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• W2009779043 abstract "The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response. The ataxia telangiectasia-mutated and Rad3-related (ATR) kinase is a master checkpoint regulator safeguarding the genome. Upon DNA damage, the ATR-ATRIP complex is recruited to sites of DNA damage by RPA-coated single-stranded DNA and activated by an elusive process. Here, we show that ATR is transformed into a hyperphosphorylated state after DNA damage, and that a single autophosphorylation event at Thr 1989 is crucial for ATR activation. Phosphorylation of Thr 1989 relies on RPA, ATRIP, and ATR kinase activity, but unexpectedly not on the ATR stimulator TopBP1. Recruitment of ATR-ATRIP to RPA-ssDNA leads to congregation of ATR-ATRIP complexes and promotes Thr 1989 phosphorylation in trans. Phosphorylated Thr 1989 is directly recognized by TopBP1 via the BRCT domains 7 and 8, enabling TopBP1 to engage ATR-ATRIP, to stimulate the ATR kinase, and to facilitate ATR substrate recognition. Thus, ATR autophosphorylation on RPA-ssDNA is a molecular switch to launch robust checkpoint response. ATR is phosphorylated at Thr 1989 after DNA damage ATR autophosphorylates Thr 1989 in trans independently of TopBP1 TopBP1 engages phosphorylated ATR via BRCT domains 7 and 8 Binding of TopBP1 to ATR facilitates ATR stimulation and substrate recognition ATR, ataxia telangiectasia-mutated (ATM), and DNA-PKcs (DNA-dependent protein kinase) are three members of the phosphoinositide-3-kinase-like protein kinase (PIKK) family and key regulators of DNA damage signaling and DNA repair. Although all these PIKKs are activated by DNA damage, their DNA damage specificities are distinct, and their functions are not identical. ATM and DNA-PKcs are activated by double-stranded DNA breaks (DSBs), whereas ATR responds to a broad spectrum of DNA damage that induces single-stranded DNA (ssDNA) (Ciccia and Elledge, 2010Ciccia A. Elledge S.J. The DNA damage response: making it safe to play with knives.Mol. Cell. 2010; 40: 179-204Abstract Full Text Full Text PDF PubMed Scopus (2561) Google Scholar, Cimprich and Cortez, 2008Cimprich K.A. Cortez D. ATR: an essential regulator of genome integrity.Nat. Rev. Mol. Cell Biol. 2008; 9: 616-627Crossref PubMed Scopus (1222) Google Scholar, Flynn and Zou, 2010Flynn R.L. Zou L. ATR: a master conductor of cellular responses to DNA replication stress.Trends Biochem. Sci. 2010; 36: 133-140Abstract Full Text Full Text PDF PubMed Scopus (188) Google Scholar). This unusual versatility of ATR enables it to play a particularly important role in the cellular responses to intrinsic genomic stresses during cell proliferation. Unlike ATM and DNA-PKcs, ATR is essential for cell survival even in the absence of extrinsic genomic insults (Brown and Baltimore, 2000Brown E.J. Baltimore D. ATR disruption leads to chromosomal fragmentation and early embryonic lethality.Genes Dev. 2000; 14: 397-402PubMed Google Scholar, Cortez et al., 2001Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar). Elucidation of the mechanism by which ATR is activated is central to understanding how genomic integrity is maintained in humans. In response to DNA damage, ATR, ATM, and DNA-PKcs are regulated by distinct DNA damage sensors. ATM is recruited and activated by the Mre11-Rad50-Nbs1 (MRN) complex (Berkovich et al., 2007Berkovich E. Monnat Jr., R.J. Kastan M.B. Roles of ATM and NBS1 in chromatin structure modulation and DNA double-strand break repair.Nat. Cell Biol. 2007; 9: 683-690Crossref PubMed Scopus (354) Google Scholar, Lee and Paull, 2005Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (995) Google