Oncogene (2009) 28, 1366–1378 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc ORIGINAL ARTICLE The cell cycle checkpoint CHK2 mediates DNA damage-induced stabilization of TTK/hMps1

Y-H Yeh1,3, Y-F Huang1,2, T-Y Lin1 and S-Y Shieh1

1Institute of Biomedical Sciences, Academia Sinica, Taipei, Taiwan and 2Institute of Molecular and Cellular Biology, National Tsing Hua University, Hsinchu, Taiwan

Cell cycle progression is monitored constantly to ensure phorylation and dephosphorylation. These proteins faithful passage of genetic codes and genome stability. We include damage recognition sensors, adapters or media- have demonstrated previously that, upon DNA damage, tors, signal transducers and effectors that mediate TTK/hMps1 activates the checkpoint kinase CHK2 by cellular responses such as growth arrest, DNA repair phosphorylating CHK2 at Thr68. However, it remains to or apoptosis (Branzei and Foiani, 2005; Niida and be determined whether and how TTK/hMps1 responds to Nakanishi, 2006; Bartek and Lukas, 2007). DNA damage. In this report, we present evidence that One of the effector is CHK2, a mammalian TTK/hMps1 can be induced by DNA damage in normal homolog of the budding yeast (Saccharomyces cerevi- human fibroblasts. Interestingly, the induction depends on siae) Rad53 and the fission yeast (Schizosaccharomyces CHK2 because CHK2-targeting small interfering RNA or pombe) Cds1 (Allen et al., 1994; Murakami and a CHK2 inhibitor abolishes the increase. Such induction Okayama, 1995; Antoni et al., 2007). In yeast, Rad53 is mediated through phosphorylation of TTK/hMps1 at is activated by both DNA damage and replication Thr288 by CHK2 and requires the CHK2 SQ/TQ cluster block, whereas Cds1 functions mainly in the replication domain/forkhead-associated domain. In cells, TTK/ checkpoint pathway. In mammals, a variety of stress hMps1 phosphorylation at Thr288 is induced by DNA signals trigger the activation of CHK2 directly or damage and forms nuclear foci, which colocalize partially indirectly through two related kinases ataxia telangiec- with c-H2AX. Reexpression of TTK/hMps1 T288A tasia mutated (ATM) and Rad3-related kinase (ATR; mutant in TTK/hMps1-knockdown cells causes a defect Hurley and Bunz, 2007). Upon sensing the damage in G2/M arrest, suggesting that phosphorylation at this signals, ATM and ATR phosphorylate CHK2 on site participates in the proper checkpoint execution. Our Thr68, which then leads to activation of CHK2. Once study uncovered a regulatory loop between TTK/hMps1 activated, CHK2 phosphorylates its downstream targets and CHK2 whereby DNA damage-activated CHK2 such as p53, Cdc25 and FOXM1, leading to growth may facilitate the stabilization of TTK/hMps1, therefore arrest, apoptosis, or DNA repair (Iliakis et al., 2003; maintaining the checkpoint control. Antoni et al., 2007). Oncogene (2009) 28, 1366–1378; doi:10.1038/onc.2008.477; TTK/hMps1/PYT is a human homolog of the yeast published online 19 January 2009 Mps1 family of protein kinases that are highly expressed in proliferating cells (Mills et al., 1992; Lindberg et al., Keywords: checkpoint; CHK2; TTK/hMps1 1993; Winey and Huneycutt, 2002). It was first discovered through screening expression libraries with antiphosphotyrosine antibodies, and later shown to be a dual specificity protein kinase that phosphorylates Introduction tyrosine or serine/threonine residues (Mills et al., 1992). TTK/hMps1 is a cell cycle-regulated kinase that Genome stability is maintained through an intricate displays maximum expression and activity in the M network of signaling pathways, notably cell cycle phase (Hogg et al., 1994). The Mps1 family of kinases checkpoints, which delay or stop the progression of has been described in several other species including the cell cycle in the event of DNA damage or replication mouse (esk/mMps1; Douville et al., 1992), S. cerevisiae block. Many proteins are involved, and these form (Mps1p; Winey et al., 1991), S. pombe (Mph1p; He cascades of signaling networks, mostly through phos- et al., 1998), Xenopus laevis (xMps1; Abrieu et al., 2001), zebrafish (mps1/ncp; Poss et al., 2002) and Drosophila Correspondence: Dr S-Y Shieh, Institute of Biomedical Sciences, (Mps1; Fischer et al., 2004). In budding yeast (S. Academia Sinica, 128 Section 2, Academia Road, Taipei 115, Taiwan. cerevisiae), this kinase is required for spindle pole E-mail: [email protected] duplication and spindle assembly checkpoint, whereas 3Current address: Graduate Institute of Microbiology, College of in mammals, although involved in spindle checkpoint, Medicine, National Taiwan University, 1 Section 1, Jen-Ai Road, Taipei 100, Taiwan. the kinase is thought to be involved in centrosome Received 11 July 2008; revised 1 December 2008; accepted 15 December duplication, although this is debated (Weiss and Winey, 2008; published online 19 January 2009 1996; Fisk and Winey, 2001; Stucke et al., 2002; Fisk TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1367 et al., 2003). During mitosis, Mps1 localizes to Increased expression and autophosphorylation of kinetochores and is required for proper TTK/hMps1 upon coexpression of CHK2 in 293T cells alignment and segregation as well as the recruitment of To dissect the mechanisms underlying the DNA other mitotic checkpoint proteins in the event of damage-induced increase in TTK/hMps1 expression, unattached chromosome or uneven tension at the plasmids expressing HA-tagged TTK/hMps1 and myc- kinetochores (Abrieu et al., 2001; Stucke et al., 2004; tagged CHK2 were cotransfected into 293T cells. Vigneron et al., 2004; Jelluma et al., 2008). The list of Coexpression of wild type (WT) but not kinase deficient Mps1 functions has been expanded recently to (KD) myc-CHK2 markedly increases HA-TTK/hMps1, include meiosis (Straight et al., 2000; Poss et al., 2004), regardless of whether the latter was WT or KD cytokinesis (Fisk et al., 2004), development (Poss et al., (Figure 2a). Such increase was partly contributed by 2002) and the response to DNA damage (Wei et al., 2005). increased autophosphorylation of WT TTK/hMps1. In our previous report (Wei et al., 2005), we This was evidenced by a significant increase in a slower demonstrated the physical interaction between TTK/ migrating, hyperphosphorylated form, which could be hMps1 and CHK2, and we established a functional reduced to the faster migrating form by l-phosphatase relationship between the two proteins. Like ATM and treatment (Figure 2b). These results are consistent with ATR, TTK/hMps1 phosphorylates CHK2 at Thr68. our observations on endogenous proteins (Figure 1) and Downregulation of endogenous TTK/hMps1/hMps1 by suggest the existence of a positive regulatory role of small interfering RNA (siRNA) or dominant-negative CHK2 on TTK/hMps1. competition by overexpression of a kinase-defective To determine whether the increase was a result of TTK/hMps1 mutant compromises DNA damage-in- stabilization of the TTK/hMps1 protein by CHK2, duced CHK2 Thr68 phosphorylation, and results in cycloheximide (CHX) was added in the coexpression defective cell cycle arrest (Wei et al., 2005). These results experiment to examine the half-life of TTK/hMps1. In suggest that TTK/hMps1, like ATM, functions up- the presence of CHX, which inhibits de novo protein stream of CHK2 in the response to genotoxic assaults. synthesis, transfected TTK/hMps1 was more stable Many questions remain unanswered. For example, if when coexpressed with WT CHK2 than with KD TTK/hMps1 plays the role of a CHK2 activator, the CHK2, indicating that the half-life of TTK/hMps1 mechanism underlying the activation of TTK/hMps1/ was significantly shorter when CHK2 was compromised hMps1 or its induction by DNA damage becomes an (Figures 2c and d). To exclude the possibility that the imminent issue to resolve. Here we describe our efforts observation might be an artifact from overexpression, to address this issue. We present evidence that TTK/ we also examined the stability of endogenous TTK with hMps1 is induced by DNA damage in normal human or without CHK2 ablation after IR. Similarly, the half- fibroblasts and that such response is under reciprocal life of endogenous TTK/hMps1 in CHK2-knockdown regulation by CHK2 phosphorylation at one critical MRC5 cells was also reduced, from about 6 h in residue, Thr288, in the N-terminal domain of TTK/ control siRNA-transfected cells to 3 h in CHK2-ablated hMps1. cells (Figures 2e and f). Taken together, these results demonstrate that DNA damage induces stabilization of TTK/hMps1 and that this effect is mediated through Result CHK2.

