Research Paper 1 A human homologue of the checkpoint kinase Cds1 directly inhibits Cdc25 phosphatase Alessandra Blasina*, Inez Van de Weyer†, Marc C. Laus†, Walter H.M.L. Luyten†, Andrew E. Parker†‡ and Clare H. McGowan* Background: In human cells, the mitosis-inducing kinase Cdc2 is inhibited by phosphorylation on Thr14 and Tyr15. Disruption of these phosphorylation sites abrogates checkpoint-mediated regulation of Cdc2 and renders cells highly sensitive to agents that damage DNA. Phosphorylation of these sites is controlled by the opposing activities of the Wee1/Myt1 kinases and the Cdc25 phosphatase. The regulation of these enzymes is therefore likely to be crucial for the operation of the G2–M DNA-damage checkpoint. Results: Here, we show that the activity of Cdc25 decreased following exposure to ionizing radiation. The irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia. We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is novel and is homologous to the Cds1/Rad53 family of checkpoint kinases in yeast. Human Cds1 was found to be activated in response to DNA damage. Addresses: *Department of Molecular Biology, The Scripps Research Institute, La Jolla, California 92037, USA. †Department of Experimental Molecular Biology, Janssen Research Foundation, Turnhoutseweg 30, B-2340 Beerse, Belgium. ‡Present address: Zeneca Pharmaceutical, Alderley Edge, Cheshire, UK. Correspondence: Clare H. McGowan E-mail: chmcg@scripps.edu Received: 11 September 1998 Revised: 27 November 1998 Accepted: 27 November 1998 Published: 17 December 1998 Current Biology 1999, 9:1–10 http://biomednet.com/elecref/0960982200900001 © Elsevier Science Ltd ISSN 0960-9822 Conclusions: These results suggest that, in human cells, the DNA-damage checkpoint involves direct inactivation of Cdc25 catalyzed by Cds1 and/or Chk1. Background In response to DNA damage, eukaryotic cells use a system of checkpoint controls to delay cell-cycle progression. Checkpoint delays provide time for repair of damaged DNA prior to its replication in S phase and prior to segregation of chromatids in M phase [1]. In many cases, the DNA-damage response pathways cause arrest by inhibiting the activity of the cyclin-dependent kinases that drive cell-cycle progression [2]. A delay in G1 results from the activation of p53 and consequent transcriptional induction of the cyclin-dependent kinase inhibitor p21 [3–5]. In human cells, the DNA-damage-induced G2 delay has been shown to be largely dependent on inhibitory phosphorylation of Cdc2 [6–8] and is therefore likely to result from a change in the activity of the opposing kinases and phosphatases that act on Cdc2. Nevertheless, evidence that the activity of these enzymes is substantially altered in response to DNA damage is lacking [9]. In this paper, we report that the Cdc25-dependentdephosphorylation of the Cdc2–Cyclin B complex was significantly reduced following γ-irradiation. This irradiation-induced decrease in Cdc25 activity was suppressed by wortmannin, an inhibitor of phosphatidylinositol (PI) 3-kinases, and was dependent on the function of the gene that is mutated in ataxia telangiectasia (AT) — the AT-mutated (ATM) gene. We also identified two human kinases that phosphorylate and inactivate Cdc25 in vitro. One is the previously characterized Chk1 kinase. The second is a novel kinase with homology to the Cds1/Rad53 family of checkpoint kinases in yeast. In contrast to the situation in fission yeast, we found that human Cds1 is activated in response to DNA damage. Taken together, these data support a model for the human DNAdamage checkpoint that involves the direct inactivation of Cdc25, catalysed by Cds1 and/or Chk1. Results and discussion Dephosphorylation of Cdc2 is inhibited by DNA damage The possibility that dephosphorylation of Cdc2 is downregulated in the presence of DNA damage was investigated. Three distinct Cdc25 proteins are expressed in human cells [10–12]. Microinjection of specific antibodies and the observation of distinct patterns of activation suggest that Cdc25A has a role primarily in the G1–S transition [13,14], and that Cdc25B and Cdc25C have roles primarily in the G2–M transition [15–17]. The exact contribution of Cdc25B and Cdc25C to M-phase progression is not known. Therefore, an assay that allows direct analysis of the phosphatases acting on Cdc2–Cyclin B was used to determine how Cdc2 is maintained in its phosphorylated state following irradiation. In the presence of EDTA, the activity of Cdc2–Cyclin B from asynchronous HeLa cell extracts was found to increase with time (Figure 1a). 2 Current Biology, Vol 9 No 1 Figure 1 Kinase activity (a) 100 Control Irradiated Anti-Cdc25 antibody + Vanadate 80 60 40 20 0 0 10 (b) 20 Time (min) 30 Control + Cdc25 Irradiated p34 Cdc2 Time (min) 0 30 30 0 + Cdc25 30 30 + Cdc25 (c) Cdc25C 1 2 CurrentBiology Cdc25 is downregulated in response to DNA damage. (a) Cultures of asynchronous HeLa cells were divided and either mock-treated (control) or irradiated with 10 Gray, 1 h before harvesting. Cell lysates were prepared and the dephosphorylation reaction was initiated by addition of 10 mM EDTA. At the indicated times, Cdc2–Cyclin B was immunoprecipitated and histone H1 kinase activity was assayed [6]. Complete dephosphorylation and activation was obtained by addition of GST–Cdc25C purified from baculovirus-infected insect cells. Histone H1 kinase activity was normalized to the plus GST–Cdc25 control (+ Cdc25). Results are the average of three data sets and are representative of 10 separate experiments. (b) The phosphorylation state of Cdc2 was confirmed by immunoblotting of Cyclin B immunoprecipitates. (c) Cell lysates from asynchronous (lane 1) and irradiated (lane 2) cells were examined for the presence of Cdc25C by western analysis. No loss of Cdc25 protein from the soluble fraction was seen following irradiation and no change in the electrophoretic mobility (due to phosphorylation of Ser216) was detected. Activation correlated with loss of the inhibited phosphorylated form of Cdc2, visualized as the slower migrating species on SDS–PAGE (Figure 1b). There was no increase in the levels of Cdc2 or Cyclin B protein, and phosphorylation by Wee1 and Myt1 was blocked by the presence of 10 mM EDTA (data not shown). Activation was prevented by vanadate, an inhibitor of Cdc25 and other tyrosine phosphatases. Furthermore, immunodepletion with Cdc25C-specific antisera showed that activation of Cdc2–Cyclin B was dependent on the presence of Cdc25 (Figure 1a). Thus, activation of Cdc2 is the result of its dephosphorylation and is a measure of total Cdc25 activity in the extract. In lysates of asynchronous HeLa cells, the endogenous Cdc25 phosphatase activity was sufficient to dephosphorylate and activate more than 80% of the available Cdc2–Cyclin B within 30 minutes (Figure 1a,b). Analysis of lysates