A Proteomic Analysis of Ataxia Telangiectasia-mutated (ATM)/ATM-Rad3-related (ATR) Substrates Identifies the Ubiquitin-Proteasome System as a Regulator for DNA Damage Checkpoints

ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G1 cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control. ATM (ataxia telangiectasia-mutated) and ATR (ATM-Rad3-related) are proximal checkpoint kinases that regulate DNA damage response (DDR). Identification and characterization of ATM/ATR substrates hold the keys for the understanding of DDR. Few techniques are available to identify protein kinase substrates. Here, we screened for potential ATM/ATR substrates using phospho-specific antibodies against known ATM/ATR substrates. We identified proteins cross-reacting to phospho-specific antibodies in response to DNA damage by mass spectrometry. We validated a subset of the candidate substrates to be phosphorylated in an ATM/ATR-dependent manner in vivo. Combining with a functional checkpoint screen, we identified proteins that belong to the ubiquitin-proteasome system (UPS) to be required in mammalian DNA damage checkpoint control, particularly the G1 cell cycle checkpoint, thus revealing protein ubiquitylation as an important regulatory mechanism downstream of ATM/ATR activation for checkpoint control. DNA damage response (DDR) 3The abbreviations used are: DDR, DNA damage response; ATM, ataxia telangiectasia-mutated; ATR, ATM-Rad3-related; HU, hydroxyurea; MS, mass spectrometry; siRNA, small interfering RNA; UPS, ubiquitin-proteasome system; Gy, gray; CldU, chlorodeoxyuridine; IdU, iododeoxyuridine. 3The abbreviations used are: DDR, DNA damage response; ATM, ataxia telangiectasia-mutated; ATR, ATM-Rad3-related; HU, hydroxyurea; MS, mass spectrometry; siRNA, small interfering RNA; UPS, ubiquitin-proteasome system; Gy, gray; CldU, chlorodeoxyuridine; IdU, iododeoxyuridine. coordinates cell cycle checkpoints, DNA repair and apoptosis. G1, intra-S, and G2/M checkpoints operate to block cells at different cell cycle stages (1Lydall D. Weinert T. Science. 1995; 270: 1488-1491Crossref PubMed Scopus (356) Google Scholar). G1 checkpoint prevents G1 cells from entering S phase with damaged DNA (2Bartek J. Lukas J. Curr. Opin. Cell Biol. 2001; 13: 738-747Crossref PubMed Scopus (461) Google Scholar, 3Lukas J. Lukas C. Bartek J. DNA Repair (Amst.). 2004; 3: 997-1007Crossref PubMed Scopus (304) Google Scholar), which operates in two distinct phases in response to ionizing radiation (IR): an initial rapid response, typically within 4 h after IR (4Agami R. Bernards R. Cell. 2000; 102: 55-66Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar) and a sustained response that operates from 4 to 24 h after IR (2Bartek J. Lukas J. Curr. Opin. Cell Biol. 2001; 13: 738-747Crossref PubMed Scopus (461) Google Scholar). While the sustained G1 arrest is mediated by p53-dependent transcription, the rapid G1 arrest is p53-independent and is poorly understood. While many proteins that regulate intra-S and G2/M checkpoints have been identified, the number of proteins known to participate in G1 checkpoint is very limited. Central to DDR are the two checkpoint kinases, ATM and ATR, that phosphorylate the effector kinases Chk1 and Chk2 and other downstream effectors (5Shiloh Y. Nat. Rev. Cancer. 2003; 3: 155-168Crossref PubMed Scopus (2125) Google Scholar). ATM/ATR substrates execute DDR directly or indirectly and are the keys for the understanding of this response. The identification of p53, NBS1, BRCA1, Chk1/Chk2, and SMC1 as substrates of ATM/ATR has significantly advanced our understanding of DDR (6Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1684) Google Scholar, 7Lim D.S. Nature. 2000; 404: 613-617Crossref PubMed Scopus (670) Google Scholar, 8Matsuoka S. Huang M. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1070) Google Scholar, 9Chehab N.H. Malikzay A. Appel M. Halazonetis T.D. Genes Dev. 2000; 14: 278-288PubMed Google Scholar, 10Yazdi P.T. Wang Y. Zhao S. Patel N. Lee E.Y. Qin J. Genes Dev. 2002; 16: 571-582Crossref PubMed Scopus (406) Google Scholar, 11Kitagawa R. Bakkenist C.J. McKinnon P.J. Kastan M.B. Genes Dev. 2004; 18: 1423-1438Crossref PubMed Scopus (346) Google Scholar, 12Kim S.T. Xu B. Kastan M.B. Genes Dev. 2002; 16: 560-570Crossref PubMed Scopus (408) Google Scholar). A thorough understanding of DDR in mammalian cells awaits the identification of new ATM/ATR substrates. Unfortunately, few techniques are available today to systematically identify in vivo substrates of protein kinase. ATM and ATR kinases preferentially phosphorylates the S/TQ motif as a consensus sequence (13Kim 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 (636) Google Scholar). It has been observed that a phospho-specific antibody raised against a phosphopeptide is not strictly site or sequence specific; it also recognizes other proteins (cross-reacting proteins) with similar sequences flanking the phosphorylation site. It has been recently realized that these cross-reacting proteins could also be substrates of the same kinase (14Shi Y. Dodson G.E. Mukhopadhyay P.S. Shanware N.P. Trinh A.T. Tibbetts R.S. J. Biol. Chem. 2007; 282: 9236-9243Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar, 15Livingstone M. Ruan H. Weiner J. Clauser K.R. Strack P. Jin S. Williams A. Greulich H. Gardner J. Venere M. Mochan T.A. DiTullio Jr., R.A. Moravcevic K. Gorgoulis V.G. Burkhardt A. Halazonetis T.D. Cancer Res. 2005; 65: 7533-7540Crossref PubMed Scopus (50) Google Scholar, 16Cortez D. Glick G. Elledge S.J. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 10078-10083Crossref PubMed Scopus (256) Google Scholar, 17Ziv Y. Bielopolski D. Galanty Y. Lukas C. Taya Y. Schultz D.C. Lukas J. Bekker-Jensen S. Bartek J. Shiloh Y. Nat. Cell Biol. 2006; 8: 870-876Crossref PubMed Scopus (542) Google Scholar). Thus, the phospho-antibodies can be used to find potential kinase substrates. See supplemental material I for “Experimental Procedures” (18Wang Y. Cortez D. Yazdi P. Neff N. Elledge S.J. Qin J. Genes Dev. 2000; 14: 927-939Crossref PubMed Scopus (94) Google Scholar, 19Jung S.Y. Malovannaya A. Wei J. O'Malley B.W. Qin J. Mol. Endocrinol. 2005; 19: 2451-2465Crossref PubMed Scopus (90) Google Scholar, 20Carpenter A.E. Jones T.R. Lamprecht M.R. Clarke C. Kang I.H. Friman O. Guertin D.A. Chang J.H. Lindquist R.A. Moffat J. Golland P. Sabatini D.M. Genome Biol. 2006; 7: R100Crossref PubMed Scopus (3178) Google Scholar). To screen for putative ATM/ATR substrates, we used phospho-antibodies raised against known ATM/ATR substrates that recognize pSQ motifs to immunoprecipitate potential new substrates. HeLa nuclear extracts were prepared from cycling cells and from those treated with IR and hydroxyurea (HU), conditions that activate ATM and ATR, respectively. Each lane of Coomassie Blue-stained gels was divided into 12 regions and proteins in each region were identified with mass spectrometry (MS). As shown as an example in Fig. 1a, structure maintenance of chromosomes 1 and 3 (SMC1/3) migrating at 170 and 150 kDa, respectively, are the most abundant proteins recognized by the ATM-pS1981 antibody (21Bakkenist C.J. Kastan M.B. Nature. 2003; 421: 499-506Crossref PubMed Scopus (2635) Google Scholar). Many proteins were identified from cycling cells and IR- or HU-treated cells (supplemental Table I). Similar experiments were carried out with antibodies against phospho-BRCA1 (pS1387, pS1423, and pS1457), and the results are summarized in supplemental Table I. It is possible that some proteins identified using this approach were co-precipitated by association with phosphorylated proteins. To minimize precipitation of associated proteins, we used radioimmunoprecipitation buffer containing high detergent concentration to wash the immunoprecipitates extensively to disrupt the weak interactions. Thus, proteins identified by this method represent both potential ATM/ATR substrates and core components of the complex that associate with the substrates and have survived the stringent wash. For example, SMC1 was co-precipitated by its strong interaction with SMC3, the substrate recognized by ATM-pS1981 antibody (Fig. 