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• W2029736072 abstract "ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser345 phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser345 phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G2 arrest and Cdk1-Tyr15 phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic β-isoform PP2A(Cβ) and the A regulatory α-isoform PP2A(Aα) are involved in the G2 induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr15 and Chk1-Ser345. In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser345 phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated γH2AX-Ser139 phosphorylation and foci formation, but down-regulation of PP2A(Aα) or PP2A(Cβ) did not decrease γH2AX-Ser139 phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aα/Cβ)-mediated G2 arrest and Chk1-Ser345 phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cβ) or PP2A(Aα) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aα/Cβ) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr. ATM and Rad3-related (ATR) is a regulatory kinase that, when activated by hydroxyurea, UV, or human immunodeficiency virus-1 Vpr, causes cell cycle arrest through Chk1-Ser345 phosphorylation. We demonstrate here that of these three agents only Vpr requires protein phosphatase type 2A (PP2A) to activate ATR for Chk1-Ser345 phosphorylation. A requirement for PP2A by Vpr was first shown with the PP2A-specific inhibitor okadaic acid, which reduced Vpr-induced G2 arrest and Cdk1-Tyr15 phosphorylation. Using small interference RNA to down-regulate specific subunits of PP2A indicated that the catalytic β-isoform PP2A(Cβ) and the A regulatory α-isoform PP2A(Aα) are involved in the G2 induction, and these downregulations decreased the Vpr-induced, ATR-dependent phosphorylations of Cdk1-Tyr15 and Chk1-Ser345. In contrast, the same down-regulations had no effect on hydroxyurea- or UV-activated ATR-dependent Chk1-Ser345 phosphorylation. Vpr and hydroxyurea/UV all induce ATR-mediated γH2AX-Ser139 phosphorylation and foci formation, but down-regulation of PP2A(Aα) or PP2A(Cβ) did not decrease γH2AX-Ser139 phosphorylation by any of these agents or foci formation by Vpr. Conversely, H2AX down-regulation had little effect on PP2A(Aα/Cβ)-mediated G2 arrest and Chk1-Ser345 phosphorylation by Vpr. The expression of vpr increases the amount and phosphorylation of Claspin, an activator of Chk1 phosphorylation. Down-regulation of either PP2A(Cβ) or PP2A(Aα) had little effect on Claspin phosphorylation, but the amount of Claspin was reduced. Claspin may then be one of the phosphoproteins through which PP2A(Aα/Cβ) affects Chk1 phosphorylation when ATR is activated by human immunodeficiency virus-1 Vpr. To ensure accurate transmission of genetic information, eukaryotic cells have developed an elaborate network of checkpoints to monitor the successful completion of every cell cycle step and to respond to cellular abnormalities such as DNA damage and replication inhibition as they arise during cell proliferation. The ATR kinase, a member of the phosphoinositide-3-kinase-related kinase family (1Shechter D. Costanzo V. Gautier J. DNA Repair. 2004; 3: 901-908Crossref PubMed Scopus (167) Google Scholar), is a central regulator for the replication checkpoint that is induced by treatment with DNA replication inhibitor hydroxyurea (HU). 