of HeLa cells in which the DNA had been damaged by exposure to 10 Gray γirradiation, 1 hour before harvesting, showed a significant reduction in the rate of activation of Cdc2, such that less than 25% of the available Cdc2–Cyclin B was activated during the 30 minute incubation. The amount of Cdc2–Cyclin B in complex was not significantly altered and it was activated to the same extent as control Cdc2–Cyclin B by addition of a glutathione-S-transferase (GST) Cdc25C fusion protein (GST–Cdc25C; Figure 1a). Irradiation with 10 Gray led to more than threefold reduction in the rate of Cdc2 dephosphorylation in the 10 time courses examined. The possibility that loss of Cdc25 activity could be due to a reduction in the amount of Cdc25 protein present in the lysates was considered. Western analysis of the supernatants used for the above assays (that had been centrifuged at 14,000 × g) showed that the extracts prepared from asynchronous and irradiated cells contained equal quantities of Cdc25 (Figure 1c). Thus, the reduced rate of Cdc2 dephosphorylation following irradiation is not due to loss of Cdc25 protein from the extracts. Cdc25 has previously been shown to be phosphorylated on Ser216 in asynchronous cultures [18] and phosphorylation of this site causes a decrease in electrophoretic mobility of Cdc25 [19]. As shown in Figure 1c, Cdc25 from extracts of asynchronous cells runs as a closely spaced doublet with the majority of the protein running as the upper, Ser216-phosphorylated species [19]. The proportion of Cdc25 migrating at the lower unphosphorylated position was not changed in response to irradiation, suggesting that the reduced rate of Cdc2 dephosphorylation following irradiation does not depend on increased phosphorylation of Ser216. Radiation-induced inactivation of Cdc25 requires the activity of kinases related to PI 3-kinases If the inactivation of Cdc25 measured above is part of the DNA-damage checkpoint response in human cells, then experimental conditions that override the DNA-damage checkpoint might be expected to block radiation-induced inhibition of Cdc25. Genetic data from a number of organisms has identified a family of related kinases that are required for DNA-damage responses [20]. Structurally, these enzymes are related to the PI 3-kinases, and two members of the family — DNA–protein kinase and the product of the ATM gene — have been shown to be sensitive to wortmannin in vitro [21,22]. The possibility that a wortmannin-sensitive kinase is upstream of the radiationinduced delay in M-phase entry was therefore tested [23]. HeLa cells can be arrested in M phase by nocodazole, and irradiation causes cells to delay in G2 prior to the Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al. 3 Figure 2 (b) (a) 60 40 30 20 10 Control Irradiated 80 60 40 20 0 C R 0 C R Wortmannin Control (c) 0 γ-irradiated 15 Time (min) 30 + Cdc25 UV-irradiated 100 ATM-minus Kinase activity 100 ATM-plus 80 60 40 80 60 40 20 20 0 Wortmannin 100 Kinase activity Mitotic index 50 kinase activity Radiation-induced inactivation of Cdc25 requires the activity of kinases related to PI 3-kinases. (a) Wortmannin overrides the radiation-induced delay in entry to M phase. Cells were cultured in in the absence or presence of 10 µM wortmannin for 1 h prior to either mock treatment (C) or irradiation with 10 Gray (R); 100 ng/ml nocodazole was added and, 14 h later, cells were fixed in 70% ethanol, stained with Hoechst DNA stain and mitotic index was scored. (b) Wortmannin prevents the irradiation-induced downregulation of Cdc25. Cells were cultured in the presence of 10 µM wortmannin for 1 h prior to either mock treatment (control) or irradiation; extracts were prepared and assayed as in Figure 1. (c) The ATM gene is required for the downregulation of Cdc25 activity following γ-irradiation but not UV irradiation. Kinase activity was assayed as described for Figure 1a. The data shown are the average of two data sets and are representative of three independent experiments. 0 15 Time (min) 30 + Cdc25 0 0 15 Time (min) 30 + Cdc25 Current Biology nocodazole-sensitive M-phase block point. Thus, by scoring the mitotic index of cells that are cultured in nocodazole, it is possible to determine whether entry into mitosis has been delayed. Control cells cultured in the presence of nocodazole for 14 hours contained 60% mitotic cells; the presence of wortmannin had little effect on this number (Figure 2a). However, irradiation reduced the number of cells that reached the nocodazole block point to 10%. By contrast, irradiation in the presence of wortmannin had only a modest effect on the number of cells that reached the nocodazole block point (Figure 2a). These results demonstrate that wortmannin overrides the DNA-damage G2 checkpoint in HeLa cells. The effects of wortmannin on the radiation-induced inactivation of Cdc25 were therefore tested. Wortmannin had a minor effect on the activation of Cdc2–Cyclin B in extracts prepared from non-irradiated cultures, although it greatly diminished the irradiation-induced decrease in Cdc25 activity (Figure 2b). A second experimental condition in which the radiationinduced G2 checkpoint is overridden is provided by cell lines derived from patients with the genetic disorder AT. ATM cells are defective in both the G1 and G2 checkpoints following exposure to many, but not all, agents that damage DNA [24–26]. The failure of ATM cells to delay in G1 correlates with a failure to upregulate the tumor suppressor gene p53 [27] and with a failure to phosphorylate and activate the proto-oncoprotein c-Abl [28,29]. The molecular basis of the failure to delay in G2 is not known. ATM cells show greatly reduced responses to agents that generate chromosomal breaks such as ionizing γ rays. Remarkably, ATM cells have near-normal responses following the base damage that is generated by irradiation with an ultraviolet (UV) radiation source [26,30,31]. Therefore, the effects of UV and γ-irradiation on the Cdc25 activity of normal (ATM-plus) and ATM (ATM-minus) SV40-transformed human fibroblast cell lines were investigated (Figure 2c). ATM-minus cells respond to UV irradiation with a robust reduction in the rate at which Cdc2 is dephosphorylated. In contrast, γ-irradiation had only a modest effect on the rate of dephosphorylation of Cdc2. In ATM-plus cells, the rate of dephosphorylation of Cdc2 was significantly reduced following either ionizing radiation or UV radiation. Samples in which Cdc2–Cyclin B was activated by addition of exogenous GST–Cdc25 showed that both cell types contained similar quantities of Cdc2–Cyclin B following irradiation. These data show that the ATM gene product is required for the efficient inactivation of Cdc25 following γ-irradiation and demonstrate a correlation between inactivation of Cdc25 and delayed entry into M phase following DNA