1, A and B). Since interaction between two protein complexes is usually transient, the identification of protein complexes from different functional categories suggests that one or more proteins in each category are recognized by the phospho-antibodies as potential ATM/ATR substrates, and other core components in each protein complex come along with the substrate. We used the following criteria to validate the identified proteins as ATM/ATR substrates. First, they must be phospho-proteins; second, they are phosphorylated in an IR/HU-inducible and ATM/ATR-dependent manner; and third, they must contain sequences similar to the SQ peptides used to raise the phospho-antibodies. As shown in Fig. 1B, when the lysate was treated with calf intestine phosphatase (CIP), no band was recognized by ATM-pS1981 antibody by Western blotting, demonstrating that all bands correspond to phospho-proteins. Furthermore, most of the proteins were phosphorylated in an IR- and HU-inducible manner (Fig. 1, B and C). To determine the identity of the protein band and to test whether the phospho-Western blot signal is ATM-dependent, we transfected siRNAs against ATM and the candidate protein that has a similar molecular weight to the protein band identified by immunoprecipitation/MS (supplemental Table 1). The cell lysate was analyzed by Western blotting of duplicate blots with either the phospho-antibody or a specific antibody against the candidate protein. As shown in Fig. 2A, the knockdown of either ATM or ARFBP1 reduced the p480 phospho-signal, suggesting that p480 is ARFBP1 and that this protein is phosphorylated in an ATM-dependent manner; this is confirmed by the reduction of the Western blotting signal of ARFBP1 on the duplicate membrane. Using this approach, we found that ARFBP1, USP34, KIAA1794, SMC3, TAOK3, and TDP1 are phosphorylated in an IR-and HU-inducible and ATM/ATR-dependent manner. Analyses of amino acid sequences of these proteins reveal that they often contain a stretch of at least 4 amino acid residues or multiple stretches of 3 amino acid resides surrounding the SQ site that are identical to the phosphopeptide antigen, suggesting that these sequences are recognized by the phospho-antibodies employed in this study. Thus, these proteins are most likely to be ATM/ATR substrates (Table 1). The protein band p250 is identified as TRAP220, but its phosphorylation is neither dependent on ATM/ATR nor IR/HU-inducible. Thus, some proteins cross-reacting to phospho-antibodies are not ATM/ATR substrates.TABLE 1Comparison of phospho-specific antibody epitopes with sequences within the identified cross-reacting proteins (S/T, D/E, and I/L are treated as the same residue in the analysis)ProteinATM, pS1981, EGSQSTBRCA1, pS1387, LSSQSDBRCA1, pS1457, LTSQKSRad18HFSQSKRNF20LQSQSS, LSSQSSLQSQSS, LSSQSSRNF40LKSQVD, LRSQALRFWD3VSSQGV, ISSQATARFBP1/ HUWEWGSQLG, CSSQSS, ESSQSELSSQEM, CSSQSS, ESSQSELSSQEM, ATSQAG, VASQKREDD1LGSQPQ, LISQAQ, SSSQSQ, SSSQSS, SSSQSD, LASQSSZBTB2LASQGA, TSSQQEBTBD12CSSQTQ, LASQTY, LSSQSSSRSQKS, LASQTY, LSSQSSUSP15ENSQSEUSP28SSSQDV, SSSQDY, STSQEPSSSQDV, SSSQDY, STSQEPUSP34DASQTT, QGSQESVCPSLSQSNIVSQLL, SLSQSNIVSQLLBAT3LSSQTS, LDSQTRLDSQTR, LSSQTSLDSQTR, LSSQTSKIAA1794LSSQEE, SSSQCS, ASSQAT, QCSQSL, DFSQST,SMC3GGSQSS,GGSQSSGGSQSSTAOK3TGSQSS,TGSQSS, LESQKK,TDP1SGSQED, QGSQKD, AGSQEPRIF1LESQESTRAP220GHSQST, LNSQSQ, SQSQSG, ESSQSG, NSSQSG, SGSQGP, GSSQSKESSQSG, NSSQSG YSSQGS, GSSQSK Open table in a new tab Some of the candidate proteins identified from the immunoprecipitation/MS analysis are not recognized by the same phospho-antibody in Western blots. To demonstrate that they are potential ATM/ATR substrates, we immunoprecipitated these proteins with the phospho-antibody and Western blotted with antibodies specific to these proteins. Using this method, we found that RFWD3 is phosphorylated in an ATR-dependent manner in response to HU (Fig. 2B), and RIF1 and VCP are phosphorylated in an ATM-dependant manner in response to IR (Fig. 2C). Substrates validated by this method could be associated proteins, but since they have survived the