The p53-Induced Oncogenic Phosphatase PPM1D ... - Cell Press

Molecular Cell, Vol. 15, 621–634, August 27, 2004, Copyright 2004 by Cell Press

The p53-Induced Oncogenic Phosphatase PPM1D Interacts with Uracil DNA Glycosylase and Suppresses Base Excision Repair Xiongbin Lu,1 Dora Bocangel,1 Bonnie Nannenga,2 Hiroshi Yamaguchi,3 Ettore Appella,3 and Lawrence A. Donehower1,2,* 1 Department of Molecular Virology and Microbiology Baylor College of Medicine Houston, Texas 77030 2 Department of Molecular and Cellular Biology Baylor College of Medicine Houston, Texas 77030 3 Laboratory of Cell Biology National Cancer Institute Bethesda, Maryland 20892

Summary The wild-type p53-induced phosphatase PPM1D (or Wip1) is a serine/threonine phosphatase that is transcriptionally upregulated by p53 following ultraviolet and ionizing radiation. PPM1D is an oncogene in transformation assays and is amplified or overexpressed in several human tumor types. Here, we demonstrate that PPM1D interacts with the nuclear isoform of uracil DNA glycosylase, UNG2, and suppresses base excision repair (BER). Point mutations that inactivate PPM1D phosphatase activity abrogate BER suppression, indicating that dephosphorylation by PPM1D is important for BER inhibition. We have identified UNG2 phosphorylation sites at threonines 6 and 126 that exhibit enhanced phosphorylation following UV irradiation. The UV-induced phosphorylated forms of UNG2 are more active than nonphosphorylated forms in mediating uracil-associated DNA cleavage. PPM1D dephosphorylation of UNG2 at phosphothreonine 6 is associated with reduced UNG2 activity. Thus, PPM1D may inhibit BER by dephosphorylating UNG2 to facilitate its inactivation after completion of DNA repair. Introduction The p53 tumor suppressor protein is critically important in mediating the cellular response to DNA damage (Giaccia and Kastan, 1998; Appella and Anderson, 2001; Sharpless and DePinho, 2002). Following DNA damage, p53 is activated and can transactivate or repress a number of cell cycle and apoptosis-associated genes (Vogelstein et al., 2000; Yu et al., 1999; Zhao et al., 2000a). The outcome is often cell cycle arrest or apoptosis. Much of the p53 activation is due to phosphorylation by DNA damage-associated kinases (Giaccia and Kastan, 1998; Appella and Anderson, 2001). Among the kinases targeting p53, p38 MAP kinase phosphorylates p53 at Ser33 and Ser46 (Bulavin et al., 1999; Appella and Anderson, 2001). Among the many p53 transcriptional targets is the wild-type p53-induced phosphatase, PPM1D (also *Correspondence: [email protected]

known as Wip1). PPM1D was initially identified in a screen for ionizing radiation-induced transcripts in Burkitt lymphoma cells that carry a wild-type p53 gene (Fiscella et al., 1997). PPM1D is induced by ionizing radiation in a p53-dependent manner. PPM1D protein has Mg⫹2dependent phosphatase activity in vitro and is insensitive to the inhibitor okadaic acid, characteristics of type 2C phosphatases (PP2Cs) (Fiscella et al., 1997). PP2Cs have been implicated in stress response signaling pathways (Cohen, 1989; Choi et al., 2000). p38 MAP kinase was identified as the first PPM1D target by Takekawa et al. (2000). UV irradiation causes phosphorylation of p38 on threonine 180 and tyrosine 182 by UV-responsive MKK dual specificity kinases (Raingeaud et al., 1995; Derijard et al., 1995). The phosphorylated p38 can in turn phosphorylate p53 on Ser 33 and Ser 46 and increase p53 activity (Bulavin et al., 1999). PPM1D is induced by many genotoxic stresses and inhibits p38 activation through dephosphorylation at threonine 180 (Takekawa et al., 2000). Thus, PPM1D may be a mediator (via p38) of a p53 negative feedback regulatory loop following its stress-induced activation. The ability of PPM1D to inhibit p53 activity suggests that it could behave as an oncogene, in a manner analogous to that of Mdm2, which facilitates p53 degradation and is amplified and overexpressed in human tumors (Michael and Oren, 2002; Alarcon-Vargas and Ronai, 2002; Momand et al., 1998). Indeed, Bulavin et al. (2002) and Li et al. (2002) have shown that the PPM1D gene at chromosomal region 17q23 is at the epicenter of an amplified DNA region in 11%–16% of human breast cancers. The PPM1D gene is amplified or overexpressed in breast cancers, prostate cancers, neuroblastomas, and ovarian clear cell adenocarcinomas (Bulavin et al., 2002; Li et al., 2002; Saito-Ohara et al., 2003; Hirasawa et al., 2003). Virtually all of the breast tumors with PPM1D overexpression were wild-type for p53, suggesting that PPM1D may functionally inactivate p53 and reduce selection for p53 structural alterations (Bulavin et al., 2002). PPM1D also complemented the oncogenes Ras, Myc, and Neu for transformation of wild-type mouse embryo fibroblasts (MEFs) (Bulavin et al., 2002). Recently, it was shown that PPM1D may enhance proliferation through suppression of p19ARF as well as p16INK4a (Bulavin et al., 2004; Bernards, 2004). p53 regulates DNA excision repair pathways, including nucleotide excision repair (NER) and base excision repair (BER) (Smith and Seo, 2002; Offer et al., 1999, 2001; Zhou et al., 2001). BER acts to repair DNA containing abasic sites, deaminated bases, or small other base lesions (Mitra et al., 2002; Smith and Seo, 2002; Hoeijmakers, 2001). A BER repairosome complex of four to six proteins includes a DNA glycosylase that recognizes a specific type of damaged base and cleaves the base from the deoxyribose (Hang and Singer, 2003). A common lesion in mammalian cell DNA is uracil formed either by deamination of cytosine or misincorporation of dUMP during replication. Mammalian cells contain at least four uracil DNA glycosylases (Aravind and Koonin,

