CHK2 Is Negatively Regulated by Protein Phosphatase 2A

University of South Florida Scholar Commons

Graduate Theses and Dissertations Graduate School

5-31-2010

CHK2 is Negatively Regulated by Protein Phosphatase 2A

Alyson Freeman University of South Florida

Follow this and additional works at: https://scholarcommons.usf.edu/etd

Part of the American Studies Commons, and the Biology Commons

Scholar Commons Citation Freeman, Alyson, "CHK2 is Negatively Regulated by Protein Phosphatase 2A" (2010). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/3443

This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected].

CHK2 is Negatively Regulated by Protein Phosphatase 2A

Alyson K. Freeman

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Cancer Biology College of Arts and Sciences University of South Florida

Major Professor: Alvaro N. A. Monteiro, Ph.D. Gary W. Litman, Ph.D. Gary W. Reuther, Ph.D. Kenneth L. Wright, Ph.D.

Date of Approval: January 14, 2010

Keywords: DNA damage response, phosphorylation, kinase, ionizing radiation, DNA double-strand break

DEDICATION

To my husband Scott N. Freeman, my parents James P. and Carolyn C. Fay, and my

sister Erin M. Fay.

ACKNOWLEDGEMENTS

I first and foremost would like to thank my mentor, Dr. Alvaro Monteiro. I could not have asked for a better experience in graduate school and I am grateful for all that I have learned from him. I would also like to thank my committee members Dr. Gary

Litman, Dr. Gary Reuther, and Dr. Kenneth Wright for their guidance and expertise over the years. I thank Dr. Fred Bunz for taking the time to serve as the outside chairperson for my defense. Thank you to former lab members Marcelo Carvalho, Virna Dapic,

Sylvia Marsillac, Jonathan Rios-Doria and current members Huey Nguyen, Melissa

Price, Michael Sweet, Aneliya Velkova, and Nicholas Woods for their help, the great scientific discussions, and for making the lab a enjoyable place to be. I thank Cathy

Gaffney for her support and all the work she does for the Cancer Biology Program. I

want to extend a special thank you to my husband, Scott Freeman, who has given me

endless support and advice as someone who went through this exact process two years

before me.

TABLE OF CONTENTS

LIST OF FIGURES iv

LIST OF ABBREVIATIONS vi

ABSTRACT viii

INTRODUCTION 1

DNA Damage Response Pathway 1 Sensors - Mre11-Rad50-Nbs1 and Rad9-Rad1-Hus1 Complexes 4 Signal Transducing Kinases - ATM and ATR 4 Effector Kinases - CHK1 and CHK2 5 Mediators - H2AX, BRCA1, MDC1, and 53BP1 7 Effector Proteins - Cdc25, E2F1, and p53 8

Checkpoint Kinase CHK2 10 CHK2 Structure and Activation 10 CHK2 in Cancer 13 CHK2 Phenotype 15

Protein Serine/Threonine Phosphatases 17 PP2A Structure and Regulation 17 PP2A Function 21 PP2A in Cancer 22

Protein Serine/Threonine Phosphatases in the DNA Damage Response 23 DNA Damage Response Proteins that are Negatively Regulated 23 ATM and ATR 25 RPA 26 CHK1 26 CHK2 28 H2AX 31 P53 32 DNA Damage Response Proteins Whose Activities are Enhanced 35 ATM 35 ATR 36 DNA-PK 36 BRCA1 37 P53 38 i

Summary and Rationale 38

MATERIALS AND METHODS 40

Cloning 40 Yeast Two-Hybrid 40 Cell Culture, Transfection, and Reagents 41 Immunoprecipitations 41 Immunoblotting 42 Kinase Activity Assay 43 Subcellular Fractionation 44 Phosphatase Activity Assay 44 Protein Stability 45

RESULTS 46

CHK2 Binds to the B'α Subunit of PP2A 46 CHK2 Interacts with the B'α Subunit of PP2A in Yeast 46 CHK2 and B'α Bind in Mammalian Cells 51

Binding Between CHK2 and PP2A is Modulated by DNA Damage 54 B'α and CHK2 Dissociate upon IR-induced DNA Damage 54 CHK2 Binds to Many PP2A Subunits 58 B'α and CHK2 are Dissociated After DNA Damage Caused by IR and Doxorubicin, but not B'α and CHK1 61

Analysis of the IR-induced Dissociation of CHK2 and B'α 63 Dissociation is an Early Event and Occurs in a Dose-dependent Manner 63 Re-Association of CHK2 and B'α does Not Correlate With Resolution of DNA Damage 67 The Activity of ATM, but not PP2A, is Necessary for the Dissociation of CHK2 and B'α 71 Mutation of CHK2 Serines 33 and 35 Affects Binding to B'α 75

Analysis of the Effects of CHK2 on PP2A 77 CHK2 can Phosphorylate B'α in vitro 77 PP2A Localization is Not Influenced by IR 79 PP2A Activity is Unaffected by IR and is Not Influenced by CHK2 82

Analysis of the Effects of PP2A on CHK2 87 Changes in CHK2 Phosphorylation Due to IR 87 Changes in CHK2 Kinase Activity after IR 91 Mutation of CHK2 Serines 33 and 35 Affects Protein Stability 94 ii

DISCUSSION 97

LIST OF REFERENCES 103

ABOUT THE AUTHOR END PAGE

iii

LIST OF FIGURES

Figure 1. The DNA damage response pathway. 3

Figure 2. The structure of CHK2. 12

Figure 3. PP2A holoenzyme structure. 20

Figure 4. Protein serine/threonine phosphatases in the DNA damage response pathway. 24

Figure 5. B'α binds to CHK2 amino acids 1-107. 49

Figure 6. CHK2 binds to B'α amino acids 89-322. 50

Figure 7. CHK2 binds to FLAG-B'α in mammalian cells. 52

Figure 8. Endogenous CHK2 and endogenous B'α bind in mammalian cells. 53

Figure 9. Binding between CHK2 and B'α is dissociated after DNA damage. 56

Figure 10. Dissociation of CHK2 and B'α is verified using another CHK2 antibody. 57

Figure 11. CHK2 binds to many PP2A B' subunits and they are dissociated after IR. 59

Figure 12. Endogenous PP2A C and CHK2 can bind and they are dissociated after IR. 60

Figure 13. Dissociation of CHK2 and B'α is caused by DNA damage induced by IR and doxorubicin. 62