Scholar, Uziel et al., 2003Uziel T. Lerenthal Y. Moyal L. Andegeko Y. Mittelman L. Shiloh Y. Requirement of the MRN complex for ATM activation by DNA damage.EMBO J. 2003; 22: 5612-5621Crossref PubMed Scopus (785) Google Scholar), whereas DNA-PKcs is recruited and activated by the Ku70-Ku80 heterodimer (Smith and Jackson, 1999Smith G.C. Jackson S.P. The DNA-dependent protein kinase.Genes Dev. 1999; 13: 916-934Crossref PubMed Scopus (750) Google Scholar). ATR, through ATRIP, recognizes RPA-ssDNA at sites of DNA damage or stressed replication forks (Ball et al., 2005Ball H.L. Myers J.S. Cortez D. ATRIP binding to replication protein A-single-stranded DNA promotes ATR-ATRIP localization but is dispensable for Chk1 phosphorylation.Mol. Biol. Cell. 2005; 16: 2372-2381Crossref PubMed Scopus (175) Google Scholar, Costanzo et al., 2003Costanzo V. Shechter D. Lupardus P.J. Cimprich K.A. Gottesman M. Gautier J. An ATR- and Cdc7-dependent DNA damage checkpoint that inhibits initiation of DNA replication.Mol. Cell. 2003; 11: 203-213Abstract Full Text Full Text PDF PubMed Scopus (282) Google Scholar, Namiki and Zou, 2006Namiki Y. Zou L. ATRIP associates with replication protein A-coated ssDNA through multiple interactions.Proc. Natl. Acad. Sci. USA. 2006; 103: 580-585Crossref PubMed Scopus (70) Google Scholar, Zou and Elledge, 2003Zou L. Elledge S.J. Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes.Science. 2003; 300: 1542-1548Crossref PubMed Scopus (1905) Google Scholar). In contrast to ATM and DNA-PKcs, ATR-ATRIP is not fully activated by the sensor-DNA complex, RPA-ssDNA (MacDougall et al., 2007MacDougall C.A. Byun T.S. Van C. Yee M.C. Cimprich K.A. The structural determinants of checkpoint activation.Genes Dev. 2007; 21: 898-903Crossref PubMed Scopus (171) Google Scholar). The full activation of ATR-ATRIP requires additional regulators including Rad17, the Rad9-Rad1-Hus1 (9-1-1) “checkpoint clamp,” and TopBP1 (Kumagai et al., 2006Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. TopBP1 activates the ATR-ATRIP complex.Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar, Liu et al., 2006Liu S. Bekker-Jensen S. Mailand N. Lukas C. Bartek J. Lukas J. Claspin operates downstream of TopBP1 to direct ATR signaling towards Chk1 activation.Mol. Cell. Biol. 2006; 26: 6056-6064Crossref PubMed Scopus (130) Google Scholar, Zou et al., 2002Zou L. Cortez D. Elledge S.J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (421) Google Scholar). Thus, ATR is a unique PIKK that is recruited and activated by different factors through a multistep process. A major gap in our understanding of ATR activation is how the ATR-ATRIP kinase is activated on RPA-ssDNA. Even in the absence of DNA and other proteins, TopBP1 directly stimulates the ATR-ATRIP kinase in vitro (Kumagai et al., 2006Kumagai A. Lee J. Yoo H.Y. Dunphy W.G. TopBP1 activates the ATR-ATRIP complex.Cell. 2006; 124: 943-955Abstract Full Text Full Text PDF PubMed Scopus (527) Google Scholar). The ATR-activating domain (AAD) of TopBP1 interacts with ATR-ATRIP in vitro, but this interaction is weak and not regulated by DNA damage (Mordes et al., 2008Mordes D.A. Glick G.G. Zhao R. Cortez D. TopBP1 activates ATR through ATRIP and a PIKK regulatory domain.Genes Dev. 2008; 22: 1478-1489Crossref PubMed Scopus (239) Google Scholar). How the stimulation of ATR-ATRIP by TopBP1 is regulated by DNA damage in vivo is still poorly understood. TopBP1 interacts with Rad9, a 9-1-1 component, through two constitutively phosphorylated residues at its C terminus (Delacroix et al., 2007Delacroix S. Wagner J.M. Kobayashi M. Yamamoto