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2000). Of these, UNG2 is the dominant enzyme for uracil removal in nuclear DNA (Kavli et al., 2002). After binding and removal of uracil by UNG2, the AP endonuclease initiates a strand incision adjacent to the baseless site. The damaged nucleotide is removed and replaced by repair polymerases and the remaining nick is sealed by DNA ligases. p53 interacts with the AP endonuclease and stimulates its activity (Gaiddon et al., 1999; Zhou et al., 2001). Thus, activated p53 stimulates BER. Here, we show that BER activity is suppressed by PPM1D. UNG2 was identified as a PPM1D interactor in a two- hybrid screen. Increased levels of PPM1D suppress BER, while reduction of PPM1D enhances BER activity. UNG2 is phosphorylated in a UV-dependent manner and one of these UNG2 phosphorylation sites is a target for PPM1D dephosphorylation. PPM1D dephosphorylation of UNG2 is correlated with reduced UNG2 activity on uracil-containing templates. Thus, PPM1D may have a homeostatic role in restoring UNG2 to basal levels after its activation by DNA damage-induced phosphorylation.

Results UNG2 Is a PPM1D-Interacting Protein To identify other potential PPM1D interactors we performed bacterial two-hybrid assays. The human PPM1D cDNA was placed into a bait vector and the target vector contained a cDNA library from Hela cells. Following cotransformation of bacterial cells with the bait and target vectors, an interaction between the PPM1D bait and one of the target gene products results in carbenicillin resistance and ␤-galactosidase-mediated blue staining in the presence of X-gal (Figure 1A). After two-hybrid screening, we identified a target with identity to the human nuclear uracil DNA glycosylase UNG2. UNG2 initiates the process of BER by binding and removing an inappropriate uracil base in nuclear DNA (Mitra, 2001; Krokan et al., 2002). To confirm the interaction of PPM1D with UNG2, we performed in vitro translation/coimmunoprecipitation and cell-based interaction studies. When in vitro translated FLAG-PPM1D and UNG2 were mixed, immunoprecipitation with FLAG antibodies pulled down both FLAG-PPM1D and UNG2 while not pulling down a noninteracting luciferase protein (Figure 1B). To determine which domains of PPM1D are required for UNG2 binding, we mixed in vitro translation reactions containing intact UNG2 with various N- and C-terminal truncated and point mutant forms of murine PPM1D. The point mutations were in those highly conserved residues known to be important for catalytic phosphatase activity in Type 2C phosphatases (Das et al., 1996). In vitro phosphatase assays confirmed that these PPM1D point mutants were deficient in phosphatase activity, whereas the wild-type and C-terminal truncation mutant ⌬C(376598) had high levels of phosphatase activity (data not shown). The PPM1D mutants in each extract were V5 tagged and were immunoprecipitated with anti-V5 antibody followed by SDS polyacrylamide gel electrophoresis and autoradiography (Figure 1C). All point mutants and two truncation mutants, ⌬N(1-49) and ⌬C(376-598), were able to bind UNG2, while two other truncation

mutants, ⌬N(1-101) and ⌬N(1-156), showed no binding to UNG2. The truncation mutants that did bind UNG2 did not delete the conserved Type 2C phosphatase domain of PPM1D (amino acids 65-370), while those that did not bind were missing significant portions of the phosphatase domain. These results indicate that UNG2 binds to PPM1D within its phosphatase domain. To demonstrate PPM1D-UNG2 interactions in intact cells, Saos-2 and U2OS cells transfected with a FLAGPPM1D expression construct were lysed and FLAGimmunoprecipitated followed by Western blotting with a UNG2-specific antibody. Endogenous UNG2 was detected in the immunoprecipitate from FLAG-PPM1Dtransfected Saos-2 and U2OS cells, but not untransfected control cells. (Figure 1D). PPM1D Suppresses BER The interaction of PPM1D with UNG2 implicates PPM1D as a potential regulator of BER. To test this, we performed BER assays on mouse fibroblasts derived from PPM1D⫹/⫹, PPM1D⫹/⫺, and PPM1D⫺/⫺ embryos. MEFs were transfected with a heat and acid treated luciferase expression plasmid. The heat and acid treatment randomly hydrolyzes the bases in DNA and is repaired primarily through BER. After transfection of the damaged plasmids, luciferase activity was measured in cell lysates taken 24 hr later. PPM1D⫺/⫺ MEFs exhibited a roughly 6-fold increase in BER activity compared to PPM1D⫹/⫹ MEFs (Figure 2A). In a second in vitro assay, we purified nuclear extracts from MEFs and incubated the heat and acid treated DNA template with 32P-dGTP for 15 min and measured 32 P incorporation into the damaged DNA template. Reduction of PPM1D dosage led to a 91% and 15% increase in BER levels in PPM1D⫺/⫺ and PPM1D⫹/⫺ MEFs, respectively, compared to their PPM1D⫹/⫹ counterparts (Figure 2B). The results implicate PPM1D as an inhibitory factor for BER. PPM1D Suppression of BER Is Not p53 Dependent Recent evidence indicates that p53 stimulates BER (Offer et al., 1999; Zhou et al., 2001; Hanawalt, 2001; Smith and Seo, 2002). Since PPM1D is induced by p53 and has negative feedback effects on p53 activity, it is possible that the increased BER in PPM1D null MEFs is merely due to increased activity of p53 in the absence of PPM1D and not to direct effects of PPM1D. To determine whether PPM1D regulation of BER is p53 independent, we studied the effects of overexpressed PPM1D on BER in Saos-2 cells, a human osteosarcoma line null for p53. We cotransfected Saos-2 cells with heat and acid-damaged luciferase plasmid DNA and an expression construct encoding either human PPM1D or mouse PPM1D. At 24 hr post-transfection, the cells were monitored for luciferase activity as an indicator of BER activity (Figure 2C). High levels of PPM1D expression in the transfected cells were confirmed by immunoblots (Figure 2C, right panel). Saos-2 cells transfected with 0.1 ␮g or 1 ␮g human PPM1D both had over 43% reduction of BER activity compared to the cells transfected with an empty expression vector. Mouse PPM1D also suppressed BER activity after transfection (Figure 2C, left panel).