Induction of TTK/hMps1 after DNA damage requires CHK2 CHK2 SCD and FHA domains, in addition to the kinase In an attempt to investigate how TTK/hMps1 is activity, are essential in the induction of TTK/hMps1 regulated by DNA damage, we examined the levels of To identify the regions in CHK2 that are required to TTK/hMps1 before and after ionizing radiation (IR) in induce TTK/hMps1, several CHK2 mutants were made normal human fibroblasts, IMR90 and MRC5 cells (Figure 3a), all with reported compromised CHK2 (Figures 1a and b). In these cells, TTK/hMps1 was activity in vitro (Xu et al., 2002; Figure 3a). These transiently increased between 1 and 4 h after IR in mutants and WT CHK2 were cotransfected with HA- IMR90 and between 0.5 and 3 h after IR in MRC5, at TTK/hMps1 into 293T cells, and the lysates were which times CHK2 Thr68 phosphorylation was also analysed by western blotting. In contrast to WT induced. A similar increase was observed in IMR90 after CHK2, neither the forkhead-associated (FHA) domain camptothecin treatment (Figure 1c), except that the deletion mutant (DFHA, amino acids 76–213 deleted) response lasted longer. The ATM-CHK2 pathway nor the SQ/TQ cluster domain (SCD) deletion mutant mediates the checkpoint response after IR (Hurley and (DSCD, amino acids 19–75 deleted) was capable of Bunz, 2007). To investigate the possibility of reciprocal inducing TTK/hMps1 (Figure 3b, lanes 1–9). A CHK2 regulation of TTK/hMps1 by CHK2, CHK2 in MRC5 mutant with Thr68 changed to Ala-induced TTK/ was downregulated with siRNA before irradiation. As hMps1 at reduced levels (Figure 3b, lanes 10–12). To demonstrated in Figure 1d, whereas TTK/hMps1 was seek correlation with their kinase activity, myc-tagged induced by IR in control siRNA-transfected cells, its WT CHK2 or mutants were immunoprecipitated from levels remained unchanged in CHK2-ablated cells, transfected 293T cells, and the kinase reactions were suggesting that CHK2 is required for a proper response performed in vitro using recombinant GST-Cdc25A as of TTK/hMps1 to radiation damage. the substrate. Unexpectedly, although DFHA mutant

