Replication Protein A2 Phosphorylation After DNA Damage by the Coordinated Action of Ataxia Telangiectasia-Mutated and DNA-Dependent Protein Kinase 1

[CANCER RESEARCH 61, 8554–8563, December 1, 2001] Replication Protein A2 Phosphorylation after DNA Damage by the Coordinated Action of Ataxia Telangiectasia-Mutated and DNA-dependent Protein Kinase 1

Hongyan Wang, Jun Guan, Huichen Wang, Ange Ronel Perrault, Ya Wang, and George Iliakis2 Department of Radiation Oncology, Kimmel Cancer Center, Jefferson Medical College, Philadelphia, Pennsylvania 19107

ABSTRACT RPA2 becomes hyperphosphorylated after exposure to IR, UV, certain chemotherapeutic agents, or inhibitors of DNA replication, Replication protein A (RPA, also known as human single-stranded implicating RPA modification in the cellular responses to DNA dam- DNA-binding protein) is a trimeric, multifunctional protein complex in- age (9–17). Identification of the kinases that phosphorylate RPA2 volved in DNA replication, DNA repair, and recombination. Phosphoryl- ation of the RPA2 subunit is observed after exposure of cells to ionizing after DNA damage as well as throughout the cell cycle is important radiation (IR) and other DNA-damaging agents, which implicates the for an understanding of the functions and the putative regulatory modified protein in the regulation of DNA replication after DNA damage properties of the protein. Evidence exists that certain cdk-cyclin or in DNA repair. Although ataxia telangiectasia-mutated (ATM) and complexes mediate some of the modifications observed during the DNA-dependent protein kinase (DNA-PK) phosphorylate RPA2 in vitro, progression of cells through the cell cycle (6, 7, 18–22). Other kinases their role in vivo remains uncertain, and contradictory results have been have been implicated in the phosphorylation of RPA2 after DNA reported. Here we show that RPA2 phosphorylation is delayed in cells damage. deficient in one of these kinases and completely abolished in wild-type, A kinase with particularly high activity in phosphorylating RPA2 is ATM, or DNA-PK-deficient cells after treatment with wortmannin at a the DNA-PK (23, 24). Conformational changes occurring on binding concentration-inhibiting ATM and DNA-PK. Caffeine, an inhibitor of of RPA to ssDNA allow a more efficient phosphorylation of RPA2 by ATM and ATM-Rad3 related (ATR) but not DNA-PK, generates an ataxia-telangiectasia-like response in wild-type cells, prevents completely DNA-PK (1, 2). In vitro, DNA-PK has been purified as the principal RPA2 phosphorylation in DNA-PKcs deficient cells, but has no effect on kinase phosphorylating RPA2, and extracts of cells deficient in ataxia-telangiectasia cells. These observations rule out ATR and implicate DNA-PK do not phosphorylate RPA2 (25). RPA-DNA-PK complexes both ATM and DNA-PK in RPA2 phosphorylation after exposure to IR. are present in unstressed cells but are disrupted on treatment with UCN-01, an inhibitor of protein kinase C, Chk1, and cyclin-dependent camptothecin, an agent that is able to induce DNA DSBs (13, 26). kinases, has no effect on IR-induced RPA2 phosphorylation. Because Although these results point to a primary role for DNA-PK in RPA2 UCN-01 abrogates checkpoint responses, this observation dissociates phosphorylation, the situation in vivo appears more complex. Thus, RPA2 phosphorylation from checkpoint activation. Phosphorylated RPA irradiation still induces phosphorylation of RPA2 in cells deficient in has a higher affinity for nuclear structures than unphosphorylated RPA DNA-PK, although the extent and the kinetics of this phosphorylation suggesting functional alterations in the protein. In an in vitro assay for are altered (25, 27). These results suggest that a kinase other than DNA replication, DNA-PK is the sole kinase phosphorylating RPA2, indicating that processes not reproduced in the in vitro assay are required DNA-PK also phosphorylates RPA2 in vivo. for RPA2 phosphorylation by ATM. Because RPA2 phosphorylation ki- RPA2 has been shown to be a phosphorylation target for immuno- netics are distinct from those of the S phase checkpoint, we propose that precipitates specific for the ATM protein kinase (28), and RPA DNA-PK and ATM cooperate to phosphorylate RPA after DNA damage colocalizes with ATM on synapsed chromosome nodules in mouse to redirect the functions of the protein from DNA replication to DNA cells during the meiotic prophase (29, 30). Furthermore, the IR- repair. induced phosphorylation of RPA2 is delayed in ATM cells (9, 12, 14), indicating that either an ATM-mediated pathway or ATM kinase INTRODUCTION activity itself plays a role in RPA2 phosphorylation. Consistent with this notion, Mec1, a yeast ATM homologue, is responsible for RPA2 RPA3 (also known as human ssDNA binding protein) is a trimeric phosphorylation in irradiated Saccharomyces cerevisiae (11). Thus, it protein complex involved in many cellular processes including DNA is possible that both ATM and DNA-PK contribute to RPA2 phos- replication initiation and elongation, DNA repair, and recombination phorylation, but this possibility has not been studied in detail. (1, 2). Human RPA is a heterotrimer composed of M 70,000 (RPA1), r Here we report experiments designed to investigate the role of M 29,000 (RPA2), and M 14,000 (RPA3) subunits (3, 4). RPA2 is a r r DNA-PK and ATM in RPA2 phosphorylation after DNA damage. For phosphoprotein that becomes differentially phosphorylated through- this purpose we combined genetics with the use of kinase inhibitors. out the cell cycle. Phosphorylation of RPA2 is first observed at the The results indicate a role for both ATM and DNA-PK in the phos- G -S transition and is maintained through late mitosis (5, 6). In vitro, 1 phorylation of RPA2 and provide information relevant to the func- phosphorylation of RPA2 occurs during SV40 DNA replication, and tions of the protein. binding of RPA to ssDNA stimulates this modification (7, 8).