Figure 14. Dissociation of CHK2 and B'α occurs in a dose-dependent manner. 65

Figure 15. Dissociation of CHK2 and B'α is and early event after IR. 66

Figure 16. CHK2 and B'α are able to re-associate hours after 6 Gy IR. 69

Figure 17. CHK2 and B'α are able to re-associate hours after 50 Gy IR. 70 iv

Figure 18. PP2A phosphatase activity is not required for CHK2 binding to or dissociating from B'α. 73

Figure 19. Inhibition of ATM and DNA-PK kinase activity prevents the IR- induced dissociation of B'α and CHK2. 74

Figure 20. Mutation of CHK2 serines 33 and 35 affects binding to B'α. 76

Figure 21. CHK2 can phosphorylate B'α in vitro. 78

Figure 22. B'α localization is not changed by IR. 80

Figure 23. Localization of endogenous PP2A proteins do not change after IR. 81

Figure 24. Total PP2A phosphatase activity does not change after IR. 84

Figure 25. Phosphatase activity of PP2A containing B'α does not change after IR. 85

Figure 26. Total PP2A activity does not differ in HCT116 wild-type and CHK2-/- cells. 86

Figure 27. Inhibition of PP2A activity increases CHK2 activity. 89

Figure 28. Mutation of CHK2 serines 33 and 35 affects CHK2 phosphorylation at other sites. 90

Figure 29. B'α overexpression negatively affects CHK2 kinase activity. 92

Figure 30. Mutation of CHK2 serines 33 and 35 affects CHK2 kinase activity. 93

Figure 31A. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability. 95

Figure 31B. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability. 96

Figure 32. Model of PP2A regulation of CHK2 through binding and dissociation. 102

LIST OF ABBREVIATIONS

4-NQO 4-nitroquinoline-N-oxide

9-1-1 Rad9-Rad1-Hus1

AT Ataxia-telangiectasia

ATLD AT-like disorder

ATM Ataxia telangiectasia mutated

ATR ATM-Rad3-related kinase

ATRIP ATR interacting protein

BRCA1 Breast cancer susceptibility 1

CHK2 Checkpoint kinase 2

CHK1 Checkpoint kinase 1

CPT Camptothecin

DDR DNA damage response

DNA-PK DNA-dependent protein kinase

DOX Doxorubicin

DSB DNA double-strand breaks

ES Embryonic stem

HR Homologous recombination

HU Hydroxyurea

IB Immunoblot

IP Immunoprecipitation vi

IR Ionizing radiation

MEF Mouse embryonic fibroblast

MMC Mitomycin C

MMS Methylmethane sulfonate

MRN Mre11-Rad50-Nbs1

NBS Nijmegen Breakage Syndrome

NCS Neocarzinostatin

NHEJ Non-homologous end joining

NLS Nuclear localization signal

OA Okadaic acid

PCNA Proliferating cell nuclear antigen

PIKK PI-3K like kinase

PP2A Protein phosphatase 2A

PPP Phosphoprotein phosphatase

PPM Metal-dependent protein phosphatase

PSP Protein serine/threonine phosphatase

RDS Radioresistant DNA synthesis

RFC Replication factor C

RPA Replication protein A

SSB Single-strand breaks

VPR Viral protein R

vii

CHK2 IS NEGATIVELY REGULATED BY PROTEIN PHOSPHATSE 2A

Alyson K. Freeman

ABSTRACT

Checkpoint kinase 2 (CHK2) is an effector kinase of the DNA damage response

pathway and although its mechanism of activation has been well studied, the attenuation

of its activity following DNA damage has not been explored. Here, we identify the B'α

subunit of protein phosphatase 2A (PP2A), a major protein serine/threonine phosphatase of the cell, as a CHK2 binding partner and show that their interaction is modulated by

DNA damage. B'α binds to the SQ/TQ cluster domain of CHK2, which is a target of

ATM phosphorylation. CHK2 is able to bind to many B' subunits as well as the PP2A C

subunit, indicating that it can bind to the active PP2A enzyme. The induction of DNA

double-strand breaks by ionizing radiation (IR) as well as treatment with doxorubicin causes dissociation of the B'α and CHK2 proteins, however, it does not have an effect on the binding of B'α to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'α can re-associate hours after DNA damage and this is not dependent upon the repair of the DNA. Dissociation is dependent on ATM activity and correlates with an increase in the ATM-dependent phosphorylation of CHK2 at serines 33 and 35 in the SQ/TQ region. Indeed, mutating these sites to mimic phosphorylation increases the dissociation after IR. CHK2 is able to phosphorylate B'α in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization.

viii

Alternatively, PP2A negatively regulates CHK2 phosphorylation at multiple sites, as well as its kinase activity and protein stability. These data reveal a novel mechanism for

PP2A to keep CHK2 inactive under normal conditions while also allowing for a rapid release from this regulation immediately following DNA damage. This is followed by a subsequent reconstitution of the PP2A/CHK2 complex in later time points after damage, which may help to attenuate the signal. This mechanism of CHK2 negative regulation by

PP2A joins a growing list of negative regulations of DNA damage response proteins by protein serine/threonine phosphatases.

INTRODUCTION

DNA Damage Response Pathway

Maintenance of genomic integrity is an essential part of cellular physiology.

Genotoxic insults that induce DNA breaks must be repaired in order to prevent the

propagation of mutations that can contribute to malignant transformation. DNA damage

occurs via multiple mechanisms including ionizing radiation (IR), ultraviolet radiation

(UV), chemicals from the environment, replication stress, and reactive oxygen species

that are made as a byproduct of cellular metabolism. The primary role of the DNA

damage response (DDR) is to induce cell cycle arrest, repair the incurred DNA damage,

or, in cases where the extent of damage is beyond repair, signal for the initiation of

apoptosis or cellular senescence (214).

The different types of DNA damage as well as each repair mechanism were

extensively reviewed recently (153). Here, we will focus primarily on the DNA damage

induced by IR, which causes mainly double-strand breaks (DSB), UV which causes single-strand breaks (SSB), and hydroxyurea (HU) which causes replication stress. DSB can be repaired through non-homologous end joining (NHEJ) and homologous recombination (HR) (60). The exact repair mechanism for stalled replication forks and

SSB in mammals has not been elucidated, but in yeast, it involves a special class of DNA polymerases called translesion polymerases (139).

After a DNA break, the DDR must arrest the cell cycle to allow time for the DNA to be repaired. Distinct cell cycle checkpoints include G1/S or G1, intra-S-phase, and 1

G2/M or G2. In the case that the damage cannot be repaired, the cell cycle arrest can lead to senescence or the cell can signal through the apoptotic pathway (108, 130). Signaling through the DDR occurs through a distinct pathway that includes sensors, signal transducing proteins, effector kinases, mediators, and effector proteins (Figure 1).

