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• W2154051092 abstract "The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase. The integrity of the human genome is preserved by signal transduction pathways called checkpoints, which delay progression through the cell cycle when DNA damage is present. Three checkpoint proteins, hRad9, hRad1, and hHus1, form a proliferating cell nuclear antigen-like, heterotrimeric complex that has been proposed to function in the initial detection of DNA structural abnormalities. hRad9 is highly modified by phosphorylation, in a constitutive manner and in response to both DNA damage and cell cycle position. Here we present evidence that Thr292 of hRad9 is subject to Cdc2-dependent phosphorylation in mitosis. Furthermore, our data are also consistent with four other hRad9 phosphorylation sites (Ser277, Ser328, Ser336, and Thr355) being regulated in part by Cdc2. We also identify Ser387 as a novel site of hRad9 constitutive phosphorylation and show that phosphorylation at Ser387 is a prerequisite for one form of DNA damage-induced hyperphosphorylation of hRad9. Characterization of nonphosphorylatable mutants has revealed that hRad9 phosphorylation plays a critical role in checkpoint signaling. Overexpression of these mutants blocks the interaction between hRad9 and the DNA damage-responsive protein TopBP1 and impairs the cellular response to DNA damage during S phase. Cell cycle checkpoints are signal transduction pathways that maintain the proper order of cell cycle events (1Hartwell L.H. Weinert T.A. Science. 1989; 246: 629-634Crossref PubMed Scopus (2423) Google Scholar). Several checkpoints preserve the integrity of DNA by sensing genetic anomalies and delaying progression through the cell cycle so that enough time is provided for these anomalies to be corrected. The contribution of checkpoints to human health is illustrated by a growing list of checkpoint genes that are mutated in cancer and cancer predisposition syndromes (2Kastan M.B. Onyekwere O. Sidransky D. Vogelstein B. Craig R.W. Cancer Res. 1991; 51: 6304-6311PubMed Google Scholar, 3Kuerbitz S.J. Plunkett B.S. Walsh W.V. Kastan M.B. Proc. Natl. Acad. Sci. U. S. 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Jaspers N.G.J. Taylor A.M.R. Arlett C.F. Miki T. Weissman S.M. Lovett M. Collins F.S. Shiloh Y. Science. 1995; 268: 1749-1753Crossref PubMed Scopus (2372) Google Scholar, 8Xu Y. Baltimore D. Genes Dev. 1996; 10: 2401-2410Crossref PubMed Scopus (350) Google Scholar). The hRad9 protein is the human homologue of Schizosaccharomyces pombe Rad9, a member of the checkpoint Rad family of proteins. In fission yeast, the checkpoint rad genes (rad1 +, rad3 +, rad9 +, rad17 +, rad26 +, and hus1 +) are required for the S phase (DNA replication) and G2 (DNA damage) checkpoints (9al-Khodairy F. Carr A.M. EMBO J. 1992; 11: 1343-1350Crossref PubMed Scopus (367) Google Scholar, 10Enoch T. Carr A.M. Nurse P. Genes Dev. 1992; 6: 2035-2046Crossref PubMed Scopus (308) Google Scholar, 11al-Khodairy F. Fotou E. Sheldrick K.S. Griffiths D.J. Lehmann A.R. Carr A.M. Mol. Biol. Cell. 1994; 5: 147-160Crossref PubMed Scopus (317) Google Scholar, 12Rowley R. Subramani S. Young P.G. EMBO J. 1992; 11: 1335-1342Crossref PubMed Scopus (187) Google Scholar, 13Marchetti M.A. Kumar S. Hartsuiker E. Maftahi M. Carr A.M. Freyer G.A. Burhans W.C. Huberman J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7472-7477Crossref PubMed Scopus (63) Google Scholar). Yeasts lacking these genes fail to inactivate Cdc2 and enter premature, lethal mitosis when challenged with agents that inhibit DNA synthesis or damage DNA (14Enoch T. Nurse P. Cell. 1990; 60: 665-673Abstract Full Text PDF PubMed Scopus (343) Google Scholar, 15Rhind N. Furnari B. Russell P. Genes Dev. 1997; 11: 504-511Crossref PubMed Scopus (223) Google Scholar). Like its yeast counterpart, hRad9 forms a ring-shaped, heterotrimeric complex with the hRad1 and hHus1 proteins (16Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 17St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar, 18Griffith J.D. Lindsey-Boltz L.A. Sancar A. J. Biol. Chem. 2002; 277: 15233-15236Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Each member of the hRad9-hRad1-hHus1 complex (also known as the 9-1-1 complex), shares sequence homology with PCNA, 1The abbreviations used are: PCNA, proliferating cell nuclear