stringent wash and all contain sequences similar to the peptide antigen, thus it is equally possible that they are kinase substrates. Using both approaches demonstrated in Fig. 2, we validated nine ATM/ATR substrates among our collection of candidates. We conclude that the above approach is capable of finding ATM/ATR substrates. The large number of potential ATM/ATR substrates identified in the proteomic screen poses a significant challenge for functional characterization. When we categorized the proteins into functional modules based on the known function or functional motifs in these proteins, we found many proteins involved in DDR, DNA repair, chromosomal cohesion, and transcriptional regulation, which are expected from ATM/ATR substrates (supplemental Table I). We also found that proteins in the ubiquitin-proteasome system (UPS) are overrepresented (Table 1). This suggests the involvement of protein ubiquitylation in DNA damage response, a notion that is well known in yeast and begins to be appreciated in mammalian cells. The UPS proteins include E3 ubiquitin ligases (Rad18, RNF20, RNF40, RFWD3, ARFBP1, EDD1), BTB domain proteins (ZBTB2 and BTBD12) that may recruit protein degradation targets to E3 ubiquitin ligase complexes (22Honda Y. Tojo M. Matsuzaki K. Anan T. Matsumoto M. Ando M. Saya H. Nakao M. J. Biol. Chem. 2002; 277: 3599-3605Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 23Xu L. Wei Y. Reboul J. Vaglio P. Shin T.H. Vidal M. Elledge S.J. Harper J.W. Nature. 2003; 425: 316-321Crossref PubMed Scopus (377) Google Scholar, 24Chen D. Kon N. Li M. Zhang W. Qin J. Gu W. Cell. 2005; 121: 1071-1083Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar, 25Zhong Q. Gao W. Du F. Wang X. Cell. 2005; 121: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (660) Google Scholar, 26Kim J. Hake S.B. Roeder R.G. Mol. Cell. 2005; 20: 759-770Abstract Full Text Full Text PDF PubMed Scopus (216) Google Scholar), ubiquitin hydrolases (USP15, USP28, and USP34) (27Baker R.T. Wang X.W. Woollatt E. White J.A. Sutherland G.R. Genomics. 1999; 59: 264-274Crossref PubMed Scopus (62) Google Scholar, 28Zhang D. Zaugg K. Mak T.W. Elledge S.J. Cell. 2006; 126: 529-542Abstract Full Text Full Text PDF PubMed Scopus (246) Google Scholar), a polyubiquitin-binding protein (VCP), and a ubiquitin-like domain containing protein (BAT3) (29Boeddrich A. Gaumer S. Haacke A. Tzvetkov N. Albrecht M. Evert B.O. Muller E.C. Lurz R. Breuer P. Schugardt N. Plassmann S. Xu K. Warrick J.M. Suopanki J. Wullner U. Frank R. Hartl U.F. Bonini N.M. Wanker E.E. EMBO J. 2006; 25: 1547-1558Crossref PubMed Scopus (127) Google Scholar, 30Banerji J. Sands J. Strominger J.L. Spies T. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 2374-2378Crossref PubMed Scopus (110) Google Scholar). Importantly, these proteins also contain stretches of amino acid sequences flanking the SQ sites that are similar to the phospho-antigens (Table 1), and the number of peptides detected in MS increases when cells are treated with IR or HU, suggesting that they are potential ATM/ATR substrates or at least tightly associated with the substrates. We thus focused our subproteome functional characterization on 10 UPS proteins for their roles in DNA damage response (Fig. 3). If these candidates are bode fide ATM/ATR substrates, they may participate in regulation of DNA damage checkpoint, DNA repair, or apoptosis. Ideally, we would like to map the precise phosphorylation sites of these candidates and test the loss-of-function mutants for their functions. Unfortunately, it is too labor intensive for a single lab to carry out such analysis on 10 proteins. We decided to examine whether the UPS proteins are required to regulate DNA damage checkpoints. We carried out intra-S and G2/M checkpoint assays in HeLa cells depleted of these genes using siRNA. In response to IR, the intra-S checkpoint is activated to mainly suppress late origin firing and is measured by a temporary decrease of nucleotide incorporation following IR. Three days after siRNA knockdown, we irradiated the cells with 10-Gy