Figure 1. The DNA damage response pathway. DNA breaks and replication stress signal through the sensors (MRN and 9-1-1), mediators (H2AX, BRCA1, MDC1,

53BP1), signal transducing kinases (ATM, ATR), effector kinases (CHK2, CHK1), and effector proteins (E2F1, p53, Cdc25) leading to cell cycle arrest and gene transcription.

Proteins that are phosphorylated by ATM and/or ATR are marked by a gray phosphate group and proteins that are phosphorylated by CHK2 and/or CHK1 are marked by a black phosphate group. An arrow signals activation and a "T" signals inhibition. 3

Sensors - Mre11-Rad50-Nbs1 and Rad9-Rad1-Hus1 Complexes

DNA damage is recognized by sensor proteins that initiate the activation of the

DDR. These sensors include the Mre11-Rad50-Nbs1 (MRN) complex and the Rad9-

Rad1-Hus1 (9-1-1) complex that localize to DSB or regions of replication stress and SSB, respectively (84, 139). These complexes are necessary for the activation of the signal transducing kinases.

The structure of the 9-1-1 complex resembles the proliferating cell nuclear antigen (PCNA) sliding clamp that is loaded onto DNA at points of replication (139).

The Rad17-replication factor C (RFC) complex acts as the 9-1-1 clamp loader in a process analogous to RFC acting as the clamp loader for PCNA (11).

The MRN complex binds to DNA DSB. Mre11 binds to Nbs1, DNA, and Rad50 and possesses DNA exonuclease, endonuclease, and unwinding activities (196). Rad50 may function to keep the broken ends of the DNA together. Nbs1 functions to recruit signal transducing kinases to the break site and mediates the DDR signal (215).

Signal Transducing Kinases - ATM and ATR

The localization of the MRN and 9-1-1 complexes to the sites of DNA damage signals to the signal transducing kinases to become active. These kinases are Ataxia- telangiectasia mutated (ATM) and ATM and Rad3-related (ATR), which are members of the PI-3K like kinase (PIKK) family with DNA-dependent protein kinase (DNA-PK).

Primarily in response to DSB, ATM is activated by autophosphorylation on

S1981 causing inactive dimers to dissociate into active monomers (7). Interestingly, the

ATM protein was first identified in patients with ataxia-telangiectasia (AT) (156). AT

causes genomic instability, a predisposition to cancer, immunodeficiency, and several

neurological impairments (33). Mutation of Mre11 and Nbs1 in humans causes the

similar disorders AT-like disorder (ATLD) and Nijmegen Breakage Syndrome (NBS),

respectively (172, 188). Nbs1 seemingly acts both upstream and downstream of ATM as

ATM is known to phosphorylate Nbs1 on S343 and at the same time Nbs1 and the MRN

complex have been shown to be required for full activation of ATM (81, 186, 215).

The localization of ATR to the break site and its subsequent activation is

dependent upon the 9-1-1 complex, binding between ATR and ATR-interacting protein

(ATRIP), and replication protein A (RPA). RPA coats single-strand DNA and consists of

3 subunits: RPA70, RPA32, and RPA14 (29). RPA32 is phosphorylated at T21 and S33 by ATM , ATR, and DNA-PK (12).

Although DNA-PK is not classified as a signal transducing kinase, it does play a role in the DDR. DNA-PK is comprised of a catalytic subunit, DNA-PKcs, and the targeting subunit, the Ku70-Ku80 heterodimer. It is activated upon association with

DNA (165). The primary role of DNA-PK is to initiate NHEJ to repair DNA double- strand breaks. Two major autophosphorylation clusters in DNA-PKcs (referred to as

ABCDE and PQR) regulate the ability of DNA-PK to function in DNA end processing during repair (117).

Effector Kinases - CHK1 and CHK2

The signal transducing kinases ATM and ATR signal through the effector kinases

CHK1 and CHK2 (checkpoint kinase 1 and checkpoint kinase 2) (8). CHK1 and CHK2 work alongside ATM and ATR to perpetuate the DDR signal. CHK2 is activated

primarily in response to DSB through the phosphorylation of T68 by ATM (3, 113, 119).

CHK1 is active even in unperturbed cells, but is also further activated through the phosphorylation of S317 and S345 by ATR, primarily in response to SSB and replication stress (8). Interestingly, there is also crosstalk between these two pathways. The processing of DSB in an ATM- and Mre11-dependent manner leads to RPA coating the single-stranded DNA, leading to activation of ATR and CHK1 (73). ATR can phosphorylate and activate ATM following UV treatment or replication stress (174).

CHK1 S317 phosphorylation has been shown to be dependent on ATM and Nbs1 in addition to ATR (53).

The importance of the effector proteins in development has been discovered using mouse models. CHK1-deficient mice are embryonic lethal, however, CHK2-deficienct mice are viable and do not have an increased risk for cancer (68, 100, 181, 182). This, along with the fact that CHK1 is active under normal circumstances, leads some to the belief that CHK1 acts as the "workhorse" of the cells and CHK2 functions only when needed after DNA damage.

Mediators - H2AX, BRCA1, MDC1, and 53BP1

The mediator proteins H2AX, BRCA1, MDC1, and 53BP1 work to coordinate the

localization of various factors in the DDR, promote their activation, and regulate

substrate accessibility (176).

The histone variant H2AX comprises 10-15% of total cellular histone H2A, as

revealed through mouse studies (19). H2AX is phosphorylated by ATM, ATR, and

DNA-PK on S139 and is commonly referred to as γ-H2AX (148). γ-H2AX forms foci

both proximal and distal to the DSB site and is dephosphorylated following repair of the

DNA (reviewed in a following section), thus it is an excellent marker for the presence of

DSB (147). γ-H2AX is required for the foci formation of Nbs1 and the other mediator proteins BRCA1, MDC1, and 53BP1, which is important for DDR signaling (9, 19, 140,

173, 178).

BRCA1 (breast cancer susceptibility 1) is phosphorylated by ATM on S1387 and by CHK2 on S988 in response to IR (21, 32, 52). In response to UV, BRCA1 is phosphorylated by ATR on S1457 (54). Phosphorylated BRCA1 forms distinct nuclear foci that co-localize with γ-H2AX, MDC1, and the MRN complex. The role of BRCA1 seems to be to direct repair through both NHEJ and HR (166, 192).