antigen; siRNA, small interfering RNA; GST, glutathione S-transferase; HU, hydroxyurea; IR, ionizing radiation. a homotrimer that encircles DNA and tethers DNA polymerase δ during DNA synthesis (19Venclovas C. Thelen M.P. Nucleic Acids Res. 2000; 28: 2481-2493Crossref PubMed Scopus (231) Google Scholar). PCNA is loaded onto DNA by the pentameric protein complex replication factor C (RFC), which is composed of one large subunit and four smaller subunits (20Tsurimoto T. Stillman B. J. Biol. Chem. 1991; 266: 1961-1968Abstract Full Text PDF PubMed Google Scholar). In a manner analogous to PCNA and RFC, 9-1-1 is loaded onto DNA by a complex between hRad17 and the four smallest subunits of RFC (21Bermudez V.P. Lindsey-Boltz L.A. Cesare A.J. Maniwa Y. Griffith J.D. Hurwitz J. Sancar A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1633-1638Crossref PubMed Scopus (264) Google Scholar). Since DNA damage induces hRad17-dependent association of 9-1-1 with chromatin, the 9-1-1 complex is believed to be involved in the direct recognition of DNA lesions during the initial stages of the checkpoint response (22Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (436) Google Scholar). Also involved in this recognition are two phosphatidylinositol 3-kinase-related kinases, ATM and ATR, that regulate several cell cycle transitions and are central components of the cell checkpoint machinery (23Abraham R.T. Genes Dev. 2001; 15: 2177-2196Crossref PubMed Scopus (1667) Google Scholar). Even though these kinases appear to respond to different types of DNA lesions, they share a long list of common checkpoint substrates, including hRad17 (24Kim S.T. Lim D.S. Canman C.E. Kastan M.B. J. Biol. Chem. 1999; 274: 37538-37543Abstract Full Text Full Text PDF PubMed Scopus (643) Google Scholar, 25Post S. Weng Y.C. Cimprich K. Chen L.B. Xu Y. Lee E.Y. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13102-13107Crossref PubMed Scopus (62) Google Scholar, 26Bao S. Tibbetts R.S. Brumbaugh K.M. Fang Y. Richardson D.A. Ali A. Chen S.M. Abraham R.T. Wang X.F. Nature. 2001; 411: 969-974Crossref PubMed Scopus (230) Google Scholar) and hRad9 (27Chen M.J. Lin Y.T. Lieberman H.B. Chen G. Lee E.Y. J. Biol. Chem. 2001; 276: 16580-16586Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In fission yeast, Rad3 (which shares homology with both ATR and ATM) requires Rad9, Rad1, Hus1, and Rad17 to phosphorylate certain substrates (28O'Connell M.J. Walworth N.C. Carr A.M. Trends Cell Biol. 2000; 10: 296-303Abstract Full Text Full Text PDF PubMed Scopus (332) Google Scholar). Similarly, in human cells, phosphorylation of hRad17 by ATR requires hHus1 (22Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (436) Google Scholar). These findings support a model in which the 9-1-1 complex recruits substrates for ATM or ATR to sites of DNA damage or stalled replication forks (29Melo J. Toczyski D. Curr. Opin. Cell Biol. 2002; 14: 237-245Crossref PubMed Scopus (399) Google Scholar). In addition to interacting with hRad1 and hHus1, hRad9 also physically interacts with TopBP1, the human orthologue of Saccharomyces cerevisiae Dpb11, and Schizosaccharomyces pombe Cut5 (30Makiniemi M. Hillukkala T. Tuusa J. Reini K. Vaara M. Huang D. Pospiech H. Majuri I. Westerling T. Makela T.P. Syvaoja J.E. J. Biol. Chem. 2001; 276: 30399-30406Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). Each of these proteins contains multiple BRCA1 carboxyl-terminal (BRCT) domains, a putative protein-protein interaction motif common in cell cycle control and DNA repair. In addition to the checkpoint rad genes, cut5 + is also required for slowing S phase and delaying mitosis when DNA replication is challenged by DNA damage (13Marchetti M.A. Kumar S. Hartsuiker E. Maftahi M. Carr A.M. Freyer G.A. Burhans W.C. Huberman J.A. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 7472-7477Crossref PubMed Scopus (63) Google Scholar, 31Saka Y. Yanagida M. Cell. 1993; 74: 383-393Abstract Full Text PDF PubMed Scopus (195) Google Scholar). A similar requirement also exists for DPB11 in budding yeast (32Araki H. Leem S.H. Phongdara A. Sugino A. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 11791-11795Crossref PubMed Scopus (243) Google Scholar), and a physical interaction between Dpb11 and Ddc1 (the budding yeast orthologue of hRad9) may play a role in this response (33Wang H. Elledge S.J. Genetics. 