K. Karnitz L.M. The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1.Genes Dev. 2007; 21: 1472-1477Crossref PubMed Scopus (333) Google Scholar, Lee et al., 2007Lee J. Kumagai A. Dunphy W.G. The Rad9-Hus1-Rad1 checkpoint clamp regulates interaction of TopBP1 with ATR.J. Biol. Chem. 2007; 282: 28036-28044Crossref PubMed Scopus (212) Google Scholar, Takeishi et al., 2010Takeishi Y. Ohashi E. Ogawa K. Masai H. Obuse C. Tsurimoto T. Casein kinase 2-dependent phosphorylation of human Rad9 mediates the interaction between human Rad9-Hus1-Rad1 complex and TopBP1.Genes Cells. 2010; 15: 761-771Crossref PubMed Scopus (32) Google Scholar). In response to DNA damage, 9-1-1 is loaded onto dsDNA by a Rad17-containing, RFC-like “clamp loader” that recognizes junctions of RPA-ssDNA and double-stranded DNA (dsDNA) (Ellison and Stillman, 2003Ellison V. Stillman B. Biochemical characterization of DNA damage checkpoint complexes: clamp loader and clamp complexes with specificity for 5′ recessed DNA.PLoS Biol. 2003; 1: E33https://doi.org/10.1371/journal.pbio.0000033Crossref PubMed Scopus (270) Google Scholar, Zou et al., 2003Zou L. Liu D. Elledge S.J. Replication protein A-mediated recruitment and activation of Rad17 complexes.Proc. Natl. Acad. Sci. USA. 2003; 100: 13827-13832Crossref PubMed Scopus (335) Google Scholar). The interaction between Rad9 and TopBP1 may help to recruit TopBP1 to sites of DNA damage and/or facilitates ATR-ATRIP activation (Delacroix et al., 2007Delacroix S. Wagner J.M. Kobayashi M. Yamamoto K. Karnitz L.M. The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1.Genes Dev. 2007; 21: 1472-1477Crossref PubMed Scopus (333) Google Scholar, Lee and Dunphy, 2010Lee J. Dunphy W.G. Rad17 plays a central role in establishment of the interaction between TopBP1 and the Rad9-Hus1-Rad1 complex at stalled replication forks.Mol. Biol. Cell. 2010; 21: 926-935Crossref PubMed Scopus (50) Google Scholar). However, it remains elusive how the 9-1-1 and TopBP1 recruited to dsDNA engage the ATR-ATRIP on RPA-ssDNA. A second major question on ATR activation is how ATR recognizes its substrates and transmits DNA damage signals. Several proteins implicated in ATR signaling, such as Rad17 and Chk1, are phosphorylated by ATR on chromatin (Smits et al., 2006Smits V.A. Reaper P.M. Jackson S.P. Rapid PIKK-dependent release of Chk1 from chromatin promotes the DNA-damage checkpoint response.Curr. Biol. 2006; 16: 150-159Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, Zou et al., 2002Zou L. Cortez D. Elledge S.J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (421) Google Scholar), suggesting that ATR functions on damaged DNA. Furthermore, Rad17, Claspin, and Chk1 are known to associate with each other via a series of ATR-orchestrated events after phosphorylation (Kumagai and Dunphy, 2003Kumagai A. Dunphy W.G. Repeated phosphopeptide motifs in Claspin mediate the regulated binding of Chk1.Nat. Cell Biol. 2003; 5: 161-165Crossref PubMed Scopus (127) Google Scholar, Wang et al., 2006Wang X. Zou L. Lu T. Bao S. Hurov K.E. Hittelman W.N. Elledge S.J. Li L. Rad17 phosphorylation is required for claspin recruitment and Chk1 activation in response to replication stress.Mol. Cell. 2006; 23: 331-341Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The role of these ATR substrates in signal transduction and the phosphorylation-mediated interactions among them suggest that ATR directs assembly of a dynamic signaling complex on DNA. Nonetheless, how ATR engages this signaling complex remains unknown. To delineate the process of ATR activation, we sought to capture ATR in its active state, to molecularly