The oncogenic phosphatase WIP1 negatively regulates nucleotide excision repair

Abstract

Nucleotide excision repair (NER) is the only mechanism in humans to repair UV-induced DNA lesions such as pyrimidine (6-4) pyrimidone photoproducts and cyclobutane pyrimidine dimers (CPDs). In response to UV damage, the ataxia telangiectasia mutated and Rad3-related (ATR) kinase phosphorylates and activates several downstream effector proteins, such as p53 and XPA, to arrest cell cycle progression, stimulate DNA repair, or initiate apoptosis. However, following the completion of DNA repair, there must be active mechanisms that restore the cell to a prestressed homeostatic state. An important part of this recovery must include a process to reduce p53 and NER activity as well as to remove repair protein complexes from the DNA damage sites. Since activation of the damage response occurs in part through phosphorylation, phosphatases are obvious candidates as homeostatic regulators of the DNA damage and repair responses. Therefore, we investigated whether the serine/threonine wild-type p53-induced phosphatase 1 (WIP1/PPM1D) might regulate NER. WIP1 overexpression inhibits the kinetics of NER and CPD repair, whereas WIP1 depletion enhances NER kinetics and CPD repair. This NER suppression is dependent on WIP1 phosphatase activity, as phosphatase-dead WIP1 mutants failed to inhibit NER. Moreover, WIP1 suppresses the kinetics of UV-induced damage repair largely through effects on NER, as XPD-deficient cells are not further suppressed in repairing UV damage by overexpressed WIP1. Wip1 null mice quickly repair their CPD and undergo less UV-induced apoptosis than their wild-type counterparts. In vitro phosphatase assays identify XPA and XPC as two potential WIP1 targets in the NER pathway. Thus WIP1 may suppress NER kinetics by dephosphorylating and inactivating XPA and XPC and other NER proteins and regulators after UV-induced DNA damage is repaired.

Figure 1

Wip1 depletion enhances nucleotide excision repair. (A) Wip1 null MEFs display increased NER activity as measured by host cell reactivation assays. A UV-damaged firefly luciferase reporter plasmid was cotransfected with an unirradiated Renilla luciferase plasmid into Wip1^+/+, Wip1^+/−, and Wip1^−/− MEFs. Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. After correcting for transfection efficiency by dividing the firefly by Renilla luciferase activity, the Wip1^+/+ MEFs cells were set to 100% and the normalized luciferase values for the other cells were reported as percent repair activity. Error bars indicate the standard error (n=6). Asterisks indicate statistical significance with P values indicated. (B) Wip1 null MEFs display enhanced kinetics of CPD repair. Kinetics of CPD repair in Wip1^+/+ and Wip1^−/− MEFs were determined by immuno slot-blot assay following 15 J/m² UVC irradiation. The percent of remaining CPD was calculated by normalizing relative CPD amounts at various timepoints to the amount of CPD at 1 hour after UV damage. Each data point represents the mean of three experiments and the error bars represent the standard error. The asterisk indicates statistical significance (p<0.05). (C) Representative western blot showing reduced cleavage of apoptosis-facilitating proteins (caspase 3 and PARP) in Wip1^−/− MEF compared to Wip1^+/+ MEF after 15 J/m² UV.

Figure 2

WIP1 inhibits NER. (A) WIP1, but not mutant murine Wip1, overexpression inhibits NER in U2-OS cells. Human WIP1, mouse (wild-type, point or truncation mutant) Wip1, or empty-CMV expression constructs were cotransfected with a UV-damaged firefly luciferase reporter plasmid and an unirradiated Renilla luciferase plasmid into U2-OS (p53 proficient cells). Twenty-four hours after transfection, the cells were harvested and luciferase activity was measured. After correcting for transfection efficiency by normalizing to firefly Renilla luciferase activity, the empty vector transfected cells were set to 100% and the normalized luciferase values for the other cells were reported as percent repair activity. Error bars indicate the standard error (n=3). Asterisks indicate statistical significance (p<0.05). (B) p53-independent inhibition of NER in Saos-2 cells by WIP1. Human wild-type or phosphatase-dead WIP1 or empty-CMV expression constructs were cotransfected with luciferase plasmids in p53 null Saos-2 cells as described above. Error bars indicate the standard error (n=4) and asterisks indicate statistical significance (p<0.05). (C) Western blot analysis of WIP1 expression in U2-OS cells transfected with human or mouse Wip1 as analyzed in (A). A monoclonal Flag or V5 antibody was used to detect human or mouse Wip1, respectively, which is the top band in each lane. β-actin was used as loading control. (D) Western blot analysis of WIP1 expression in Saos-2 cells transfected with human WIP1, as analyzed in (B). (E) WIP1 suppression of UV-damage repair is largely dependent on a functioning NER pathway. WIP1 overexpression inhibits NER in XP17BE (XPD deficient) cells complemented with an XPD plasmid. Human wild-type or phosphatase-dead WIP1 or empty-CMV expression constructs were cotransfected with luciferase plasmids in the presence or absence of exogenous XPD in XP17BE cells as described above. After correcting for transfection efficiency by dividing the firefly by Renilla luciferase activity, the empty vector + exogenous XPD transfected cells were set to 100% and the normalized luciferase values for the other cells were reported as percent repair activity. Error bars indicate the standard error (n=3). Asterisks indicate statistical significance (p<0.05).