2002; 160: 1295-1304Crossref PubMed Google Scholar). hRad9 is a unique member of the 9-1-1 complex in that it contains a C-terminal region (of about 110 amino acids) that does not share homology with PCNA and is not believed to be directly involved in association with hRad1 or hHus1. This region of the protein is both constitutively and transiently phosphorylated at several amino acid residues (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), and hence represents a potential regulatory region for the effector functions of 9-1-1. We have characterized the extensive phosphorylation of hRad9 in this region and show that it is partially regulated by Cdc2. We also demonstrate that the C-terminal phosphorylation of hRad9 has roles in regulating both hRad9 interaction with TopBP1 and the cellular response to DNA damage in S phase. Cell Culture and Transfections—HeLa cells, fibroblasts derived from an ataxia telangiectasia patient (ATM –/–) (CRL-7201; ATCC, Manassas, VA), and IMR90 fibroblasts were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Invitrogen) at 37 °C in 5% CO2 atmosphere. hTERT-RPE1 cells, a human retinal pigment epithelial cell line that stably expresses the human telomerase reverse transcriptase subunit (BD Biosciences, Mississauga, Canada), were maintained as above, only in Dulbecco's modified Eagle's medium/F-12 medium (Invitrogen). Transient transfections were performed as described previously (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). The development of the stable HeLa Tet-Off cell line was performed according to the manufacturer's instructions (BD Biosciences). This cell line was maintained in 200 μg/ml of Geneticin (G418) (Invitrogen). Transgene expression was limited by the addition of 1 μg/ml tetracycline to the culture medium. Small interfering RNA (siRNA) transfections were conducted using OligofectAMINE (Invitrogen), according to the manufacturer's protocol. Briefly, siRNA transfections were carried out in six-well plates using 4 μl of OligofectAMINE reagent and 0.1 nmol of siRNA duplex per well. siRNAs targeting hRad9 and luciferase (GL2) were purchased commercially (Dharmacon Research, Lafayette, CO). The siRNA sequence targeting hRad9 was (5′-AACCCUUGGAGGACGGGCUCU-3′). Drugs and Irradiation—Cells were irradiated using a 137Cs γ-irradiator at 0.78 grays/min. Thymidine (Sigma), hydroxyurea (Sigma), and nocodazole (Sigma) were typically administered for 18 h at concentrations of 2 mm, 1 mm, and 0.1 μg/ml, respectively. Plasmids and Site-directed Mutagenesis—All hRad9 point mutants were generated using the Transformer site-directed mutagenesis kit (BD Biosciences) according to the manufacturer's instructions. GST expression plasmids were generated by PCR subcloning segments of the hRad9 cDNA (wild-type and point mutants) into the BamHI and EcoRI restriction sites of the pGEX-2T vector. Antibodies—Rabbit polyclonal α-phospho-Ser272, α-phospho-Ser387, and α-phospho-Thr292 antibodies were raised against phospho-Ser272 (SDTDSHpSQDLGS; where pS represents phosphoserine), phospho-Ser387 (PVLAEDpSEGEG), and phospho-Thr292 (QLQAHSpTPHPDD; where pT represents phosphothreonine) hRad9 peptides, respectively (Bethyl Laboratories, Montgomery, TX). Antisera were cleared of nonspecific binding activity by passage over immobilized, nonphosphorylated peptides and then affinity-purified with immobilized phosphorylated peptides. α-hRad9 polyclonal chicken antibodies have been described previously (17St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar). α-hHus1 and α-hRad1 polyclonal chicken antibodies were generated against bacterially expressed and purified His-hHus1 and GST-hRad1, respectively (RCH Antibodies, Kingston, Canada). Antibodies were cleared of GST reactivity and then affinity-purified, as described previously (17St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar). Other antibodies used in this study were mouse monoclonal antibodies directed against TopBP1 (BD Biosciences), Myc (9E10; Santa Cruz Biotechnology, Santa Cruz, CA), PCNA (PC10; Santa Cruz Biotechnology), and Cdc2 (17St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar) (Santa Cruz Biotechnology). Flow Cytometry—HeLa cells were fixed in 50% ethanol in phosphate-buffered saline for at least 30 min on ice. Cells were then collected by centrifugation, resuspended in phosphate-buffered saline with 50 μg/ml PI and 0.1 mg/ml RNase A, and analyzed