define the state, and to dissect the biochemical events leading to this state. We found that during its activation, ATR, like ATM and DNA-PKcs, is transformed into a hyperphosphorylated state with multiple sites phosphorylated. Surprisingly, however, among the phosphorylation sites of ATR that we identified, only Thr 1989 is critical for robust ATR activation. The phosphorylation of Thr 1989 occurs in trans among the ATR-ATRIP complexes that congregate on RPA-ssDNA. Phosphorylated Thr 1989 is directly recognized by TopBP1, enabling TopBP1 to stably engage the ATR-ATRIP complex, to efficiently stimulate the kinase, and to act as a scaffold for ATR-substrate interactions. These findings reveal unexpected links among the recruitment, stimulation, and substrate recognition of ATR-ATRIP, presenting a clearer picture of how ATR is fully activated at sites of DNA damage. To determine whether ATR is phosphorylated during activation, we used mass spectrometry to analyze Flag-tagged ATR purified from hydroxyurea (HU)-treated 293E cells. Our data showed that ATR was phosphorylated at Ser 428, Ser 435, Thr 1989, and possibly Ser 436 and Ser437 (Figures 1A and see Figure S1A available online). The phosphorylation of Ser 428 was previously shown by others using an antibody from Cell Signaling (http://www.cellsignal.com/products/2853.html). The phosphorylation of Ser 435 and Thr 1989 was documented by large-scale studies on protein phosphorylation (Daub et al., 2008Daub H. Olsen J.V. Bairlein M. Gnad F. Oppermann F.S. Korner R. Greff Z. Keri G. Stemmann O. Mann M. Kinase-selective enrichment enables quantitative phosphoproteomics of the kinome across the cell cycle.Mol. Cell. 2008; 31: 438-448Abstract Full Text Full Text PDF PubMed Scopus (471) Google Scholar, Dephoure et al., 2008Dephoure N. Zhou C. Villen J. Beausoleil S.A. Bakalarski C.E. Elledge S.J. Gygi S.P. A quantitative atlas of mitotic phosphorylation.Proc. Natl. Acad. Sci. USA. 2008; 105: 10762-10767Crossref PubMed Scopus (1180) Google Scholar). To date, none of these phosphorylation sites have been functionally characterized. The location of Thr 1989 in the FAT (FRAP, ATM, TRRAP) domain, a potential regulatory element conserved among PIKKs, prompted us to focus our initial analysis on this phosphorylation site. We first asked whether the phosphorylation of T1989 is induced by DNA damage. To monitor T1989 phosphorylation in vivo, we generated phospho-specific antibodies to this site. In cells irradiated with ultraviolet (UV) light, the phospho-T1989 antibody specifically recognized Flag-tagged wild-type ATR (ATRWT), but not the T1989A mutant (ATRT1989A; Figure 1B). Endogenous ATR was also recognized by the phospho-T1989 antibody after UV irradiation in several cell lines (Figure S1B). Treatment of cell extracts with phosphatase reduced the recognition of ATR by the phospho-T1989 antibody (Figure 1C). In addition to UV, ionizing radiation (IR) and HU also induced T1989 phosphorylation (Figure 1D). Together, these results demonstrate that ATR is phosphorylated at T1989 in a DNA damage-induced manner. The phosphorylation of ATR at T1989 occurs rapidly after DNA damage. Like the ATR-mediated Chk1 phosphorylation, T1989 phosphorylation was detected within 0.5 hr after UV treatment (Figure S1C). Unlike Chk1 phosphorylation, which declined after 2 hr, T1989 phosphorylation persisted until 12 hr post UV treatment. The UV-induced T1989 phosphorylation is dose dependent. T1989 phosphorylation was readily detected in cells treated with 5 J/m2 of UV, and was maximally induced by 50 J/m2 of UV (Figure S1D). Phosphorylated ATR was coimmunoprecipitated