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Figure 1. Human and Mouse PPM1D Interact with UNG2 (A) Two-hybrid experiment shows human PPM1D-UNG2 interaction. Positive bacterial cotransformants indicative of PPM1D interactors on a carbenicillin-containing plate are shown. In the upper dish, Sector 3 expresses UNG2. Sectors 4, 5, and 6 express three other interacting proteins. Sectors 1 and 2 are positive and negative controls, respectively. The lower dish shows the same cotransformants growing on the X-gal indicator plate without carbenicillin. Only positive colonies turn blue. (B) PPM1D in vitro interactions with UNG2. A FLAG-tagged version of human PPM1D was in vitro transcribed and translated in the presence of 35S-methionine and mixed with similarly prepared lysates containing either luciferase or UNG2. Some lysates were immunoprecipitated with the FLAG antibody and subjected to SDS-polyacrylamide gel electrophoresis and autoradiography. (C) In vitro binding of UNG2 to wild-type and mutant forms of PPM1D. In vitro translated lysates containing wild-type UNG2 were mixed with V5-tagged wild-type or mutant murine PPM1D and immunoprecipitated with V5 antibody. (D) UNG2 binds to PPM1D in Saos-2 cells and U2OS cells. Saos-2 and U2OS cell lines were mock transfected or transfected with a human PPM1D-FLAG expression construct and protein lysates prepared from the transfected cells 48 hr later. Immunoprecipitations were performed with FLAG antibody followed by Western blotting with a UNG2 antibody.


To determine those domains of PPM1D that are responsible for regulating BER, we assayed the effects of our murine PPM1D mutants on cellular BER activity. We cotransfected wild-type or mutant PPM1D expression constructs into Saos-2 cells along with heat and acid treated luciferase expression constructs and monitored luciferase activity 24 hr later. Western blot analysis of the transfected cells indicated that the PPM1D mutants were expressed at a reasonably high level (Figure 2D, right panel). As before, the wild-type PPM1D showed reduced BER activity, with a 2- to 3-fold reduction when the expression plasmids were damaged with heat and acid for 45 min or 6 hr (Figure 2D, left, middle panels). Two phosphatase-dead point mutants (A95D, R266A) exhibited minimal BER suppression, indicating that the phosphatase activity of PPM1D plays an important role in BER inhibition. The C-terminal deletion mutant (⌬C376-598) of PPM1D had only a slight suppressive effect on BER, suggesting that C-terminal domains outside the phosphatase domain are involved in BER suppression. In other experiments, we reduced PPM1D expression in U2OS and Saos-2 human osteosarcoma cells by transfection with PPM1D siRNAs (Figure 2E, right panel). BER activity as measured by repair of the damaged luciferase plasmid was significantly increased in the presence of PPM1D siRNA (Figure 2E, left panel). These results indicate that BER suppression by PPM1D is at least in part a p53-independent effect. Uracil-Associated BER Is Suppressed by PPM1D To demonstrate that PPM1D directly suppresses uracil DNA glycosylase function in BER, we utilized in vitro assays that measure BER-associated incision on double-stranded oligonucleotide templates containing a single uracil residue. UNG2 recognizes the incorporated uracil and initiates BER. The uracil-containing 51-mer oligonucleotide is 32P-labeled at its 5⬘ end and cleavage next to the uracil residue after incubation with nuclear extract generates a 32P-labeled 25-mer that is monitored on a denaturing polyacrylamide gel. When Saos-2 cells

