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Threonine 68 Phosphorylation by Ataxia Telangiectasia Mutated Is Required for Efficient Activation of Chk2 in Response to Ionizing Radiation¹

Department of Hematology-Oncology, St. Jude Children’s Research Hospital, Memphis, Tennessee 38105 [J-Y. A., C. E. C.], and Department of Cell Biology and Physiology [J. K. S., H. P-W.], Howard Hughes Medical Institute [H. P-W.], Washington University School of Medicine, St. Louis, Missouri 63110

	ABSTRACT

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Eukaryotic cells activate an evolutionarily conserved set of proteins that rapidly induce cell cycle arrest to prevent replication or segregation of damaged DNA before repair is completed. In response to ionizing radiation (IR), the cell cycle checkpoint kinase, Chk2 (hCds1), is phosphorylated and activated in an ataxia telangiectasia mutated (ATM)-dependent manner. Here we show that the ATM protein kinase directly phosphorylates T68 within the SQ/TQ-rich domain of Chk2 in vitro and that T68 is phosphorylated in vivo in response to IR in an ATM-dependent manner. Furthermore, phosphorylation of T68 was required for full activation of Chk2 after IR. Together, these data are consistent with the model that ATM directly phosphorylates Chk2 in vivo and that this event contributes to the activation of Chk2 in irradiated cells.

	Introduction

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Mutations in the ATM³ gene are responsible for the pleiotropic recessive disorder AT, which is characterized by progressive cerebellar ataxia, oculocutaneous telangiectasia, immunodeficiency, predisposition to cancer, and extreme sensitivity to IR (1) . Cell lines derived from AT patients are also radiosensitive and exhibit defects in various cell cycle checkpoint responses to IR including p53-dependent G₁ cell cycle arrest and rapid p53-independent S and G₂ phase arrests (2 , 3) . ATM encodes a M_r 370,000 protein kinase, the activity of which is enhanced in response to DNA strand breaks, and targets several effectors of checkpoint control including Brca1, p95/nbs1, p53, and Mdm2 and, therefore, may be a key regulator of checkpoint responses when cells are exposed to IR (4, 5, 6, 7, 8, 9, 10, 11, 12) .

Chk2 (hCds1) is a mammalian homologue of the Saccharomyces cerevisiae Rad53 and Schizosaccharomyces pombe Cds1 checkpoint protein kinases (13, 14, 15, 16) . In response to IR, Chk2 is phosphorylated and activated in an ATM-dependent manner, and this event has been linked to the regulation of p53 stability and maintenance of G₂ cell cycle arrest (13, 14, 15, 16, 17, 18, 19) . Here we present evidence that Chk2 is a direct target of the ATM protein. Through site-directed mutagenesis of various Chk2 residues, we identified T68 as the major site of phosphorylation by the ATM kinase in vitro. In response to IR, Chk2 is specifically phosphorylated on T68 in an ATM-dependent manner consistent with a recent report from Zhou et al. (20) . In this report, we provide additional evidence that phosphorylation of T68 is necessary for efficient activation of the Chk2 kinase in irradiated cells, thus further establishing a direct link between ATM and Chk2 in checkpoint control.

	Materials and Methods

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Cell Lines, DNA Transfections, and Retroviral Transductions.
Human neuroblastoma SY5Y cells (kindly provided by G. Brodeur, The Children’s Hospital of Philadelphia, Philadelphia, PA) and 293T cells were transiently transfected with the indicated expression vectors using FuGene 6 transfection reagent (Roche Molecular Biochemicals) according to the manufacturer’s suggestions. Under these conditions, 90% transfection efficiency is obtained. A29 (p53-/-) and A38 (p53-/- ATM-/-) mouse embryo fibroblasts (21) were infected with retrovirus generated in 293T cells after transient transfection with either pBabePuro3'mycChk2 wt or T68A mutant constructs plus an ecotropic packaging construct. After 2 days, infected cells were selected in culture with 4 µg/ml Puromycin (Sigma) and used for further experimentation.