by ATRIP (Figure 1E), showing that T1989 is phosphorylated in the ATR-ATRIP complex. Furthermore, T1989 phosphorylation was detected only in the chromatin fractions, but not in the soluble fractions (Figure 1F), suggesting that ATR is phosphorylated on chromatin. These features of T1989 phosphorylation are consistent with a potential role in ATR activation. We next used the ATRT1989A mutant to investigate whether T1989 phosphorylation is implicated in ATR activation. Like ATRWT, ATRT1989A was able to phosphorylate a Rad17-derivative substrate (GST-Rad17) in vitro (Figure 2A ), showing that the T1989A mutation does not significantly alter the kinase domain. When inducibly expressed in stable cell lines, ATRT1989A, but not ATRWT, attenuated the ATR-mediated Chk1 phosphorylation after UV treatment (Figure 2B; cell-cycle distributions shown in Figure S2A). Moreover, even in the absence of UV, induction of ATRT1989A elicited H2AX phosphorylation in a large fraction of cells (Figures S2B and S2C), indicating an increase in genomic instability. These results suggest that although ATRT1989A possesses an intact kinase domain, it interferes with the function of endogenous ATR. To directly determine whether ATRT1989A is functional, we established ATRflox/--derivative cell lines (Cortez et al., 2001Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar) allowing inducible expression of Flag-HA-tagged ATRWT or ATRT1989A in cells devoid of endogenous ATR. Both ATRWT and ATRT1989A were expressed at levels similar to that of endogenous ATR in these cell lines (Figure 2C). As expected, the ATRT1989A mutant expressed in the cell line was detected by ATR antibodies but not the phospho-T1989 antibody after UV irradiation, and it retained the ability to phosphorylate GST-Rad17 in vitro (Figures S2D and S2E). In cells lacking endogenous ATR, Chk1 and Rad17 were efficiently phosphorylated by ATRWT but not ATRT1989A after UV treatment (Figure 2C; lanes 4 and 8). To rule out the possibility that the compromised checkpoint response in ATRT1989A expressing cells is due to unexpected events during cell line generation, we tested additional independently generated cell lines that express ATRWT or ATRT1989A. Consistent with the experiment above, all ATRT1989A-expressing cell lines displayed defective Chk1 activation (Figure S2F). Furthermore, similar results were obtained using ATRflox/- cells infected with Cre-expressing adenovirus (Ad-Cre) and transfected with plasmids encoding Flag-ATRWT or Flag-ATRT1989A (Figure 2D). Together, these results demonstrate that the ATRT1989A mutant is compromised in its ability to initiate robust checkpoint signaling. In marked contrast to ATRT1989A, neither ATRS428A nor ATRS435/436/437A failed to activate Chk1 (Figure 2D and Figure S2G), showing that among the phosphorylation sites of ATR that we identified, T1989 is the only one critical for ATR activation. Since ATR is critical for genomic stability in cycling cells, deletion of ATR from ATRflox/- cells resulted in loss of cell viability (Cortez et al., 2001Cortez D. Guntuku S. Qin J. Elledge S.J. ATR and ATRIP: partners in checkpoint signaling.Science. 