Figure 3

WIP1 inhibits repair of CPD. (A) Quantitative analysis of CPD repair in UV irradiated HeLa cells with or without overexpressed WIP1. The CPD fluorescence intensity (normalized to DAPI) from the mock-transfected and WIP1-transfected cells were measured by high-throughput microscopic imaging and plotted. Each data point represents the average of two (open boxes or circles) or three (filled boxes or circles) coverslips, each containing at least 3,500 independent cells. Error bars represent the standard error and the asterisk indicates statistical significance: (* = p<0.0078; ** = p<0.0072; *** = p<0.0011). (B–G) WIP1 overexpression inhibits the repair of CPD. HeLa cells transfected with no DNA (B–D) or wild-type WIP1 (E–G) were irradiated with 15 J/m² UVC 24 hours after transfection. Cells were fixed at various timepoints after irradiation and immunostained with anti-Flag (red fluorescence) and anti-CPD antibodies (green fluorescence) to assess CPD repair. DAPI staining (blue fluorescence) was used to identify nuclei. (B, E) no UV, (C, F) 8 hours post UV, (D, G) 24 hours post UV irradiation.

Figure 4

Effects of UVB exposure on Wip1 deficient mice. Skin sections from Wip1^+/+ and Wip1^−/− mice exposed to 2.5 kJ/m² UVB were collected at the times indicated. (A, B) Epidermal morphology in Wip1^+/+ (A) and Wip1^−/− (B) mice. Skin sections were stained with hematoxylin and eosin. (C) Wip1 null mice displayed increased CPD repair activity. Genomic DNA was isolated from irradiated mouse skin and CPD were detected by immuno slot-blot assay. Each data point (blue squares = Wip1^+/+, red circles = Wip1^−/−) represents an individual mouse. The green triangle represents the average of the genotype and the error bars represent the standard error. The asterisk indicates statistical significance (p<0.05). (D) Wip1 null mice are less sensitive to UV-induced apoptosis. A TUNEL apoptosis assay was performed on skin sections. TUNEL fluorescence intensity was normalized to DAPI staining and plotted. Each data point (blue squares = Wip1^+/+, red circles = Wip1^−/−) represents an individual mouse. The green triangle represents the average of the genotype and the error bars represent the standard error. The asterisk indicates statistical significance (p<0.05). (E, F) TUNEL staining of the epidermis of Wip1^+/+ (E) and Wip1^−/− (F) mice.

Figure 5

XPA and XPC phosphopeptides are dephosphorylated by WIP1 in vitro. (A) Protein sequence alignment of various orthologues of XPA, containing serines 173 and 196, indicated by arrows. Identical amino acids are highlighted with a black background while conservative amino acid substitutions are indicated with a gray background. (B, C) Protein sequence alignment of various orthologues of XPC, containing serines 350 (B) and 892 (C), indicated by arrows. (D) XPA S196 and XPC S892 phosphopeptides are dephosphorylated by WIP1 in vitro. XPA pS196 and XPC pS892 phosphopeptides were incubated with human recombinant WIP1, phosphatase-dead WIP1-D314A, or PP2Cα protein. p38 pT180 and UNG2 pT31 phosphopeptides were used as a positive and negative control, respectively. −Mg²⁺ indicates incubation in the absence of magnesium and +OA indicates incubation with the PP1α and PP2A phosphatase inhibitor, okadaic acid. Free phosphate released from the phosphopeptide was measured by malachite green phosphate assay to determine relative phosphatase activities on each phosphopeptide. Error bars represent the standard error (n=3) and the asterisk indicates statistical significance (p<0.05). (E) WIP1, but not other serine/threonine phosphatases, dephosphorylates XPA S196 and XPC S892 phosphopeptides in vitro. XPA pS196 and XPC pS892 phosphopeptides were incubated with human recombinant WIP1, PP1α, PP2A, or PP2Cα protein. Positive controls were as follows: p38 pT180 for WIP1 and PP2Cα, CHK1 pS345 for PP1α, and the generic serine/threonine phosphopeptide, RRA(pT)VA, for PP2A. Phosphatase activity was measured as described above. Error bars represent the standard error (n=3) and the asterisk indicates statistical significance (p<0.05).