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. 2016 Apr 12;7(15):20425-39.
doi: 10.18632/oncotarget.7966.

Acetylation and deacetylation of Cdc25A constitutes a novel mechanism for modulating Cdc25A functions with implications for cancer

Enerlyn M Lozada  1 , Zdenek Andrysik  1   2 , Moying Yin  1 , Nicholas Redilla  1 , Kathryn Rice  1 , Peter J Stambrook  1
Affiliations

Acetylation and deacetylation of Cdc25A constitutes a novel mechanism for modulating Cdc25A functions with implications for cancer

Enerlyn M Lozada et al. Oncotarget. .

Abstract

The dual specificity phosphatase Cdc25A is a key regulator of the cell cycle that promotes cell cycle progression by dephosphorylating and activating cyclin-dependent kinases. In response to genotoxicants, Cdc25A undergoes posttranslational modifications which contribute to its proteasome-mediated degradation and consequent cell cycle checkpoint arrest. The most thoroughly studied Cdc25A modification is phosphorylation. We now provide the first evidence that Cdc25A can be acetylated and that it directly interacts with the ARD1 acetyltransferase which acetylates Cdc25A both biochemically and in cultured cells. When acetylated, Cdc25A has an extended half-life. We have also identified the class IV histone deacetylase, HDAC11, as a Cdc25A deacetylase. We further show that DNA damage, such as exposure to methyl methanesulfonate (MMS), etoposide or arsenic, increases Cdc25A acetylation. Importantly, this acetylation modulates Cdc25A phosphatase activity and its function as a cell cycle regulator, and may reflect a cellular response to DNA damage. Since Cdc25A, ARD1, and HDAC11 are frequently dysregulated in multiple types of cancer, our findings may provide insight into a novel mechanism in carcinogenesis.

Keywords: ARD1; Cdc25A acetylation; DNA damage; HDAC11; cancer.

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Conflict of interest statement