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1368

Figure 1 CHK2-dependent induction of TTK/hMps1 by DNA damage in normal human fibroblasts, MRC5 and IMR90. Cells growing in the exponential phase were irradiated with 4 Gy of X-ray (a, b) or treated with 0.5 mM camptothecin (CPT; c), and collected at the time points indicated. Lysates were examined by SDS–polyacrylamide gel electrophoresis (PAGE) followed by western blotting using the indicated antibodies. (d) TTK/hMps1 induction by ionizing radiation (IR) is dependent on CHK2. Normal human fibroblasts, MRC5, were transfected with control small interfering RNA (siRNA) or CHK2-targeting siRNA for about 40 h and then treated with IR. Cells were harvested at 1 and 2 h after irradiation, and the total proteins were analysed as described above.

was largely defective in phosphorylating Cdc25A, the autophosphorylation of CHK2 (Figure 3e) at least DSCD mutant was only slightly defective. In addition, in vitro. the immunoprecipitated T68A mutant phosphorylated the substrate and autophosphorylated almost as well as did WT CHK2 (Figure 3c). This result indicates that the The N-terminal domain of TTK/hMps1 is responsible kinase activity of CHK2, although required, is not the for the induction by CHK2 only determinant for inducing TTK/hMps1. To identify the region in TTK/hMps1 that responds to Other factors such as direct protein–protein interac- CHK2, we constructed two TTK/hMps1 truncation tion were examined further by an in vitro GST pull- mutants: one encompassing amino acids 1–386 (TTKN) down assay using GST or GST-TTK/hMps1 fusion and and the other harboring amino acids 387–857 (TTKC). recombinant purified His-tagged WT or mutant CHK2 Similar to WT TTK/hMps1, the level of TTKN (1–386) (KD, DFHA, DSCD). Results indicated that full-length was increased significantly by WT CHK2 but not by CHK2 KD but not WT could bind TTK/hMps1 CHK2KD (Figure 4a, lanes 1–3 and 7–9). In contrast, (Figure 3d). In contrast, neither DFHA nor DSCD the TTKC mutant was not induced (Figure 4a, lanes could bind, suggesting that both the FHA and SCD 4–6). We further divided the TTKN (amino acids 1–386) domains are required for the interaction. Indeed, a mutant into two domains: one containing amino acids recombinant protein harboring these two domains 1–193 and the other containing amino acids 194–386. (SCD þ FHA, Figure 3d) was able to interact with The TTKN (194–386) construct was able to respond to TTK/hMps1. CHK2, whereas the TTKN (1–193) construct behaved The inability of the recombinant WT CHK2 to bind similarly to TTKC and remained inert to CHK2 TTK/hMps1 was intriguing, and the possibility that this (Figure 4b). The inability of TTKC and TTKN may be because of its autophosphorylation in bacteria (1–193) to respond to CHK2 was not because of was further explored. Indeed, after it was treated with defective translation, because all constructs were driven l-phosphatase, it now became capable of interacting by the same translation initiation sequence and were with GST-TTK/hMps1 almost as well as the KD N-terminally tagged with the same HA epitope as the mutant (Figure 3e). WT construct. Furthermore, both constructs, as well as On the basis of these analyses, we conclude that both WT TTK/hMps1, gave rise to products of the expected the kinase activity and protein–protein interaction are sizes in the in vitro reticulocyte translation system required for CHK2 to induce TTK/hMps1 (Figure 3d); (Supplementary Figure S1). However, TTKC and and the interaction may be negatively regulated by TTKN (1–193) were expressed consistently at levels