MDC1 is phosphorylated in an ATM- and CHK2-dependent manner, binds to γ-

H2AX and co-localizes with γ-H2AX foci, and is required for the formation of MRN,

BRCA1, and 53BP1 foci (103, 173, 177). Thus, MDC1 functions as a molecular scaffold

to mediate parts of the DDR downstream of foci formation (126).

53BP1 was first discovered based on its ability to bind to the DNA-binding domain of p53 (70). In the DDR, 53BP1 localizes to the sites of DSB, is required for cell 7

cycle checkpoints, and is required for p53 accumulation and BRCA1 foci formation

(190). It seems to be involved in ATM activation, although the precise mechanism is not

known (125).

Effector Proteins - Cdc25, E2F1, and p53

The effector proteins Cdc25, p53 and E2F1 function to activate cell cycle

checkpoints and regulate the transcription of genes whose products are important in the

end result of the DDR, whether it be DNA repair, apoptosis, or senescence.

The Cdc25 phosphatases regulate progression through the cell cycle by removing

inhibitory Y15 phosphatases from the cyclin-dependent kinases Cdk1 and Cdk2 (38).

The negative regulation of Cdc25 leads to G1/S, intra-S, and G2/M checkpoints. CHK1

phosphorylates Cdc25A on S76, leading to Cdc25A ubiquitination and degradation (74).

CHK1 and CHK2 phosphorylate Cdc25C on S216 which allows 14-3-3 binding and

subsequent sequestration of Cdc25C in the cytoplasm, away from substrates. Cdc25B1

and Cdc25B2 are phosphorylated on S309 and S323, respectively, leading to inactivation

through the same 14-3-3-mediated mechanism (38, 210).

The transcription factor p53 may be the most widely recognized tumor suppressor and has been found to be mutated in more than half of all tumor cases (62). In addition,

the multi-organ cancer syndrome, Li-Fraumeni syndrome, is caused in most cases to be

from inheritance of a mutant copy of the p53 gene, TP53, and subsequent loss of the

wild-type copy (109, 170).

P53 is stabilized in the DDR which allows it to activate the transcription of genes

whose products participate in cell cycle arrest, DNA repair, senescence, or apoptosis,

8 depending upon the cellular stress (63). Mdm2 negatively regulates p53 through ubiquitin-mediated proteosomal degradation (64, 69, 78). MdmX also negatively regulates p53 by binding to Mdm2 and enhancing Mdm2 binding to and ubiquitination of p53 (98, 143, 185). ATM, ATR, DNA-PK, CHK1 and CHK2 stabilize p53 by direct phosphorylation on S15 and S20 which prevent p53 binding to Mdm2 (5, 160, 161).

ATM and ATR inhibit Mdm2 by phosphorylating it on S395 and S407, respectively (114,

162). ATM phosphorylates MdmX on S403 which leads to its ubiquitination and degradation (141). CHK2 phosphorylates MdmX on S367 and S342 which promotes 14-

3-3 binding and degradation of MdmX (24, 82). Therefore, multiple DDR proteins coordinate to stabilize and activate p53 through phosphorylation of p53 and its negative regulators Mdm2 and MdmX.

The transcription factor E2F1 is also activated in the DDR. ATM and CHK2 phosphorylate E2F1 on S31 and S364, respectively, which leads to E2F stabilization (97,

171). E2F1 can then upregulate genes that lead to cell cycle checkpoints or apoptosis, although the specific genes that are responsible for these cellular effects have not yet been determined.

Checkpoint Kinase CHK2

The functions of CHK2 in phosphorylating and activating factors in the DDR are outlined above. This section will focus on the details of CHK2 structure and activation, its mutations found in cancer, and the pursuit of a CHK2 cellular phenotype.

CHK2 Structure and Activation

CHK2 contains three major domains (Figure 2): an SQ/TQ domain (amino acid residues 19-69), an FHA domain (amino acid residues 112-175), and a serine/threonine kinase domain (amino acid residues 220-486), all of which play a role in the activation of

CHK2. The SQ/TQ domain contains seven serine or threonine residues followed by a glutamic acid. SQ and TQ sites are preferentially phosphorylated by ATM and ATR (77,

132). The FHA domain recognizes and binds to phosphorylated proteins and therefore mediates protein-protein interactions (41). The kinase domain is the enzymatically active domain and is responsible for the phosphorylation of serine and threonine residues of

CHK2 substrates.

Under normal conditions, CHK2 exists as a monomer (2). In response to DNA damage, ATM phosphorylates CHK2 on T68 in the SQ/TQ domain (3, 113, 119). The

FHA domain of one molecule of CHK2 binds to the phosphorylated T68 of another, allowing autophosphorylation of T383 and T387 within the kinase domain (83, 200).

CHK2 then dissociates into active monomers (2). CHK2 is also phosphorylated at serines 19, 33, and 35 in the SQ/TQ domain by ATM and autophosphorylated at S516 and these events enhance CHK2 activity (16, 79, 124, 198). Phosphorylation of CHK2 at

S456 regulates CHK2 stability and autophosphorylation at S379 leads to CHK2

10 ubiquitination and positively affects CHK2 function (75, 104). Of note, a useful tool in studying CHK2 has been the D347A dominant negative kinase-dead mutant (22, 112).

The crystal structures of the FHA and kinase domains, alone and together, have been solved, which has proven extremely informative for deciphering the specific residues necessary for the oligomerization and activation of CHK2 as well as binding between CHK2 and its substrates (17, 95, 137). Attempts to crystallize full length CHK2 have been unsuccessful due to the fact that the SQ/TQ domain is highly susceptible to digestions, suggesting that it is unstructured or loosely folded (17). In order to crystallize the kinase domain of CHK2 and preserve the ability to form dimers, a small-molecular inhibitor or a kinase-dead mutant needed to be used, since active CHK2 dissociates into monomers (17, 137).

Figure 2. The structure of CHK2. The SQ/TQ domain is displayed in light gray, the

FHA domain in dark gray, and the kinase domain in black. Important CHK2 phosphorylation sites are highlighted. 12

CHK2 in Cancer

CHK2 is considered a tumor suppressor and mutations in CHEK2, the gene

encoding human CHK2, have been found in various cancers. The first mutation found in

CHEK2 was 1100delC, originally identified in families with Li-Fraumeni syndrome that

did not have mutations in TP53 (10). Later, this mutation was also found to be

significantly associated with breast cancer in familial cases without a BRCA1 or BRCA2

mutation (187). This CHEK2 allele seems to act as a low-penetrance tumor suppressor

gene and may be an adverse indicator of prognosis (34, 118). The SUM102PT breast

cancer cell line contains the 1100delC mutation (195).