damage [24–26]. Identification of a human homolog of fission yeast checkpoint kinase Cds1 To identify potential mediators of the checkpoint-dependent inactivation of Cdc25, we searched for human homologs of known yeast checkpoint genes. In view of the 4 Current Biology, Vol 9 No 1 Figure 3 (a) HsCds1 SpCds1 Rad53 Dun1 1 1 1 1 HsCds1 SpCds1 Rad53 Dun1 47 23 29 22 P NS S QS S A K QV V N GE N I V C QRS NK P - HS - - - - HsCds1 SpCds1 Rad53 Dun1 93 55 61 54 WA - - - - QDG - - - - - - - HsCds1 SpCds1 Rad53 Dun1 138 76 83 72 T YS RVS RLS D I S KKH FR N FH FE NKH FQ T FHAE I F RE V GP K NS - - - Y I I YQGHRNDS DE S E NV I L L GE - - DGN - - - - F H L L Q MD V D N F Q R N L A V L I Y I FL L L NV EDHS HDHS ND I S I DKS HsCds1 SpCds1 Rad53 Dun1 181 122 122 117 RP T I QL Y I NNNSE S NGDE S QGDE K NGDR I I I I I - F - I - - - - - NDK SSS - - - - F - D L TV - - - F L CQV P V F K QC L E QNK V TD I ENDDEKV HsCds1 SpCds1 Rad53 Dun1 209 157 168 160 QS - - KN - - KN TSK KNDDE A N TG AA I I - - - RK - - - NK HsCds1 SpCds1 Rad53 Dun1 226 173 204 206 L L V L K K K K I SK I NK I SK FHA HsCds1 SpCds1 Rad53 Dun1 272 218 243 247 TE RE RE EE I E I I D I LEV TN I HsCds1 SpCds1 Rad53 Dun1 312 259 284 293 KV FL FV R I V I A V HsCds1 SpCds1 Rad53 Dun1 358 305 330 339 QE E D - - DDP TRRE HsCds1 SpCds1 Rad53 Dun1 387 332 358 384 TP T T MG TLA TPS YL YL YV YV HsCds1 SpCds1 Rad53 Dun1 423 371 404 417 G YP AS I GH L GFP P P P P HsCds1 SpCds1 Rad53 Dun1 469 416 449 463 V I V L KAR F T TEE EKR I SESE NNRS TAAK DE R YN I DE HsCds1 Rad53 Dun1 515 495 509 P S T S R K R P R E G E A E G A E T T K R P A V CA A V L E N M D D A Q Y E F V K A Q R K L Q M E Q Q L Q E Q D Q E D Q D G K I Q G F K I P A H A P I 540 E LSCL Rad53 Rad53 Rad53 Rad53 Rad53 Rad53 Rad53 541 587 633 679 725 771 817 R Y T QP K S I E A E T RE QK L L HS NN T E NV K S S K K K GNGR F L T L K P L P DS I I QE S L E I QQGV NP F F I GRS E DCNCK I E DNR L S RV HC F I F K K RHA V GK S MYE S P A QG L DD I WYCH T G T NV S Y L NNNRM I QG T K F L L QDGDE I K I I WD K N N K F V I G F K V E I N D T T G L F N E G L G M L Q E Q R V V L K Q T A E E K D L V K K L T Q MMA A Q R A N Q P S A S S S S MS A K K P P V S D T N N N G N N S V L N D L V E S P I NA N T GN I L K R I HS V S L S QS Q I DP S K K V K RA K L DQ T S K GP E N LQFS MS ME ME MS RE - N I - - R - L L L L SDV - - T - - - - L WA - - - - - - - I V L - E - - - AS FK A - P K GS GA CGE V GS G T F A V V GQGA F A T V GA GH YA L V DP NP DP NP L L L L KK L KS L QK L MR V GNK R L A NGS I A HGA V RK TC L - - - NP A A A A FS FA FS FS QQS - - - - - - S - S - HGS - - - - - - - A - CS - - - - QP EP QP - - HGS - - T - - - - FA - - - - N - L - E - CV - V - S - - N D N YW H N G FWG I K K VWT - - NV T T A LS L R I GL T V GV V F GK SRNKV FV GV P K DE I GV E S D I L SCS F L FK F S S L YA - - GL PQ - - TS I S S A YP K KSE ANK T TS K K K K A A A A L L K E V - G T L S S L E T V S T QE L N E N V F MK L V M T R M L RV I C T T GQ I P I RD L S S E Y T C L GH L V N L I NHP HHP NHP QHP - - TA TS S S SSSS HVS EK FS TDSS TS TM - QN I QE Q I FKRQ 46 22 28 21 YS I P E DQE DGK T E V I P S A D I S QV L P GK E QK V E P - E - E - P - TP - - - - 92 54 60 53 L - K - R - TDK - - - - - - - F GRDK S F GRHK S F GRNP A I GRS RS I I V V C I GV R I N I DAED NDDE D TES EP I SK S - - - QV Q L I V V V P P P P E E E E L L I L VSV KSK RGK TKK K I KNFF QCHE I CE R L K G F YE N L LDS FV Y F YQML L LKQL L I SRQ I L L FKQL L - - - - - - - - - - - - G P WD E G D - - - - DE - - - NV TSV - - - E HR T QV S L K DQ S S - S QA K C I E L GS - T QDQ L YK Q DQ L GP P S L K E Q A A A A - - V - M I S MV QNA V S T QS QGS S S QS QG I - E E A T QA T QE A P L - QQS T QA T QR F L I - - L S T K RE HS GDV FERK TCKKVA V E V NS GKWYA I E R T T GK T F A K NK K T GQQV A KEA TCK L DE QDCK P GE DA GRE RQDE S K A V V V V S - L L L L I - NL SP - I I I I LAV E TL TA I TGL I I I I QY LH K Y K Y L L I L S L CE YC CEVV CD YH CDV I GNG SNG TNG RNG P L K I E DQE T TD EKR TNR - FDEP L NGP L GN I LSEP T FV TFL TW L TF I - - - - - - TRP V S L S - N TE LV N FER L NGQK V NGNR L RK RK RK QQ FA - I - V - - I GS A RE L L TSSE I GN - - NDDQK K Q - Y - L - Y QK Y I V F I V Y MV YLV HE HK HS HE I NG I QGV MG I QN I LE ME ME LE I HRD THRD SHRD I HRD L I L I A K - 225 172 203 205 A LNVE TEMFQ MD G V T NKQFR 271 217 242 246 L L L L L I I L SS TN EQ N I 357 304 329 338 L MR FLE F MK F TN T T T T LCG FCG FCG LCG 386 331 357 383 CL ML I L CL S T T C 422 370 403 416 KK L L NRML DS L L SNL L V E Q V 468 415 448 462 QV L A Q NSSS QQK L L P K T YS 514 460 494 508 - D DE - - E - TAG - GG RNE - - G YNRA YDDK YS S L YTSK V V V V D CWS D I WS D MW S D L WS D MK R K F Q D L L S E E N E S THEHR TPPSSSEHE P L GS QS YGD F S Q I S L QQQS S V S L E L QR L Q I T T V E ENV EN I DN I EN I I L - GE T S V I HGTGT V Q - GNGS F T - G E MQ RHPWL QDE QHPWF Y T V N H P W I K MS NHPWFND I MS K - - R I DE L GK I I I L P P P P K K K K SEK SEE SEE DDS 208 156 167 159 K K K K F GHS FGLA FGLA FGLA V WA E V LENE I KD FR I YWD K I - - - RDSQ RSN L SRS Y 311 258 283 292 TD SD TD AD E L L P 180 121 121 116 GK NS NS - K FD MD MD FE I I I I YN F I P YP I E P YHE GP YA F YS 137 75 82 71 L L L L K K K K T S GK S K GA GRGS L QA K YR - - - - L ME G G E YV E GGD F V S GGD K I DDGE DC L I - FH L - V LV D I QV - - YE - - DP RA - - - P - R R N D GK AK EK VK DD KHS DR I SSE LRDE Y S H YE I FKD FS F FDK Y A - L GV I L GCV MG C L A GV I A LD G I D ARD V LH L L V L LV L I F I L I S TA LP A TEQL S QS L S TDNK I F I YV YV YV 586 632 678 724 770 816 2.2 kb– H LHe 60 L C a M M L KO 5 Ra LT-4 62 ji SW A5 480 4 G 9 36 1 Sp l Th een ym Pr us o Te stat s e O tis va Sm ry C all i ol nt PB on est in L e (b) 2.2 kb– Current Biology similarity of mitotic control mechanisms in fission yeast and mammals, our attention focused on two kinases, Chk1 and Cds1, that are required for the DNA-damage and replication checkpoint in fission yeast [32,33]. An expressed sequence tag (EST) with significant homology to the cds1 gene of Schizosaccharomyces pombe was identified. The human cDNA predicts a translation product of 543 amino acids with a molecular weight of 61 kDa. The Identification of a human homolog of Cds1. (a) A human cDNA encoding an open reading frame of 534 amino acids with similarity to checkpoint kinases from S. pombe and S. cerevisiae was identified. Alignment of Cds1 homologs was generated using the CLUSTALW program. Amino-acid identities are boxed, conservative changes are shaded. Putative forkhead-associated domains are underlined. HsCds1 is human Cds1; SpCds1 is a checkpoint kinase from the fission yeast S. pombe; Rad53 and Dun1 are checkpoint kinases from the budding yeast S. cerevisiae. (b) Northern analysis of HsCds1. Blots containing 2 µg polyadenylated RNA from the indicated tissues or cell lines were probed with an HsCds1 probe. PBL, peripheral blood leukocytes. predicted human Cds1 (HsCds1) protein is 28% identical to the Cds1 protein