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

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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).
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