Received 6/12/01; accepted 9/28/01. MATERIALS AND METHODS The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with Cell Culture. HeLa cells were grown in Joklik’s modification of MEM 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by National Cancer Institute Grants 1RO1 CA56706, CA76203, and P30 containing 5% iron-supplemented bovine calf serum (Sigma Chemical Co.) CA56036 awarded from NIH and Department of Health and Human Services. and antibiotics. Cells were maintained in the logarithmic phase of growth by 2 To whom requests for reprints should be addressed, at Institute of Medical Radiation subculturing every 4 days at an initial concentration of 106 cells/100-mm tissue Biology, University of Essen, Hufenlandstrasse 55, D45122-Essen, Germany. Phone: culture dish. For experiments, 2 ϫ 106 cells were seeded in 100-mm dishes and 49-201-723-4153; Fax: 49-201-723-5966; E-mail: [email protected]. 3 The abbreviations used are: RPA, replication protein A; ATM, ataxia telangiectasia- allowed to grow for 3 days in a humidified incubator at 37°C, in an atmosphere mutated; DNA-PK, DNA-dependent protein kinase; PKc, protein kinase c; IR, ionizing of 5% CO2 and 95% air. radiation; ssDNA, single-stranded DNA; cdk, cyclin-dependent kinase; DSB, double M059-J cells (kindly provided by Dr. Joan Allalunis-Turner, University of strand break; PMSF, phenylmethylsulfonyl fluoride; CE, cytoplasmic extract; NE, nuclear Alberta, Edmonton, Alberta, Canada) were derived from a human malignant extract; PI3k, phosphatidylinositol 3Ј-kinase; HRR, homologous recombination repair; NHEJ, nonhomologous end-joining; DDT, dithiothreitol; CDK1, cyclin-␤ dependent glioma as described previously and found to be deficient in DNA-PKcs kinase 1; ATR, ATM-Rad3 related; TAg, SV40 large T antigen. (31–33). They were grown in DMEM supplemented with 10% fetal bovine 8554

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. RADIATION-INDUCED RPA2 PHOSPHORYLATION serum, 1% nonessential amino acids, and 1% L-glutamine, at 37°Cina and transferred to polyvinylidene difluoride membrane. Western blot analysis humidified incubator, in an atmosphere of 5% CO2 and 95% air. Cells were is performed using enhanced chemiluminescence according to the manufac- maintained in a phase of nearly logarithmic growth by subculturing every 4 turer (Amersham) and is visualized using the Storm (Molecular Dynamics). days at an initial concentration of 0.5 ϫ 106 cells/100-mm tissue culture dish. RPA antibody (p34–20) was generously provided by Dr. Gilbert Hurwitz, The same cells were also used to seed cultures for experiments at 0.5 ϫ 106 London Health Sciences Centre, Ontario, Canada. Quantitation of Western cells/100-mm dish, which were allowed to grow for 3 days. The growth blots is carried out using the ImageQuant software (Molecular Dynamics) and medium was changed the day before the experiment. At this point, cells is shown mainly for the purpose of facilitating comparison between the results reached a density of ϳ1.5 ϫ 106/dish and were irradiated to determine RPA2 obtained after different treatments or with different cell lines. phosphorylation. Typically, 6–7 ϫ 106 cells were collected per sample. In In Vitro DNA Replication Assay. The SV40-based in vitro DNA replica- some experiments we used as a control for M059-J cells M059J/Fus1 cells tion assay was described previously (37). Briefly, reaction mixtures (25 ␮l)