were transfected with wild-type PPM1D expression constructs, nuclear lysates derived from these PPM1Dtransfected cells showed a significant dose-dependent inhibition of uracil-associated incision compared to lysates derived from empty vector-transfected cells (Figure 3A). Lysates transfected with phosphatase-dead PPM1D mutants or the C-terminal truncation mutant showed little incision inhibition in Saos-2 cells (Figure 3B). These results indicate that the phosphatase activity of PPM1D and domains of PPM1D other than its phosphatase domain are both important in inhibiting the subsequent uracil-associated cleavage of the DNA strand backbone. Overexpression of PPM1D suppresses uracil-associated BER incision. We also generated nuclear lysates from PPM1D-deficient MEFs to determine whether reduction or absence of PPM1D affects the uracil incision reaction. PPM1D⫺/⫺ MEF lysates show significantly higher levels of uracil-associated incision than their PPM1D⫹/⫹ counterparts (Figure 3C). PPM1D⫹/⫺ MEFs showed a slightly higher level of incision compared to PPM1D⫹/⫹ MEFs, but this difference was not statistically significant. In BER, once the uracil is removed, it is replaced with the correct nucleotide by repair polymerases and ligated to form an intact strand. We attempted to measure the effects of PPM1D on this aspect of BER by performing a 32 P-dCTP incorporation reaction on the uracil-containing oligonucleotide template. In this case, the template is unlabeled, but successful replacement of the uracil with the correct cytosine results in 32P incorporation in the 51-mer. Nuclear lysates from U2OS and Saos-2 cells transfected with PPM1D expression vectors both produced significant decreases in labeled 51-mer compared to lysates from their empty vector transfected counterparts (Figures 3D, 3E, and data not shown). When the PPM1D mutants were assessed for suppression of incorporation, three phosphatase dead point mutants were as efficient as the wild-type PPM1D (Figure 3E). This outcome was surprising given that these same mutants failed to inhibit uracil BER incision. Such results suggest that only the early phases but not the later

Figure 2. PPM1D Suppresses BER (A) PPM1D null fibroblasts show increased BER activity following transfection with a damaged DNA marker plasmid. A luciferase expression construct treated with heat and acid for 6 hr was transfected into PPM1D⫹/⫹, PPM1D⫹/⫺, and PPM1D⫺/⫺ MEFs. Luciferase activity as an index of BER activity in the transfected cells was measured 24 hr after transfection. (B) In vitro BER activity in PPM1D⫹/⫹, PPM1D⫹/⫺, and PPM1D⫺/⫺ MEF nuclear extracts. Undamaged pUC18 plasmid DNA was used as a negative control. pGL3-CMV treated with heat and acid for 45 min was incubated with nuclear extracts and 32P-dGTP. The upper left panel shows linearized plasmid DNA separated by 1% agarose gel electrophoresis. Incorporation of 32P-dGTP into damaged pGL3-CMV plasmid DNA is shown in the lower left panel. Quantitation of BER activity in PPM1D⫹/⫹, PPM1D⫹/⫺, and PPM1D⫺/⫺ MEF extracts is shown in the right panel. (C) p53-independent suppression of BER activity in Saos-2 cells by human and murine PPM1D. Expression constructs of human FLAG-tagged wild-type human PPM1D and murine wild-type V5-tagged PPM1D cDNAs and empty vector DNAs (control) were transfected into p53 null Saos-2 cells after a 6 hr heat and acid treatment. Luciferase activities were measured in the transfected cell lysates (left panel). Lysates from the transfected and mock transfected cells were subjected to Western blot analysis with the indicated FLAG or V5 antibody (right panel). (D) Mutant forms of murine PPM1D fail to suppress BER in Saos-2 cells. Human and murine wild-type and mutant murine V5-tagged PPM1D expression constructs were transfected into Saos-2 cells along with acid and heat treated luciferase expression vectors and monitored for luciferase activity. Luciferase constructs were acid/heat treated for 45 min (left panel) or 6 hr (middle panel). In the right panel, expression levels of the wild-type and mutant PPM1D proteins in the transfected cells are indicated. (E) Reduction of PPM1D mRNA by PPM1D siRNA results in increased BER. Saos-2 and U2OS cells were transfected with PPM1D siRNA or control RNA and a damaged luciferase expression construct. Forty eight hours post-transfection, lysates were prepared from the transfected cells and luciferase assays performed (left panel). White bars represent control siRNA transfected cells and black bars represent PPM1D siRNA transfected cells. Total RNA was purified from transfected cells 48 hr post-transfection and equal amounts of RNA from each line were subjected to RT-PCR with PPM1D-specific primers, followed by visualization of the PCR products (right panel).

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Figure 3. Effects of PPM1D on Uracil BER In Vitro (A) Effect of increasing PPM1D on uracil-incision activity. Saos-2 cells were transfected with increasing amounts of PPM1D cDNA and lysates tested for uracil incision. Incision assays were performed by allowing Saos-2 nuclear extracts to react with a 32P-labeled uracil- or cytosine-