2The abbreviations used are: HU, hydroxyurea; PP2A, protein phosphatase type 2A; OA, okadaic acid; siRNA, specific small interference RNA; Adv, adenovirus vector; NOC, nocodazole; PBS, phosphate-buffered saline; RT, reverse transcription; p.i., post-infection; p.t., post-transfection; Cdk, cyclindependent kinase; ATR, ATM and Rad3-related; RPA, replication protein A. When ATR is activated, it initiates a regulatory cascade of phosphorylation events. One of the intermediate steps in this cascade is the activating phosphorylation of the Chk1 effector kinase (2Zhao H. Piwnica-Worms H. Mol. Cell. Biol. 2001; 21: 4129-4139Crossref PubMed Scopus (866) Google Scholar). This activated Chk1 in turn phosphorylates and inactivates the Cdc25 phosphatases. Cdc25 phosphatases control the activity of cyclin-dependent kinases by removing the inhibitory phosphates on Tyr15 and Thr14 (1Shechter D. Costanzo V. Gautier J. DNA Repair. 2004; 3: 901-908Crossref PubMed Scopus (167) Google Scholar). Specifically for the G2 to M transition, which is controlled by activation of Cdk1, activation of ATR leads to inactivation of the Cdc25 phosphatases, in particular Cdc25C (3Sanchez Y. Wong C. Thoma R.S. Richman R. Wu Z. Piwnica-Worms H. Elledge S.J. Science. 1997; 277: 1497-1501Crossref PubMed Scopus (1124) Google Scholar), ultimately resulting in the persistence of the phosphorylated Tyr15 and Thr14 on Cdk1, which causes a G2 arrest (4Boutros R. Dozier C. Ducommun B. Curr. Opin. Cell Biol. 2006; 18: 185-191Crossref PubMed Scopus (346) Google Scholar). ATR can be activated by many agents including HU, which blocks replication, and DNA damaging agents such as UV and ionizing radiation. The agents capable of activating ATR share the common property of generating long single-stranded DNA regions either by blocking DNA replication, such as occurs with HU, to give stalled replication forks or by nuclease processing of the initial DNA damage (5Bartek J. Lukas C. Lukas J. Nat. Rev. Mol. Cell Biol. 2004; 5: 792-804Crossref PubMed Scopus (596) Google Scholar). The signal thought to activate ATR comes from the single-stranded DNA-binding protein RPA (replication protein A), which coats these abnormally long regions of single-stranded DNA (6Zou L. Elledge S.J. Science. 2003; 300: 1542-1548Crossref PubMed Scopus (2050) Google Scholar). A number of other factors, such as ATRIP, Rad1, Rad9, Hus1, and Rad17, are thought to play various roles in activating ATR at these RPA-coated regions (1Shechter D. Costanzo V. Gautier J. DNA Repair. 2004; 3: 901-908Crossref PubMed Scopus (167) Google Scholar, 5Bartek J. Lukas C. Lukas J. Nat. Rev. Mol. Cell Biol. 2004; 5: 792-804Crossref PubMed Scopus (596) Google Scholar). The actual mechanism of ATR activation does not appear to involve phosphorylation or any other covalent modification of ATR, and in vitro assays of immunoprecipitated ATR before and after activation generally do not show an increase in activity (7Bakkenist C.J. Kastan M.B. Cell. 2004; 118: 9-17Abstract Full Text Full Text PDF PubMed Scopus (417) Google Scholar). Instead of a covalent modification, most models propose that the principal mechanism of activation is the formation of macromolecular assembles bringing substrates in close proximity to ATR. If these macromolecular assemblies are large enough, they can be visualized as foci in the nucleus by immunofluorescence. For example, ATR and RPA form nuclear foci in response to HU or UV (8Zhang J. Bao S. Furumai R. Kucera K.S. Ali A. Dean N.M. Wang X.F. Mol. Cell. Biol. 2005; 25: 9910-9919Crossref PubMed Scopus (65) Google Scholar), and these foci may represent the initial formation of macromolecular complexes where substrates are brought to be phosphorylated by ATR. Recently an alternative to this model of ATR activation by relocalization has been proposed. Kumagai et al. (9Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar) showed that