Many other mutations in CHK2 have also been identified, but the clinical

significance of most are not yet clear. I157T and 1422delT were found in Li-Fraumeni

families lacking a TP53 mutation (10). R180H, R117G, R137Q, and delE161 were

identified in breast cancer patients with familial history (167, 168). Y424H and S428F

were found in Ashkenazi Jewish families with a high risk of breast cancer (80, 159).

R145W was identified in the HCT15 colon carcinoma cell line (10). The nucleotide

mutation T908A was found in the UACC812 breast cancer cell line (195). In addition,

multiple germline CHEK2 mutations have been found in prostate cancer patients and families with familial prostate cancer (37). CHK2 transcriptional downregulation due to

promoter methylation has also been identified in human cancers (191, 212).

The 1100delC mutation causes a frameshift at amino acid T366 and the 1422delT

causes a frameshift at amino acid R475, both of which result in an early stop codon (10).

These mutants have no kinase activity in vitro (199). The T908A mutation also causes an

early stop codon at L303 (195). R145W has reduced kinase activity in vitro, is not

phosphorylated at T68, cannot be activated in response to IR, and cannot bind to p53 in

vivo (46, 199). The R145W mutation also leads to a destabilization and degradation of

the CHK2 protein (85). I157T cannot bind to p53 in vivo, has reduced kinase activity in vitro, and has deficiencies in CHK2 oligomerization and autophosphorylation (46, 158,

199). This is most likely due to the fact that this site plays an integral role in the FHA-

kinase domain interface during CHK2 activation (17). 1100delC and E161del are unstable and not efficiently phosphorylated at T68 following IR whereas R117G is phosphorylated at T68 but does not display a mobility shift via Western blot, suggesting it is not phosphorylated fully at other sites that are necessary for the activation of CHK2

(169). S428F could not complement a Rad53 (CHK2 homologue) deletion in S. cerevisiae and therefore is thought to abrogate CHK2 function (159). R117G, R145W, and E161del are likely to be deleterious to CHK2 function based on a bioinformatics analysis of CHK2 peptides from nine species (169).

The germline mutations 1100delC and R145W are associated with loss of the

wild-type allele (85). The HCT15 cell line, which harbors the R145W mutation on one

CHEK2 allele, has heterozygous inactivation of CHK2 (85). Using reverse transcriptase-

PCR, the only detectable CHEK2 gene in the HCT15 cell line harbored the mutation

which causes the expression of the R145W protein and therefore, HCT15 cells lack

functional CHK2 (46). The SUM102PT and UACC812 breast cancer cell lines also lack

detectable CHK2 protein levels (195).

In summary, mutations in CHK2 have been found that affect protein function in a

number of ways. Many mutations have been identified in Li-Fraumeni families, primary

human cancers, and cancer cell lines, although more work needs to be done to elucidate

the effects on CHK2 function as well as the clinical significance of these mutations.

CHK2 Phenotypes

Two CHK2-/- mouse models have given much information about the importance

of CHK2 in specific aspects of the DDR. Surprisingly, given the many actions of CHK2,

it was not found to be necessary for organism development, whereas CHK1-null mice are

embryonic lethal (68, 100, 181, 182). It is possible that since CHK2 and CHK1 share

many targets, the absence of CHK2 may be compensated for by CHK1. Here, the

phenotypes of cells lacking CHK2 will be discussed.

In one CHK2 mouse model, CHK2-/- MEFs (mouse embryonic fibroblasts) and

ES (embryonic stem) cells in a C57BL/6 background displayed a normal G2/M

checkpoint following IR. Initiation of the G1/S checkpoint was intact but cells were

deficient in maintenance of the checkpoint (181). In contrast, in another mouse model,

-/- thymocytes from CHK2 mice in the same background displayed a decrease in G1/S

arrest, G2/M arrest, and apoptosis following IR (68).

Using HCT15 cells expressing HA-tagged CHK2, CHK2 was shown to play an integral role in the intra-S-phase checkpoint (47). Cells lacking functional CHK2

continued to replicate DNA after DSB instead of activating an intra-S-phase checkpoint

in a phenotype called radioresistant DNA synthesis (RDS). This is due to the inability to

induce the degradation of Cdc25A.

Surprisingly, CHK2 was found to be not required for the p53-mediated DDR

response. Knockdown of CHK2 in HCT116 cells inhibited Cdc25C phosphorylation at

S216 but had no effect on p53 stabilization or phosphorylation on S20 following

neocarzinostatin (NCS) treatment, which causes DSB (1). CHEK2 in the HCT116 cell

line was disrupted using gene targeting (71). HCT116 CHK2-/- cells displayed normal

p53 accumulation and phosphorylation at S20 in response to IR, intact G1/S and G2/M

checkpoints following IR, and an appropriate apoptotic response to 5-fluorouracil when

compared to the HCT116 wild-type cells. Gene targeting was also used to generate

CHK2-deficient chicken DT40 cells (144). These cells prematurely entered mitosis

following IR and therefore were defective in the G2/M checkpoint.

These often contradictory results demonstrate the difficulties in clearly demonstrating a cellular phenotype for cells lacking CHK2. CHK2 function appears to be dependent on cell type and mechanism of induction of DNA damage. In the literature, the function of CHK2 in cells is often measured by RDS, apoptosis, and the G1/S and

G2/M checkpoints.

Protein Serine/Threonine Phosphatases

There are two major families of protein serine/threonine phosphatases (PSP). The phosphoprotein phosphatases (PPP) include protein phosphatases (PP) PP1, PP2A, PP2B,

PP4, PP5, PP6, and PP7 and the metal-dependent protein phosphatases (PPM) include

PP2C.

Four isoforms of PP1 are derived from three genes and PP1 is regulated by interactions with many proteins (189). PP2A consists of an A structural subunit, a C catalytic subunit, and one of many B regulatory subunits, described in detail in the following section. PP4 and PP6 are closely related to PP2A C. The holoenzyme of PP6 is proposed to consist of the PP6 catalytic subunit, a SAPS subunit and an ankyrin repeat subunit (123). PP2B, also known as PP3 and calcineurin, and PP7 are stimulated by

Ca2+. PP2B consists of an A catalytic subunit and a B subunit that is related to

calmodulin. PP7 contains EF-hand domains (127). PP5 is unique in the fact that it has

three TRP domains, which mediate protein-protein interactions, and a peptidyl-prolyl cis-

trans isomerase-like domain that are not present in any other PPP (67).