phases of BER may be suppressed by the phosphatase activity of PPM1D. Also unexpected was the lack of suppression of 32P-incorporation produced by the PPM1D C-terminal truncation mutant 376-T, suggesting that PPM1D domains outside of the phosphatase domain may be relevant in the late phases of BER. To extend the above BER incorporation results, we performed similar assays with nuclear lysates from PPM1D⫹/⫹, PPM1D⫹/⫺, and PPM1D⫺/⫺ MEFs. The PPM1D⫺/⫺ MEF lysates produced a significant increase in 32P-dCTP incorporation compared to the PPM1D⫹/⫹ lysates, consistent with the data observed in the incision assays (Figure 3F). Identification of Potential PPM1D Phosphorylation Sites on UNG2 The binding of the PPM1D phosphatase to UNG2 suggested that part of its role in inhibiting BER could be through dephosphorylation of phosphoserines or phosphothreonines on UNG2. To identify potential PPM1Dtargeted phosphorylation sites on UNG2, we synthesized UNG2-derived phosphopeptides that showed similarities to the one known PPM1D target site on p38 MAP kinase. This site, TDDEMpTGpYVAT, contains the TXY motif that is the target for the dual specificity MKKs that phosphorylate p38. There are two TXY motifs on UNG2 and we generated phosphopeptides from these sites and from a series of other sites containing only threonines (Figure 4A). These UNG2 phosphopeptides were incubated with recombinant PPM1D phosphatase in an in vitro phosphatase assay. Release of free phosphate from the phosphopeptides was measured and compared for each phosphopeptide. The phosphopeptide containing the p38 MAP kinase MKK target site and known PPM1D target site at threonine 180 was efficiently dephosphorylated by recombinant PPM1D in vitro whether or not tyrosine 182 was phosphorylated (Figure 4B). The UNG2 phosphopeptide 1-11, containing a dual phosphorylated TXY motif, was strongly dephosphorylated by PPM1D. If only the threonine 6 was phosphorylated, the peptide was still efficiently dephosphorylated by PPM1D. In contrast, the threonine on UNG2 121-132 phosphopeptide was only dephosphorylated efficiently by PPM1D when both threonine and tyrosine were phosphorylated. If the tyrosine was nonphosphorylated, PPM1D did not dephosphorylate threonine 126. No other UNG2 threonine phosphopeptides showed ap-

preciable PPM1D-induced phosphate release, making UNG2 threonines 6 and 126 the best candidates for PPM1D target sites in vivo. Effects of PPM1D on UV-Induced Phosphorylation of UNG2 To determine whether threonines 6 and 126 were true in vivo targets for PPM1D, we generated polyclonal antibodies to UNG2 phosphothreonine peptides 1-11 and 121-131. These antibodies were used to probe Western blots of cell lysates from empty vector-transfected and PPM1D vector-transfected unstressed and UV-treated U2OS cells. The antibody specific for phosphothreonine 6 (6pT) on UNG2 produced low-intensity bands on the Western blot for both empty vector transfected and PPM1D vector transfected U2OS cells (Figure 5A, left panel). When these same transfected U2OS cells were subjected to UV irradiation, the band intensity corresponding to phosphorylated 6pT UNG2 dramatically increased in the absence of exogenous PPM1D. However, increasing amounts of transfected PPM1D decreased the levels of phospho-UNG2. These results indicate that phosphothreonine 6 on UNG2 is a UV-responsive target for phosphorylation and that it is dephosphorylated by PPM1D. Similar Western blot experiments utilizing the UNG2 121-132 phosphothreonine peptide antibody were performed. In unstressed cells, 126pT UNG2 levels were relatively low regardless of transfected PPM1D levels (Figure 5A, right panel). However, following UV radiation, levels of 126pT UNG2 were significantly increased and remained increased even in the presence of high levels of PPM1D. This result indicates that phospho-threonine 126 is also a UV responsive target for phosphorylation, but is not a significant in vivo PPM1D target. To further show that PPM1D directly dephosphorylates UNG2 at phosphothreonine 6, we immunoprecipitated UNG2 from UV-irradiated human cells and incubated the UNG2 with increasing amounts of purified FLAG-PPM1D in an in vitro phosphatase assay (Figure 5B). We then subjected the reactions to SDS-PAGE and immunoblotting with the phosphospecific UNG2 6pT antibody. We found that increasing amounts of purified FLAG-PPM1D resulted in increased dephosphorylation of UNG2 at phosphothreonine 6 (Figure 5B). Moreover, removal of Mg⫹2 from the reaction inhibited PPM1D dephosphorylation (data not shown), consistent with the

containing 51-mer duplex DNA. Bar diagram summarizes quantitation of at least three independent experiments ⫾ standard error. (B) Effects of wild-type and mutant PPM1D on uracil-incision activity. Saos-2 cells were transfected with empty vector, cDNA for wild-type (WT), point mutant, or C-terminal truncation (376-T) variants of PPM1D. Controls (lanes 1 and 2), empty vector (lane 3), WT PPM1D (lane 4), and mutant PPM1D (lanes 5–8) are indicated. Bar diagram shows quantitation of three independent experiments ⫾ standard error. (C) Increased in vitro uracil BER incision activity in PPM1D null cells. Nuclear extracts of MEFs from PPM1D⫹/⫹, PPM1D⫹/⫺, or PPM1D⫺/⫺ mice were tested for uracil incision activity as in (A) and (B) (top panel). Bar diagram (bottom panel) summarizes results of at least three independent experiments. (D) In vitro uracil-BER incorporation assay. Nuclear extracts from Saos-2 cells transfected with plasmids containing an empty vector (lanes 2 and 3) or wild-type PPM1D cDNA (lane 4) were isolated and allowed to react with a nonlabeled uracil-containing 51-mer duplex DNA in standard repair synthesis reactions containing labeled [␣-32P]-dCTP. Lane 1 is a no lysate negative control. (E) Bar diagram summarizing results for 32P-dCTP incorporation into uracil-containing templates after incubation with nuclear extracts from Saos-2 cells transfected with empty vector, WT PPM1D, and point mutant or a C-terminal truncation mutants of PPM1D. Bar diagram summarizes results of at least three independent experiments ⫾ standard error. (F) In vitro incorporation assay using uracil-containing templates incubated with nuclear extracts from wild-type and PPM1D-deficient MEFs. The incorporation assay was performed as in (D) (upper panel). The lower panel quantitates the results of three independent experiments.