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1369

Figure 2 CHK2 enhances the stability of TTK/hMps1. (a) Increased expression of TTK upon coexpression of CHK2 in 293T cells. HA-tagged wild-type (WT) or kinase-defective (KD) TTK/hMps1 (D647A) was cotransfected with either WT or KD myc-tagged CHK2 into 293T cells. Protein expression was detected by anti-HA or anti-myc antibodies. (b) Hyperphosphorylation of TTK/hMps1. Lysates coexpressing WT TTK/hMps1 and WT CHK2 were treated with l-phosphatase in the presence (lane 3) or absence (lane 2) of the inhibitor orthovanadate. The slower migrating form of TTK/hMps1 was converted to a faster migrating form, an indication of protein phosphorylation. (c) TTK/hMps1 is more stable when cotransfected with WT than with KD CHK2. Transfection experiments were performed in 293T cells with HA-TTK and myc-CHK2. Cycloheximide (CHX) was added at a final concentration of 20 mg/ml. Levels of TTK/hMps1 from three experiments were quantified and normalized to those of actin and shown in (d). (e) The half-life of TTK/hMps1 in ionizing radiation (IR)-treated, CHK2-depleted cells is reduced. MRC5 cells were transfected with control or chk2 small interfering RNA (siRNA), and irradiated with IR 40 h after transfection. One hour after IR, CHX was added, and cells were collected 2, 4 or 6 h later. The results from three experiments were quantified and shown in (f). lower than the WT construct (Supplementary Figure result, Ser281, Thr288 and Ser291 were identified as the S1), perhaps reflecting the intrinsic instability of these probable sites of phosphorylation. Figure 4d illustrates two truncated proteins. one analysis identifying the Ser281 and Thr288 sites. Ser281 and Thr288 were then mutated to Ala in the context of TTKN (194–290), and their roles in the Mutation of TTK/hMps1 Thr288 to Ala mitigates response to CHK2 were examined by cotransfection CHK2-mediated TTK/hMps1 stabilization experiments using 293T cells. Compared with the WT Whether TTKN (194–386) can be phosphorylated by construct, the S281A mutant was less inducible, and the CHK2 and therefore be stabilized was investigated using T288A mutant was the least induced, by CHK2 an in vitro kinase assay with bacteria expressed, purified (Figure 4e). In the context of the full-length TTK/ His-CHK2 as the kinase and recombinant His-TTKN hMps1 protein, however, only the T288A mutant (194–386) as the substrate. As demonstrated in showed diminished induction by CHK2 (Figure 4f). Figure 4c, TTK/hMps1N (194–386) was phosphorylated The level of induction with the S291A mutant was efficiently by CHK2 in a time-dependent manner. indistinguishable from that of the WT (data not shown). We then subjected the products of the in vitro kinase Consistently, the half-life of the T288A mutant was reaction to trypsin digestion, followed by liquid significantly shorter than that of the WT when they were chromatography and tandem mass spectrometry to coexpressed with CHK2 (Figure 4g). These results identify the phosphorylated amino-acid residues. As a suggest that Thr288 is the critical CHK2 phosphorylation

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1370

Figure 3 Induction of TTK/hMps1 by CHK2 requires the SQ/TQ cluster domain (SCD)/forkhead-associated (FHA) domain of CHK2. (a) Schematic representations of the full-length (wild type (WT)) and various mutants of CHK2. DSCD, amino acids 19–75 deleted; DFHA, amino acids 77–213 deleted; SCD þ FHA, amino acids 19–175. (b) Full-length CHK2, but not the FHA or SCD domain-deleted mutant, induces TTK/hMps1. HA-TTK and myc-CHK2, either WT or the indicated mutants, were cotransfected into 293T cells as described in Figure 2. Cell lysates were prepared 40–44 h after transfection and analysed by western blotting. CHK2 T68A was a point mutant of CHK2 with Thr68 mutated to Ala. (c) Kinase activities of exogenously expressed WT and mutant CHK2. Lysates from (b) were immunoprecipitated with anti-myc, and the activity of CHK2 in the immunoprecipitate was assessed by an in vitro kinase assay using GST-Cdc25A (amino acids 101–140) as the substrate. (d) TTK/hMps1–CHK2 interaction requires the FHA and the SCD domains of CHK2. GST pull-down assays were performed with the indicated purified His-tagged CHK2 proteins and either GST or GST-TTK. After washing, bound proteins were analysed on SDS–polyacrylamide gel electrophoresis (PAGE) followed by western blotting using anti-His. GST proteins were detected by Ponceau S stain. (e) Phosphorylation of CHK2 interferes with the interaction with TTK/hMps1. Bacteria-expressed, His-tagged WT CHK2 was first treated with l-phosphatase then used in a GST pull- down assay as described in (d). Phosphatase treatment enhanced the interaction between WT CHK2 and TTK/hMps1 to the level comparable to that seen with kinase-defective (KD) CHK2.

site that plays an important role in CHK2-mediated pull-down assay, the T288A mutant interacted with TTK/hMps1 stabilization. CHK2 almost as well as the WT construct (Figure 4i). As the level of induction is at least partly attributable We conclude that the T288A mutant is intrinsically less to TTK/hMps1 autophosphorylation (Figures 2a and stable, most likely because of the absence of a critical b), we next compared the kinase activity of the T288A CHK2 phosphorylation site, Thr288. mutant and WT. In the in vitro kinase assay, the T288A mutant was fully active in phosphorylating the sub- Phosphorylation at TTK/hMps1 Thr288 is enhanced strates, His-p53 and CHK2KD (Figure 4h), and at by CHK2 in vitro and in vivo after IR times, performed better than the WT. Therefore, the As Thr288 phosphorylation was identified through inability to be stabilized by CHK2 cannot be attributed in vitro assay, whether it is a physiological site then to its intrinsic kinase activity. Furthermore, in the GST becomes a critical issue. To determine if Thr288 is a

