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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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Effect of nutlin-3 on APE1 ubiquitination(A) Cells were treated with nutlin-3 for 16 h before the ubiquitin assay. Immunoblot assays with anti-APE1 (Top), anti-p53 (FL-393, Middle), and anti-MDM2 (Bottom). (B) Ubiquitination on the endogenous APE1. HCT116 cells were transfected with His-ubi for overnight. Cells were lysed after 4 h incubation with (lane 2) or without (lane 1) 10 μM nutlin-3, and ubiquitinated APE1 was purified by NTA resin. (C) Effect of downmodulation of MDM2 on APE1 protein levels. A control siRNA or MDM2-siRNA were co-transfected with His-ubi, APE1, and ND21 cDNA in the HCT116 cells with or without 10 μM nutlin-3. Cells were lysed after 30 h for immunoblot assay using anti-APE1 antibody. (D) Effect of nutlin-3 on the level of endogenous APE1. HCT116 (p53+/+ or p53-/-) were treated with DMSO (lanes 1 and 3) or 10 μM nutlin-3 for 6 h (lanes 2 and 4), in the presence of a deubiquitinase inhibitor mixture and 10 μM MG132 (Materials and Methods).
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Figure 6: Effect of nutlin-3 on APE1 ubiquitination(A) Cells were treated with nutlin-3 for 16 h before the ubiquitin assay. Immunoblot assays with anti-APE1 (Top), anti-p53 (FL-393, Middle), and anti-MDM2 (Bottom). (B) Ubiquitination on the endogenous APE1. HCT116 cells were transfected with His-ubi for overnight. Cells were lysed after 4 h incubation with (lane 2) or without (lane 1) 10 μM nutlin-3, and ubiquitinated APE1 was purified by NTA resin. (C) Effect of downmodulation of MDM2 on APE1 protein levels. A control siRNA or MDM2-siRNA were co-transfected with His-ubi, APE1, and ND21 cDNA in the HCT116 cells with or without 10 μM nutlin-3. Cells were lysed after 30 h for immunoblot assay using anti-APE1 antibody. (D) Effect of nutlin-3 on the level of endogenous APE1. HCT116 (p53+/+ or p53-/-) were treated with DMSO (lanes 1 and 3) or 10 μM nutlin-3 for 6 h (lanes 2 and 4), in the presence of a deubiquitinase inhibitor mixture and 10 μM MG132 (Materials and Methods).

Mentions: MDM2 is transcriptionally activated by p53. In turn, the accumulated MDM2 degrades p53 via polyubiquitination, forming a negative feedback loop (Vousden and Lane, 2007). Nutlin, a potent inhibitor of MDM2 for p53 ubiquitination, specifically interferes with the interaction of MDM2 with p53 (Vassilev et al., 2004). An interesting possibility was that nutlin might also inhibit APE1 ubiquitination. If indeed this were the case, the nutlin-binding domain of MDM2 would be required for APE1 ubiquitination. APE1 might thus compete with p53 for MDM2 interaction. However, when HCT116 cells (p53+/+) were treated with nutlin-3, APE1 ubiquitination was significantly increased (Fig. 6A). Therefore, nutlin-3 did not interfere with APE1 ubiquitination via MDM2. The higher ubiquitination activity in the presence of nutlin-3 can be explained by the fact that nutlin-3 causes p53 stabilization which increases the MDM2 level via transcriptional activation (Fig. 6A). Consistently, cells deficient in p53 (HCT116 p53-/-) did not respond to nutlin-3 by increasing APE1 ubiquitination (Fig. 6A). To detect ubiquitination on endogenous APE1, we transiently introduced the His-ubi alone to HCT116 which was then treated with nutlin-3. We observed the ubiquitinated form of endogenous APE1 (Fig. 6B), indicating that ubiquitination of endogenous APE1 can also occur depending on the intracellular level of MDM2.


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

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

Effect of nutlin-3 on APE1 ubiquitination(A) Cells were treated with nutlin-3 for 16 h before the ubiquitin assay. Immunoblot assays with anti-APE1 (Top), anti-p53 (FL-393, Middle), and anti-MDM2 (Bottom). (B) Ubiquitination on the endogenous APE1. HCT116 cells were transfected with His-ubi for overnight. Cells were lysed after 4 h incubation with (lane 2) or without (lane 1) 10 μM nutlin-3, and ubiquitinated APE1 was purified by NTA resin. (C) Effect of downmodulation of MDM2 on APE1 protein levels. A control siRNA or MDM2-siRNA were co-transfected with His-ubi, APE1, and ND21 cDNA in the HCT116 cells with or without 10 μM nutlin-3. Cells were lysed after 30 h for immunoblot assay using anti-APE1 antibody. (D) Effect of nutlin-3 on the level of endogenous APE1. HCT116 (p53+/+ or p53-/-) were treated with DMSO (lanes 1 and 3) or 10 μM nutlin-3 for 6 h (lanes 2 and 4), in the presence of a deubiquitinase inhibitor mixture and 10 μM MG132 (Materials and Methods).
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Figure 6: Effect of nutlin-3 on APE1 ubiquitination(A) Cells were treated with nutlin-3 for 16 h before the ubiquitin assay. Immunoblot assays with anti-APE1 (Top), anti-p53 (FL-393, Middle), and anti-MDM2 (Bottom). (B) Ubiquitination on the endogenous APE1. HCT116 cells were transfected with His-ubi for overnight. Cells were lysed after 4 h incubation with (lane 2) or without (lane 1) 10 μM nutlin-3, and ubiquitinated APE1 was purified by NTA resin. (C) Effect of downmodulation of MDM2 on APE1 protein levels. A control siRNA or MDM2-siRNA were co-transfected with His-ubi, APE1, and ND21 cDNA in the HCT116 cells with or without 10 μM nutlin-3. Cells were lysed after 30 h for immunoblot assay using anti-APE1 antibody. (D) Effect of nutlin-3 on the level of endogenous APE1. HCT116 (p53+/+ or p53-/-) were treated with DMSO (lanes 1 and 3) or 10 μM nutlin-3 for 6 h (lanes 2 and 4), in the presence of a deubiquitinase inhibitor mixture and 10 μM MG132 (Materials and Methods).
Mentions: MDM2 is transcriptionally activated by p53. In turn, the accumulated MDM2 degrades p53 via polyubiquitination, forming a negative feedback loop (Vousden and Lane, 2007). Nutlin, a potent inhibitor of MDM2 for p53 ubiquitination, specifically interferes with the interaction of MDM2 with p53 (Vassilev et al., 2004). An interesting possibility was that nutlin might also inhibit APE1 ubiquitination. If indeed this were the case, the nutlin-binding domain of MDM2 would be required for APE1 ubiquitination. APE1 might thus compete with p53 for MDM2 interaction. However, when HCT116 cells (p53+/+) were treated with nutlin-3, APE1 ubiquitination was significantly increased (Fig. 6A). Therefore, nutlin-3 did not interfere with APE1 ubiquitination via MDM2. The higher ubiquitination activity in the presence of nutlin-3 can be explained by the fact that nutlin-3 causes p53 stabilization which increases the MDM2 level via transcriptional activation (Fig. 6A). Consistently, cells deficient in p53 (HCT116 p53-/-) did not respond to nutlin-3 by increasing APE1 ubiquitination (Fig. 6A). To detect ubiquitination on endogenous APE1, we transiently introduced the His-ubi alone to HCT116 which was then treated with nutlin-3. We observed the ubiquitinated form of endogenous APE1 (Fig. 6B), indicating that ubiquitination of endogenous APE1 can also occur depending on the intracellular level of MDM2.

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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