direct binding of TopBP1 was sufficient to activate ATR and proposed that this transient association with TopBP1 is the initial step in activation of ATR (9Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (564) Google Scholar). HIV-1 Vpr protein has recently been shown to be another agent that induces a G2 arrest through ATR (10Roshal M. Kim B. Zhu Y. Nghiem P. Planelles V. J. Biol. Chem. 2003; 278: 25879-25886Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar). Early results showed that Vpr induces cell cycle G2 arrest through inhibitory Tyr15 phosphorylation of Cdk1 in mammalian and yeast cells, suggesting that this viral protein exerts a highly conserved effect on a basic cellular function (11He J. Choe S. Walker R. Di Marzio P. Morgan D.O. Landau N.R. J. Virol. 1995; 69: 6705-6711Crossref PubMed Scopus (0) Google Scholar, 12Re F. Braaten D. Franke E.K. Luban J. J. Virol. 1995; 69: 6859-6864Crossref PubMed Google Scholar, 13Zhao Y. Cao J. O'Gorman M.R. Yu M. Yogev R. J. Virol. 1996; 70: 5821-5826Crossref PubMed Google Scholar). Consistent with an ATR pathway, the inhibitory Tyr15 phosphorylation of Cdk1 is achieved by inhibition of Cdc25 phosphatase (14Elder R.T. Yu M. Chen M. Zhu X. Yanagida M. Zhao Y. Virology. 2001; 287: 359-370Crossref PubMed Scopus (78) Google Scholar, 15Bartz S.R. Rogel M.E. Emerman M. J. Virol. 1996; 70: 2324-2331Crossref PubMed Google Scholar), although activation of Wee1 kinase may also be involved (14Elder R.T. Yu M. Chen M. Zhu X. Yanagida M. Zhao Y. Virology. 2001; 287: 359-370Crossref PubMed Scopus (78) Google Scholar, 16Yuan H. Kamata M. Xie Y.M. Chen I.S. J. Virol. 2004; 78: 8183-8190Crossref PubMed Scopus (41) Google Scholar). Roshal et al. (10Roshal M. Kim B. Zhu Y. Nghiem P. Planelles V. J. Biol. Chem. 2003; 278: 25879-25886Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar) demonstrated that ATR has a major role in Vpr-induced G2 arrest through phosphorylation and activation of Chk1. Further studies have shown numerous similarities between the ATR pathway activated by Vpr and by other agents such as HU and UV. These similarities include a requirement for Rad17 and Hus1 (17Zimmerman E.S. Chen J. Andersen J.L. Ardon O. Dehart J.L. Blackett J. Choudhary S.K. Camerini D. Nghiem P. Planelles V. Mol. Cell. Biol. 2004; 24: 9286-9294Crossref PubMed Scopus (110) Google Scholar), the induction of phosphorylation on Chk1 (10Roshal M. Kim B. Zhu Y. Nghiem P. Planelles V. J. Biol. Chem. 2003; 278: 25879-25886Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 17Zimmerman E.S. Chen J. Andersen J.L. Ardon O. Dehart J.L. Blackett J. Choudhary S.K. Camerini D. Nghiem P. Planelles V. Mol. Cell. Biol. 2004; 24: 9286-9294Crossref PubMed Scopus (110) Google Scholar), and the formation of nuclear foci by RPA, 53BP1, BRCA1, and γH2AX (17Zimmerman E.S. Chen J. Andersen J.L. Ardon O. Dehart J.L. Blackett J. Choudhary S.K. Camerini D. Nghiem P. Planelles V. Mol. Cell. Biol. 2004; 24: 9286-9294Crossref PubMed Scopus (110) Google Scholar, 18Lai M. Zimmerman E.S. Planelles V. Chen J. J. Virol. 2005; 79: 15443-15451Crossref PubMed Scopus (76) Google Scholar). The variant histone H2AX is a protein commonly used to monitor the nuclear foci formed during DNA damage and replication checkpoints. In response to replication arrest or many forms of DNA damage, H2AX is phosphorylated on Ser139 to form γH2AX foci, and this phosphorylation is dependent on one or more phosphoinositide-3-kinase-related kinase including ATR (19Stucki M. Jackson S.P. DNA Repair. 