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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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Involvement of p53 in APE1 ubiquitination(A) p53-dependent APE1 ubiquitination. HCT116 p53+/+ and p53-/- cells were transfected with APE1 and His-ubi, and 24 h later the cells were treated for 4 h with the DNA-damaging reagents as indicated. His-ubi-enriched fractions as well as the total fractions were analyzed in immunoblot assays with anti-APE1. (B) APE1 ubiquitination by MDM2. Ubiquitination assays were performed by co-transfecting a control vector (lanes 1 and 3) or the MDM2-encoding plasmids (lanes 2 and 4) in addition to the His-ubi and APE1. The cells were lysed in a non-denaturing condition, and the total fractions (lanes 1 and 2) or the ubiquitin-enriched fractions (lanes 3 and 4) were analyzed with anti-APE1 antibody. Positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), and truncated APE1 (*, due to degradation).
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Figure 2: Involvement of p53 in APE1 ubiquitination(A) p53-dependent APE1 ubiquitination. HCT116 p53+/+ and p53-/- cells were transfected with APE1 and His-ubi, and 24 h later the cells were treated for 4 h with the DNA-damaging reagents as indicated. His-ubi-enriched fractions as well as the total fractions were analyzed in immunoblot assays with anti-APE1. (B) APE1 ubiquitination by MDM2. Ubiquitination assays were performed by co-transfecting a control vector (lanes 1 and 3) or the MDM2-encoding plasmids (lanes 2 and 4) in addition to the His-ubi and APE1. The cells were lysed in a non-denaturing condition, and the total fractions (lanes 1 and 2) or the ubiquitin-enriched fractions (lanes 3 and 4) were analyzed with anti-APE1 antibody. Positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), and truncated APE1 (*, due to degradation).

Mentions: Upon stress, p53 activates a number of genes that result in apoptosis, cell cycle arrest, or senescence (Shmueli and Oren, 2004). Based on the genetic link between p53 and APE1 (Meira et al., 1997), we examined the effects of DNA-damaging reagents, such as etoposide (a topoisomerase II inhibitor) and H2O2, on APE1 ubiquitination, because these reagents affect the intracellular p53 level. When HCT116 (p53+/+ or p53-/-) cells expressing the His-ubi and APE1 were treated with H2O2 and etoposide, APE1 ubiquitination was significantly increased in the p53+/+ but not in the p53-/- cells (Fig. 2A). These findings suggest that APE1 ubiquitination is dependent on the level of p53 and induced in response to cellular stress.


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

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

Involvement of p53 in APE1 ubiquitination(A) p53-dependent APE1 ubiquitination. HCT116 p53+/+ and p53-/- cells were transfected with APE1 and His-ubi, and 24 h later the cells were treated for 4 h with the DNA-damaging reagents as indicated. His-ubi-enriched fractions as well as the total fractions were analyzed in immunoblot assays with anti-APE1. (B) APE1 ubiquitination by MDM2. Ubiquitination assays were performed by co-transfecting a control vector (lanes 1 and 3) or the MDM2-encoding plasmids (lanes 2 and 4) in addition to the His-ubi and APE1. The cells were lysed in a non-denaturing condition, and the total fractions (lanes 1 and 2) or the ubiquitin-enriched fractions (lanes 3 and 4) were analyzed with anti-APE1 antibody. Positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), and truncated APE1 (*, due to degradation).
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Related In: Results  -  Collection

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Figure 2: Involvement of p53 in APE1 ubiquitination(A) p53-dependent APE1 ubiquitination. HCT116 p53+/+ and p53-/- cells were transfected with APE1 and His-ubi, and 24 h later the cells were treated for 4 h with the DNA-damaging reagents as indicated. His-ubi-enriched fractions as well as the total fractions were analyzed in immunoblot assays with anti-APE1. (B) APE1 ubiquitination by MDM2. Ubiquitination assays were performed by co-transfecting a control vector (lanes 1 and 3) or the MDM2-encoding plasmids (lanes 2 and 4) in addition to the His-ubi and APE1. The cells were lysed in a non-denaturing condition, and the total fractions (lanes 1 and 2) or the ubiquitin-enriched fractions (lanes 3 and 4) were analyzed with anti-APE1 antibody. Positions are indicated for intact APE1 (open arrow), monoubiquitinated APE1 (filled arrow), and truncated APE1 (*, due to degradation).
Mentions: Upon stress, p53 activates a number of genes that result in apoptosis, cell cycle arrest, or senescence (Shmueli and Oren, 2004). Based on the genetic link between p53 and APE1 (Meira et al., 1997), we examined the effects of DNA-damaging reagents, such as etoposide (a topoisomerase II inhibitor) and H2O2, on APE1 ubiquitination, because these reagents affect the intracellular p53 level. When HCT116 (p53+/+ or p53-/-) cells expressing the His-ubi and APE1 were treated with H2O2 and etoposide, APE1 ubiquitination was significantly increased in the p53+/+ but not in the p53-/- cells (Fig. 2A). These findings suggest that APE1 ubiquitination is dependent on the level of p53 and induced in response to cellular stress.

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.

Show MeSH