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1371 genuine in vivo CHK2 phosphorylation site, we gener- Taken together, these results demonstrate that TTK/ ated a phospho-Thr288-specific antibody by immunizing hMps1 Thr288 is a CHK2 target site after IR. rabbits with a synthetic phosphopeptide carrying phosphorylation at Thr288. As shown in Figure 5a, The TTK/hMps1 Thr288 to Ala mutant (T288A) the antibody recognized WT TTK/hMps1 but not the is defective in G2/M checkpoint control T288A mutant immunoprecipitated from transfected Because TTK/hMps1 is required for proper activation 293T cells (Figure 5a, lanes 3–8). The signal was of CHK2 in the G2/M checkpoint (Wei et al., 2005) and, enhanced when coexpressed with WT CHK2, whereas as demonstrated above, CHK2 may in turn control it was reduced in the presence of KD CHK2 (Figure 5a, the stability of TTK/hMps1, we wondered whether this lanes 3–5). In addition, the antibody did not recognize feedback control is essential in the G2/M arrest induced the GST-TKN (amino acids 268–308) fusion protein by IR. To address this issue, we first established Tet-off from bacteria unless it was coexpressed with WT but not cell lines that inducibly express HA-tagged, siRNA- with KD CHK2 (Figure 5b). Taken together, these data resistant WT TTK/hMps1 or the T288A mutant. As indicate that the antibody is phopho-Thr288-specific depicted in Figure 6a, these cells expressed comparable in vivo and that CHK2 can phosphorylate this site . amounts of HA-TTK (WT or T288A) upon withdrawal We examined further whether Thr288 phosphory- of doxycycline (Doxy). Following siRNA-mediated lation is inducible by DNA damage in cells. Because depletion of endogenous TTK/hMps1, the response of TTK/hMps1 also autophosphorylated at Thr288, at these cells to IR was analysed by flow cytometry. Escherichia coli least when expressed in (data not Ablation of endogenous TTK/hMps1 led to compro- shown), we investigated the DNA damage effect in G1 mised G2/M arrest after IR (Wei et al., 2005; Figures 6b cells where CHK2 is functional and the kinase activity and c, upper rows). However, whereas the induction of et al of TTK/hMps1 is minimal (Hogg ., 1994). TTK/ RNAi-resistant WT TTK/hMps1 could compensate for hMps1 was first immunoprecipitated from G1-synchro- the lost endogenous protein and increased the G2/G1 nized HeLa cells treated with or without IR, followed by ratio by 67%, the T288A mutant could do so by only western analysis using the phopho-Thr288-specific anti- 16% (Figures 6b and c, right columns). Cdc2 Tyr15 body. Increased Thr288 phosphorylation was detected phosphorylation, which accumulates in G2/M-arrested between 30 and 60 min after IR, and the signal decreased cells, was higher in WT than in T288A cells (Figure 6d). after 90 min in G1 phase HeLa cells (Figure 5c). In agreement, in another pair of cell lines, cyclin B, Increased Thr288 phosphorylation was also observed which accumulates in G2/M-arrested cells, was also in G1-synchronized normal human fibroblast IMR90 as higher in WT than in T288A cells (Supplementary revealed by the pT288/TTK ratios (Figure 5d). Phos- Figure S3). Of note, the CHK2 level was reproducibly phorylation was enhanced by increasing doses of IR up lower in T288A cells than in WT cells after IR. The to 4 Gy (Figure 5e). In both of these cells, the induction molecular basis of such reduction remains unclear. was either abolished (Figure 5c) or delayed (Figure 5d) These observations were reproduced in additional clones when cells were pretreated with CHK2 inhibitor before (Supplementary Figure S3), indicating that the defect IR, indicating that CHK2 is responsible for the increase was not a result of clonal selection. We conclude that or induction after IR. Similarly, caffeine, which inhibits the feedback phosphorylation of TTK/hMps1 Thr288 ATM-CHK2 signaling, also dampened the induction in by CHK2 is important not only for maintaining the G1 phase HeLa cells (Supplementary Figure S2). As a stability of TTK/hMps1, but also for proper G2/M positive control, BRCA1 Ser988 phosphorylation, a arrest after IR. et al CHK2-specific event (Lee ., 2000), was similarly As TTK/hMps1 was shown to be required for proper deregulated as the phosphorylation at TTK Thr288 mitotic progression (Fisk et al., 2003), whether the (Figure 5c; Supplementary Figure S2), indicating that feedback regulation would affect long term cell survival indeed CHK2 was effectively inhibited in these assays. was assessed by clonogenic assay. Consistent with the We noted that the basal phosphorylation at TTK previous report (Fisk et al., 2003), knockdown of TTK Thr288 and at BRCA1 Ser988 was enhanced upon severely hampered cell survival as revealed by the treatment either with CHK2 inhibitor (Figures 5c and d) numbers of colonies formed after 2 weeks of culture or with caffeine (Supplementary Figure S2), suggesting (Figure 6e). This defect was rescued by reexpression of that another kinase with similar substrate specificity WT but not T288A TTK in the Tet-off cells (Figure 6e), might have been activated when CHK2 function was suggesting that the CHK2-TTK feedback control and compromised. TTK Thr288 phosphorylation are important for cell Many checkpoint proteins, including CHK2, form survival. phosphorylation-specific nuclear foci upon DNA da- mage (Ward et al., 2001). After IR, phospho-Thr288 foci were also detected in the nuclei by immunofluores- cence microscopy (Figure 5f). The number and intensity Discussion of the foci increased significantly 30 min after IR, and these colocalized partially with g-H2AX. The foci were It is widely accepted that members of the PI3K-related reduced by CHK2 inhibition, indicating that the kinase family of kinases, such as ATM and ATR, act as activity of CHK2 was responsible for the increase after initiating kinases and are responsible for the triggering IR (Figure 5g). of the DNA damage response (DDR) in response to

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1372 various types of stress. Despite this consensus, we have interacts with and activates CHK2 in DDR (Wei et al., demonstrated previously the involvement of an unre- 2005). In this report, we present evidence that TTK/ lated kinase TTK/hMps1 (Wei et al., 2005). Indeed, hMps1, in addition to its role in centrosome duplication TTK/hMps1, known as a mitotic checkpoint kinase, and spindle assembly checkpoint, can respond to DNA