2006; 5: 534-543Crossref PubMed Scopus (327) Google Scholar). The γH2AX foci have been shown to retain DNA repair factors at the site of DNA damage, and it has been suggested that one of the roles of γH2AX is to concentrate DNA repair factors at sites of DNA damage (20Fernandez-Capetillo O. Lee A. Nussenzweig M. Nussenzweig A. DNA Repair. 2004; 3: 959-967Crossref PubMed Scopus (802) Google Scholar, 21Furuta T. Takemura H. Liao Z.Y. Aune G.J. Redon C. Sedelnikova O.A. Pilch D.R. Rogakou E.P. Celeste A. Chen H.T. Nussenzweig A. Aladjem M.I. Bonner W.M. Pommier Y. J. Biol. Chem. 2003; 278: 20303-20312Abstract Full Text Full Text PDF PubMed Scopus (387) Google Scholar). Recently, protein phosphatase 2A (PP2A) has been shown to be recruited to DNA damage foci by binding to γH2AX (22Chowdhury D. Keogh M.C. Ishii H. Peterson C.L. Buratowski S. Lieberman J. Mol. Cell. 2005; 20: 801-809Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Chowdhury et al. (22Chowdhury D. Keogh M.C. Ishii H. Peterson C.L. Buratowski S. Lieberman J. Mol. Cell. 2005; 20: 801-809Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar) proposed that the role of the PP2A at the γH2AX foci was to dephosphorylate γH2AX during recovery after DNA repair had been completed. PP2A is one of the major Ser/Thr phosphatases implicated in the regulation of many cellular processes including regulation of signal transduction pathways, cell cycle progression, DNA replication, gene transcription, and protein translation (23Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar, 24Zolnierowicz S. Biochem. Pharmacol. 2000; 60: 1225-1235Crossref PubMed Scopus (173) Google Scholar, 25Virshup D.M. Curr. Opin. Cell Biol. 2000; 12: 180-185Crossref PubMed Scopus (292) Google Scholar, 26Janssens V. Goris J. Van Hoof C. Curr. Opin. Genet. Dev. 2005; 15: 34-41Crossref PubMed Scopus (365) Google Scholar). The PP2A holozyme is composed of a 36-kDa catalytic C subunit (PP2A(C)), a 65-kDa scaffolding A subunit (PP2A(A) or PR65), and a regulatory B subunit (PP2A(B)). The core enzyme consists of PP2A(C) and PP2A(A), and one of the many B regulatory subunits associates with this core enzyme (A/C) to form a holoenzyme with specific properties and substrates (27Kremmer E. Ohst K. Kiefer J. Brewis N. Walter G. Mol. Cell. Biol. 1997; 17: 1692-1701Crossref PubMed Scopus (151) Google Scholar). There are two isoforms of the catalytic core of PP2A, i.e. PP2A(Cα) and PP2A(Cβ), which share 97% identity in their primary amino acid sequences (28Arino J. Woon C.W. Brautigan D.L. Miller Jr., T.B. Johnson G.L. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4252-4256Crossref PubMed Scopus (117) Google Scholar, 29Stone S.R. Hofsteenge J. Hemmings B.A. Biochemistry. 1987; 26: 7215-7220Crossref PubMed Scopus (121) Google Scholar, 30Green D.D. Yang S.I. Mumby M.C. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 4880-4884Crossref PubMed Scopus (81) Google Scholar). PP2A(Cα) is more abundant than PP2A(Cβ). PP2A(A) is also present in two isoforms in mammalian cells, α and β, which share 86% sequence identity (31Hemmings B.A. Adams-Pearson C. Maurer F. Muller P. Goris J. Merlevede W. Hofsteenge J. Stone S.R. Biochemistry. 1990; 29: 3166-3173Crossref PubMed Scopus (193) Google Scholar). PP2A(Aβ) is less abundant than PP2A(Aα), and it does not bind strongly to the catalytic C and regulatory B subunits (32Hendrix P. Turowski P. Mayer-Jaekel R.E. Goris J. Hofsteenge J. Merlevede W. Hemmings B.A. J. Biol. Chem. 1993; 268: 7330-7337Abstract Full Text PDF PubMed Google Scholar, 33Zhou J. Pham H.T. Ruediger R. Walter G. Biochem. J. 2003; 369: 387-398Crossref PubMed Scopus (71) Google Scholar). Much of the diversity of PP2A holoenzymes comes from the four major classes of