PPM proteins require Mg2+ or Mn2+ for enzymatic activity. There are at least 18

genes encoding PP2C proteins. PP2Cδ is also known as Wip1 or PMDD1. Wip1,

PP2Cα, PP2Cβ, and PP2Cγ have regions of high similarity (107).

PP2A Structure and Regulation

The active PP2A holoenzyme is comprised of three subunits: a structural A subunit (PR65), a catalytic C subunit, and a B subunit (Figure 3). There are two forms of the A subunit, α and β, that have 87% identity (66). There are also two forms of the C

subunit, α and β, which share 97% identity (175). The B subunits can be divided into four major families: the B (PR55), B' (PR61/B56), B'' (PR72), and B'''

(PR93/PR110/striatins). These regulatory subunits are transcribed from 15 genes that encode at least 26 differentially spliced isoforms (42). The B subunits are thought to

confer to PP2A its functional specificity (189).

The crystal structure of the holoenzyme containing PP2A Aα, Cα, and B'γ1 was

solved, revealing the active enzyme structure as well as binding sites between the three

subunits (25, 201). The Aα subunit contains 15 HEAT repeats of antiparallel α-helices

and forms a horseshoe shape. The Cα subunit forms a compact ellipsoidal structure. The

B'γ1 subunit forms a shape similar to the Aα subunit and is comprised of 18 α-helices

and 8 pseudo-HEAT repeats.

The full B' family was originally cloned in 1995 and is comprised of five genes,

two of which are differentially spliced to form three isoforms each (116). The names of

all of the B' family members are as follows: B'α, B'β, B'ε, B'δ1, B'δ2, B'δ3, B'γ1, B'γ2,

and B'γ3. Although this nomenclature is the most commonly used, some laboratories use

older nomenclature for certain B' proteins, using the same Greek letters, but these names

do not match up with the current naming system. Therefore, the literature of the B'

family can often be confusing. In this document, we will only refer to the most common

naming for uniformity and therefore it may differ from the terminology used in the

original publications.

Various mechanisms of PP2A activity regulation have been discovered.

Phosphorylation of PP2A C at Y307 results in inactivation of the enzyme (23).

Methylation of the PP2A C subunit at L309 regulates binding to the B family members, 18 but not B' or B'' proteins, which likely affects PP2A substrate specificity (102). The serine/threonine kinase PKR phosphorylates B'α and this increases PP2A activity in vitro

(202). Therefore, in addition to substrate specificity based on the B subunit in the holoenzyme, post-translational modifications can affect PP2A activity.

Figure 3. PP2A holoenzyme structure. The A structural subunit binds to the C catalytic subunit and one of many B regulatory subunits to form a holoenzyme complex. 20

PP2A Function

The PP2A C subunit is an abundant protein, making up 0.1% of total cellular

protein (189). PP2A participates in diverse cellular processes such as differentiation, cell

cycle regulation, and signal transduction (72). A few key roles of PP2A, focusing on the

role of the B' family, will be discussed here, however, an exhaustive list of proteins that

have been shown to bind to PP2A directly can be found in a recent review (42). The

specific roles of PP2A in the DDR are detailed in a following section.

Several PP2A mouse models have illuminated the importance of PP2A in cell

growth and embryonic development. Knockout of the gene encoding PP2A Cα results in

embryonic lethality (56). Transgenic mice overexpressing B'γ in the lung die neonatally

and lack proper lung development (45). A dominant negative PP2A A mutant that lacks

the ability to bind to the B subunits causes dilated cardiomyopathy and often premature

death in transgenic mice expressing the protein in muscle (14).

C-Myc is transcription factor that regulates genes involved in cell growth,

differentiation, and apoptosis. The overexpression of c-Myc has been found in many

human tumors (31). PP2A containing B'α binds to c-Myc and dephosphorylates it at

S62, resulting in decreased c-Myc protein levels and decreased c-Myc transcriptional

activity (6).

PP2A has a negative effect on the G2/M transition through the negative regulation

of Cdc25 (30, 86). In order to achieve full activation of Cdc25C for entry into mitosis, it

is phosphorylated at T138, which causes removal of 14-3-3. PP2A containing B'δ

counteracts this by dephosphorylating T138 in Cdc25C. Chk1 phosphorylates PP2A B'δ at S37, enhancing B'δ binding to the A and C subunits. Therefore, Chk1 keeps the cell 21

from progressing into mitosis by enhancing dephosphorylation of T138, keeping 14-3-3 bound (110).

The apoptotic agent ceramide was shown to induce apoptosis through mitochondrial PP2A dephosphorylation of Bcl2 at S70, which enhances its activation

(152). This is mediated by the B'α subunit and ceramide promotes translocation of B'α to the mitochondrial membrane (151).

PP2A in Cancer

Several independent lines of evidence suggest a role for the PP2A holoenzyme as a tumor suppressor and indicate the importance of inhibiting PP2A function in cellular transformation. The SV40 small T antigen can bind to the PP2A A-C complex in place of a B subunit, inhibiting PP2A activity, and this interaction is required for SV40- mediated transformation (61, 138, 204). Okadaic acid (OA), a known PP2A inhibitor,

was also found to promote tumor formation (179).

Somatic mutations in the gene encoding the PP2A Aβ subunit, PPP2R1B, were

found in 15% of primary lung tumors, 15% of primary colon tumors, and 6% of lung

tumor-derived cell lines (194). Five missense mutations in PPP2R1B leading to amino

acid substitutions (G15A, L499I, V498E, V500G, and S365P) were found in four colon

carcinomas (180). The PP2A Aα mutations E64D in a lung carcinoma, E64G in a breast

cancer, R418W in a melanoma cell line, and a frameshift mutation at amino acid F170 in

a breast cancer were recently identified (18). These mutations prevent binding to the B'

or B and C subunits (44, 150). In addition, several cancer cell lines have reduced

expression of the A subunit as compared to primary normal epithelial cells (216).

Protein Serine/Threonine Phosphatases in the DNA Damage Response

Phosphorylation plays an integral role in the positive and negative regulation of the DNA damage response pathway signaling, as described above. Interestingly, phosphatases are increasingly being shown to play integral roles in the positive and negative regulation of the cellular signaling in response to different DNA damages. This section will review the recent literature implicating the importance of phosphatases in the

DDR alongside kinases.