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1373 damage. Our data also show that this response requires then the next question is why and how, under that CHK2 and is mediated through phosphorylation of situation, CHK2 is dysregulated. Further investigation is Thr288. Upon DNA damage, TTK/hMps1 phospho- needed to unravel the underlying mechanisms. rylates CHK2 at Thr68 (Wei et al., 2005), and, as shown Many kinases have been reported to regulate various in this study, the activated CHK2 in turn phospho- activities of Mps1. For example, MAPK regulates rylates TTK/hMps1 at Thr288 and stabilizes the kinase, kinetochore localization of Mps1 in Xenopus extract thus forming a positive regulatory loop. (Zhao and Chen, 2006). Cdc28 of S. cerevisiae As demonstrated in this report, this relationship phosphorylates Mps1 on Thr28 to mediate spindle pole between TTK/hMps1 and CHK2 requires not only the duplication (Jaspersen et al., 2004). Likewise, murine kinase activity of CHK2 but also the interaction Cdk2 activity is required for mMps1 stability and between the two proteins. Peculiarly, bacteria-expressed phosphorylates mMps1 in vitro (Fisk and Winey, CHK2 KD but not WT binds TTK/hMps1 (Figure 3d). 2001). In addition, autophosphorylation of TTK/hMps1 Kinases are known to autophosphorylate themselves at Thr676 enhances the kinase activity (Mattison et al., when overproduced in bacteria. Consistently, treating 2007) and is required for the spindle checkpoint (Kang WT CHK2 with phosphatase restored its interaction et al., 2007). Notably, in addition to CHK2, two other with TTK (Figure 3e), suggesting that the binding is in kinases are also implicated in regulating the stability of fact regulated by the phosphorylation state of CHK2. TTK/hMps1. Cdk2/Cyclin A regulates the stability of One can imagine that, once activated by DNA damage, hMps1 at the centrosome by phosphorylating Thr468 autophosphorylation of CHK2 ensues; this then serves (Kasbek et al., 2007); and oncogenic B-Raf stabilizes as a means to downregulate its interaction with TTK/ hMps1 through ERK (Cui and Guadagno, 2008), hMps1 and to turn off the DDR of TTK/hMps1. The although the responsible phosphorylation site has not transient nature of the pT288 damage foci (Figure 5f) been identified. How these phosphorylation events lead seems to support this hypothesis. A definitive view of the to stabilization of hMps1 is still unclear. Although regulation may await side-by-side comparison of the Cdk2-specific siRNA-mediated downregulation of kinetics of TTK Thr288 phosphorylation and the hMps1 at the centrosome is reversed by the proteasome various phosphorylation events on CHK2. inhibitor MG115, levels of whole cell hMps1 are not Theimportanceofsuchfeedbackregulationwas altered significantly by either treatment (Kasbek et al., further revealed in cells where endogenous WT TTK/ 2007). Similarly in our study, the effect of CHK2 hMps1 was replaced with a mutant that cannot be knockdown on the stability of hMps1 was not rescued phosphorylated at Thr288 (T288A). Although the mutant by the proteasome inhibitor MG132, and treatment of possesses an intrinsic kinase activity comparable to that cells with MG132 did not appear to affect the level of of the WT (Figure 4h), it is defective in the proper hMps1 (data not shown). Collectively, these observa- maintenance of G2/M arrest after IR. Intriguingly, a tions imply that the stability of hMps1, except at the marked reduction in CHK2 was also observed in these centrosome, is regulated by mechanisms other than T288A cells after IR, suggesting that Thr288 phosphor- proteasome-mediated degradation. On a related note, ylation may regulate CHK2 in some way. Several we were unable to observe the DNA damage-induced possibilities may account for the decrease in CHK2. increase in hMps1 in several cancer cell lines, unless One possible explanation is that T288A is less able to hMps1 was first downregulated by siRNA (data not interact with and phosphorylate CHK2 at Thr68 in vivo, shown) or when cells were enriched in G1 phase by which is unlikely in light of the results presented in synchronization (Figure 5c). The cause for this is Figures 4h, i and 6d. Another possibility is that T288A unclear, but we suspect that this may relate to oncogenic may not localize properly, for example to the damage mutation (for example, B-Raf as mentioned above) or foci, or that its localization may be poorly maintained elevated Cdk activity observed frequently in tumors because of the lack of phosphorylation at Thr288. If so, (Enders and Maude, 2006; de Ca´ rcer et al., 2007). It is