of S. pombe, 28% identical to Rad53 and 27% identical to the Dun1 kinase of Saccharomyces cerevisiae. Sequence alignment of these homologs (Figure 3a) shows several regions of homology outside the kinase domain, including conservation of the forkheadassociated domain [34]. The human protein shows the same overall structure as Cds1 and Dun1 in that it lacks the long carboxy-terminal extension found in Rad53. Northern blot analysis with HsCds1 identified a single transcript of ~2.2 kb expressed in testis and in eight human cancer samples examined (Figure 3b). Cds1 and Chk1 fusion proteins phosphorylate and inhibit Cdc25 in vitro To facilitate biochemical analysis, amino-terminal histidine6 (His6)-tagged Cds1 and Chk1 fusion proteins were expressed in insect cells, affinity purified and incubated in extracts of HeLa cells in the presence of an ATP-regenerating system. After 30 minutes at 30°C, EDTA was added to inhibit kinases in the extract, and the rate of dephosphorylation and activation of Cdc2–Cyclin B was monitored as in Figure 1. Both His6–Cds1 and His6–Chk1 were found to significantly reduce the activation of Cdc2–Cyclin B in these assays (Figure 4a). The reduced activation of Cdc2 was dose dependent and required ATP (data not shown). Confirmation that Cdc2 was not irreversibly inhibited by His6–Chk1 or His6–Cds1 was shown by the activation that resulted when excess GST–Cdc25C was added after kinase treatment. Thus, both His6–Cds1 and His6–Chk1 can mimic the radiation-induced downregulation of Cdc25 seen in extracts. These experiments used HeLa cell lysates that had been clarified by centrifugation; therefore it seemed unlikely that changes in subcellular localization could account for the inactivation of Cdc25. Indirect mechanisms of inhibition could, however, not be excluded by this assay. We therefore used affinity-purified reagents (Figure 4c) to determine whether His6–Cds1 or His6–Chk1 can directly phosphorylate and inhibit GST–Cdc25 activity. GST–Cdc25 was incubated with either His6–Cds1, mock beads or His6–Chk1 in the presence of [γ-32P]ATP for 15 minutes at 30°C. Proteins were resolved by SDS–PAGE and visualized by autoradiography Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al. 5 Figure 4 (a) No ATP + ATP His6–Chk1 His6–Cds1 (d) 100 kDa 197– 97– Untreated 20 43– 0 0 30 30 0 30 30 0 30 30 0 30 30 Time (min) – – + – – + GST–Cdc25 +– – +– – 20 0 68– 40 40 28– GST (b) (e) GST–Cdc25 + His6–Chk1+PP2A 60 60 GST–Cdc25 + His6–Cds1 GST–Cdc25 + His6–Cds1+PP2A GST–Cdc25 + His6–Chk1 80 80 GST–Cdc25 100 Kinase activity Kinase activity G 120 ST – H Cdc is 25 6– H Ch is k1 6– C ds 1 (c) His6–Chk1 Cdc2 GST–Cdc25 0 5 10 10 10 10 10 His6–Cds1 His6–Chk1 10 GST–Cdc25 His6–Cds1 Cdc2 + – + + – – + + – – – + – GST–Cdc25 + His6–Cds1 – His –Chk1 6 5 10 10 10 10 10 10 10 GST–Cdc25 Current Biology His6–Cds1 and His6–Chk1 phosphorylate and inhibit Cdc25 in vitro. (a) His6–Cds1 and His6–Chk1 downregulate the dephosphorylation of Cdc2 in HeLa cell extracts. Recombinant His6–Cds1 or His6–Chk1 purified from insect cells was incubated with HeLa cell extracts in the presence of an ATP-regenerating system for 30 min at 30°C [57]. Control extracts incubated at 30°C in the presence of ATP (+ ATP) or maintained on ice for 30 min (no ATP) show similar kinetics of activation. Dephosphorylation and activation of Cdc2–Cyclin B was initiated by addition of excess EDTA at the zero time point. Data are the average of three experiments. (b) GST–Cdc25 purified from insect cells was incubated with either His6–Cds1, mock beads or His6–Chk1 in the presence of [γ-32P]ATP for 15 min at 30°C. GST–Cdc25 bound to GSH–sepharose was washed three times prior to addition of SDS. Proteins were resolved by SDS–PAGE and visualized by autoradiography. The authenticity of each band was confirmed by reprecipitation of the labeled bands with the relevant sera. (c) Affinitypurified GST–Cdc25, His6–Chk1 and His6–Cds1 were resolved by SDS–PAGE and visualized by Coomassie blue staining. (d) Both His6–Cds1 and His6–Chk1 inhibit purified GST–Cdc25. GST–Cdc25c from insect cells was incubated with His6–Cds1, His6–Chk1 or buffer in the presence of 1 mM ATP for 30 min at 30°C. The sample was split and either incubated alone or with PP2A for 30 min at 30°C. GST–Cdc25 was washed three times with phosphatase buffer containing 0.4 µM microcystin prior to incubation with Cdc2–Cyclin B immunoprecipitates from asynchronous HeLa cells. The histone H1 kinase activity of Cdc2–Cyclin B was determined. (e) Reduced GST–Cdc25 activity after phosphorylation by His6–Chk1 or by His6–Cds1. GST–Cdc25 was assayed by its ability to convert Cdc2 to the faster-migrating dephosphorylated form. In the presence of GST–Cdc25, the majority of Cdc2 is converted to the lower band of the triplet. Prior phosphorylation of GST–Cdc25 by His6–Chk1 or His6–Cds1 reduced the appearance of the dephosphorylated form. GST–Cdc25 was phosphorylated with increasing quantities of each kinase. The numbers refer to relative quantities of proteins added and are arbitrary. The results are representative of five independent experiments. (Figure 4b). As previously shown [35], GST–Cdc25 was phosphorylated by His6–Chk1. GST–Cdc25 was also phosphorylated by His6–Cds1 in vitro. GST–Cdc25 by His6–Cds1 or by His6–Chk1 inhibited the ability of GST–Cdc25 to activate Cdc2–Cyclin B (Figure 4d). Inhibition was dependent on the presence of ATP, was seen at a molar ratio of kinase to GST–Cdc25 of 1:100, and was reversed by treatment of GST–Cdc25 with protein phosphatase 2A (PP2A; Figure 4d). Experiments were performed to determine if Cdc25 phosphatase activity was effected by phosphorylation. In one set of experiments, GST–Cdc25 was assayed by its ability to activate the histone H1 kinase activity of Cdc2–Cyclin B immunoprecipitates. Phosphorylation of These results were somewhat surprising in the light of previous reports that Chk1 does not inactivate Cdc25 [19] 6 Current Biology, Vol 9 No 1 dc 25 activity [37,38] and extend them by showing that the negative regulation involves inactivation of the phosphatase activity and that a second checkpoint kinase also phosphorylates and inactivates Cdc25. ST –C C 6– Increased 14-3-3 binding is not required for inhibition of Cdc25 in vitro G is H H eL a ce ll l y sa te (a) hk 1 Figure 5 –14-3-3 – – (b) – + – – + + + His6–Chk1 (–ATP) – HeLa (–ATP) –GST–Cdc25 –14-3-3 – – Cdc2 – S 1 2 3 4 5 Current Biology Inhibition of GST–Cdc25 correlates with Chk1 phosphorylation and not with increased binding of 14-3-3 protein. (a) Purified GST–Cdc25 (~1 µg) and ~1 µg purified His6–Chk1 (see Figure 4) were probed with a broad specificity anti-14-3-3 rabbit antiserum; 2 µg HeLa cell lysate was run alongside for comparison. (b) GST–Cdc25C purified from Sf9 cells was not treated (lane 1), mock phosphorylated (lanes 2,3), or phosphorylated with