is an essential DNA repair/gene regulatory protein in mammals of which intracellular level significantly affects cellular sensitivity to genotoxicants. The apurinic/apyrimidinic endonuclease 1 (APE1) functions are altered by phosphorylation and acetylation. We here report that APE1 is also modified by ubiquitination. APE1 ubiquitination occurred specifically at Lys residues near the N-terminus, and was markedly enhanced by mouse double minute 2 (MDM2), the major intracellular p53 inhibitor. Moreover, DNA-damaging reagents and nutlin-3, an inhibitor of MDM2-p53 interaction, increased APE1 ubiquitination in the presence of p53. Downmodulation of MDM2 increased APE1 level, suggesting that MDM2-mediated ubiquitination can be a signal for APE1 degradation. In addition, unlike the wild-type APE1, ubiquitin-APE1 fusion proteins were predominantly present in the cytoplasm. Therefore, monoubiquitination not only is a prerequisite for degradation, but may also alter the APE1 activities in cells. These results reveal a novel regulation of APE1 through ubiquitination.

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