(kindly provided by Dr. Cordula U. Kirchgessner, University of Stamford, contain 40 mM HEPES (pH 7.5); 8 mM MgCl2; 0.5 mM DTT; 3 mM ATP; 200 Stamford, CA) grown under similar conditions. These cells have been derived ␮M each CTP, GTP, and UTP; 100 ␮M each of dATP, dGTP, and dTTP; 40 ␮M from M059-J by cell fusion with irradiated Scid/hu8 cells containing the dCTP; 40 mM creatine phosphate; 1.25 ␮g of creatine phosphokinase; 0.15 ␮g human chromosome 8 and retain a fragment of the human chromosome 8 of superhelical plasmid DNA; 100–200 ␮g of CE; and 0.5 ␮g of SV40 large containing the gene for DNA-PKcs (34). M059-J/Fus1 cells express DNA- T antigen (TAg). The reaction mixture was incubated at 37°Cfor1h. PKcs and show partial correction for the radiosensitive phenotype of M059-J Reactions were terminated by addition of 20 mM EDTA. TAg was prepared as cells. described earlier (37). DNA-PK was purified as described (38). AT5BIVA cells (kindly provided by Dr. Colin Arlett, Brunel University, Uxbridge, Middlesex, UK), generated by immortalizing AT5BI, were grown in MEM supplemented with 10% fetal bovine serum and antibiotics. Cells were RESULTS maintained by routinely subculturing every 4 days at an initial density of 0.5 ϫ 106 cells/100-mm dish. For experiments, cells were seeded at an initial Phosphorylation of RPA2 in Irradiated HeLa Cells. We inves- density of 0.5 ϫ 106 cells and were allowed to grow for 3 days at 37°Cina tigated IR-induced RPA2 phosphorylation in exponentially growing humidified incubator in an atmosphere of 5% CO2 and 95% air. The growth HeLa cells exposed to 50 Gy of X-rays. Levels and phosphorylation medium was changed the day before the experiment. All of the chemicals were status of RPA2 were separately analyzed in CEs and NEs, because in from Sigma Chemical Co. unless indicated otherwise. UCN-01 was obtained nonirradiated cells RPA is present in both fractions, and we were from the drug development branch of the National Cancer Institute. interested to investigate whether the fractionation pattern changes in Irradiation. Cells were irradiated using a Pantak X-ray machine operated irradiated cells. An additional rationale for this protocol was that the at 310 kV, 10 mA with a 2-mm Al filter (effective photon energy ϳ90 kV), at in vitro SV40 DNA replication assay, which we and others use to a distance of 50 cm and a dose rate of 2.7 Gy/min. Dosimetry was performed with a Victoreen dosimeter that was used to calibrate an in-field ionization study the regulation of DNA replication after DNA damage uses monitor. exclusively CE (39–41). Because in such studies RPA surfaces as a Extract Preparation. CEs or NEs were used for the analyses described in candidate regulatory factor (10, 15, 42, 43), it was important to this work as indicated in the individual experiments. The characterization of examine its fractionation characteristics after DNA damage. the extract as cytoplasmic or nuclear is based on the procedure of preparation. Extracts were fractionated by SDS-PAGE and RPA2 detected by It does not reflect in a strict sense the localization of proteins in the functioning Western blotting. Fig. 1A shows the results obtained. In NEs of cell. The procedures used for CE preparation have been described in detail (35, nonirradiated cells there is a main band at 0 and 24 h and faint bands 36) and are presented here only briefly. Cells were trypsinized and collected by of slower mobility reflecting phosphorylated RPA2 probably deriving centrifugation, washed in PBS and hypotonic buffer, and resuspended for 15 from cells late in the cell cycle (6, 44). Exposure to IR causes strong min in three packed cell volumes of hypotonic buffer solution containing 10 phosphorylation of RPA2, which in NEs becomes clearly visible at 1 h mM HEPES (pH 7.8) at 4°C, 1.5 mM MgCl 5mM KCl, 0.2 mM PMSF, and 2, and peaks at 4 h after irradiation. Phosphorylation is gradually re- 0.5 mM DTT. Cells were disrupted by three cycles of freeze (Ϫ80°C) and thaw (37°C). Subsequently, 0.11 volumes of high salt buffer containing 10 mM duced at later times and approaches background 24 h after irradiation HEPES (pH 7.8) at 4°C, 500 mM KCl (unless indicated otherwise), and 1.5 mM (Fig. 1, A and E). A quantitative description of the kinetics of RPA2 ϫ MgCl2 was added and the homogenate was spun at 3,300 g for 20 min to phosphorylation is depicted in Fig. 1E. Plotted is the mean and SE of precipitate the nuclei. The nuclear pellet is used for the preparation of NE (see the relative phosphorylation calculated by image analysis using results below), whereas the supernatant, which is operationally defined as the CE, is from five independent experiments. It is thought that this transient collected and spun once again at 14,000 ϫ g for 5 min. When indicated, the phosphorylation of RPA2 reflects a response of irradiated cells to resulting CE is dialyzed overnight in 25 mM Tris-HCl (pH 7.5) at 4°C, 10% DNA damage. glycerol, 20% sucrose, 50 mM NaCl, 1 mM EDTA, 0.5 mM DTT, and 0.2 mM Although phosphorylation of RPA2 is clearly observed after irra- ϫ PMSF and centrifuged at 14,000 g for 5 min to remove precipitated protein. diation in CE as well (Fig. 1A), the level is low when compared with Protein concentration is determined using the Bradford assay (Bio-Rad), and the corresponding NE. Thus, phosphorylated RPA2 fractionates pref- aliquots are snap-frozen and stored at Ϫ80°C. To prepare NE, the nuclear pellet obtained after cell disruption and addition erentially in the nuclear fraction. Similar to observations with NEs, of salt is washed with hypotonic buffer containing 50 mM KCl and is resus- phosphorylation approaches background levels 24 h after irradiation pended in equal volume (packed nuclear volume) of low salt buffer [20 mM (Fig. 1A). This reduction in the levels of phosphorylated RPA2 cannot

HEPES (pH 7.9) at 4°C, 1.5 mM MgCl2,20mM KCl, 0.2 mM EDTA, 0.2 mM be attributed to selective loss of highly damaged cells, because cell PMSF, and 0.5 mM DTT] containing 800 mM KCl to achieve a final KCl detachment at this time is rather limited. concentration of 400 mM. Nuclei are carefully resuspended and protein ex- Extracts analyzed in the above-described experiment were dialyzed tracted for 30 min at 4°C under gentle shaking. After this incubation, extracted in the absence of phosphatase inhibitors before analysis. A compari- ϫ nuclei are spun for 15 min at 14,000 g, the supernatant collected, spun, son between these results and those obtained with nondialyzed sam- Ϫ snap-frozen, and stored in small aliquots at 80°C. Where indicated, the NE ples (last four lanes in Fig. 1A) indicates that the phosphorylation is dialyzed for 10 min in low salt buffer containing 20 mM HEPES (pH 7.9) at levels remain unaffected by dialysis. This is relevant for SV40 in vitro 4°C, 20% glycerol, 20% sucrose, 100 mM KCl, 0.2 mM EDTA, 0.2 mM PMSF, DNA replication experiments where extract dialysis is carried out and 0.5 mM DTT. Western Blot Analysis. Protein (10 ␮g) from CE and 5 ␮g from NE are before assembling reactions. Results shown below were obtained resuspended in 2 ϫ SDS loading buffer [50 mM Tris-HCl (pH 6.8), 2% using nondialyzed NEs or CEs unless stated otherwise. ␤-mercaptoethanol, 2% SDS, 0.2% bromphenol blue, and 10% glycerol]. Cell The preferential fractionation of phosphorylated RPA2 in the nu- lysate is boiled for 5 min. Proteins are separated by SDS-PAGE on a 12% gel clear fraction suggested a tight association with chromatin or the 8555