His6–Chk1 (lanes 4,5). Beads were washed three times and either not treated (lane 1), or treated with HeLa cell extracts in the presence of an ATP regenerating system for 30 min at 30°C (lanes 2,4), or in the presence of 10 mM EDTA (lanes 3,5). Samples were washed three times and divided into two parts for the Cdc25 assay and for western analysis. The uppermost panel shows that a constant amount of GST–Cdc25 was recovered in each sample. The middle panel shows increased binding of 14-3-3 protein in samples that were exposed to HeLa cell extracts. The left and right hand sides of these panels have been cropped from a single gel to reflect the same order as the lowermost panel, which shows that dephosphorylation of Cdc2 is inhibited by Chk1 phosphorylation but not by increased 14-3-3 binding. S corresponds to the starting substrate. The data are representative of three separate experiments. and that Cds1 phosphorylates Wee1 in fission yeast [36]. We therefore used a second assay system in which dephosphorylation of Cdc2 was monitored by the disappearance of the slower migrating species of Cdc2 on gelmobility analysis (Figure 4e). In these assays, Cdc25 activity was measured in the presence of 10 mM EDTA and in the absence of ATP, conditions that eliminate the possibility of His6–Chk1 or His6–Cds1 phosphorylating Cdc2 or Cyclin B. GST–Cdc25 catalysed a reduction in the slower migrating phosphorylated forms of Cdc2. Prior phosphorylation of GST–Cdc25 by His6–Chk1 or His6–Cds1 led to a dose-dependent reduction in GST–Cdc25 activity (Figure 4e). These data confirm genetic predictions that Chk1 negatively regulates Cdc25 The 14-3-3 protein has been implicated in the regulation of Cdc25 by Chk1 [19,35]. Therefore, the possibility that 14-3-3 binding is required for inactivation of Cdc25 was tested. The GST–Cdc25 and the His6–Chk1 preparations used in these experiments were examined for the presence of 14-3-3 proteins by western analysis. As shown in Figure 5a, 14-3-3 protein was readily detectable in a sample containing ~1 µg affinity-purified GST–Cdc25 but not in the adjacent lane which contained ~1 µg His6–Chk1; 2 µg whole HeLa cell lysate was run on the same blot for comparison. The 14-3-3 proteins bind with high affinity to specific phosphoserine-containing proteins [39,40]; the absolute requirement for phosphorylation of a serine residue within the 14-3-3-binding motif suggested that GST–Cdc25 expressed in insect Sf9 cells is, at least partially, phosphorylated at the 14-3-3-binding site. In contrast, 14-3-3 proteins were not detected in the preparation of His6–Chk1. This finding suggested that inactivation of GST–Cdc25 occurs in the presence of a constant amount of 14-3-3 protein. GST–Cdc25 was also purified from bacteria free of 14-3-3 proteins; however, the very low specific activity of this protein made activity assays unreliable (data not shown). To test further the requirement for 14-3-3 binding in the inactivation of GST–Cdc25, we took advantage of the observation that GST–Cdc25 becomes phosphorylated on Ser216 and binds 14-3-3 protein on incubation in eukaryotic cell extracts ([18,41] and data not shown). As shown in Figure 5b, approximately fivefold more 14-3-3 protein was found in association with GST–Cdc25 following incubation in HeLa cell lysates in the presence of ATP, and no decrease in Cdc25 activity was detected in these samples. By contrast, GST–Cdc25 that was phosphorylated using purified His6–Chk1 was inhibited whether or not increased 14-3-3 protein was bound. Thus, inactivation of Cdc25 correlates with Chk1 phosphorylation and not with increased binding of 14-3-3 protein. The observations presented here — that irradiation does not lead to increased phosphorylation of Ser216 and that in vitro inactivation of Cdc25 does not require increased 14-33 binding — suggest that inactivation of Cdc25 may result from phosphorylation of sites other than Ser216. Previous in vitro mapping studies using mammalian Chk1 and Cdc25C showed that Ser216 is the major in vitro site of phosphorylation of Cdc25C by Chk1 [35]. Mapping experiments in vitro, using Chk1 and Cdc25 from other species, showed that phosphorylation of 14-3-3-binding sites by Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al. 7 Figure 6 (a) His6–Cds1– –p64Cds1 Cds1 is activated in response to DNA damage Having determined that His6–Cds1 inactivates Cdc25 in vitro and that Cdc25 is inactivated in vivo following DNA damage, we were interested in determining whether DNA damage might lead to modification or activation of human Cds1. To facilitate analysis of endogenous human Cds1, polyclonal antisera were raised against His6–Cds1. The specificity of the antisera was tested by immune-precipitation kinase assay in the presence of [γ-32P]ATP. The immunoprecipitates were examined by autoradiography following SDS–PAGE. A signal corresponding to His6–Cds1 was readily detected in immune-precipitation kinase assays from extracts of Sf9 cells infected with baculovirus encoding His6–Cds1 (Figure 6a), but not in cells infected with virus encoding His6–Chk1 nor in uninfected cells. A very weak signal corresponding to HsCds1 was detected in the immunoprecipitate from asynchronous HeLa cells; increased phosphorylation of HsCds1 was seen following irradiation. That this band represents Cds1 5 6 8 9 le – + – + H yd ro xy az o id in e Th ym As yn ch ro – + 7 ur ea 4 oc od 3 no us (b) – + γ-irradiation p64Cds1– GST–Cdc25– (c) 100 G2/M S G1 80 60 40 Hydroxyurea Hydroxyurea (irradiated) Nocodazole Nocodazole (irradiated) Thymidine Thymidine (irradiated) Asynchronous (irradiated) Asynchronous 20 0 Chk1 is highly conserved [42,43]. Nevertheless, Cdc25 is phosphorylated by Chk1 at a number of sites in Xenopus and fission yeast [42,43]. Future experiments will be necessary to determine whether inactivation of human Cdc25 is the result of phosphorylation of novel sites that are not readily detected by in vitro mapping experiments. Although these data indicate that 14-3-3 binding is not required for inactivation of Cdc25, an important role for 143-3 binding in determining the localization or sequestration of Cdc25 [19,44] is not precluded by this in vitro analysis. 