PP2A(B) regulatory subunits, PR55/B, PR61/B′, PR72/B′′, and PR93/PR110/B′′′. Each exists in at least four isoforms leading to many different holoenzymes, which partially explains the multiple and diverse cellular functions of PP2A (23Janssens V. Goris J. Biochem. J. 2001; 353: 417-439Crossref PubMed Scopus (1542) Google Scholar, 34Mayer-Jaekel R.E. Hemmings B.A. Trends Cell Biol. 1994; 4: 287-291Abstract Full Text PDF PubMed Scopus (156) Google Scholar). A role for PP2A in Vpr-induced G2 arrest was originally suggested by studies with the inhibitor okadaic acid in mammalian and yeast cells (12Re F. Braaten D. Franke E.K. Luban J. J. Virol. 1995; 69: 6859-6864Crossref PubMed Google Scholar, 13Zhao Y. Cao J. O'Gorman M.R. Yu M. Yogev R. J. Virol. 1996; 70: 5821-5826Crossref PubMed Google Scholar). Further evidence came from fission yeast where deletion of the gene for a catalytic subunit (ppa2) or a regulatory subunit (pab1) of PP2A reduced Vpr-induced G2 arrest (14Elder R.T. Yu M. Chen M. Zhu X. Yanagida M. Zhao Y. Virology. 2001; 287: 359-370Crossref PubMed Scopus (78) Google Scholar, 35Masuda M. Nagai Y. Oshima N. Tanaka K. Murakami H. Igarashi H. Okayama H. J. Virol. 2000; 74: 2636-2646Crossref PubMed Scopus (62) Google Scholar). However, specific involvement of PP2A in Vpr-induced G2 arrest in mammalian cells is controversial. A report showing Vpr induces G2 arrest in mammalian cells by interacting with the B55 regulatory subunit of PP2A (36Hrimech M. Yao X.J. Branton P.E. Cohen E.A. EMBO J. 2000; 19: 3956-3967Crossref PubMed Scopus (50) Google Scholar) was retracted (37Hrimech M. Yao X.J. Branton P.E. Cohen E.A. EMBO J. 2002; 213918Crossref PubMed Scopus (6) Google Scholar). In this study we investigated the role of PP2A in the activation of ATR by Vpr during the induction of cell cycle G2 arrest. We measured the Vpr-induced phosphorylation of Chk1 and cell cycle G2 arrest when PP2A enzymatic activity was downregulated either by the potent PP2A inhibitor okadaic acid (OA) or by specific small interference RNA (siRNA) to PP2A. We show here that the Cβ and Aα subunits of PP2A, but not the Cα subunit, are required for the Vpr-induced and ATR-dependent phosphorylation of Chk1. However, depletion of the Cβ and Aα subunits did not decrease the extent of nuclei with γH2AX foci or the amount of γH2AX formed even though these events are ATR-dependent. In contrast to the PP2A(Aα/Cβ) dependence of Chk1 phosphorylation when ATR is activated by Vpr, depletion of the Aα or Cβ subunit of PP2A has no effect on the ATR-dependent phosphorylation of Chk1 when ATR is activated by HU or UV. Thus, PP2A has a positive role in the ATR-dependent phosphorylation of Chk1 activated by Vpr but not when ATR is activated by HU or UV. Cell Culture—HeLa cells were grown in Dulbecco's modified Eagle's medium (Cellgro) supplemented with 10% fetal bovine serum (Invitrogen). The DL-3 cell line is a derivative of the HEK293VE-632 cell line stably transfected with an inducible vpr expression plasmid (pZH-vpr) (38Zhou Y. Ratner L. Virology. 2001; 287: 133-142Crossref PubMed Scopus (16) Google Scholar). DL-3 cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, 100 μg/ml zeocin, and 200 μg/ml hygromycin (Invitrogen). The expression of vpr is induced by 1 μm muristerone A (Invitrogen) as previously described (38Zhou Y. Ratner L. Virology. 