Fig. 1. RPA2 phosphorylation after exposure of HeLa cells to IR. A, logarithmically growing cultures of HeLa cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C. NEs and CEs were prepared at various times thereafter and analyzed for RPA2 phosphorylation by Western blotting before or after dialysis. B, logarithmically growing HeLa cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C for 4 h before processing for extract preparation. Different amounts of KCl were added after homogenization and before the final extraction with 400 mM KCl. RPA2 was analyzed by Western blotting. CE, material obtained in first step of fractionation. C, logarithmically growing HeLa cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C for 4 h before processing for extract preparation. Cells were homogenized and protein extracted with 0.05, 0.4, and 2 M KCl. RPA2 was analyzed by Western blotting. D, logarithmically growing HeLa cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C for different periods of time as indicated before processing for extract preparation. In one set 20 ␮M wortmannin was added 1 h before irradiation and was allowed to act for the entire period of incubation. In a second set of samples (repeat)20␮M wortmannin was added 1 h before, as well as 3 h and 7 h after irradiation. Arrows, approximate times of wortmannin addition. RPA2 was analyzed by Western blotting. E, quantitative analysis of RPA2 phosphorylation in NEs. Percentage of phosphorylated RPA2 as a function of time was calculated by analyzing Western blots using the Storm (Molecular Dynamics). Mean is plotted from 3 to 5 experiments; bars, Ϯ SE. For the calculation, material above the unphosphorylated band was included in the phosphorylated component (72). Some variation in levels of phosphorylation was observed in different experiments, which is re- flected in the size of the error bars.

nuclear matrix. To investigate this phenomenon in greater detail, we not shown), PI3k can be ruled out as a candidate RPA2 kinase. Fig. prepared extracts by gradually increasing the concentration of KCl 1D shows results obtained after treatment of HeLa cells with 20 ␮M during the step where the cytoplasmic fraction is generated. Fig. 1B of wortmannin given 1 h before irradiation together with the corre- shows the results obtained. An increase in KCl concentration from 0 sponding samples of untreated cells. Wortmannin reduces RPA2 to 280 mM increases the amount of phosphorylated RPA2 in the phosphorylation in NEs to background levels for Յ2 h after irradia- cytoplasmic fraction. These results suggest that phosphorylated RPA tion. However, at later times RPA2 phosphorylation resumes and associates tightly with nuclear structures and can be extracted only by reaches a maximum 6 h after irradiation. high salt concentrations. Because wortmannin is unstable in aqueous solution, we reasoned To examine in greater detail the association of phosphorylated RPA that the phosphorylation observed after 2 h may coincide with drug with nuclear structures, we evaluated the level of RPA by Western inactivation and the generation of new pools of wortmannin-sensitive blotting after extraction at 0.05, 0.4, and 2 M of KCl 4 h after exposure kinases. To investigate this possibility we administered 20 ␮M of to IR. The results in Fig. 1C indicate that the majority of phospho- wortmannin in three repeats given 1 h before as well as3hand7h rylated RPA2 is released from the nucleus at 0.4 M and that tightly after irradiation (indicated by the arrows in the lower gel of Fig. 1D). bound RPA2 tends to be in a phosphorylated form after IR. This holds It is evident that repeated treatment with wortmannin reduces to true even for the very small residual fraction remaining on chromatin undetectable levels IR-induced RPA2 phosphorylation suggesting that (“Resid.” in Fig. 1C) after extraction with 2 M KCl. This finding is in RPA2 phosphorylation depends, directly or indirectly, on wortman- line with earlier observations (45) suggesting that phosphorylated nin-sensitive kinases other than PI3k. Because wortmannin binds RPA2 binds preferentially to chromatin in nonirradiated cells. In the covalently to the ATP binding site and inactivates irreversibly target experiments described below NEs were prepared after release of the kinases, the return of phosphorylation 4 h after a single application cytoplasmic component at 50 mM KCl. indicates not only the resynthesis of the target kinases but also the To obtain information on the kinases responsible for RPA2 phos- persistence of the activating signal. The activating signal seems to phorylation after IR, we treated cells with wortmannin. Wortmannin decay after ϳ10 h, because only weak phosphorylation is observed at inhibits the PI3k at nanomolar concentrations and other kinases of the 24 h in HeLa cells treated with wortmannin 1 h before as well as 3 h PI3 family of kinases such as DNA-PK, ATM, and ATR at 1000-fold and 7 h after irradiation (Fig. 1D). higher concentrations (46–50). Because concentrations of wortman- From the pool of wortmannin-sensitive kinases, DNA-PK, ATM, nin Յ2 ␮M did not affect IR-induced RPA2 phosphorylation (results and to a lesser degree ATR, are inhibited at the drug concentrations 8556