Figure 4 The effect of CHK2 on TTK/hMps1 is mediated through phosphorylation by CHK2 at Thr288 in the N-terminal domain of TTK/hMps1. (a) The N-terminal domain of TTK/hMps1 is responsible for CHK2-dependent induction. HA-TTK full-length or the N- (amino acids 1–386) or the C- (amino acids 387–857) domain was coexpressed with myc-CHK2 in 293T cells, and the level of proteins was examined by western blotting using the antibodies indicated. (b) A domain encompassing amino acids 194–386 of TTK/ hMps1 is responsible for the induction by CHK2. The N terminus of TTK/hMps1 was divided further at amino acid 193 into two regions, and each was coexpressed with myc-CHK2 as in (a). Only the C-terminal region (194–386) responded to CHK2 in the coexpression assay. (c) CHK2 phosphorylates TTKN (194–386). The in vitro kinase assay was conducted with purified recombinant His-tagged proteins as indicated, and phosphorylation was detected by autoradiography. (d) Mass spectrometry showing the identification of a fragment that was doubly phosphorylated at Ser281 and Thr288. (e, f) Mutation of TTK/hMps1 at Thr288 but not Ser281 to Ala mitigates CHK2-mediated TTK/hMps1 stabilization. 293T cells were transfected with myc-tagged CHK2 and HA- tagged TTKN (amino acids 194–290; e) or full-length TTK (f) carrying an Ala substitution at Ser281 or Thr288. Lysates were prepared 40 h after transfection and analysed by western blotting for expression using the antibodies indicated. (g) The TTK/hMps1 T288A mutant is less stable than wild type (WT). Transfection of 293T cells was conducted as described above and the half-lives of proteins were measured by the addition of cycloheximide (CHX). (h) The kinase activity of the TTK T288A mutant is comparable to that of WT. The in vitro kinase assay was performed with recombinant purified GST-TTK WT or T288A using His-CHK2KD or His-p53 as the substrate. (i) TTK/hMps1 T288A binds CHK2 as efficiently as does WT TTK/hMps1. The GST pull-down assay was performed as described in Figure 3d using GST, GST-TTK WT, or T288A and His-CHK2. The pull-down proteins were detected by western using an anti-His antibody.

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1374 possible that, as a result of these alterations, the Mps1 phosphorylation at Thr288 may carry an additional kinase is fairly stable in cancer cells, and additional function because replacing the endogenous Mps1 with a phosphorylation by CHK2, as would occur upon DNA kinase-competent T288A mutant in HeLa cells could not damage, may be unable to cause further discernible restore full G2 arrest (Figure 6c). Further investigation changes. Nevertheless, our results also indicate that, in on the significance of Thr288 phosphorylation is addition to regulating hMps1 stability, CHK2-mediated warranted to reveal its other faces of regulation.

Figure 5 DNA damage-induced phosphorylation at Thr288 is dependent on CHK2 in vivo.(a) TTK/hMps1 Thr288 phosphorylation is enhanced by wild-type (WT) CHK2 but dampened by kinase-defective (KD) CHK2 in cotransfected 293T cells. HA-TTK was first immunoprecipitated from cell lysates, and the level of Thr288 phosphorylation was detected by western blotting using phospho-Thr288-specific antibody (pT288). Note that the antibody did not recognize the T288A mutant, demonstrating the specificity of the antibody. (b) CHK2 phosphorylates TTK at Thr288 in vivo. Flag-tagged CHK2, WT or KD, was coexpressed in Escherichia coli with GST or GST fused to TTK amino acids 268–308 (GST-TKN). Lysates were then prepared, and level of Thr288 phosphorylation was determined by western analysis following GST pull-down. Three different amounts of lysates were loaded for the comparison. (c) CHK2-dependent induction of Thr288 phosphorylation in G1-synchronized HeLa cells after ionizing radiation (IR). HeLa cells were arrested in prometaphase with nocodazole (50 ng/ ml) and irradiated with X-rays 7 h after release when most of cells were in G1, with or without pretreatment with 2 mM of CHK2 inhibitor II (Calbiochem) for 1 h. Cell extracts were prepared at the indicated times, and Thr288 phosphorylation was examined by first immunoprecipitation with anti-TTK followed by western analysis with the phospho-Thr288-specific antibody (pT288). BRCA1 Ser988 phosphorylation, a CHK2-specific event, was used as a control for CHK2 inhibition. (d) Increase in Thr288 phosphorylation in G1- synchronized IMR90 cells. Near confluent IMR90 cells were starved for 3 days before they were replated in normal medium. Seven hours after replating, cells were irradiated with IR (8 Gy), and whole cell lysates were prepared for western analysis using pT288 antibody. (e) Dose- dependent increase in Thr288 phosphorylation. G1-synchronized IMR90 cells were irradiated with increasing doses of IR. Cell lysates were collected 1 h after IR and analysed by western blotting using pT288 antibody. (f) Phospho-Thr288 damage foci are induced by IR. HeLa cells enriched in G1 were irradiated with X-rays (8 Gy), fixed at the indicated time after brief permeabilization and then processed for indirect immunofluorescence microscopy using pT288 and g-H2AX antibodies. The nuclei were stained with 40-6-diamidino-2-phenylindole (DAPI). (g) Phospho-Thr288 damage foci are diminished upon inhibition of CHK2. Cells as described in (f) were pretreated with CHK2 inhibitor II for 1 h before IR, then processed for immunofluorescence microscopy. Foci at 30 min after IR were compared.

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1375

Figure 5 Continued.

Materials and methods against TTK/hMps1 (sc-540), p-CHK2 (Thr68; sc-16297-R), p-BRCA1 (Ser988; sc-12888-R), myc (sc-40), His-tag (sc-803) Cell culture and treatments and GST (sc-138) were from Santa Cruz Biotechnology To grow tetracycline-repressible stable HeLa cells expressing (Santa Cruz, CA, USA). Monoclonal antibody against CHK2 HA-TTK/hMps1, G418 (300 mg/ml), puromycin (0.4 mg/ml) (K0087) was purchased from MBL (Nagoya, Japan), and Doxy (1 mg/ml) were included in the medium. IR treatment and polyclonal antiactin antibody (A2066) was from Sigma was performed as described previously (Wei et al., 2005). (St Louis, MO, USA).