DNA Damage Response Proteins that are Negatively Regulated

Since much of the signaling of the DNA damage response pathway is regulated by serine and threonine phosphorylation, it is intuitive that PSPs would negatively regulate these phosphorylation events. Indeed, there are many proteins that are negatively regulated in this manner (Figure 4).

Figure 4. Protein serine/threonine phosphatases in the DNA damage response pathway. The positive and negative regulation of DDR proteins via PSPs is shown. An arrow indicates activation and a "T" indicates inhibition. 24

ATM and ATR

The first evidence that PP2A might play a role in the ATM-dependent DDR came from the finding that the PP2A B subunit dissociates from the PP2A heterotrimer in the nucleus in an ATM-dependent manner after IR (58). It was later found that inhibition of

PP2A with OA or expression of a dominant-negative PP2A C increased ATM autophosphorylation at S1981 under normal conditions, but did not affect ATM activity.

ATM binds to the A and C subunits, but this binding is dissociated after IR, partially in an ATM-dependent manner (55). PP2A was able to dephosphorylate ATM S1981, but not as efficiently as Wip1, described below (163).

The PP2A interacting protein TIP binds directly to PP2A C and inhibits PP2A activity. PP2A was found to oppose the ATM and ATR phosphorylation of a 32kD protein and TIP prevented this function of PP2A (115). In X. laevis nucleoplasmic extracts, DSB inhibit replication by inhibiting the loading of Cdc45, RPA, and polymerase α onto DNA. However, PP2A and caffeine, an inhibitor of ATM and ATR, was able to reverse the inhibition of replication and restore loading of the necessary factors (142).

Two different Wip1 mouse models have shown a role for Wip1 in negatively regulating ATM. E1A+Ras expressing Wip1-null MEFs were seen to have an increase in

ATM phosphorylation at S1987 (human S1981) and ATM activity under normal conditions as well as after IR. Phosphorylation of p53 S18 (human S15), the ATM phosphorylation site, was increased slightly as well (163). Splenocytes from Wip1+/- and

Wip1-/- mice in an Eµ-myc background displayed an increase in ATM phosphorylation

and p53 S18 phosphorylation (164). Wip1+/- and Wip1-/- mice were more resistant to

25 tumor formation and this was dependent on ATM and p53 (164). Wip1 was found to dephosphorylate ATM at S1981, S367 and S1893 in vitro (163, 164).

In human cells, the overexpression of Wip1 decreased ATM autophosphorylation and ATM-dependent CHK2 phosphorylation after IR. The induction of Wip1 protein levels in control cells correlated with the decrease in phosphorylated ATM after IR.

ATM phosphorylation also remained longer after IR in cells that had decreased Wip1.

Wip1 could dephosphorylate ATM S1981 in vitro (163). Another study, however, used a tetracycline-inducible Wip1 and showed that the expression of Wip1 after IR did not have an effect on S1981 phosphorylation (106). This difference may be due to the different cell lines and conditions used.

RPA

After treatment with and removal of HU, RPA32 is dephosphorylated at S21 and

S33. Inhibition of PP2A with OA or knockdown via siRNA results in persistence of RPA phosphorylation and foci. An RPA mutant that could not be dephosphorylated resulted in reduced cell viability following UV or HU treatment and reduced DNA repair following replication stress (48). This implicates PP2A in the recovery of the DDR.

CHK1

CHK1 has been shown to be negatively regulated by multiple PSPs. In S. pombe, the PP1 homologue Dis2 negatively regulates CHK1. Cells lacking Dis2 have a prolonged G2 arrest following treatment with the double-strand break inducer MMS

(methylmethane sulfonate) or the UV mimetic 4-NQO (4-nitroquinoline-N-oxide) but

DNA repair is not affected. The overexpression of Dis2 caused a decrease in CHK1

activation following UV (35). CHK1 S345 could be dephosphorylated in vitro by Dis2

or human PP1 (35, 106).

In human cells, the inhibition of PP2A induced CHK1 phosphorylation in the

absence of DNA damage and also prevented CHK1 dephosphorylation after HU removal.

The knockdown of PP2A increased CHK1 phosphorylation on S317 and S345 and in

vitro, PP2A was able to directly dephosphorylate CHK1 (88). In X. laevis egg extracts,

the addition of PP2A C reversed CHK1 phosphorylation at S344 (human S345) after

activation by DSB. The inhibition of PP2A also enhanced CHK1 phosphorylation

following activation by DSB (142).

Human CHK1 is also regulated by Wip1. Wip1 could dephosphorylate CHK1

primarily at S345 and slightly at S317 in vitro. In vivo, the overexpression of Wip1

resulted in the elimination of CHK1 phosphorylation at S345 and S317, a decrease of

Cdc25C phosphorylation at S216, a decrease in Cdk1 phosphorylation at Y15, and a

decrease in the S-phase and G2/M checkpoints, whereas the knockdown of Wip1 via siRNA caused the reverse effects. Breast cancer cell lines with overexpression of Wip1

(MCF-7, BT474, and MDAMB231) showed only a marginal increase in CHK1 S345 phosphorylation after UV as compared to the control cells (HEK and U2OS) (106).

Interestingly, Wip1-/- MEFs in a 129/sv-C57BL6 background showed a greater increase

in phosphorylation at S345 in CHK1 after IR than wild-type MEFs whereas Wip1+/+ and

Wip1-/- splenocytes expressing Eµ-myc did not show any difference in Chk1 S345 phosphorylation, possibly indicating a cell type specific or DNA damage specific regulation of CHK1 via Wip1 (106, 164).

In summary, CHK1 phosphorylation at S317 and S345 and CHK1 activity

following DNA damage are regulated by PP1, PP2A, and Wip1. This seems to play an

important role in recovery from the DDR checkpoints.

CHK2

Much of the work examining the negative regulation of CHK2 has been done

using the S. cerevisiae CHK2 homologue Rad53. The PP2C phosphatases Ptc2 and Ptc3

as well as the type 2A phosphatase Pph3 have all been shown to negatively regulate

Rad53 under different circumstances. To study double-strand breaks in yeast, a system

that induces the HO endonuclease to create a single double-strand break at a specific

locus that cannot be repaired by homologous recombination was utilized (57). A single

break will cause a G2/M arrest and then adaptation to the checkpoint. Cells lacking Ptc2

and/or Ptc3 were defective in adaptation to an HO-induced G2/M arrest. Ptc2 and Ptc3 were required for recovery from the checkpoint as measured by Rad53 dephosphorylation and release from the G2/M checkpoint. Ptc2 and Ptc3 bind to the FHA1 domain of Rad53

and protein kinase CK2 phosphorylation of Ptc2 at T376 is necessary for this interaction

(57, 87). Mutation of this site prevented adaptation and recovery of the G2/M checkpoint

(57). Therefore, Ptc2- and Ptc3-mediated dephosphorylation of Rad53 results in recovery

from the G2/M checkpoint.