using a flow cytometer (Beckman/Coulter EPICS ALTRA, Mississauga, Canada). Immunoprecipitations and Immunoblotting—For immunoprecipitation reactions, cells were lysed in 250 mm NaCl, 1 mm EDTA, 20 mm Tris (pH 8.0), 0.5% Nonidet P-40, and 10% glycerol, supplemented with 20 μg/ml aprotinin, 4 μg/ml leupeptin, 2 mm sodium orthovanadate, 20 mm β-glycerophosphate, and 0.2 mm sodium fluoride. Lysates were incubated on ice for 30 min and centrifuged at 13,000 × g. Supernatants were precleared with α-chicken IgY-agarose (Promega, Madison WI) or with protein G Sepharose (Amersham Biosciences) for 30 min at 4 °C prior to immunoprecipitation. The immunoprecipitation was performed with 1 μg of antibodies directed against the Myc epitope and 20 μl of protein G-Sepharose or antibodies directed against hRad9 and α-chicken IgY-agarose for 2 h at 4 °C. Immune complexes were washed four times with 1 ml of lysis buffer and then resuspended in SDS-PAGE loading buffer. Immunoblotting was performed essentially as previously described (17St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar). Protein Purification—GST and His-tagged protein expression was induced in logarithmically growing BL21 bacteria, with 0.1 mm isopropyl-1-thio-β-d-galactopyranoside for 3 h at 37 °C. GST fusion proteins were batch-purified from bacterial lysates with glutathione-Sepharose (Promega) using standard techniques. Bound proteins were eluted with 20 mm reduced glutathione in 50 mm HEPES, 10 mm MgCl2, and 1 mm dithiothreitol (final pH of 7.4). His-hHus1 was purified under denaturing conditions (6 m guanidine HCl) on a nickel column. Kinase Assays—HeLa cells, arrested in mitosis with nocodazole or in S phase with thymidine, were lysed in 1 ml each of kinase lysis buffer (50 mm Tris (pH 7.4), 1 mm EDTA (pH 8.0), 25 mm NaCl, and 0.1% Nonidet P-40). The Cdc2 kinase was immunoprecipitated from these extracts using 4 μg of mouse monoclonal antibody directed against Cdc2. Immune complexes were washed three times with 1 ml of phosphate-buffered saline and then two times with 1 ml of kinase reaction buffer (50 mm HEPES (pH 7.4), 10 mm MgCl2, 1 mm dithiothreitol, 50 μm ATP) Reactions were carried out in 60 μl of kinase reaction buffer in the presence of 4 μCi of [γ-32P]ATP, and 3 μg of GST fusion substrate for 30 min at 30 °C. Reactions were stopped by the addition of 30 μl of 3× SDS-PAGE loading buffer. 20 μl from each reaction was subjected to SDS-PAGE (12%), and proteins were stained with Coomassie Brilliant Blue. Gels were then dried and exposed to x-ray film (Eastman Kodak Co.) for 24 h. hRad9 Is Phosphorylated at Thr292 during Mitosis—We had previously identified four amino acid residues in hRad9 that contribute to the constitutive phosphorylation of the protein (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Phosphorylation at Ser277, Ser328, Ser336, and Thr355 results in a significant reduction in the electrophoretic mobility of hRad9 derived from undamaged, asynchronous cells. We had also previously shown that a T292A mutation prevented hyperphosphorylation of hRad9 in mitosis (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To further characterize the nature of Thr292 phosphorylation, phosphospecific antibodies were raised against a phospho-Thr292 hRad9 peptide. As shown in Fig. 1A, these antibodies (α-p292) recognize wild-type (but not T292A) hRad9 from cells that were arrested in mitosis by treatment with the microtubule inhibitor nocodazole (+NOC). The α-p292 antibodies did not recognize hRad9 from asynchronous cultures (–NOC), indicating that hRad9 is indeed hyperphosphorylated at Thr292 in mitosis. We had also previously reported that mutation of Ser277 (a constitutively phosphorylated residue in hRad9) to an alanine reduced the efficiency of hRad9 hyperphosphorylation in mitosis (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). To determine whether Thr292 phosphorylation required prior phosphorylation at Ser277 or any of the other constitutively phosphorylated residues above, the S277A, S328A, S336G, and T355A mutants were also immunoblotted with the α-p292 antibodies. We found that whereas Thr292 phosphorylation was readily detectable in S328A, S336G, and T355A mutants derived from mitotic extracts, it was severely reduced in the S277A mutant (Fig. 1A). Similar results were obtained when hRad9 proteins harboring multiple mutations were analyzed (Fig. 