Cell transfection and western blot analysis RNA interference 293T and HeLa cells were transfected by calcium phosphate The sequences targeted by TTK/hMps1 and CHK2 siRNA are precipitation and Lipofectamine 2000 (Invitrogen, Carlsbad, 50-tgaacaaagtgagagacat-30 and 50-aatgtgtgaatgacaactact-30, re- CA, USA), respectively. Lysis of cells and western analysis were spectively. All siRNAs were synthesized by and purchased conducted as described previously (Wei et al., 2005). Antibodies from Qiagen (Valencia, CA, USA). Transfection of siRNA

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1376

Figure 6 The TTK/hMps1 T288A mutant is compromised in restoring G2/M arrest in TTK-knockdown cells. (a) Expression of small interfering RNA (siRNA)-resistant HA-TTK/hMps1 in two of the stable HeLa cell lines wild type (WT)-r-9 and T288A-r-16 upon withdrawal of doxycycline (Doxy). These Tet-off cells were transfected with TTK siRNA in the presence or absence of Doxy, and irradiated 2 days later with X-rays (8 Gy; b, c). Cells were collected at the times indicated and processed for flow cytometry. Numbers indicate the G2/G1 ratios. (d) Impaired accumulation of Cdc2 Tyr15 phosphorylation in cells expressing T288A-r. Tet-off cells were transfected with TTK siRNA in the absence of Doxy and processed as described in (b, c). Cells were lysed 14 h after irradiation and analysed by western blotting using the antibodies indicated. Nucleolin was used as a loading control. (e) Reexpression of WT TTK but not the T288A mutant rescued the survival of the TTK-ablated cells. Colony survival assays were performed with TTK siRNA- transfected WT-r-9 and T288A-r-16 cells in the presence or the absence of Doxy. Colonies from one representative assay were shown on the left. Results from three duplicated experiments were averaged and shown on the right.

was conducted using Oligofectamine (Invitrogen) at a final The construct for E. coli expression of His-CHK2 concentration of 0.15–0.2 mM. SCD þ FHA (amino acids 19–175) was generated by PCR using appropriate primers and cloned into the KpnI and EcoRI sites of pRSET B (Invitrogen). For expression of GST-TTK/ Plasmid construction hMps1 and His-TTKN (amino acids 194–386), the vectors Plasmids for mammalian expression of the myc-tagged WT pGEX-4T (Amersham Biosciences, Piscataway, NJ, USA) and and KD (D347A) CHK2 were described previously (Shieh pRSET (Invitrogen) were used, respectively, for cloning. et al., 2000). DFHA (amino acids 77–213 deleted) was For coexpression of CHK2 and GST-TTKN (268–308) in generated by removing an internal Bsu36I fragment from the E. coli, CHK2 was recloned into the SalIandNotI sites of pET29b cDNA followed by religation. T68A and DSCD (amino acids (Novagen, Gibbstown, NJ, USA) and expressed as a C-terminal 19–75 deleted) mutants were constructed by plasmid-based Flag-tagged protein. site-directed mutagenesis (Wang et al., 2006). The TTK/hMps1 truncation mutants were generated by PCR and cloned into pcDNA-HA for expression. The TTK/hMps1 S281A and Phospho-Thr288-specific antibody T288A point mutants were made by PCR-based site-directed Rabbit polyclonal antiphospho-Thr288 antibody was raised mutagenesis and confirmed by sequencing. against a phosphopeptide H-CDVKpTDDSV-OH conjugated TTK/hMps1 siRNA-resistant constructs (TTK/hMps1-r) to ovalbumin by LTK Biolaboratories (Touyuan, Taiwan). were made by introducing silent mutations within the TTK/ The antibody was purified by binding to phospho-Thr288 hMps1 siRNA-targeted region (nt 50–68; 50-tgaacaaagtGAGA peptide, followed by passing the eluted antibodies through an gacat-30 to 50-tgaacaaagtACGCgacat-30) by PCR-based site- unphosphorylated peptide column to remove those that directed mutagenesis. cross-reacted with unphosphorylated epitopes. All peptides

Oncogene TTK/hMps1 as a CHK2 substrate Y-H Yeh et al 1377 were synthesized by Kelowna International Scientific Inc. Immunofluorescence microscopy (San Chung, Taipei, Taiwan). G1 phase HeLa cells grown on coverslips were first ex- tracted on ice for 2 min with 0.5% Triton X-100 in cytoskeleton buffer, then fixed and processed for immuno- Cell synchronization fluorescence staining as described previously (Dimitrova and Exponentially growing HeLa cells were first arrested in the M Gilbert, 2000). phase by overnight (16–18 h) incubation in 50 ng/ml nocodazole, andthenwashedandreleasedintofreshmedium.Cellswere irradiated at 7 h after replating when most of the cells were in G1. Acknowledgements For IMR90 cells, near confluent monolayer cells were incubated in medium containing 0.05% serum for 3 days. Cells We thank the LC/MS/MS core of the Institute of Biomedical were then trypsinized and replated. Irradiation was performed Sciences for the expert assistance in mass spectrometry. This at 6–7 h after replating. work was supported by grants from Academia Sinica and G1 phase synchronization was confirmed by flow cytometry. National Science Council of Taiwan to S-Y Shieh.

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Supplementary Information accompanies the paper on the Oncogene website (http://www.nature.com/onc)

Oncogene