2001; 294: 1713-1716Crossref PubMed Scopus (709) Google Scholar). Expression of ATRWT, but not ATRT1989A, suppressed the growth defects of Ad-Cre-infected ATRflox/- cells in both cell proliferation and colony formation assays (Figure 2E and Figure S2H). These results suggest that T1989 is critical not only for the activation of ATR by extrinsic DNA damage but also for its essential function in cycling cells. While the ATRT1989A mutant is defective for checkpoint response, the phosphomimetic ATRT1989D mutant is fully functional in Chk1 activation after DNA damage (Figure S2I). Furthermore, we note that ATRT1989D did not induce Chk1 phosphorylation in the absence of DNA damage, suggesting that T1989 phosphorylation is necessary but not sufficient for initiating robust checkpoint signaling. As described below, phosphorylated ATR functions in concert with other DNA damage sensors and TopBP1 to activate checkpoint response. We next investigated which kinase is responsible for T1989 phosphorylation. T1989 is followed by a Pro residue, raising the possibility that it is a substrate of CDKs. However, inhibitors of various CDKs did not affect T1989 phosphorylation after UV (Figure S3A). Treatment of cells with 50 μM of roscovitine for 14 hr completely abolished the CDK-dependent Mcm2 phosphorylation (Montagnoli et al., 2006Montagnoli A. Valsasina B. Brotherton D. Troiani S. Rainoldi S. Tenca P. Molinari A. Santocanale C. Identification of Mcm2 phosphorylation sites by S-phase-regulating kinases.J. Biol. Chem. 2006; 281: 10281-10290Crossref PubMed Scopus (156) Google Scholar) but did not alter the UV-induced T1989 phosphorylation (Figure 3A ). In marked contrast, T1989 phosphorylation was clearly diminished by caffeine, a pan-inhibitor of ATR and ATM (Figure 3B). To pinpoint the PIKK responsible for T1989 phosphorylation, we tested the effects of specific ATM and DNA-PKcs inhibitors. Even when used in combination at high concentrations, ATM and DNA-PKcs inhibitors did not eliminate T1989 phosphorylation (Figure 3B and Figure S3B). These results suggest that ATR, rather than ATM and DNA-PKcs, is likely responsible for the UV-induced T1989 phosphorylation. If T1989 is autophosphorylated by ATR, one would expect that the kinase-deficient ATR mutant (ATRKD) is not phosphorylated at T1989. To test this possibility, we transiently expressed Flag-ATRWT and Flag-ATRKD in cells lacking endogenous ATR (Figure 3C). In the absence of endogenous ATR, Flag-ATRWT but not Flag-ATRKD was phosphorylated at T1989 after UV treatment, showing that T1989 phosphorylation is dependent upon ATR activity. Inhibition of Chk1 did not alter T1989 phosphorylation, suggesting a direct role of ATR in this phosphorylation event (Figure 3B). To test whether T1989 is directly phosphorylated by ATR, we generated a GST-fusion protein that contains a peptide encompassing T1989 and its surrounding residues (GST-T1989). Like GST-Rad17, GST-T1989 was significantly phosphorylated by ATR (Figure 3D). This phosphorylation of T1989 by ATR was specific because the kinase did not phosphorylate GST-T1989A, and the phosphorylation of T1989 was compromised when ATRKD was used. Together, these results suggest that T1989 is a direct substrate of ATR in vitro. To pinpoint the role of T1989 phosphorylation in ATR activation, we asked if this event is dependent upon TopBP1. Knockdown of TopBP1 with siRNA dramatically reduced UV-induced Chk1 phosphorylation but did not affect T1989 phosphorylation (Figure 4A ). On the other hand, in the absence of endogenous ATR and the presence of ATRT1989A, UV-induced TopBP1 phosphorylation at T1062 was compromised (Figure 4B). Together these results show that T1989 phosphorylation is independent of TopBP1 but that the phosphorylation of TopBP1 requires T1989 phosphorylation. Thus, T1989 phosphorylation likely functions upstream of TopBP1 during ATR activation. In response to UV damage, ATR was coimmunoprecipitated by TopBP1 from the chromatin fractions (Figure 4C). This damage-induced interaction of ATR and TopBP1 was abolished by phosphatase treatment of extracts (see Figure 