Plasmids.
To generate NH₂-terminal FLAG-tagged Chk2 expression constructs, wt and kd Chk2 cDNA fragments were excised from GST-Chk2 and GST-Chk2 (D347A; kindly provided by S. Elledge, Howard Hughes Medical Institute, Baylor College of Medicine, Houston, TX) and ligated into a derivative of the mammalian expression vector, pSG5 (Stratagene) such that an NH₂-terminal FLAG-epitope would be linked to Chk2. A T68A derivative of FLAG-Chk2 was then generated using the QuickChange Site Directed Mutagenesis kit (Stratagene). COOH-terminal myc-tagged Chk2 wt, kd, and T68A mutant expression constructs were generated by PCR amplifying each respective cDNA using Pfu polymerase (Stratagene) and ligating PCR products into a pSG5 vector derivative such that a COOH-terminal myc epitope would be linked to Chk2. 3' myc Chk2 cDNAs were then transferred to the retroviral pBabePuro vector. A prokaryotic GST-Chk2_1–80 fusion protein expression vector was made by linking the first 80 amino acids of Chk2 to GST of pGEX-3X (Pharmacia). Various mutants of pGEX-Chk2_1–80 (S19A, S26A, T28A, S33A, S35A, S50A, and T68A) were made using the QuickChange Site-Directed Mutagenesis protocol. The T68A derivative of pGEX2TN-hCds1 kd (D368N) was generated as discussed above using site-directed mutagenesis.

In Vitro Kinase Assays and Immunoblot Analysis.
FLAG-tagged wt and kd ATM were immunoprecipitated from transiently transfected 293T cells and subjected to an in vitro kinase assay as described (10 , 22) using 1 µg of each respective GST-Chk2 (hCds1) substrate purified from bacteria (22) . For Chk2 kinase assays, SY5Y cells transiently expressing FLAG-tagged Chk2 were lysed in 20 mM Tris (pH 7.5), 150 mM NaCl, 0.5% NP40, 0.5% Tween 20, 1 mM NaF, 1 mM Na₃VO_4, 1 mM phenylmethylsulfonyl fluoride, 2 µg/ml pepstatin A, 5 µg/ml leupeptin, 10 µg/ml aprotinin, and 1 mM DTT. FLAG-tagged proteins were immunoprecipitated with anti-FLAG M2 monoclonal antibody-agarose affinity gel (Sigma). Kinase reactions contained immunoprecipitated FLAG-tagged Chk2, 1 µg of GST-Cdc25C motif, 20 mM HEPES (pH 7.5), 10 mM MgCl₂, 1 mM NaF, 1 mM Na₃VO₄, 1 mM DTT, 10 µM ATP, and 10 µCi [-³²P]ATP for 15 min at 30°C. Kinase reactions were then subjected to SDS-PAGE and transferred to nitrocellulose membrane. Radiolabeled proteins were visualized by PhosphorImager analysis and quantitated using ImageQuant software (Molecular Dynamics). Two dimensional phosphoamino acid analysis of hydrolyzed, radiolabeled GST-Chk2_1–80 was carried out using the Hunter System (C.B.S. Scientific) as described (23) . Antibodies used for immunoblot analysis were anti-FLAG M5 (Sigma) and anti-GST (Roche Molecular Biochemicals). Phosphothreonine-containing proteins were detected with rabbit anti-phosphothreonine antibodies (New England Biolabs). The c-myc antibody (9E10) used for both immunoprecipitation and Western blotting was obtained from Roche Molecular Biochemicals.