2 N 1 Percentage of cells Activation of HsCds1 in response to DNA damage. (a) Anti-Cds1 antisera immunoprecipitates prepared from 100 µg (unless otherwise indicated) Sf9 cell extract were incubated in the presence of 10 µCi [γ-32P]ATP for 15 min at 30°C. Proteins were resolved by SDS–PAGE and visualized by autoradiography. Lane 1: uninfected Sf9 cells; lane 2: cells infected with recombinant virus encoding His6–Chk1; lane 3: cells infected with recombinant virus encoding His6–Cds1; lanes 4,5: 400 µg irradiated HeLa cell extract (lane 4) and asynchronous HeLa cell extract (lane 5); lanes 6,7: 400 µg irradiated HeLa cell extract boiled in 4% SDS, diluted 10-fold in phosphate-buffered saline containing 2% Triton X-100 and reprecipitated with anti-Cds1 antiserum in the absence (lane 6) or the presence (lane 7) of ~2 µg purified His6–Cds1; lanes 8,9: 5 and 10%, respectively, of the initial immune-complex kinase assay. (b) Anti-Cds1 antibody immunoprecipitates prepared from 400 µg HeLa cell extract were incubated in the presence of 10 µCi [γ-32P]ATP for 15 min at 30°C. The upper panel shows the phosphorylation of Cds1. The lower panel shows phosphorylation of GST–Cdc25 by Cds1 immunoprecipitates. GST–Cdc25 here corresponds to the first 145 amino acids of fission yeast Cdc25 expressed as a GST fusion protein in bacteria. No phosphorylation of GST–Cdc25 was detected when pre-immune serum was used (data not shown). Cells were accumulated with unreplicated DNA by addition of 2 mM thymidine for 17 h, or by addition of 2 mM hydroxyurea for 17 h. Cells were accumulated in M phase by growth in the presence of 100 ng/ml nocodazole for 18 h. Where indicated, cultures were exposed to 10 Gray ionizing radiation, 1 h prior to harvesting. (c) Cell-cycle position was determined by flow cytometric analysis of DNA content. Current Biology and not an associated protein was confirmed as we could reprecipitate the protein following denaturation in 4% SDS in the absence, but not in the presence, of excess purified His6–Cds1 (Figure 6a). The phosphorylation of Cds1 in vitro most likely represents autophosphorylation, in which case the increased signal reflects an increase in activity following irradiation, and the increased autophosphorylation should be paralleled by increased phosphorylation of an exogenous substrate. Cds1 was therefore immunoprecipitated and assayed both for autophosphorylation activity and for the ability to phosphorylate an exogenous substrate. For practical reasons, fission yeast Cdc25 fused to GST and expressed in bacteria provided a convenient substrate (data not shown). As shown in Figure 6b, Cds1 was activated by irradiation of asynchronous cells. The increased phosphorylation of p64Cds1 suggests that, like Rad53 and Dun1 [45–47], HsCds1 is activated in response to DNA damage. Fission yeast Cds1 has recently been shown to be activated in response to incompletely replicated DNA [36,48]. We therefore examined the consequences of arresting DNA synthesis on the activity of HsCds1. No significant increase in autophosphorylation or phosphorylation of GST–Cdc25 8 Current Biology, Vol 9 No 1 Figure 7 γ-irradiation ATM Cds1/Chk1 Cdc25 Cdc2–Cyclin B Inactive Cdc2–Cyclin B Active Current Biology Model of the DNA-damage G2 checkpoint in human cells. The product of the ATM gene is required for the efficient inactivation of Cdc25 in response to γ-irradiation. By analogy with model systems, the ATM protein is assumed to be upstream of Chk1 and Cds1 [2]. Phosphorylation of Cdc25 by Cds1 and/or Chk1 inhibits its activity and thereby prevents dephosphorylation and activation of Cdc2–Cyclin B. was seen in response to thymidine or hydroxyurea (Figure 6b). Both agents blocked DNA replication and led to the accumulation of cells with a 2N content of DNA (Figure 6c). Increased phosphorylation of p64Cds1 and GST–Cdc25 was detected following irradiation of thymidine-arrested or hydroxyurea-arrested cells. Finally, the effect of damaging DNA in cells that have been predominantly arrested outside S phase was tested. Cells were cultured in the presence of nocodazole for 20 hours prior to irradiation. Again a weak, but detectable, signal was seen in the non-irradiated sample, whereas irradiation of nocodazole-arrested cells led to increased activity (Figure 6b). In all circumstances tested, the increased in vitro phosphorylation of HsCds1 was closely paralleled by increased phosphorylation of the exogenous substrate. These data strongly suggest that Cds1 is activated in response to DNA damage. The possibility that the signal is due to an associated kinase, which also phosphorylates GST–Cdc25, cannot be totally excluded. Nevertheless, the observation that HsCds1 is modified or activated in response to DNA damage, but not in the presence of unreplicated DNA, contrast with the situation in fission yeast, suggesting a role for human Cds1 in the DNA-damage checkpoint rather than in the replication checkpoint. This study was based, in part, on the premise that checkpoint control proteins are conserved between organisms. That assumption has facilitated the cloning of a number of human checkpoint genes ([35] and this work). Despite the structural homology, some these proteins may not be true functional homologs; for example, in fission yeast, chk1 mutants are highly sensitive to DNA damage and are resistant to agents that block DNA replication [49], whereas mutants in the homologous Drosophila gene, grapes, respond normally to DNA damage but are sensitive to delayed DNA synthesis [50,51]. Likewise, for Cds1, fission yeast mutants are sensitive to agents that block replication but have normal responses to DNA damage [32], whereas the budding yeast homologs, Rad53 and Dun1, have functions in both the replication and DNAdamage responses [45,52]. In this study, we have defined a mechanism by which both human Cds1 and Chk1 might contribute to the G2–M checkpoint (Figure 7). The possibility that Cds1 carries out some of the other checkpoint/repair functions undertaken by its structural homologs remains to be explored. Materials and methods Cell lines Normal (GM637G) and AT-deficient (GM5849C) SV40-transformed human fibroblasts obtained from Coriell Institute for Medical Research, (Camden, NJ) were grown in DMEM supplemented with 15% fetal bovine serum, 100 µg/ml penicillin and streptomycin. For γ-irradiation experiments, cells were irradiated with a cesium137 source in a Gamma Cell 1000 at a rate of 3.8 Gray/min. For UV irradiation experiments, cells were washed once with PBS and irradiated at 254 nm in a UV Stratalinker 1800. Assays and western analysis Cells were lysed in ice-cold lysis buffer (50 mM Tris pH 7.4 containing 2 mM MgCl2, 1 mM phenylmethylsulphonyl fluoride, and 5 µg/ml leupeptin, pepstatin and aprotinin). Lysates were cleared by centrifugation at 14,000 × g for 10 min and the protein concentration of the supernatants determined using the Lowry assay. Dephosphorylation of Cdc2 was initiated by addition of 10 mM EDTA and incubation at 30°C. At the indicated time, the activity of Cdc2–Cyclin B was assayed by measuring the histone H1 kinase activity present in anti-Cyclin B immunoprecipitates [6]. For immunoblots, 400 µg cell lysate was immunoprecipitated using anti-Cyclin B antibody and resolved on 11% acrylamide–SDS gels. Monoclonal antibody against the PSTAIRE motif of Cdc2 was used to detect the different phospho-forms of Cdc2 (gift of S. Reed, TSRI). Polyclonal anti-14-3-3 serum (k-19) was from Santa Cruz Biotechnology. For