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. RADIATION-INDUCED RPA2 PHOSPHORYLATION used (50). To obtain additional insight into the actual role of these RPA2 is observed in nonirradiated cells. In NEs, exposure to 50 Gy kinases in RPA2 phosphorylation, we examined other inhibitors with X-rays induces phosphorylation that is detectable 4 h after irradiation. a different spectrum of specificity. Caffeine has been reported recently The levels of phosphorylation progressively increase at later times, to inhibit ATM and ATR but not DNA-PK (51–54). Therefore, we and there is no evidence for decay in phosphorylation even in samples examined the effect of 4 mM of caffeine on RPA2 phosphorylation. analyzed 24 h after irradiation. On the other hand, results obtained The results obtained using NEs are shown in Fig. 2A together with a with M059-J/Fus1 cells, generated from M059-J by cell fusion to set of parallel controls from untreated cells. A quantitative analysis of express DNA-PK (see “Materials and Methods”), show RPA2 phos- the results is shown in Fig. 2C. Caffeine delays and reduces the phorylation patterns similar to those of HeLa cells. overall levels of RPA2 phosphorylation implying a role for ATM or Compared with the results of M059-J/Fus1 cells and those shown in ATR in this response. However, clear phosphorylation of RPA2 is still Fig. 1 for HeLa cells, the results with M059-J cells suggest that observed suggesting the operation of a caffeine-resistant but wort- DNA-PK deficiency causes a delay in RPA2 phosphorylation. Al- mannin-sensitive kinase such as DNA-PK. These results and those though a reduction in the overall levels of phosphorylation is also shown above suggest that more than one kinase is responsible for occasionally observed, the quantitation shown in Fig. 1E (includes the RPA2 phosphorylation. results from four experiments with M059-J cells) suggests that these Although the above results implicate ATM and ATR in RPA2 differences are not statistically significant. The response in the CE is phosphorylation, it is not clear whether the effect is direct or indirect. qualitatively similar (Fig. 3A), but as with experiments using HeLa Chk1 is a kinase operating downstream of ATR (55, 56) and is a cells, the vast majority of the phosphorylated RPA2 fractionates in the candidate kinase for the ATR-dependent component of RPA2 phos- NE. It is notable that in M059-J cells the abundance of phosphorylated phorylation. Therefore, we examined the effect of 8-hydroxystauros- RPA2 in the cytoplasmic component is lower than in HeLa cells (see porine (UCN-01), an inhibitor of Chk1 (57), on RPA2 phosphoryla- Fig. 1), suggesting a leaky nuclear envelope in the latter cell line, as tion. The results in Fig. 2, B and D, show no measurable changes in suggested by other experiments as well (62). IR-induced RPA2 phosphorylation after treatment of cells with 1 ␮M RPA2 phosphorylation is only modestly reduced after a single of UCN-01 and suggest that PKc, Chk1, and UCN-01-sensitive cdks application of 20 ␮M of wortmannin in M059-J cells (Fig. 3A). (58–60) are not the primary RPA2 kinases after DNA damage. However, three repeat-applications of 20 ␮M of wortmannin given RPA2 Phosphorylation in Cells Deficient in DNA-PK. The according to the protocol outlined in Fig. 1D additionally reduce above results in aggregate point to ATM, ATR, and DNA-PK as RPA2 phosphorylation to nearly undetectable levels (Fig. 3B). Thus, candidate RPA2 kinases. For additional insight regarding the potential in line with previous observations (25) and despite the absence of role and the interplay between these kinases in RPA2 phosphorylation active DNA-PK in M059-J cells, RPA2 phosphorylation is clearly we studied M059-J cells. These cells are radiosensitive to killing by observed. IR and have no detectable DNA-PK activity (33, 61). The results We probed whether the wortmannin-sensitive kinase operating in obtained are shown in Fig. 3A. It is notable that no phosphorylation of M059-J cells is ATM or ATR by treating cells with 4 mM of caffeine.

Fig. 2. Effects of caffeine and UCN-01 on RPA2 phosphorylation. A, logarithmically growing cultures of HeLa cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C in the presence or absence of 4mM caffeine. NEs were prepared at various times thereafter and analyzed for RPA2 phosphorylation by Western blotting. B, same as A but for cells incubated in the presence or absence of 1 ␮M UCN-01. The drug was given to cells 30 min before irradiation. C, quantitative analysis of results in A. D, quantitative analysis of results in B. In quantitation, background phosphorylation observed in nonirradiated cells has been subtracted.

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Fig. 3. RPA2 phosphorylation in cells deficient in DNA-PK. A, logarithmically growing cultures of M059-J cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C. In one set of samples 20 ␮M wortmannin was added 1 h before irradiation and left to act for the remainder of the incubation period. NEs and CEs were prepared at various times thereafter and analyzed for RPA2 phosphorylation by Western blotting. As control, results of M059-J/Fus1 cells are shown. These cells have been derived from M059-J and express DNA-PKcs. B, same as A for NE but for cells treated with 4 mM caffeine or three repeat-applications of 20 ␮M wortmannin.