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Figure 4. PPM1D In Vitro Phosphatase Assays on UNG2 Phosphopeptides (A) List of phosphopeptides tested in the in vitro phosphatase assay. Control phosphopeptides 1 and 2 are from p38 MAP kinase and are known to be dephosphorylated by PPM1D. The remaining phosphopeptides are derived from UNG2. Phosphothreonines and phosphotyrosines are indicated in bold. (B) Three phosphopeptides from two regions of UNG2 are dephosphorylated in vitro by recombinant PPM1D. An in vitro phosphatase assay that detects free phosphate released from the fifteen p38 and UNG2 phosphopeptides incubated with recombinant PPM1D was used to determine relative phosphatase activities. Numbers along the X axis refer to the phosphopeptides in (A). Gray bars show the phosphatase activity of PPM1D on p38 phosphopeptides, while black bars show PPM1D activity on UNG2 phosphopeptides.

magnesium dependence of type 2C phosphatases (Fiscella et al., 1997). UNG2 Activity Is Enhanced by Phosphorylation and Is Inhibited by PPM1D UNG2 is phosphorylated at threonine residues 6 and 126 and PPM1D dephosphorylates phosphothreonine residue 6 in vivo. BER activity in general and UNG2 activity in particular are inversely correlated with PPM1D dosage. To more directly show that UNG2 phosphoryla-

tion enhances its activity and that PPM1D dephosphorylation inhibits UNG2 activity, we performed an in vitro uracil template incision assay. The uracil-containing 51mer duplex was incubated with nuclear extracts derived from unirradiated and UV-irradiated cells. UV irradiation resulted in a roughly 2-fold increase in uracil incision activity (Figure 6). When the UV-irradiated extracts were pre-cleared with an antibody to UNG2, the uracil incision activity was reduced to about 40% of the UV-treated extract containing UNG2 (Figure 6A). This indicates that


Figure 5. PPM1D Suppresses UNG2 Phosphorylation at Threonine 6 (A) UNG2 phosphorylation in U2OS cells transfected with PPM1D cDNA. Western blot analysis of nuclear extracts from U2OS cells transfected with empty vector, or with increasing amounts of WT PPM1D cDNA, using antibodies that recognize phosphorylated threonine at positions 6 (left panel), or position 126 (right panel). Results were standardized by Western blot with UNG2 and ␤-actin antibodies (lower panels). Bar diagram summarizes results of at least three independent experiments ⫾ standard error. (B) In vitro phosphatase assays on intact phospho-UNG2 protein by purified PPM1D-FLAG. Immunoprecipitated UNG2 proteins from UVradiated HCT116 cells were dephosphorylated in vitro by increasing (0–300 ng) amounts of purified FLAG-PPM1D. After the assay, the reaction mix was immunoblotted and the level of threonine 6 phosphorylated UNG2 was detected by the UNG2(p6T) phospho-specific antibody.

the majority of the uracil-associated incision activity in the nuclear extracts is due to UNG2. When the UVtreated nuclear extracts are pre-cleared with phosphospecific UNG2 6pT antibody or phosphospecific UNG2 126pT antibody, there was a moderate decrease in uracil-associated incision activity (Figure 6A). When both phospho-specific antibodies were used together to preclear the UV-treated extracts, the reduction in uracil incision activity was nearly as great as that exhibited by the UNG2 precleared extracts (Figure 6A). Since there was still unphosphorylated UNG2 in these extracts, this

suggests that the phosphorylated forms of UNG2 are more active in uracil incision than unphosphorylated forms. To demonstrate that PPM1D directly affects the uracil-associated incision activities of UNG2, we added purified human PPM1D protein to the UV-treated nuclear extracts in the uracil incision assay. PPM1D addition reduced uracil incision activity by about 40%, whereas addition of inactivated PPM1D had no effect (Figure 6B). When the UV-treated extracts were precleared of UNG2, again there was a 60% reduction in uracil incision activ-

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Figure 6. UNG2-Associated Uracil Incision Activity Is Increased by UV-Induced Phosphorylation and Is Decreased by PPM1D Dephosphorylation (A) UV treatment increases UNG2 phosphorylation and uracil incision activity. Nuclear extracts from unirradiated (lane 3) or UV-irradiated (lanes 4–8) Saos-2 cells were incubated with a uracil-containing 32P-end-labeled 51 bp duplex. Prior to incubation, some extracts were precleared by treatment with antibodies to UNG2 protein (lane 5) or to phospho-specific UNG2 6pT (lane 6) or 126pT (lane 7) or both phosphospecific antibodies (lane 8). Controls were uracil duplexes not incubated with lysates (lane 1) and non-uracil containing duplexes incubated with lysates (lane 2). Following incubations, the labeled duplex DNAs were subjected to polyacrylamide gel electrophoresis and visualized by autoradiography. Formation of a 25-mer band indicated incision adjacent to the uracil moiety. The relative incision results were quantitated by phosphorimaging and presented in graph form in the second panel. An equal portion of each nuclear extract was also subjected to SDSpolyacrylamide gel electrophoresis and immunoblotting with antibodies to UNG2, phospho-specific UNG2 6pT, PPM1D, and ␤-actin (lower four panels). (B) PPM1D dephosphorylates UNG2 and reduces its uracil-associated incision activity. Reaction conditions are similar to those in (A) and extract treatments are indicated above each lane. Purified PPM1D, either intact (lanes 5 and 8) or inactivated (lane 6), were added to the extracts. Extracts were from cells treated with UV, except those in lanes 2 and 3, and extracts in lanes 7 and 8 were pre-cleared with UNG2 antibody. Controls in lanes 1 and 2 are as in (A), as are quantitations (second panel) and UNG2, UNG2(p6T), and ␤-actin protein amounts (lower three panels).