	Results and Discussion

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Structurally, Chk2 protein is composed of three domains: one rich in SQ/TQ motifs, one fork head-associated domain, and a kinase domain (Fig. 1 ; Refs. 13, 14, 15, 16 ). ATM has been shown to preferentially phosphorylate SQ/TQ motifs, and the SQ/TQ-rich domain was, therefore, used as a substrate for ATM in vitro (22) . Immunoprecipitated FLAG-tagged, wt ATM phosphorylated GST-Chk2_1–80 protein, whereas catalytic-inactive (kd) FLAG-tagged ATM did not confirming that the activity observed here is intrinsic to ATM (Fig. 2A) . To map the site of ATM phosphorylation, each of the seven potential serines or threonines adjacent to a glutamine within GST-Chk2_1–80 were individually substituted with alanine. Analysis of these mutated GST-Chk2_1–80 substrates revealed that T68 is the major target of ATM in vitro (Fig. 2A) . Further phosphoamino acid analysis demonstrated that GST-Chk2_1–80 was predominantly phosphorylated on threonine residues, although some serine phosphorylation was also seen, suggesting that ATM may target an additional SQ motif on GST- Chk2_1–80 (Fig. 2B) . Full-length Chk2 (hCds1) protein fused to GST was also predominantly phosphorylated on T68 by ATM in vitro (Fig. 2C) .

View larger version (10K): [in this window] [in a new window]   Fig. 1. Domain structure of human Chk2 showing the placement of the SQ/TQ-rich domain with respect to the fork head-associated (FHA) and kinase domain. Potential ATM phosphorylation sites within the SQ/TQ region are underlined and in bold.

View larger version (19K): [in this window] [in a new window]   Fig. 2. The ATM kinase directly phosphorylates the SQ/TQ rich domain on T68 in vitro. A, immunoprecipitated wt or catalytic-inactive (kd) ATM were subjected to in vitro kinase assays with GST-Chk21–80 wt and SQ/TQ mutant proteins. Equal amounts of ATM and GST-fusion proteins contained in each reaction were confirmed by Western analysis with anti-FLAG or anti-GST antibodies. B, GST-Chk21–80 wt protein phosphorylated by wt ATM was subjected to two-dimensional phosphoamino acid analysis. The positions of unlabeled serine, threonine and tyrosine standards are circled. C, full-length GST-hCds1 and T68A mutant proteins were tested as substrates for ATM. Because Chk2/hCds1 exhibits a relatively high level of autophosphorylation, catalytic-inactive D368N GST-hCds1 or D368N/T68A GST-hCds1 proteins were used.

View larger version (27K): [in this window] [in a new window]   Fig. 3. IR-induced T68 phosphorylation of Chk2 by ATM is required for optimal activation. A, A29 (p53-/- ATM+/+) and A38 (p53-/- ATM-/-) mouse embryo fibroblasts were infected with retrovirus encoding either myc-tagged Chk2 wt or T68A. Cells were exposed to 0–6 Gy IR and harvested 1 h later. Chk2 proteins were immunoprecipitated with anti-myc antibody and Western blotted with anti-phosphothreonine antibody (bottom panel) or anti-myc (top panel). B, FLAG-tagged wt, kd, or T68A mutant Chk2 proteins were transiently expressed in SY5Y cells. Cells were exposed to 0–6 Gy IR. Chk2 was then immunoprecipitated 1 h later with anti-FLAG and subjected to in vitro kinase assays using GST-Cdc25C200–256 as a substrate (bottom panel). Total levels of Chk2 contained in FLAG immunoprecipitates were determined with anti-FLAG (top panel). C, the ratio of 32P incorporation into GST-Cdc25C200–256 by FLAG-Chk2 immunoprecipitated from irradiated versus unirradiated cells is shown graphically. Background 32P incorporation into GST-Cdc25C represented by kd Chk2 immunoprecipitates was subtracted prior to ratio calculations. Data are expressed as the mean fold activation from three independent experiments; bars, SE.

In conclusion, these results suggest that ATM may directly phosphorylate Chk2 at T68 in vivo, and that this event appears to be necessary for complete activation of Chk2 in irradiated cells. Chk2 has been implicated as an ATM effector in several DNA damage response pathways. Both ATM and Chk2 are required for efficient stabilization of p53 in response to IR (19 , 24) . Chk2 and p53 have recently been genetically linked with the discovery of Chk2 germ-line mutations among several Li-Fraumeni syndrome patients that failed to show the typical mutations in the p53 gene (25) . Chk2 may regulate the stability of p53 by phosphorylating serine 20 of p53, which in turn interferes with Mdm2 binding and associated p53 protein degradation (17 , 19 , 26 , 27) . Chk2 also associates with and phosphorylates the Brca1 breast cancer gene product and may participate in the regulation of the G₂-M checkpoint pathway by targeting the cdc25C phosphatase (13, 14, 15, 16 , 19 , 20 , 28) . Future studies will be directed at understanding the involvement of Chk2 in mediating the various functions of ATM.