The results obtained are shown in Fig. 3B together with the corre- is without effect on RPA2 phosphorylation (Fig. 5, B and C). This sponding controls of cells incubated in the absence of caffeine. Al- result suggests that the target of caffeine in this type of experiment is though the overall levels of RPA2 phosphorylation are lower in this ATM and renders unlikely a contribution by ATR and other caffeine- experiment, it is evident that caffeine completely prevents RPA2 sensitive kinases. ATR remains active in AT cells. This result is also phosphorylation. in line with the lack of RPA2 phosphorylation in vitro by ATR (54) In contrast to the strong effect of caffeine on IR-induced RPA2 and the observation that inhibition of Chk1, a downstream target of phosphorylation, UCN-01 is without effect (Fig. 4), suggesting that ATR (55), has no effect on RPA2 phosphorylation (see below). The the family of kinases targeted by this inhibitor is not contributing above results suggest that a wortmannin-sensitive but caffeine-resis- measurably to RPA2 phosphorylation in M059-J cells. This is indi- tant kinase phosphorylates RPA2 in AT cells. From the candidate cated by the results of the gels shown in Fig. 4A but also by the kinases DNA-PK satisfies these requirements. quantitative analysis in Fig. 4B. Fig. 6 shows the effect of UCN-01 on RPA2 phosphorylation in AT Thus, a wortmannin- and caffeine-sensitive kinase phosphorylates cells together with results of parallel samples of untreated cells. It is RPA2 in M059-J cells in a somewhat delayed but persistent manner. Kinases that satisfy these properties include ATM and ATR. RPA2 Phosphorylation in ATM-deficient Cells. The above results prompted us to examine the role of ATM in RPA2 phosphorylation. AT cells were exposed to 50 Gy X-rays and RPA2 analyzed in CEs and NEs at different times thereafter. Fig. 5, A and B, show the results obtained with CEs and NEs, respectively. A quantitative analysis of selected results is given in Fig. 5C as well as in Fig. 1E.As reported previously by us and others (9, 14, 16), ATM defects lead to delayed phosphorylation of RPA2 compared with normal cells. When results from four experiments are compiled, the kinetics shown in Fig. 1E are obtained indicating that ATM deficiency results in RPA2 phosphorylation kinetics comparable with that seen in cells deficient in DNA-PK. However, in contrast with the results with DNA-PK- deficient cells, background RPA2 phosphorylation is in AT cells at levels similar to those of wild-type cells. RPA2 phosphorylation is significantly stronger in NE (Fig. 5B) than in CE (Fig. 5A) indicating the rather strong association of phosphorylated RPA with nuclear structures in AT cells as well. It is notable that here again RPA2 phosphorylation does not show signs of decay and persists even 24 h after irradiation. This trend is maintained even when the dose of radiation is reduced to 20 Gy (lowest gel in Fig. 5B). Repeat-treatment with 20 ␮M of wortmannin reduces RPA2 phosphorylation to undetectable levels up to 12 h after irradiation. The phosphorylation observed at 24 h probably reflects drug degradation Fig. 4. Effect of UCN-01 on RPA2 phosphorylation in cells deficient in DNA-PK. A, and partial regeneration of the wortmannin-sensitive kinases, and same as in Fig. 3B but for cells treated with 1 ␮M UCN-01. Drug was given to the cells 30 min before irradiation. B, quantitative analysis of the results in A shown after indicates the persistence of the activating signal in AT cells as subtraction of the background of phosphorylated RPA2 measured in nonirradiated cells. compared with HeLa cells. Interestingly, in AT cells caffeine at 4 mM Note, however, that this background was very low in M059-J cells (see Fig. 1E). 8558

cubation with 1 mM of caffeine or 1 ␮M of UCN-01 has no detectable effect on RPA2 phosphorylation (Fig. 7, Lanes 4 and 5) suggesting that, under these in vitro conditions, inhibition of ATM and possibly ATR, as well as Chk1 and possibly other kinases, leaves unchanged RPA2 phosphorylation. Low but detectable levels of ATM are present in the extract (results not shown). There is no phosphorylation in reactions assembled with extracts of M059-J cells under any of the conditions examined, suggesting that phosphorylation observed in HeLa cells relies on DNA-PK. This is additionally confirmed by the observation that addition of purified DNA-PK restores RPA2 phosphorylation (Fig. 7, Lanes 10 and 11). However, extracts of M059-J cells effectively support SV40 DNA replication (Ref. 15; results not shown) confirming that RPA2 phosphorylation is not required for DNA replication (8). In AT cells RPA2 phosphorylation is sensitive to wortmannin, and, similar to results obtained with HeLa cells, incubation with caffeine or UCN-01 has no effect on the level of RPA2 phosphorylation. To mimic the in vivo DNA damage conditions in this in vitro assay, we assembled reactions in which we included 0.1 ␮g of blunt-end DNA obtained by digesting pUC18 with HaeIII. The presence of DNA in the reactions did not qualitatively alter RPA2 phosphorylation in any of the extracts tested. In summary, the results of the in vitro studies identify DNA-PK as the kinase-phosphorylating RPA2. DNA-PK functions in this system even in the absence of double- stranded DNA. Dependence of RPA2 phosphorylation from the ATM kinase could not be demonstrated under the in vitro conditions used.

DISCUSSION ATM and DNA-PK as RPA2 Kinases. The results presented above suggest that RPA2 phosphorylation is mediated through the direct or indirect action of two members of the PI3 family of kinases,