2001; 287: 133-142Crossref PubMed Scopus (16) Google Scholar). Drug Treatment—OA, a potent PP2A inhibitor, was purchased from Sigma. OA also inhibits other phosphatases (PPases) such as PP1, PP2B, and PP2C at high concentration (39Bialojan C. Takai A. Biochem. J. 1988; 256: 283-290Crossref PubMed Scopus (1515) Google Scholar). The range of OA concentrations (10–25 nm) used here should specifically inhibit PP2A (40Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar). To maintain proper final concentrations of OA during experiments, the same concentration of OA was added again 8 h after the first treatment. Cell treatment with HU or nocodazole (NOC) have been described previously (17Zimmerman E.S. Chen J. Andersen J.L. Ardon O. Dehart J.L. Blackett J. Choudhary S.K. Camerini D. Nghiem P. Planelles V. Mol. Cell. Biol. 2004; 24: 9286-9294Crossref PubMed Scopus (110) Google Scholar). Briefly, cells were incubated with 10 mm HU for 2 h or 100 ng/ml NOC for 24 h before harvest. Antibodies—Rabbit polyclonal anti-phospho-Ckd1-Tyr15, rabbit monoclonal anti-phospho-Chk1-Ser345 (133D3), and mouse monoclonal anti-PP2A(A) (4G7) antibodies were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Mouse monoclonal anti-Chk1 (G-4) and rabbit polyclonal anti-PP2A(C) (FL-309) antibodies were purchased from Santa Cruz Biotechnology, Inc. Mouse monoclonal anti-Cdk1 (Ab-2) and rabbit polyclonal anti-ATR (Ab-2) antibodies were from Calbiochem. Mouse monoclonal anti-β-actin (AC-15) antibody was from Sigma-Aldrich. Rabbit polyclonal anti-Claspin (BL73) antibodies were from Bethyl Laboratories Inc. (Montgomery, TX), and mouse monoclonal anti-phosphohistone γ-H2AX (Ser139) antibody was from Upstate, Inc. (Charlottesville, VA). Texas Red® goat anti-mouse IgG (H + L) secondary antibody was purchased from Molecular Probes, Inc (Eugene, OR). Goat anti-mouse IgG (H + L) horseradish peroxidase (HRP) conjugate and goat anti-rabbit IgG (H + L) HRP conjugate secondary antibodies were from Bio-Rad. Rabbit polyclonal anti-Vpr serum was custom generated through the Proteintech Group Inc. (Chicago, IL). Immunofluorescence Staining—Cells were fixed with 2% paraformaldehyde in PBS for 35 min at 4 °C on Labtek II slides 48 h post-transfection. After washing 3 times for 5 min in PBS, cells were blocked and permeabilized for 20 min in blocking buffer (3% bovine serum albumin, 0.2% Triton X-100, 0.01% skim milk in PBS). Cells were then incubated with primary antibody with proper dilutions in the incubation buffer (1% bovine serum albumin and 0.02% Triton X-100 in PBS) for 45 min. After washing, cells were incubated with secondary antibody at the suggested dilutions in the incubation buffer for 35 min. After washing, cells were mounted with FluorSave reagent and visualized on a Leica DM4500B microscope (Leica Microsystems) with Openlab software (Improvision, Lexington, MA). Western Blotting—Cells were lysed with lysis buffer (50 mm Tris, pH 7.5, 150 mm NaCl, 2 mm EDTA, 1% Triton X-100) on ice for 30 min, and the debris was removed by centrifugation at 13,000 rpm for 1 min. The protein concentrations of supernatants were measured by BCA protein assay kit (Pierce). After boiling, 50 μg of protein was loaded on Criterion Precast Gels (Bio-Rad) for electrophoretic separation. Proteins were transferred to the Trans-blot® nitrocellulose membranes and blocked with 5% skim milk in TBST buffer (10 mm Tris, pH 8.0, 150 mm NaCl, 0.1% Tween 20) for 1 h at room temperature. Primary antibodies were then applied overnight at 4 °C. After washing 3 times in TBST for 10 min each time, the membranes were incubated with secondary antibody for 1 h at room temperature. Membranes were washed again, and proteins were detected with Supersignal® West Pico chemiluminescent substrate (Pierce). siRNA Transfection—SMARTpool™ combination of specific siRNAs, which were pre-designed commercially for PP2A(Aα) (PPP2R1A, #M-010259), Claspin (#M-005288), and ATR-2 pub. siRNA Duplex (#P-002090), were purchased from Dharmacon (Chicago, IL). Pre-designed PP2A(Cα) siRNA mix (PPP2CA, #16704) was purchased from Ambion (Lafayette, CO), PP2A(Cβ) (#RI-4682, RI-4683, and RI-4684) were purchased from Molecula (Columbia, MD), and the control nonsilencing siRNA (#1022083) was from Qiagen (Valencia, CA (10Roshal M. Kim B. Zhu Y. Nghiem P. Planelles V. J. Biol. Chem. 2003; 278: 25879-25886Abstract Full Text Full Text PDF PubMed Scopus (173) Google Scholar, 41Recher C. Ysebaert L. Beyne-Rauzy O. Mansat-De Mas V. Ruidavets J.B. Cariven P. Demur C. Payrastre B. Laurent G. Racaud-Sultan C. Cancer Res. 2004; 64: 3191-3197Crossref PubMed Scopus (131) Google Scholar)). The sequences of the three siRNAs against PP2A Cα were 5′-GGGATACCGTTTAATTTAA-3′,5′-GGCTAAAGAAATCCTGACA-3′, and 5′-GGTTCGATGTCCAGTTACT-3′. The sequences of the three siRNAs against Cβ were 5′-ATGTGCAAGAGGTTCGTTG-3′,5′-TGTCTGCGAAAGTATGGGA-3′, and 5′-TTGGTGTCATGATCGGAAT-3′. Each siRNA mixture was transfected at a concentration of 100 nm into ∼5 × 105 dividing HeLa cells by using 15 μl of Oligofectamine following the manufacturer's instructions (Invitrogen). Measurement of transfection efficiency of siRNAs by using rhodamine-labeled siRNA indicated >90% transfection efficiency. A semiquantitative RT-PCR assay for Cα and Cβ mRNA used the primers 5′-ACCAAGGAGCTGGACCAGTG-3′ and 5′-CCATGCACATCTCCACAGAC-3′ with SuperScript™ one-step RT-PCR system (#10928-034, Invitrogen). These primers are perfect matches to both the Cα and Cβ sequences and give a 161-bp PCR product. When digested with the TaqI restriction enzyme, the Cα PCR product gives a 101-bp band, whereas Cβ gives a 136-bp band. Adenoviral Infection—Adenoviral vector (Adv) and Adenoviral vector inserted with vpr gene (Adv-Vpr) were kindly provided by Dr. L. J. Zhao (42Zhao L.J. Jian H. Zhu H. Gene (Amst.). 2003; 316: 137-141Crossref PubMed Scopus (76) Google Scholar). The viral stocks were produced and titrated as described previously (43Zhang S. Feng Y. Narayan O. Zhao L.J. Gene (Amst.). 2001; 263: 131-140Crossref PubMed Scopus (73) Google Scholar). Approximately 1 × 106 dividing HeLa cells pretreated with siRNA for 24 h were infected with Adv or Adv-Vpr viruses. Cells were harvested for cell cycle analysis or Western blot analysis 48 h post-infection (p.i.). Infections were performed at a multiplicity of infection of 0.5 with 10 μg/ml Polybrene. These conditions were shown to achieve more greater than 90% efficiency with these viral stocks. Cell Cycle Analysis—At 48 h post-transfection (p.t.), cells were collected by trypsinization. Cells were then washed twice with 2 ml of 5 mm EDTA/PBS and centrifuged at 1500 rpm. After resuspension in 1 ml of 5 mm EDTA/PBS, cells were fixed with 2.5 ml of 95–100% cold ethanol and kept at 4 °C overnight. After centrifugation, fixed cells were washed twice with 2 ml of 5 mm EDTA/PBS and centrifuged again at 1500 rpm. After resuspension again in 0.5 ml of PBS, cells were incubated with RNase A (10 μg/ml) at 37 °C for 30 min and then at 0 °C with the addition of propidium iodine (10 μg/ml) for 1 h. Cells were then filtered before analysis of DNA content by FACScan flow cytometry (Becton Dickinson). The cell cycle profiles were modeled by use of the ModFit software (Verity Software House, Inc.). Inhibition of PP2A Enzymatic Activity Reduces Vpr-induced G2 Arrest—OA, a specific and potent inhibitor of PP2A, was first used to determine whether PP2A is involved in Vpr-induced G2 arrest. A vpr-inducible system in the HEK293 cell line (38Zhou Y. Ratner L. Virology. 