	ACKNOWLEDGMENTS

 
We thank S. Elledge for the Chk2 cDNA constructs; M. Kastan and C. Bakkenist for helpful discussions and comments on the manuscript; and A. Rose and B. Xu for excellent technical assistance.

Note Added in Proof: Similar results showing that ATM-dependent T68 phosphorylation of Chk2 is required for activation in response to IR have been reported recently by Matsuoka et al. (Proc. Natl. Acad. Sci. USA, 97: 10389–10394, 2000) and Melchionna et al. (Nat. Cell Biol., 2: 762–765, 2000).

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ This work was supported by the American Lebanese Syrian Associated Charities of the St. Jude Children’s Research Hospital (to C. E. C.) and the NIH (to H. P-W.). H. P-W. is an Investigator of the Howard Hughes Medical Institute.

² To whom requests for reprints should be addressed, at Department of Hematology-Oncology, St. Jude Children’s Research Hospital, 332 North Lauderdale Street, DTRT 1034, Memphis, TN 38105. E-mail: christine.canman{at}stjude.org

³ The abbreviations used are: ATM, ataxia telangiectasia mutated; IR, ionizing radiation; wt, wild type; kd, kinase dead; MEF, mouse embryo fibroblast; GST, glutathione S-transferase; Chk, checkpoint kinase.

Received 6/14/00. Accepted 9/19/00.

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				D. I. Beardsley, W.-J. Kim, and K. D. Brown N-Methyl-N'-nitro-N-nitrosoguanidine Activates Cell-Cycle Arrest through Distinct Mechanisms Activated in a Dose-Dependent Manner Mol. Pharmacol., October 1, 2005; 68(4): 1049 - 1060. [Abstract] [Full Text] [PDF]

				N. Shirata, A. Kudoh, T. Daikoku, Y. Tatsumi, M. Fujita, T. Kiyono, Y. Sugaya, H. Isomura, K. Ishizaki, and T. Tsurumi Activation of Ataxia Telangiectasia-mutated DNA Damage Checkpoint Signal Transduction Elicited by Herpes Simplex Virus Infection J. Biol. Chem., August 26, 2005; 280(34): 30336 - 30341. [Abstract] [Full Text] [PDF]

				J.-S. Kim, T. B. Krasieva, H. Kurumizaka, D. J. Chen, A. M. R. Taylor, and K. Yokomori Independent and sequential recruitment of NHEJ and HR factors to DNA damage sites in mammalian cells J. Cell Biol., August 1, 2005; 170(3): 341 - 347. [Abstract] [Full Text] [PDF]

				C.-R. Chen, W. Wang, H. A. Rogoff, X. Li, W. Mang, and C. J. Li Dual Induction of Apoptosis and Senescence in Cancer Cells by Chk2 Activation: Checkpoint Activation as a Strategy against Cancer Cancer Res., July 15, 2005; 65(14): 6017 - 6021. [Abstract] [Full Text] [PDF]

				J.-H. Wei, Y.-F. Chou, Y.-H. Ou, Y.-H. Yeh, S.-W. Tyan, T.-P. Sun, C.-Y. Shen, and S.-Y. Shieh TTK/hMps1 Participates in the Regulation of DNA Damage Checkpoint Response by Phosphorylating CHK2 on Threonine 68 J. Biol. Chem., March 4, 2005; 280(9): 7748 - 7757. [Abstract] [Full Text] [PDF]