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1. Cdc25A and ARD1 interact in vivo and in vitro
(A) Cdc25A co-immunoprecipitates with ARD1 in vitro. Purified FLAG-Cdc25A was incubated with anti-GFP beads, either alone or in combination with GFP-ARD1A. Immunoprecipitated products were subjected to SDS-PAGE and probed with anti-GFP and anti-FLAG antibodies. (B) ARD1 and Cdc25A associate after co-transfection. FLAG-ARD1A was co-transfected into HEK 293T cells with either GFP-Cdc25A or the GFP vector alone. After 24 hours, cell lysates were subjected to immunoprecipitation using anti-GFP antibody. Precipitated proteins were examined by Western blot. (C) Co-immunoprecipitation of Cdc25A and ARD1 in cultured cells. HEK 293T cell lysates were subjected to immunoprecipitation using antibody to Cdc25A or mouse IgG. Immunoprecipitates were analyzed with anti-ARD1 and anti-Cdc25A antibodies. (D) and (E) Cdc25A-ARD1 interact directly. Purified ARD1A (D), Cdc25A (E), and a BSA control were immobilized on nitrocellulose membranes at the concentrations indicated above each panel. Membranes were incubated in buffer containing (D) Cdc25A (2400 ng) or (E) ARD1A (720 ng). Immunodetection of bound protein was performed with anti-Cdc25A (D) or anti-GFP (E) antibodies.
Figure 2
Figure 2. ARD1 acetylates Cdc25A in vitro and in cells
(A) ARD1 mediates Cdc25A acetylation in vitro. Purified GFP-ARD1A was incubated at 4°C, or 37°C, with purified FLAG-Cdc25A. The reactions were analyzed using anti-acetyl lysine antibody, followed by chemiluminescent detection. (B) Cdc25A is endogenously acetylated. Cdc25A was immunoprecipitated from HEK 293T cell lysates and its acetylation status was analyzed by Western blot using an acetyl-specific antibody. (C) ARD1 overexpression increases Cdc25A acetylation in cells. Cdc25A was immunoprecipitated from HEK 293T cell lysates transfected with FLAG-ARD1A and the Cdc25A acetylation status was analyzed by Western blot using an antibody specific for acetylated lysines.
Figure 3
Figure 3. ARD1 expression affects Cdc25A protein stability by modulating its ubiquitination
(A) ARD1 knockdown efficiency. HEK 293 cells were transfected with siRNA against ARD1A/B or with scrambled siRNA as control to establish ARD1 knockdown. (B) ARD1A/B knockdown induces a reduction in Cdc25A level, but not of Cdc25B or Cdc25C. (C) Overexpression of ARD1 in HEK 293 cells increases the level of Cdc25A but not of Cdc25B, and Cdc25C. (D) ARD1 increases Cdc25A half-life. HEK 293 cells were transfected with GFP vector of GFP-ARD1A followed by treatment of cells with CHX for the indicated time. Cell lysates were analyzed by Western Blot with anti-Cdc25A, anti-GFP, and anti-β-actin antibodies. (E) ARD1 decreases Cdc25A ubiquitination. Cell lysates from HEK 293T were incubated with HA-Ub, His-Cdc25A, and/or Myc-DDK-ARD1. After immunoprecipitation with an anti-HA antibody, the immonoprecipitates were subjected to Western blot analysis using an anti-Cdc25A antibody.
Figure 4
Figure 4. DNA damage increases endogenous Cdc25A acetylation
(A) HEK 293T cells were treated with MMS, sodium arsenite, etoposide, or HU as described, or left untreated. The acetylation status of Cd25A was assessed by its immunoprecipitation followed by western blot using an acetyl-specific antibody. (B) Bar graph from data of Cdc25A acetylation from Panel A (mean ± SEM of 4 independent experiments; *p < 0.05 when compared with untreated cells).
Figure 5
Figure 5. Cdc25A acetylation modulates its phosphatase activity
(A) ARD1 induces a reduction in Cdc25A phosphatase activity. Phosphatase activity was measured spectrophotometrically based on Cdc25A catalysis of 6, 8-difluoro-4-methylumbelliferyl phosphate (DiFMUP) hydrolysis to 6, 8-difluoro-4-methylumbelliferone. Cdc25A phosphatase activity was measured in the absence or presence of ARD1. (B) Cdc25A acetylation is associated with accumulation of cells in the S and G2/M phases of the cell cycle. Cell cycle profiles of untransfected cells (GFP negative) or cells overexpressing GFP-ARD1A (GFP Positive) were compared by flow cytometry.
Figure 6
Figure 6. Influence of HDACs and Sirtuins in Cdc25A acetylation levels
(A) HEK 293T cells were incubated with increasing concentrations of TSA for 4 h. Cdc25A was immunoprecipitated from cell lysates and the immunoprecipitates were subjected to SDS-PAGE and western blotting with anti-acetyl lysine antibody (upper panel). Each cell lysates (100 μg) was analyzed by Western blotting with anti-Cdc25A antibody. (B) HEK 293T cells were treated with or without 10 mM Sodium-Butyrate, 5 mM Nicotinamide, and/or 10 μM TSA for 4 h. Cell lysates were subjected to immunoprecipitation with antibody to Cdc25A and immunoprecipitates were subjected to SDS-PAGE and western blotting with anti-acetylated lysine antibody. (C) Quantification for Cdc25A acetylation (mean ± SD of 3 independent experiments. * = P < 0.05 when compared with control untreated cells).
Figure 7
Figure 7. Cdc25A interacts directly with HDAC11
(A) and (B) Interaction between endogenous Cdc25A and HDAC11. Cdc25A (A) or HDAC11 (B) was immunoprecipitated from HEK 293T cell lysates. Immunoprecipitates were subjected to SDS-PAGE and probed with anti-HDAC11 and anti-Cdc25A antibodies. (C) Cdc25A binds directly to HDAC11. Purified His-HDAC11 and BSA (control) were immobilized on nitrocellulose membrane at the indicated concentrations and subsequently incubated in buffer containing FLAG-Cdc25A (800 ng). Cdc25A bound to His-HDAC11 was detected using anti-FLAG antibodies. (D) HDAC11 binds directly to Cdc25A. The reciprocal experiment used purified FLAG-Cdc25A and BSA immobilized on a nitrocellulose membrane which was incubated in buffer containing His-HDAC11 (800 ng). Bound His-HDAC11 was detected using anti-His antibodies.
Figure 8
Figure 8. Acetylated Cdc25A is a substrate for deacetylation by HDAC11
(A) HDAC11 mediates Cdc25A deacetylation in vitro. Purified His-Cdc25A was incubated with increasing amounts of purified HDAC11. The reactions were analyzed using antibody to acetylated lysine followed by chemiluminescent detection. (B) Endogenous Cdc25A is deacetylated by HDAC11. Cdc25A was immunoprecipitated from HEK 293T cell lysates and then incubated with or without HDAC11. The enzyme's acetylation status was analyzed by western blot using an acetyl-specific antibody.
Figure 9
Figure 9. Acetylation as a novel regulatory layer for modulating Cdc25A activity and cell cycle control
Following DNA damage, Cdc25A becomes phosphorylated by multiple kinases in preparation for proteasome-mediated degradation. Consistent with data in this report, Cdc25A is also acetylated by ARD1, which is antagonistic to its ubiquitination and degradation. When cells are not challenged, the homeostatic acetylation status of Cdc25A is maintained by the HDAC11 deacetylase. Phosphorylation of Cdc25A promotes its association with the SCFβTrCP E3 ubiquitin ligase, its ubiquitination and subsequent degradation. Acetylation may compete for the same residue as that targeted for ubiquitination (dashed arrow), thereby ameliorating the extent of Cdc25A degradation. Alternatively, Acetylation may occur at distant sites and inhibit ubiquitination via steric interference or conformational changes (elbow dashed arrow). In either case, the consequence of posttranslational modification is disruption of the cell cycle.
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