Fig. 5. RPA2 phosphorylation in ATM-deficient cells. A, logarithmically growing cultures of AT5BIVA cells were exposed to 50 Gy X-rays or left unirradiated and returned to 37°C. One set was left untreated, one was incubated with 4 mM caffeine, and one with 20 ␮M wortmannin. CEs were prepared at various times thereafter and analyzed for RPA2 phosphorylation by Western blotting. B, same as in A but for NEs. Results shown here for wortmannin were obtained after repeat application of the drug 1 h before and3hand7h after irradiation. Shown are also results of an experiment in which cells were exposed to 20 Gy X-rays. C, quantitative analysis of the results obtained with nontreated and caffeine or wortmannin treated cells in B after subtraction of the background RPA2 phosphorylation measured in nonirradiated cells. evident that UCN-01 does not inhibit RPA2 phosphorylation. Quan- titative analysis of the results in Fig. 6A indicates (Fig. 6B) that in the presence of UCN-01, RPA2 phosphorylation accelerates and reaches kinetics similar to those measured in wild-type cells (compare with the results shown in Fig. 1E). However, the overall level of phosphorylation remains unaffected. Phosphorylation of RPA2 in Vitro. The above results in aggregate point to ATM and DNA-PK as the RPA2 kinases in vivo and indicate that deficiency in anyone of them leads to delayed phosphorylation that persists longer than in wild-type cells. We were interested to investigate whether the suggested contribution of these two kinases in RPA2 phosphorylation can be reproduced under in vitro DNA replication conditions. Extracts prepared from nonirradiated cells were used to assemble SV40-based DNA replication reactions and the phosphorylation status of RPA2 examined after1hofincubation. Fig. 7 shows the results obtained. Low levels of RPA2 phosphorylation are observed in the absence of TAg in extracts of HeLa cells (Fig. 7, Lane Fig. 6. Effect of UCN-01 on RPA2 phosphorylation in ATM-deficient cells. A, logarithmically growing cultures of AT5BIVA cells were exposed to 50 Gy X-rays or left 1). Phosphorylation increases significantly in complete reactions (Fig. unirradiated and returned to 37°C in the presence or absence of 1 ␮M UCN-01. The drug 7, Lane 2), suggesting the activation of an RPA2 kinase by ongoing was given to the cells 30 min before irradiation. NEs were prepared at various times ␮ thereafter and analyzed for RPA2 phosphorylation by Western blotting. B, quantitative DNA replication. This kinase is sensitive to 2 M of wortmannin as analysis of the results in A after subtraction of the background RPA2 phosphorylation preincubation abolishes RPA2 phosphorylation (Fig. 7, Lane 3). In- measured in nonirradiated cells. 8559

ever, expression in AT cells of ATM fragments of the kinase domain of the protein do not correct the RPA2 phosphorylation defect, suggesting that functions in addition to kinase activity are required for this end point (12). Such functions may be related to protein-protein or protein-DNA interactions that determine ATM localization and function in the intact cell. Similar functional requirements may lead to the ATM-independent RPA2 phosphorylation under in vitro DNA replication conditions, either in the presence or absence of DNA fragments (Fig. 7), but the possibility that low levels of ATM in the extract prevented manifestation of the effect should be left open. It may be relevant for these observations that ATM associates with chromatin (28), and that the same holds true for a fraction of the cellular RPA (Fig. 1; Ref. 45). Thus, compartmentalization of these proteins may play an important role in their activity and function in the cell. However, it is also possible that ATM-induced RPA2 phosphorylation is indirect and actually mediated by a kinase interacting with Fig. 7. Phosphorylation of RPA2 under in vitro SV40 DNA replication conditions. ATM in vivo but not in vitro. Reactions were assembled using extracts of HeLa, AT5BIVA, and M059-J cells in the presence or absence of TAg, Caffeine, wortmannin, UCN-01, and HaeIII digested (blunt) Although the persistent phosphorylation of RPA2 after exposure to DNA as indicated and incubated at 37°C for 1 h. RPA2 phosphorylation in the completed IR of cells deficient in DNA-PK could be attributed to the persistence reaction was measured by Western blotting. The indicated amounts of purified DNA-PK of unrejoined DNA DSBs in these cells (61), such an interpretation is were added in reactions assembled with extracts of M059-J cells (Lanes 10 and 11). not tenable for AT cells, because in these cells, rejoining of DNA DSBs follows nearly normal kinetics (67, 68). Thus, if the presence of DNA-PK and ATM. The lack of caffeine effect on AT cells suggests phosphorylated RPA2 is the result of persistent signaling from DNA that ATR is not contributing to a measurable extent to RPA2 phos- damage, the signal is unlikely to originate from the initially induced phorylation. Also, the general inability of UCN-01 to modulate IR- DNA DSBs (69, 70). Cells deficient in DNA-PK also show persistent induced RPA2 phosphorylation in all of the cell lines examined here inhibition of DNA replication after exposure to IR, but the relevance, renders unlikely that Chk1 or other kinases targeted by this agent if any, of this observation to the persistence in RPA2 phosphorylation contribute significantly. Because UCN-01 is able to abrogate IR- remains unclear (71). induced checkpoints including the DNA replication checkpoint (63), Characteristics of RPA2 Phosphorylation. RPA2 can be phos- the results presented here dissociate RPA phosphorylation from phorylated at several sites localized in the extreme NH2-terminal checkpoint activation, in line with earlier observations (12). It is region of the protein. Two consensus cyclin-cdk sites are present at notable that a defect in either ATM or DNA-PK leads to a delay in Ser-23 and Ser-29 (6), and mutation of these residues to alanine reaching the maximum level of RPA2 phosphorylation. On the basis greatly reduces RPA2 phosphorylation in mouse cells (6), in crude of this observation we propose that IR-induced RPA2 phosphorylation extracts of human cells (18), or of recombinant RPA by cyclin is mediated by the coordinated action of ATM and DNA-PK and that B-dependent kinase 1 (cdk1) (20, 22). both activities are required for rapid phosphorylation. The mechanism For DNA-PK, the primary phosphorylation site has been localized underlying the putative coordinated action of ATM and DNA-PK to Thr-21 and Ser-33 (22, 72), but DNA-PK is likely to also phos- cannot be anticipated on the basis of the results presented here. phorylate other sites in this region of the protein (72). As could be However, it is possible that the delayed phosphorylation observed predicted, mutation of Ser-23 and Ser-29 does not affect RPA2 after IR in cells defective in either kinase reflects a dampening of the phosphorylation by DNA-PK (18, 20), suggesting that cell cycle and DNA damage-sensing mechanisms and/or a defect in the pathway that DNA damage-mediated phosphorylation probably serves different removes such damage. Cooperation between ATM and DNA-PK is functions. Phosphopeptide maps of RPA2 phosphorylated in vivo and also inferred by the fact that cells lacking DNA-PKcs have low ATM in vitro either with cyclin-cdk or with DNA-PK are identical, verify- kinase levels (64) as well as by the observation that DNA ends ing that the sites determined from in vitro phosphorylation studies are activate both kinases (24, 65, 66). the same as those modified in vivo (6, 72). We assume that RPA2 The effects noted here with DNA-PKcs-deficient cells are in line phosphorylation as observed here reflects modification of Thr-21 and with the very low level of phosphorylation observed 2 h after expo- Ser-33, but direct evidence is presently lacking. It is additionally sure of scid cells to doses in the range between 0 and 20 Gy (27). They assumed that the sites of RPA2 phosphorylation by DNA-PK and are also in line with results obtained with M059-J cells (25) demon- ATM are identical (28). Direct evidence for this has been presented strating a delay in RPA2 phosphorylation and a persistence of this recently (16). effect up to 6 h after irradiation, the last time point included in the The Functional Significance of RPA2 Phosphorylation. The latter report. Differences in RPA2 phosphorylation observed in DNA- role of RPA2 phosphorylation in the functions of the holoenzyme, PKcs-deficient cells in the above reports may partly reflect differences particularly after DNA damage, remains unclear. Some reports are in in the sampling times. line with the hypothesis that phosphorylation affects the ability of A delay in RPA2 phosphorylation was noted earlier in lymphoblastoid RPA to support DNA replication. Thus, extracts of human cells cells derived from AT patients (9) and was confirmed in a wide range of exposed to IR or UV, treatments that lead to RPA2 hyperphosphory- fibroblasts homozygous for the ATM mutation (14); a smaller effect was lation, are deficient in supporting SV40 DNA replication in vitro but also observed in fibroblasts heterozygous for the ATM mutation (14). In can be restored to the activity of nontreated cell extracts by the the latter study, persistence of RPA2 phosphorylation was seen in AT addition of purified RPA (10, 15). Furthermore, a mutant recombinant cells exposed to IR. It is interesting that an AT-like phenotype in terms of RPA lacking the NH2-terminal 33 residues of RPA2 is inactive in RPA2 phosphorylation is also generated when wild-type cells are treated DNA replication in the presence of DNA-PK (73). More recent with caffeine (Fig. 1), an inhibitor of ATM (52–54). Furthermore, ex- studies indicate that RPA phosphorylation by DNA-PK or mutation of pression of ATM dominant-negative fragments in normal cells causes a the RPA2 phosphorylation sites to aspartic acid reduces RPA-TAg delay in IR-induced phosphorylation of RPA2 (12). Interestingly, how- complex formation and prevents RPA from physically interacting and 8560