				A. Kudoh, M. Fujita, L. Zhang, N. Shirata, T. Daikoku, Y. Sugaya, H. Isomura, Y. Nishiyama, and T. Tsurumi Epstein-Barr Virus Lytic Replication Elicits ATM Checkpoint Signal Transduction While Providing an S-phase-like Cellular Environment J. Biol. Chem., March 4, 2005; 280(9): 8156 - 8163. [Abstract] [Full Text] [PDF]

				A. W. Adamson, D. I. Beardsley, W.-J. Kim, Y. Gao, R. Baskaran, and K. D. Brown Methylator-induced, Mismatch Repair-dependent G2 Arrest Is Activated through Chk1 and Chk2 Mol. Biol. Cell, March 1, 2005; 16(3): 1513 - 1526. [Abstract] [Full Text] [PDF]

				I. H. Ismail, S. Nystrom, J. Nygren, and O. Hammarsten Activation of Ataxia Telangiectasia Mutated by DNA Strand Break-inducing Agents Correlates Closely with the Number of DNA Double Strand Breaks J. Biol. Chem., February 11, 2005; 280(6): 4649 - 4655. [Abstract] [Full Text] [PDF]

				J. Kang, D. Ferguson, H. Song, C. Bassing, M. Eckersdorff, F. W. Alt, and Y. Xu Functional Interaction of H2AX, NBS1, and p53 in ATM-Dependent DNA Damage Responses and Tumor Suppression Mol. Cell. Biol., January 15, 2005; 25(2): 661 - 670. [Abstract] [Full Text] [PDF]

				E. U. Kurz, P. Douglas, and S. P. Lees-Miller Doxorubicin Activates ATM-dependent Phosphorylation of Multiple Downstream Targets in Part through the Generation of Reactive Oxygen Species J. Biol. Chem., December 17, 2004; 279(51): 53272 - 53281. [Abstract] [Full Text] [PDF]

				T. D. McSherry and P. R. Mueller Xenopus Cds1 Is Regulated by DNA-Dependent Protein Kinase and ATR during the Cell Cycle Checkpoint Response to Double-Stranded DNA Ends Mol. Cell. Biol., November 15, 2004; 24(22): 9968 - 9985. [Abstract] [Full Text] [PDF]

				E. Tosti, L. Waldbaum, G. Warshaw, E. A. Gross, and R. Ruggieri The Stress Kinase MRK Contributes to Regulation of DNA Damage Checkpoints through a p38{gamma}-independent Pathway J. Biol. Chem., November 12, 2004; 279(46): 47652 - 47660. [Abstract] [Full Text] [PDF]

				J. Wang, T. Wiltshire, Y. Wang, C. Mikell, J. Burks, C. Cunningham, E. S. Van Laar, S. J. Waters, E. Reed, and W. Wang ATM-dependent CHK2 Activation Induced by Anticancer Agent, Irofulven J. Biol. Chem., September 17, 2004; 279(38): 39584 - 39592. [Abstract] [Full Text] [PDF]

				L. A. Parsels, J. D. Parsels, D. C.-H. Tai, D. J. Coughlin, and J. Maybaum 5-Fluoro-2'-Deoxyuridine-Induced cdc25A Accumulation Correlates with Premature Mitotic Entry and Clonogenic Death in Human Colon Cancer Cells Cancer Res., September 15, 2004; 64(18): 6588 - 6594. [Abstract] [Full Text] [PDF]

				K. Cerosaletti and P. Concannon Independent Roles for Nibrin and Mre11-Rad50 in the Activation and Function of Atm J. Biol. Chem., September 10, 2004; 279(37): 38813 - 38819. [Abstract] [Full Text] [PDF]

				X. Xu, J. Lee, and D. F. Stern Microcephalin Is a DNA Damage Response Protein Involved in Regulation of CHK1 and BRCA1 J. Biol. Chem., August 13, 2004; 279(33): 34091 - 34094. [Abstract] [Full Text] [PDF]

K. Tanaka and P. Russell Cds1 Phosphorylation by Rad3-Rad26 Kinase Is Mediated by Forkhead-associated Domain Interaction with Mrc1 J. Biol. Chem., July 30, 2004; 279(