5A ), showing its dependence on phosphorylation. In cells expressing ATRWT or ATRT1989A, only ATRWT but not ATRT1989A was efficiently coprecipitated by TopBP1 (Figure 4C). Furthermore, TopBP1 was efficiently coprecipitated by ATRWT, but not ATRT1989A (Figure 4D). These results suggest that T1989 phosphorylation is important for the interaction between ATR and TopBP1 after DNA damage. To understand how TopBP1 interacts with phosphorylated T1989 (Figures 4C, 4D, and 5A), we generated two biotinylated peptides that contain phosphorylated or unphosphorylated T1989 and its surrounding residues. Only the phospho-T1989 peptide, but not the unphosphopeptide, captured endogenous TopBP1 from extracts (Figure 5B). Using this binding assay and Flag-tagged TopBP1 fragments, we mapped the phospho-T1989-binding motif of TopBP1 to its BRCT domains 7 and 8 (Figures 5C and 5D and Figure S4A), which are required for activation of the ATR pathway (Gong et al., 2010Gong Z. Kim J.E. Leung C.C. Glover J.N. Chen J. BACH1/FANCJ acts with TopBP1 and participates early in DNA replication checkpoint control.Mol. Cell. 2010; 37: 438-446Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). When overexpressed in human cells, a TopBP1 fragment containing only BRCT 7-8 associated with endogenous ATR (Figure S4B). Furthermore, TopBP1 fragments lacking BRCT 1-6, expressed and purified from E. coli, directly bound to the phospho-T1989 peptide (Figure 5E and Figure S4C). These results demonstrate that TopBP1 directly engages phosphorylated ATR via BRCT 7-8. To confirm the specificity of the interaction between BRCT 7-8 and phospho-T1989, we characterized the interaction using point mutants of both binding partners. When the phosphate-binding pocket of BRCT 7-8 was disrupted by the S1273A, R1280Q, or K1317M mutations (Leung et al., 2011Leung C.C. Gong Z. Chen J. Glover J.N. Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control.J. Biol. Chem. 2011; 286: 4292-4301Crossref PubMed Scopus (36) Google Scholar), the interaction between purified BRCT 7-8 and phospho-T1989 was compromised (Figure S4C). We recently showed that the binding of phospho-T1133 of BACH1 to BRCT 7-8 depends on its neighboring residues at the +3/+4 positions (Leung et al., 2011Leung C.C. Gong Z. Chen J. Glover J.N. Molecular basis of BACH1/FANCJ recognition by TopBP1 in DNA replication checkpoint control.J. Biol. Chem. 2011; 286: 4292-4301Crossref PubMed Scopus (36) Google Scholar). Similarly, Ala substitutions of the +3 Glu and +5 Lys residues of T1989, which are highly conserved among the ATR orthologs in mammals (see Figure S6D), significantly reduced the binding of phospho-T1989 to BRCT 7-8 (Figure S4D). These results suggest that both phospho-T1989 and the +3/+5 residues contribute to the specific binding to BRCT 7-8. T1989 phosphorylation is induced by DNA damage, and it occurs on chromatin. ATRT1989A colocalized with RPA at DNA damage-induced foci (Figure S5), suggesting that T1989 is not required for the localization of ATR to sites of DNA damage. In cells treated with ATRIP or RPA1 siRNA, UV-induced T1989 phosphorylation was diminished (Figures 6A and 6B ). In contrast, knockdown of Rad17, a regulator of ATR that does not affect the recruitment of ATR-ATRIP to RPA-ssDNA (Zou et al., 2002Zou L. Cortez D. Elledge S.J. Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin.Genes Dev. 2002; 16: 198-208Crossref PubMed Scopus (421) Google Scholar), did not alter T1989 phosphorylation (Figure 6C). These results suggest that T1989 phosphorylation may be directly regulated by the recruitment of ATR-ATRIP to RPA-ssDNA. The recruitment of ATR-ATRIP by RPA-ssDNA may bring multiple ATR-ATRIP complexes together and promote ATR autophosphorylation in trans. Consistent with this possibility, purified Flag-tagged ATR-ATRIP pulled down increased amounts of GFP-ATR from extracts in the presence of RPA-ssDNA (Figure 6D), suggesting that multiple ATR-ATRIP complexes congregate on RPA-ssDNA. To test whether ATR can phosphorylate T1989 in trans, we generated an ATR mutant lacking the kinase domain at the C terminus (ATRΔC). In the presence of endogenous ATR, ATRΔC was efficiently phosphorylated at T1989 after UV treatment (Figure 6E). This result, although it does not exclude the possibility of ATR cis autophosphorylation, demonstrates that ATR can indeed autophosphorylate T1989 in trans after DNA damage. The crosstalk among ATR molecules on RPA-ssDNA raised the possibility that the defect of ATRT1989A might be complemented in trans. To assess this possibility, we generated an ATRKD,T1989D double mutant. Since ATRKD,T1989D is inactive as a kinase, it does not directly contribute to ATR substrate phosphorylation. However, the phosphomimetic mutation of ATRKD,T1989D may allow it to bring in TopBP1 and facilitate activation of neighboring ATRT1989A molecules on RPA-ssDNA. Indeed, coexpression of ATRKD,T1989D and ATRT1989A in cells lacking endogenous ATR partially rescued UV-induced Chk1 activation (Figure 6F). This result strongly suggests that the defect of ATRT1989A stems from its compromised ability to interact with TopBP1, and this defect can be partially complemented in trans by the phosphomimetic ATRKD,T1989D mutant. The results above suggest that T1989 phosphorylation is a crucial event linking ATR-ATRIP recruitment to TopBP1-mediated ATR-ATRIP activation. To directly test whether T1989 is important for stimulation of the specific kinase activity of ATR-ATRIP, we purified ATRWT-ATRIP and ATRT1989A-ATRIP complexes from 293E cells and GST-tagged TopBP1 from E. coli. To ensure that the in vitro kinase assay measures the effect of TopBP1 on ATR-ATRIP activity rather than substrate binding, we used GST-Rad17, which only contains a short peptide from Rad17, as substrate. Compared to ATRWT-ATRIP, ATRT1989A-ATRIP was stimulated by full-length TopBP1 (TopBP1WT) less efficiently (Figure 7A ). Furthermore, a TopBP1 fragment lacking BRCT 7-8 (TopBP1ΔBRCT7-8) stimulated ATRWT-ATRIP less efficiently than TopBP1WT (Figure S6A). Thus, the efficient stimulation of ATR-ATRIP by TopBP1 relies on the ATR-TopBP1 interaction mediated by the phospho-T1989 of ATR and the BRCT 7-8 of TopBP1. A number of proteins participating in ATR signaling are phosphorylated after DNA damage. One example of these proteins is Rad9 (Roos-Mattjus et al., 2003Roos-Mattjus P. Hopkins K.M. Oestreich A.J. Vroman B.T. Johnson K.L. Naylor S. Lieberman H.B. Karnitz L.M. Phosphorylation of human Rad9 is required for" @default.
• W2009779043 created "2016-06-24" @default.
• W2009779043 creator A5008140692 @default.
• W2009779043 creator A5009938266 @default.
• W2009779043 creator A5017369634 @default.
• W2009779043 creator A5023653054 @default.
• W2009779043 creator A5026859412 @default.
• W2009779043 creator A5026961383 @default.
• W2009779043 creator A5046996966 @default.
• W2009779043 creator A5058771826 @default.
• W2009779043 creator A5077786047 @default.
• W2009779043 date "2011-07-01" @default.
• W2009779043 modified "2023-10-14" @default.
• W2009779043 title "ATR Autophosphorylation as a Molecular Switch for Checkpoint Activation" @default.
• W2009779043 cites W1514911782 @default.
• W2009779043 cites W1595925846 @default.
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