Downloaded from cancerres.aacrjournals.org on September 26, 2021. © 2001 American Association for Cancer Research. RADIATION-INDUCED RPA2 PHOSPHORYLATION stimulating the activity of DNA polymerase ␣-DNA primase.4 Similar stration that phosphorylated RPA functions in HRR will define RPA2 effects involving the functional homologues of TAg and pol ␣- as a marker for this process. primase in vivo may facilitate a redirection of the RPA activities from Is the Phosphorylation Direct or Indirect? There is evidence in DNA replication to DNA repair. the literature supporting the notion that both ATM and DNA-PK can However, other studies question the functional significance of function as RPA2 kinases (1, 2). The results presented here are also in RPA2 phosphorylation. Thus, no significant differences are detected line with a direct phosphorylation by both kinases. However, recent between the activities of wild-type phosphorylated and nonphospho- results for direct phosphorylation in vivo, particularly by ATM, must rylated RPA in SV40 DNA replication (8, 74, 75), and deletion of the be considered preliminary at present. Whereas direct phosphorylation

NH2-terminal RPA2 residues 2–30 prevents RPA2 phosphorylation is certainly possible, it is also possible that it is initiated by repair but has no effect on the ability of RPA to support in vitro SV40 DNA processes and carried out by other kinases with sensitivities to caf- replication (18, 73). Although the origin of these discrepancies re- feine, wortmannin, and UCN-01, as shown here. mains unknown, it may well be that they derive from protein functions and interactions taking place in vivo but which are only partially preserved and reproduced in vitro. Indeed, we were unable to repro- ACKNOWLEDGMENTS duce the ATM-dependent RPA2 phosphorylation under in vitro SV40 We thank Drs. J. Allalunis-Turner and C. Arlett for cells, and Dr. G. DNA replication conditions (Fig. 7). Hurwitz for antibodies. We also thank Nancy Mott for secretarial help and for What is the role of RPA2 phosphorylation in DNA metabolism? editing the manuscript. The results presented here as well as those reported previously (12) dissociate RPA2 phosphorylation from checkpoint activation. Neither the kinetics of RPA2 phosphorylation nor the effects of compounds REFERENCES abrogating checkpoint response (see above) are compatible with such 1. Wold, M. S. Replication protein, A. A heterotrimeric, single-stranded DNA-binding a notion. The fact that RPA2 phosphorylation occurs even in cells protein required for eukaryotic DNA metabolism. Annu. Rev. Biochem., 66: 61–92, with defective checkpoints, such as AT, suggests that it is a late step 1997. 2. Iftode, C., Daniely, Y., and Borowiec, J. A. Replication protein A (RPA): the in the cellular responses to DNA damage. eukaryotic SSB. Crit. Rev. Biochem. Mol. Biol., 34: 141–180, 1999. An attractive hypothesis is that phosphorylation helps to redirect 3. Fairman, M. P., and Stillman, B. Cellular factors required for multiple stages of SV40 DNA replication in vitro. 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Hongyan Wang, Jun Guan, Huichen Wang, et al.

Cancer Res 2001;61:8554-8563.

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