flow cytometric analysis, cells were fixed in 70% ethanol prior to treatment with 100 µg/ml RNAse and 40 µg/ml propidium iodide as recommended by Becton Dickinson. Cells were analyzed on a Becton Dickinson FACSort. The proportion of cells in G1, S or G2/M was determined using CellQuest software. Recombinant baculoviruses and protein production Recombinant viruses encoding His6–Chk1, His6–Cds1, His6–Wee1, His6–Myt1, His6–Cdc2 and GST–Cdc25C were generated using the Bac-to-Bac expression system from Gibco/BRL. His6-fusion proteins were purified following the procedure described in [53]. GSH–sepharose beads were incubated for 15 min in Sf9 extracts. The beads were collected by centrifugation and washed three times with lysis buffer (50 mM Tris pH 8.0, 5 mM EDTA, 150 mM NaCl, 0.1% NP40, 5% glycerol, 0.1% β-mercaptoethanol and protease inhibitors). Beads were washed twice with kinase assay buffer (50 mM Tris pH 7.4 10 mM MgCl2) prior to phosphorylation reactions or twice with phosphatase assay buffer (50 mM imidazole pH 7.4, 5 mM EDTA and 0.1% β-mercaptoethanol) prior to phosphatase assays. Phosphorylated Cdc2 was purified from Sf9 cells that had been simultaneously infected with recombinant baculoviruses encoding His6–Cdc2, His6–Wee1, His6–Myt1 and GST–Cyclin B [54]. His6–Cdc2 complexed to Cyclin B was purified using GSH beads using the conditions described for GST–Cdc25 except that 1 mM VO4 was included in the lysis buffer. The catalytic subunit of PP2A was purified from rabbit Research Paper Checkpoint kinases directly inactivate Cdc25 Blasina et al. skeletal muscle [55]. Western analysis showed that quadruple infection resulted in phosphorylation of the majority of Cdc2–GST–Cyclin B at one or both inhibitory sites. Cloning A search for sequences similar to S. pombe cds1+ was carried out using the TBLASTN program. A human EST (No. 864164) was identified in the proprietary LifeSeq® database (Incyte Pharmaceuticals Inc., Palo Alto, USA). Sequence analysis of the 1.3 kb insert revealed an incomplete open reading frame which was highly similar to the S. pombe cds1. Approximately 650 nucleotides of novel 5′ DNA sequence were obtained by 5′ rapid amplification of cDNA ends (RACE). Termination codons were present in all three reading frames in the 120 nucleotides immediately 5′ to the putative HsCds1 initiation codon, indicating that the complete coding region had been isolated. The sequence shown here is identical to two partial sequences in the National Centre for Biotechnology Information (NCBI) databases. The EST (AA285249) and genomic sequence (H55451) most likely encode the same protein product. The EMBL accession number for HsCds1 is AJ131197. A human EST encoding a protein with homology to S. pombe Chk1 [33] and to Drosophila Grapes [51] was identified by BLAST analysis of the NCBI database. Clones were retrieved from ATCC (IMAGE Clone ID 663485) and sequenced. The human Chk1 homolog used here is identical to the one described by Sanchez et al. [35], with the exception of three amino-acid substitutions (H/N at amino acid 64, R/H at 141 and N/M at 410 using single-letter amino-acid code). Antibodies Antibodies to HsCds1 were generated by immunizing a rabbit with His6–Cds1 purified from Sf9 cells according to [56]. The resulting sera immunoprecipitates an active kinase of the expected molecular weight from Sf9 cells infected with His6–Cds1 virus but not from uninfected Sf9 cells or from cells infected with His6–Chk1 virus (data not shown). Acknowledgements We thank Beth Furnari and Paul Russell for providing GST–Cdc25. This work was supported by DOD grant DAMD1794-J-4227 grant to C.H.McG. References 1. Hartwell L, Weinert T: Checkpoints: controls that ensure the order of cell cycle events. Science 1989, 246:629-634. 2. Elledge S: Cell cycle checkpoints; preventing an identity crisis. Science 1997, 274:1664-1671. 3. El-Deiry WA, Tokino T, Velculescu VE, Levy DB, Parsons R, Trent JM, et al.: WAF1, a potential mediator of p53 tumor suppression. Cell 1993, 75:817-825. 4. Harper JW, Adami R, Wei N, Keyomarsi K, Elledge SJ: The p21 CDKinteracting protein Cip1 is a potent inhibitor of G1 cyclin dependent kinase. Cell 1993, 75:805-816. 5. Dulic V, Kaufmann WK, Wilson SJ, Tlsty TD, Lees E, Harper JW, et al.: p53-dependent inhibition of cyclin E/Cdk2 kinase activity in human fibroblasts during radiation-induced G1 arrest. Cell 1994, 76:1013-1023. 6. Blasina A, Paegle ES, McGowan CH: The role of inhibitory phosphorylation of CDC2 following DNA replication block and radiation induced damage in HeLa Cells. Mol Biol Cell 1997, 8:1-11. 7. Jin P, Gu Y, Morgan DO: Role of inhibitory CDC2 phosphorylation in radiation-induced G2 arrest in human cells. J Cell Biol 1996, 134:963-970. 8. Poon YC, Jiang W, Toyoshima H, Hunter T: Cyclin-dependent kinases are inactivated by a combination of p21 and Thr14/Tyr15 phosphorylation after UV-induced DNA damage. J Biol Chem 1996, 271:13283-13291. 9. Poon R, Chau M, Yamashita K, Hunter T: The role of Cdc2 feedback loop control in the DNA damage checkpoint in mammalian cells. Cancer Res 1997, 57:5168-5178. 10. Sadhu K, Reed SI, Richardson H, Russell P: Human homolog of fission yeast cdc25 mitotic inducer is predominantly expressed in G2. Proc Natl Acad Sci USA 1990, 87:5139-5143. 9 11. Nagata A, Igarashi M, Jinno S, Suto K, Okayama H: An additional homolog of the fission yeast cdc25+ gene occurs in humans and is highly expressed in some cancer cells. New Biol 1991, 3:959-968. 12. Galaktionov K, Beach D: Specific activation of cdc25 tyrosine phosphatases by B-type cyclins: evidence for multiple roles of mitotic cyclins. Cell 1991, 67:1181-1194. 13. Hoffmann I, Draetta G, Karsenti E: Activation of the phosphatase activity of human cdc25A by a cdk2-cyclin E dependent phosphorylation at the G1/S transition. EMBO J 1994, 13:4302-4310. 14. Jinno S, Nagat A, Igarashi M, Kanaoka Y, Nojima H, Okayama H: Cdc25A is a novel phosphatase functioning early in the cell cycle. EMBO J 1994, 13:1549-1556. 15. Nishijima H, Nishitani H, Seki T, Nishimoto T: A dual-specificity phosphatase Cdc25B is an unstable protein and triggers p34cdc2/Cyclin B activation in hamster BHK21 cells arrested with hydroxyurea. J Cell Biol 1997, 138:1105-1116. 16. Millar JBA, Blevitt J, Gerace L, Sadhu K, Featherstone C, Russell P: p55CDC25 is a nuclear protein required for the initiation of mitosis in human cells. Proc Natl Acad Sci USA 1991, 88:10500-10504. 17. Gabrielli BG, De Souza CP, Tonks ID, Clark JM, Hayward NK, Ellem KA: Cytoplasmic accumulation of cdc25B phosphatase in mitosis triggers centrosomal microtubule nucleation in HeLa cells. J Cell Sci 1996, 7:1081-1093. 18. Ogg S, Gabrielli B, Piwnica-Worms H: Purification of a serinekinase that associates with and phosphorylates human Cdc25C on serine 216. J Biol Chem 1994, 269:30461-30469. 