ity, but this activity was not further suppressed when removal of UNG2 was accompanied by addition of purified PPM1D (Figure 6B). This result suggests that PPM1D specifically inhibits UNG2 and not other uracil DNA glycosylases. Discussion BER is the chief protector against DNA damage due to cellular metabolism, including that initiated by reactive oxygen species, methyIation, deamination, and hydroxylation (Hoeijmakers, 2001; Mitra et al., 2002). Following repair of DNA damage, the cell must have a mechanism

to return the repair machinery to basal levels in the absence of continuing damage. Post-translational modifications such as phosphorylation and dephosphorylation could play a role in regulating repair protein activities. There is increasing evidence that DNA repair processes are modulated in this way (Zhao et al., 2000b; Araujo et al., 2000; Christmann et al., 2002). For example, UNG2 is phosphorylated on its amino terminus (MullerWeeks et al.,1998; Caradonna and Muller-Weeks, 2001). Here we show that UNG2 is phosphorylated on threonines 6 and 126 and that phosphorylation at these sites is augmented following UV irradiation. UV-induced phosphorylation of UNG2 at threonines


Figure 7. Model for PPM1D Activities in Regulating p53 and BER after UV Irradiation UV irradiation induces cyclobutane pyrimidine dimer formation. Cytosines in these dimers have very high rates of deamination and formation of uracils. After dimer bond removal, the remaining uracils are targets for UNG2 activity. UV damage also causes activation of the ATR and p38 MAP kinases. These kinases phosphorylate p53, contributing to p53 activation. In turn, p53 promotes BER activity. BER repairosomes that include UNG2 repair the damage. We hypothesize that UV-activated MKKs, while targeting p38, may also phosphorylate UNG2, increasing its activity. After initiation of BER, p53 transcriptionally upregulates PPM1D. Increased PPM1D protein eventually results in dephosphorylation and inhibition of p38 MAP kinase. Decreased p38 activity reduces p53 phosphorylation and destabilizes it. The increased PPM1D may repress uracil-BER through binding and dephosphorylation of UNG2 at threonine 6. BER repression by PPM1D helps revert DNA repair activity to basal levels after elimination of DNA damage. Solid lines indicate immediate responses to UV and dashed lines indicate more delayed responses.

6 and 126 also increases UNG2 activity as measured by an in vitro uracil-associated incision assay. This is corroborated by experiments showing that removal of the phosphorylated forms of UNG2 from the in vitro incision assays removed virtually all of the UNG2-associated incision activity. Thus, the UV-induced phosphorylation of UNG2 may be an important initiating step in UNG2-mediated BER. However, once BER is completed, and PPM1D accumulates to high levels in the cell at 8–12 hr post UV treatment (Takekawa et al., 2000), the excess PPM1D may facilitate inactivation of UNG2 through dephosphorylation at threonine 6. Presumably, other phosphatases contribute to UNG2 inactivation through dephosphorylation of threonine 126. Thus, PPM1D may perform a homeostatic role by helping to return the cell to a basal DNA repair state after correction of DNA lesions. The similarity of the TXY motifs associated with threonines 6 and 126 on UNG2 and a similar MKK target site on p38 MAP kinase implicate the dual specificity MKKs in the phosphorylation of UNG2 (Figure 7). Phosphorylation by upstream kinases could be a key factor in activating UNG2. If so, it would link the upstream MKK stress signaling pathways to downstream BER proteins that execute DNA repair.

We have presented evidence that UV-induced phosphorylation of UNG2 increases its activity on uracil-containing templates. This raises the question of why a protein that responds to uracil in DNA would be upregulated following UV, since UV is not known to directly produce uracil moieties. In fact, when pyrimidine dimers in DNA are formed by UV irradiation, those dimers containing cytosine have a dramatically increased propensity to be deaminated to uracils (Peng and Shaw, 1996; Tu et al., 1998). Thus, upregulation of UNG2 through phosphorylation may be an important component of the repair response to UV irradiation. Here we show that PPM1D, as a p53-induced protein, suppresses BER. Part of this inhibition is likely to occur through its interactions with UNG2. PPM1D binds to UNG2 and dephosphorylates it at threonine 6 in vivo. The phosphatase activity of PPM1D is important for its role in BER suppression, as PPM1D phosphatase dead mutants are defective in aspects of BER suppression in general and uracil-associated BER suppression in particular. In vitro, PPM1D inhibits events associated with UNG2 function, including uracil-mediated incision and final repair of the uracil lesion. Interestingly, the PPM1D phosphatase-dead mutants are defective for suppression of uracil proximal incision, but can sup-