1B). Whereas hRad9 protein containing the S328A, S336G, and T355A mutations was still efficiently phosphorylated at Thr292 in mitosis, the addition of one or both of the S277A and T292A mutations inhibited this phosphorylation. Hence, the mitotic phosphorylation of hRad9 at Thr292 requires prior phosphorylation at Ser277. hRad9 Phosphorylation Is Partially Dependent on Cdc2—To further characterize the mitotic phosphorylation of hRad9, we examined the mobility of endogenous hRad9 as cells entered and exited nocodazole-induced mitotic arrest. Consistent with our previous observations (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar), the predominant phospho form of hRad9 in asynchronous cultures (hRad9α) migrated at an apparent molecular mass of ∼60 kDa. The discrepancy between the apparent molecular mass of hRad9 and its predicted molecular mass of 43 kDa is largely due to phosphorylation at Ser277, Ser328, Ser336, and Thr355. Several less abundant, faster migrating phosphorylation intermediates (hRad9β forms) represent hRad9 protein that is only partially phosphorylated at these sites. When cells were blocked in mitosis by treatment with nocodazole, the abundance of both hRad9α and hRad9β forms was reduced, and the majority of hRad9 existed as an even slower migrating species, hRad9μ (see Fig. 2A). When the mobility pattern of hRad9 was examined at increasing time points following the removal of nocodazole from the culture medium, the emergence of normal hRad9 mobility coincided with the exit of cells from mitosis, as defined by flow cytometry (Fig. 2A, 120 and 400 min). The observations made in Figs. 1 and 2A, are consistent with each of Ser277, Ser328, Ser336, Thr355, and Thr292 being quantitatively phosphorylated during mitosis, although the vast majority of hRad9 is already phosphorylated at the four former residues prior to mitosis. Each of these five amino acids share the common consensus sequence ((S/T)PX(R/P)) and are thus potential targets for cyclin-dependent kinases. For these reasons, we sought to determine whether Cdc2, the cyclin-dependent kinase that controls the G2/M transition, could phosphorylate Ser277, Thr292, Ser328, Ser336, and Thr355 in vitro. To this end, we purified a series of hRad9 C-terminal peptides as GST fusion proteins that encompassed the amino acids indicated above, either left intact or mutated to alanine or glycine residues. Peptides were used as substrates for Cdc2, which was purified by immunoprecipitation from HeLa cells arrested in mitosis or S phase. These analyses revealed that Cdc2 could phosphorylate each of the five (S/T)PX(R/P) sites in vitro, with the exception of Thr292 (Fig. 2B). Phosphorylation of hRad9 and the histone-positive control was enhanced if Cdc2 was derived from mitotic rather than S phase extracts, illustrating the specificity of the α-Cdc2 antibody. Phosphorylation of Ser277 was demonstrated by introduction of the S277A mutation in GST-hRad9266–301, which abolished phosphorylation of this protein by Cdc2. Neither the S277A mutant nor the S277A/T292A double mutant were phosphorylated at levels significantly above background, indicating that Thr292 was not phosphorylated by Cdc2 in this assay. Phosphorylation of Ser328 and Ser336 was shown using GST-hRad9314–344. Whereas the S328A and S336G single mutants of GST-hRad9314–344 were still modestly phosphorylated, the S328A/S336G double mutant showed only background levels of 32P incorporation. Thr355 was more efficiently phosphorylated than any of the other hRad9 sites (see GST-hRad9348–391). There was no appreciable difference in phosphorylation of T355A and P3A (T355A/S375A/S380G), indicating that Ser375 and Ser380 are not phosphorylated by Cdc2 in vitro. This is consistent with our previous work in which we found no evidence for phosphorylation of these residues in vivo (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Furthermore, there did not appear to be any significant phosphorylation of non-Cdk consensus sites in hRad9, thus underscoring the specificity of Cdc2 toward Ser277, Ser328, Ser336, and Thr355 of hRad9 in this assay. To determine whether Cdc2 phosphorylates hRad9 in vivo, we employed the Cdk inhibitor roscovitine. Roscovitine is a selective inhibitor of both Cdc2 and Cdk2 (35De Azevedo W.F. Leclerc S. Meijer L. Havlicek L. Strnad M. Kim S.H. Eur. J. Biochem. 