19. Peng C-Y, Graves PR, Thoma RS, Wu Z, Shaw AS, Piwnica-Worms H: Mitotic and G2 checkpoint control: regulation of 14-3-3 protein binding by phosphorylation of Cdc25C on serine-216. Science 1997, 277:1501-1505. 20. Hawley RS, Friend SH: Strange bedfellows in even stranger places: the role of ATM in meiotic cells, lymphocytes, tumors, and its functional links to p53. Genes Dev 1996, 10:2383-2388. 21. Banin S, Moyal L, Shieh S, Taya Y, Anderson CW, Chessa L, et al.: Enhanced phosphorylation of p53 by ATM in response to DNA damage. Science 1998, 281:1674-1677. 22. Hartley K, Gell D, Smith G, Zhang H, Divecha N, Connelly M, et al.: DNA-dependent protein kinase catalytic subunit: a relative of phosphatidylinositol 3-kinase and the ataxia telangiectasia gene product. Cell 1995, 82:849-856. 23. Price B, Youmell MB: The phosphatidylinositol 3-kinase inhibitor wortmannin sensitizes murine fibroblasts and human tumor cells to radiation and blocks induction of p53 following DNA damage. Cancer Res 1996, 56:246-250. 24. Zampetti-Bosseler F, Scott D: Cell death, chromosome damage and mitotic delay in normal, ataxia telangiectasia and retinoblastoma fibroblasts after X-irradiation. Int J Radiat Biol 1981, 39:547-558. 25. Painter RB, Young BR: Radiosensitivity in ataxia-telangiectasia: a new explanation. Proc Natl Acad USA 1980, 77:7315-7317. 26. Canman CE, Wolff AC, Chen C-Y, Fornace JAJ, Kastan MB: The p53dependent G1 cell cycle checkpoint pathway and AtaxiaTelangiectasia. Cancer Res 1994, 54:5054-5058. 27. Kastan MB, Zhan Q, El-Deiry WS, Carrier F, Jacks T, Walsh WV, et al.: A mammalian cell cycle checkpoint pathway utilizing p53 and GADD45 is defective in Ataxia-Telangiectasia. Cell 1992, 71:587-589. 28. Baskaran R, Wood LD, Whitaker LL, Canman CE, Morgan SE, Xu Y, et al.: Ataxia telangiectasia mutant protein activates c-Abl tyrosine kinase in response to ionizing radiation. Nature 1997, 387:516-519. 29. Shafman T, Khanna KK, Kedar P, Spring K, Kozlov S, Yen T, et al.: Interaction between ATM protein and cABL in response to DNA damage. Nature 1997, 387:520-523. 30. Khanna KK, Lavin MF: Ionizing radiation and UV induction of p53 protein by different pathways in ataxia-telangiectasia cells. Oncogene 1993, 8:3307-3312. 31. Xu Y, Baltimore D: Dual roles of ATM in the cellular response to radiation and in cell growth control. Genes Dev 1996, 10:2401-2410. 32. Murakami H, Okayama H: A kinase from fission yeast responsible for blocking mitosis in S phase. Nature 1995, 374:817-819. 33. Walworth N, Davey S, Beach D: Fission yeast chk1 protein kinase links the rad checkpoint pathway to cdc2. Nature 1993, 363:368-371. 10 Current Biology, Vol 9 No 1 34. Hofmann K, Bucher P: The FHA domain: a putative nuclear signalling domain found in protein kinases and transcription factors.Trends Biochem Sci 1995, 20:347-349. 35. Sanchez Y, Wong C, Thoma RS, Richman R, Wu Z, Piwnica-Worms H, Elledge SJ: Conservation of the Chk1 checkpoint pathway in mammals: linkage of DNA damage to Cdk regulation through cdc25. Science 1997, 277:1497-1501. 36. Boddy MN, Furnari B, Mondesert O, Russell P: Replication checkpoint enforced by kinases Cds1 and Chk1. Science 1998, 280:909-912. 37. Furnari B, Rhind N, Russell P: Cdc25 mitotic inducer targeted by Chk1 DNA damage checkpoint kinase. Science 1997, 277:1495-1497. 38. Weinert T: A DNA damage checkpoint meets the cell cycle engine. Science 1997, 277:1450. 39. Yaffe MB, Rittinger K, Volinia S, Caron P, Aitken A, Leffers H, et al.: The structural basis for 14-3-3: phosphopeptide binding specificity. Cell 1997, 91:961-971. 40. Yaffe M, Schutkowski M, Shen M, Zhou X, Stukenberg P, Rahfeld J-U, et al.: Sequence-specific and phosphorylation-dependent proline isomerization: a potential mitotic regulatory mechanism. Science 1997, 278:1957-1960. 41. Peng CY, Graves PR, Ogg S, Thoma RS, Byrnes MJ 3rd, Wu Z, et al.: C-TAK1 protein kinase phosphorylates human Cdc25C on serine 216 and promotes 14-3-3 protein binding. Cell Growth Differ 1998, 9:197-208. 42. Kumagai A, Guo Z, Emami KH, Wang SX, Dunphy WG: The Xenopus chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts. J Cell Biol 1998, 142:1559-1569. 43. Zeng Y, Forbes KC, Wu Z, Moreno S, Piwnica-Worms H, Enoch T: Replication checkpoint requires phosphorylation of the phosphatase Cdc25 by Cds1 or Chk1. Nature 1998, 395:507-510. 44. Ford JC, Al-Khodairy F, Fotou E, Sheldrick KS, Griffiths JF, Carr AM: 14-3-3 protein homologs required for the DNA damage checkpoint in fission yeast. Science 1994, 265:533-535. 45. Zhou Z, Elledge SJ: DUN1 encodes a protein kinase that controls the DNA damage response in yeast. Cell 1993, 75:1119-1127. 46. Sun Z, Fay DS, Marini F, Foiani M, Stern DF: Spk1/Rad53 is regulated by Mec1-dependent protein phosphorylation in DNA replication and damage checkpoint pathways. Genes Dev 1996, 10:395-406. 47. Sanchez Y, Desany BA, Jones WJ, Liu Q, Wang B, Elledge SJ: Regulation of RAD53 by the ATM-like kinases MEC1 and TEL1 in yeast cell cycle checkpoint pathways. Science 1996, 271:357-360. 48. Lindsay HD, Griffiths DJ, Edwards RJ, Christensen PU, Murray JM, Osman F, et al.: S-phase-specific activation of Cds1 kinase defines a subpathway of the checkpoint response in Schizosaccharomyces pombe. Genes Dev 1998, 12:382-395. 49. Al-Khodairy F, Carr AM: DNA repair mutants defining G2 checkpoint pathways in Schizosaccharomyces pombe. EMBO J 1992, 11:1343-1350. 50. Sibon OCM, Stevenson VA, Theurkauf WE: DNA-replication, checkpoint at the Drosophila midblastula transition. Nature 1997, 388:93-97. 51. Fogarty P, Campbell SD, Abu-Shumays R, de Staint Phall, B, Yu KR, Uy GL, et al.: The Drosophila grapes gene is related to checkpoint gene chk1/rad27 and is required for late syncytial division fidelity. Curr Biol 1997, 7:418-426. 52. Allen JB, Zhou Z, Siede W, Friedberg EC, Elledge SJ: The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev 1994, 8:2401-2415. 53. Kumagai A, Dunphy WG: Control of the CDC2/Cyclin B complex in Xenopus egg extracts arrested at a G2/M checkpoint with DNA synthesis inhibitors. Mol Biol Cell 1996, 6:199-213. 54. Parker LL, Piwnica-Worms H: Inactivation of the p34cdc2-cyclin B complex by the human WEE1 tyrosine kinase. Science 1992, 257:1955-1957. 55. Cohen P, Alemany S, Hemmings BA, Resink TJ, Stralfors P, Tung HY: Protein phosphatase-1 and protein phosphatase-2A from rabbit skeletal muscle. Methods Enzymol 1988, 159:390-408. 56. Harlow E, Lane D: Antibodies. Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press; 1988. 57. McGowan CH, Russell P: Cell cycle regulation of human WEE1. EMBO J 1995, 14:2166-2175. Because Current Biology operates a ‘Continuous Publication System’ for Research Papers, this paper has been published on the internet before being printed. The paper can be accessed from http://biomednet.com/cbiology/cub — for further information, see the explanation on the contents page.