IR and measured nucleotide incorporation 1 h post-irradiation. Of the 10 genes tested, six exhibited radio-resistant DNA synthesis significantly above a control siRNA (siVim) transfected cells (Fig. 3A), indicating that these UPS proteins are required to regulate the intra-S phase checkpoint. The G2/M checkpoint is activated to prevent cells with damaged DNA from entering mitosis. We irradiated siRNA-transfected cells with 10-Gy IR and measured the mitotic index 1 h post irradiation. The population of histone H3-pS10 positive cells, which correspond to mitotic cells, was visualized using a microscope. Knocking down 2 out of the 10 genes leads to a profound G2/M checkpoint failure that cells accumulate more than 4 times as many mitotic cells as in control transfected cells (compare >30% with 7.5% relative mitotic index for control transfected cells). Interestingly, siRNA transfection of ZBTB2 causes a marked increase in mitotic index before IR and virtually no further decrease after IR, indicating that ZBTB2 may play an essential role in mitotic progression, which complicates the interpretation of its role in G2/M checkpoint control. The G1 checkpoint arrests cells with damaged DNA at the G1/S boundary. G1 checkpoint is the least understood checkpoint among the DNA damage checkpoints, at least in part due to the lack of an accurate and sensitive assay. Current G1 checkpoint assay, which measures G1 accumulation by flow cytometry, is to cell cycle after DNA damage We an G1 checkpoint by cells with two at times before and after DNA damage cells that have S phase after DNA damage from the cells at the of damage this for accurate of with a We have validated this by the G1 arrest of known checkpoint proteins and confirmed the of p53-independent rapid response 4 h and p53-dependent sustained arrest 4 Wang and J. for We used this to examine in whether the UPS genes are involved in both rapid and sustained G1 arrest in cells. We the cells with to the and with 4 h to the Using and we cells that were and as S phase the 4 h after IR. We the S phase as the of and cells among cells. relative S phase of S phase after and before IR in the 4 h between and h following 10-Gy IR, we the G1 checkpoint in cells depleted of genes by siRNA In control siRNA-transfected cells, relative S phase is to the 4 h after IR as a of rapid G1 checkpoint activation and is further to and from 4 h after IR. Of the 10 genes tested, genes are required to regulate the rapid G1 checkpoint 4 h after IR, and genes are required to regulate the sustained G1 arrest (Fig. to degradation has been shown to regulate the rapid G1 response (4Agami R. Bernards R. Cell. 2000; 102: 55-66Abstract Full Text Full Text PDF PubMed Scopus (316) Google Scholar), and is in this by I. G. E. EMBO J. 13: PubMed Scopus Google Scholar, N. J. Lukas C. R.G. M. Bartek J. Lukas J. Science. 2000; PubMed Scopus Google Scholar). screen has significantly the number of potential G1 checkpoint and the notion that protein degradation is an important mechanism for the rapid G1 the finding that UPS proteins are also required for sustained G1 arrest thus its It is that the UPS may regulate the sustained G1 arrest p53-dependent transcriptional as or other to be the identification of substrates of the UPS proteins. As summarized in Fig. of 10 UPS genes are required for at least one cell cycle checkpoint, and 1 ARFBP1, checkpoints. checkpoint assays siRNA that are of sequences to knockdown, the of be the high for checkpoint genes that a proteomic screen for ATM/ATR substrates with a functional screen is in protein candidates for further of the mediated by The of large number of antibodies to screen for ATM/ATR substrates has an of proteins in DNA damage response. J. When with functional and of functional for checkpoint, DNA repair or potential substrates as in supplemental Table I can be screened and into different in the DNA damage response the of DNA damage response. is an example of this to reveal the involvement of UPS among the ATM/ATR substrates in mammalian checkpoint control. We of the Qin for and with