1997; 243: 518-526Crossref PubMed Scopus (658) Google Scholar). When HeLa cells were arrested in mitosis with nocodazole and then treated with roscovitine (Ros) for 2 h, a drastic reduction in hRad9 phosphorylation was observed (Fig. 2C). Roscovitine had no effect however, on hRad9 phosphorylation in asynchronous cultures (Fig. 2C). hRad9 Is Constitutively Phosphorylated at Ser387—Site-directed mutagenesis of potentially phosphorylated residues in the C terminus of hRad9 identified a mutant, S387A, which was not normally hyperphosphorylated in response to the DNA synthesis inhibitor hydroxyurea (HU). To characterize this residue, phosphospecific antibodies were raised against a phospho-Ser387 hRad9 peptide. The quality of these antibodies was determined by testing their reactivity toward phosphorylated and dephosphorylated wild-type and S387A hRad9. Although transiently expressed wild-type and S387A hRad9 were indistinguishable in terms of their SDS-PAGE mobility, they were clearly distinct with regard to their detection when immunoblotted with α-phospho-Ser387 (α-p387) antibodies (Fig. 3A). Whereas the α-p387 antibodies recognized all differentially migrating forms of wild-type hRad9, they were completely nonreactive toward the S387A mutant (Fig. 3A, right blot). In addition, the α-p387 antibodies did not detect wild type hRad9 that had been dephosphorylated with calf intestinal phosphatase. Thus, hRad9 is phosphorylated at Ser387, and the α-p387 antibodies are effective in specifically recognizing Ser387-phosphorylated hRad9. To determine whether phosphorylation at Ser387 of hRad9 was regulated in a cell cycle- or DNA damage-dependent manner, we collected HeLa cells that were in G1, S, G2, and M phases of the cell cycle, as we have previously described (34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). HeLa cells were synchronized with a double thymidine block and then released and collected 2 h (S), 7 h (G2), and 11 h (G1) later. One hour prior to harvest, these cells were treated with 20 Gy of ionizing radiation (IR), as indicated. Cells were arrested in mitosis (M) by treating asynchronous cells with nocodazole. We also collected HeLa cells that were grown in the presence of HU for 18 h or were harvested 18 h after a 20-Gy dose of IR. At the time of harvest, these cells were arrested in early S phase and G2, respectively. In all cases, cell cycle position was confirmed using flow cytometry of propidium iodide-stained nuclei. Soluble cell lysates from these cells were subjected to immunoblotting with α-hRad9, α-p387, and phosphospecific antibodies directed against phosphorylated Ser272 of hRad9 (α-p272), a site of DNA damage-dependent phosphorylation (27Chen M.J. Lin Y.T. Lieberman H.B. Chen G. Lee E.Y. J. Biol. Chem. 2001; 276: 16580-16586Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). In agreement with several previous reports (16Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 22Zou L. Cortez D. Elledge S.J. Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (436) Google Scholar, 27Chen M.J. Lin Y.T. Lieberman H.B. Chen G. Lee E.Y. J. Biol. Chem. 2001; 276: 16580-16586Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 34St. Onge R.P. Besley B.D. Park M. Casselman R. Davey S. J. Biol. Chem. 2001; 276: 41898-41905Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 36Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar, 37Roos-Mattjus P. Vroman B.T. Burtelow M.A. Rauen M. Eapen A.K. Karnitz L.M. J. Biol. Chem. 2002; 277: 43809-43812Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), the constitutively phosphorylated hRad9 protein underwent hyperphosphorylation under various conditions (Fig. 3B, top panel). Ionizing radiation induced rapid phosphorylation of hRad9 at Ser272 regardless of cell cycle position (27Chen M.J. Lin Y.T. Lieberman H.B. Chen G. Lee E.Y. J. Biol. Chem. 2001; 276: 16580-16586Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Whereas Ser272 phosphorylation did produce a subtle mobility shift, it was more easily visualized by immunoblotting with α-p272 antibodies (Fig. 3B, middle panel). The α-p272 blot revealed that IR-induced phosphorylation at Ser272 was not only rapid but also transient and dissipa" @default.
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• W2154051092 title "A Role for the Phosphorylation of hRad9 in Checkpoint Signaling" @default.
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