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press the final replacement steps of BER. However, a C-terminal truncation mutant of PPM1D is defective for suppression of dCTP replacement. These results suggest the possibility that PPM1D, following its binding to UNG2, may recruit other factors through its C-terminal domain to maintain suppression of the latter stages of the uracil BER process. Thus, the binding of PPM1D to UNG2 may not merely be to facilitate transient catalytic dephosphorylation, but may be part of a stable interaction that further assists in BER suppression. PPM1D participates in a negative feedback autoregulatory loop for p53. Since activated p53 stimulates BER, deactivation of p53 through PPM1D may assist in downregulating BER through reduction of p53 stimulation. However, PPM1D also suppresses BER independently of p53 (in p53 null cells), and the phosphatase activity of PPM1D is important for that suppression. Thus, PPM1D may suppress BER through inactivation of multiple targets, including p53 (via p38 MAP kinase) and BER repairosome proteins (Figure 7). PPM1D has been shown to be a human oncogene (Bulavin et al., 2002; Li et al., 2002; Saito-Ohara et al., 2003; Hirasawa et al., 2003). In those tumors in which PPM1D is amplified and/or overexpressed, one possible effect is the suppression of DNA repair function. Thus, tumors with increased PPM1D levels may exhibit increased mutation rates due to decreased BER activity and some of these mutations could promote increased oncogenicity. Consistent with this, MCF-7 breast cancer cells with amplified PPM1D show 4- and 7-fold less BER activity than Saos-2 cells and U2OS cells, respectively (X.L., unpublished data). Thus, PPM1D could promote tumorigenesis through multiple p53-dependent and -independent mechanisms. Further studies may yet uncover other oncogenic roles for this phosphatase. Experimental Procedures Cell Culture Saos-2 and U2OS cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat inactivated fetal bovine serum. PPM1D⫹/⫹, ⫹/⫺ and ⫺/⫺ mouse embryonic fibroblasts (MEFs) were obtained as previously described (Choi et al., 2002) and were cultured in DMEM-15% heat-inactivated fetal bovine serum.

BER Assays The two BER assays used in this study were described previously (Offer et al., 1999, 2001). The protocols are described in the Supplemental Data. In Vitro Translation/Coimmunoprecipitation In vitro transcription/translation and coimmunoprecipitations were carried out as described in the Supplemental Data. Coimmunoprecipitation/Western Blot Analysis Saos-2 cells were seeded on 100 mm culture dishes and transfected with 3 ␮g of pCMV-PPM1D-FLAG DNA. Forty eight hours later, cells were lysed in Tris-buffered saline containing 1% Triton X-100 and protease inhibitors. Cell lysates were centrifuged at 10 K rpm for 10 min. The supernatant was used for immunoprecipitation. Ten micrograms of monoclonal FLAG antibodies was added to the cell lysate and mixed for 1 hr. 40 ␮l of protein A/G agarose beads were added and incubated at 4⬚C overnight. Immunoprecipitates were spun down and washed twice in PBS and twice in PBS-0.25% NP40. Immunoprecipitates were boiled in sample buffer for 3 min and loaded on 10% SDS-polyacrylamide gels. Proteins were transferred to PVDF membranes. UNG2 protein was detected by a UNG2 monoclonal antibody provided by Geir Slupphaug. The protein-antibody complexes were detected with an HRP-conjugated secondary antibody and enhanced chemiluminescence substrates (Pierce). SiRNA-Mediated PPM1D Silencing The PPM1D and control siRNAs were synthesized by Dharmacon. The RNAi sequences and protocols are described in the Supplemental Data. Nuclear Extract Preparation for Uracil-Associated In Vitro Repair Assays Nuclear extracts were obtained as described previously (Lee et al., 2001) with slight modifications that are indicated in the Supplemental Data. Preparation of DNA Substrates for Uracil-Associated In Vitro Repair Assays Single stranded oligonucleotides (51-mers) containing a uracil or cytosine residue at position 26 were obtained from Invitrogen. Preparation of the oligonucleotide for repair assays is described in the Supplemental Data. Uracil-Associated Incision and Incorporation Assays in Nuclear Extracts Uracil-associated BER incision assays were performed as described (Wang et al., 1997) with modifications. The incision and incorporation assays are described in the Supplemental Data.

Plasmid Construction Murine PPM1D cDNA was obtained by RT-PCR from mouse liver mRNA and cloned into the pcDNA 4.0c vector. Primer sequences for generating PPM1D cDNA constructs and derived mutants are listed in the Supplemental Data (at http://www.molecule.org/cgi/ content/full/15/4/621/DC1).

Phosphopeptide Synthesis, Purification, and Antibody Generation These methods are described in the Supplemental Data.

Screening of PPM1D Interactors by a Bacterial Two-Hybrid System The BacterioMatch Two-Hybrid system and human Hela cell cDNA library were obtained from Stratagene and two- hybrid assays were carried out according to Stratagene specifications. Details are in the Supplemental Data.

Acknowledgments

Generation of DNA Molecules Containing Damaged Bases Ten micrograms pGL3-CMV was incubated in 40 ␮l buffer containing 0.01 M sodium citrate and 0.1 M KCl (pH 5.0) at 70⬚C for 45 min or 6 hr. The heat and acid treated DNA was neutralized by addition of 10 ␮l Tris buffer (0.2 M, pH 7.8). For the in vitro BER assay, heat and acid treated pGL3-CMV served as the substrate DNA while undamaged pUC18 DNA served as a control.

In Vitro Protein Phosphatase Assays These methods are described in the Supplemental Data.

We thank Geir Slupphaug for the UNG2 antibody. We are grateful to Sankar Mitra, Tapas Hazra, and Lee Wiederhold for supplying expertise and reagents. We thank Andrew Rice for plasmids. We also thank Sal Caradonna and Dima Bulavin for helpful discussions. This work was supported by a grant from the National Cancer Institute (to L.A.D.). B.N. was supported by a Department of Defense Breast Cancer Research Program training grant. Received: December 12, 2003 Revised: May 24, 2004 Accepted: June 8, 2004 Published: August 26, 2004


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