2001; 287: 133-142Crossref PubMed Scopus (16) Google Scholar) was used to test the effect of Vpr in the presence or absence of PP2A. The expression of vpr was induced by 1 μm muristerone A, and increasing concentrations of OA in a range that specifically inhibits PP2A were added to the culture medium of vpr-expressing or vpr-repressing cells. Sixty-four hours after gene induction, cells were analyzed by flow cytometry for DNA content. The cell cycle profiles are shown in Fig. 1A, and the percentage of cells in G2/M are shown in Fig. 1B. As expected, vpr-expressing HEK293 cells showed a significant accumulation (62.1%) of cells in G2/M phase, whereas without vpr expression only 12.5% of cells were in G2/M phase (Fig. 1A). No significant differences were seen in cells with or without vpr expression treated with 10 nm OA. However, Vpr-induced G2 accumulation was significantly reduced when the concentrations of OA reached 17.5 and 25 nm. Although treatment with these higher concentrations of OA on cells without vpr expression caused small increases of cells in G2 to 12.5–21.3%, the percentage of the G2 cells decreased from 62.1 to 37.1 and 37.3% in the vpr-expressing cells treated with these higher concentrations of OA (Fig. 1B). These results suggest that the activity of PP2A is required for Vpr-induced G2 arrest. The Aα and Cβ Subunits of PP2A, but Not Cα, Are Required for the Vpr-induced Cell Cycle G2 Arrest—Even though OA has been shown to be a potent PP2A inhibitor at the concentrations used (40Favre B. Turowski P. Hemmings B.A. J. Biol. Chem. 1997; 272: 13856-13863Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar), we cannot rule out the possibility that other phosphatases might also be inhibited by OA. To further confirm the involvement of PP2A in Vpr-induced G2 arrest, we used specific siRNA to deplete PP2A. Because siRNA technology is highly specific, different subunits of PP2A can be knocked down to allow detailed dissection of the role of PP2A in Vpr-induced G2 arrest. Here we tested both isoforms of the catalytic subunits of PP2A(C), i.e. PP2A(Cα) and PP2A(Cβ), and the predominant regulatory PP2A(Aα) subunit. HeLa cells were transfected with specific siRNAs for PP2A(Cα), PP2A(Cβ), PP2A(Aα), and control siRNA. Twenty-four hours p.t., the cells were either mock-infected or infected with Adv or Adv-Vpr, and 48 h post-infection (p.i.), the cellular DNA content was analyzed by flow cytometry. All of the mock or Adv infected cells displayed near normal cell cycle profiles regardless of the siRNA treatment, indicating those siRNAs had no significant effect on the cell cycle (Fig. 2A). Consistent with the idea that Vpr induces cell cycle G2 arrest, cells infected with Adv-Vpr and either untreated or pretreated with control siRNA had a marked accumulation of cells with G2/M DNA content. The percentage of G2/M cells increased from 8.0 to 8.4% without Vpr to 71.9–87.3% with Vpr. However, Vpr-induced accumulation of G2/M cells was markedly reduced when either PP2A(Cβ) or PP2A(Aα) was depleted by siRNA (Fig. 2A, bottom panels). Only 28.4% of the cells had G2/M DNA content after PP2A(Cβ) depletion and only 32.9% for PP2A(Aα) depletion. In contrast, no significant reduction of cells in G2/M was observed in Adv-Vpr-infected cells when PP2A(Cα) was depleted (Fig. 2A, middle panel). The extent of depletion by the siRNAs was examined by immunoblots and, for the catalytic subunit, by a semiq" @default.
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• W2029736072 title "Phosphatase Type 2A-dependent and -independent Pathways for ATR Phosphorylation of Chk1" @default.
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