Also in S. cerevisiae, deletion of the PP2A-like phosphatase Pph3 caused hypersensitivity to MMS. Pph3-Psy2 binds to Rad53 and dephosphorylates it directly.

The Pph3-Psy2 complex is necessary for the dephosphorylation of Rad53 during

28 recovery from the intra-S-phase checkpoint and it promotes the resumption of normal

DNA synthesis following removal of MMS (131).

One study attempted to differentiate the roles of Pph3, Ptc2, and Ptc3 in negatively regulating Rad53. Cells lacking Pph3 again showed hyperphosphorylation of

Rad53, but Rad53 was still deactivated after MMS treatment. Cells lacking Pph3, Ptc2, and Ptc3 have impaired Rad53 deactivation following MMS treatment, although Rad53 deactivation from replication stress is only slightly delayed. Pph3 is not required for

Rad53 dephosphorylation and deactivation following replication stress. Ptc2 and Ptc3 were not required for Rad53 deactivation and dephosphorylation following genotoxic stress in S-phase via HU (184). Thus, distinct phosphatases appear to be required for the dephosphorylation and deactivation of Rad53 following various DNA damages and the phosphatase required for recovery from replication stress has not yet been identified.

In humans, CHK2 was also found to be regulated by PP2A and Wip1. Inhibition of PP2A using OA increased the phosphorylation of CHK2. Although PP2A was also found to negatively regulate ATM, the effect on CHK2 phosphorylation is ATM- independent (55). Utilizing a yeast two-hybrid system, an in vitro binding assay, and co- immunoprecipitation, CHK2 was found to bind to PP2A A, C, and many B' subunits.

CHK2 was able to phosphorylate B'γ1 and B'γ3 in vitro and this increased PP2A activity in vitro. The overexpression of B'γ3 resulted in a decrease in CHK2 phosphorylation after adriamycin treatment, which causes DNA adducts (40). Following cisplatin treatment, PP2A containing a B subunit was found to bind to CHK2. Inhibition of PP2A with OA or knockdown via siRNA caused an increase in CHK2 phosphorylation at T68.

PP2A was found to dephosphorylate CHK2 in vitro as well (96).

Wip1 and CHK2 bind in the nucleus and binding is dependent upon the CHK2

SQ/TQ domain, kinase activity, and nuclear localization signal (NLS) and the Wip1 N- terminal domain (50, 206). Endogenous Wip1 and CHK2 can bind in MCF7 cells, which have a higher expression level of Wip1 and in A549 cells (50, 206). Using GST-Wip1 in a GST pulldown assay with lysates of HCT15 cells stably expressing HA-CHK2, Wip1

was seen to bind to CHK2 only after IR, suggesting that CHK2 phosphorylation is

important for the interaction (136). In vitro, Wip1 can dephosphorylate CHK2 at S19,

S33/35, T68, T432, and S516 and this significantly reduces CHK2 kinase activity (50,

136). Overexpression of Wip1 inhibited CHK2 kinase activity in vitro, and in vivo,

decreased T68 phosphorylation, caused a delay in CHK2 activation following IR, and

caused a delay in G2/M arrest (50, 136). Knockdown of Wip1 resulted in sustained

CHK2 T68 phosphorylation and kinase activity following IR, as well as an increase in apoptosis (50). In the acute promyelocytic leukemia (APL) cell line NB4, arsenic trioxide could induce CHK2 T68 phosphorylation through the inhibition of Wip1 (205).

Finally, in stomach adenocarcinoma tumors, high Wip1 expression correlated with low

CHK2 phosphorylation (51).

In yeast, Rad53 is negatively regulated by PP2A-like and PP2C-like phosphatases. In humans PP2A and the PP2C phosphatase Wip1 have been shown to negatively regulate CHK2. These phosphatases have been demonstrated to negatively regulate the activation of CHK2 as well as dephosphorylate it to allow for recovery from

DDR checkpoints.

H2AX

PP1, PP2A and PP4 have been implicated in negatively regulating γ-H2AX. In yeast, deletion of any of the PP4-like complex HTP-C genes (Pph3, Psy2, and Ybl046w) increases the amount of cellular γ-H2AX. HTP-C can dephosphorylate γ-H2AX in vitro.

Although Pph3 deficient cells had similar rates of DSB repair and loss of γ-H2AX foci as wild-type cells, even though H2AX remained phosphorylated, Rad9 (human 53BP1) and

Rad53 (human CHK2) remained active longer and this correlated with maintenance of the G2/M checkpoint (76).

In human cells, the inhibition of PP1 partially inhibited the elimination of γ-

H2AX after DNA damage induced by IR (129). The inhibition of PP2A or knockdown with siRNA increased the amount of γ-H2AX, increased the amount of foci-positive cells, increased the intensity of the foci, and increased the amount of time the foci were maintained following treatment with the topoisomerase I inhibitor camptothecin (CPT)

(27). Although PP1 has been shown to dephosphorylate γ-H2AX in nucleosomes in vitro, PP2A is at least 25 times more active toward γ-H2AX in monomeric form or when incorporated into nucleosomes (27, 129). Indeed, PP2A C co-localizes with and binds to

γ-H2AX after CPT treatment and the binding increases with increasing DNA damage

(27).

Thus far, PP2A and PP4 have been implicated in negatively regulating γ-H2AX.

In cells with both PP4C and PP2A C knocked-down, γ-H2AX levels were higher and sustained longer after CPT as compared to control cells, although the PP2A C knockdown had a greater effect. PP2A C and PP4C had comparable abilities to

dephosphorylate γ-H2AX from mononuclosomes in vitro (28). Both PP2A and PP4, but

not PP1, can dephosphorylate γ-H2AX in vitro from acid extracted histones. Knockdown of PP4C and PP2A C caused an increase in the total levels of γ-H2AX in untreated and

irradiated cells (128).

Further examination of the specific roles of PP2A and PP4 in the regulation of γ-

H2AX has found distinct roles for each. PP4C silenced cells, but not PP2A silenced

cells, showed an increase in γ-H2AX even before DNA damage (28). PP2A C silenced

cells were slightly weakened in the ability to repair DSB induced by X-rays but PP4C silenced cells were not impaired in DNA repair. Knockdown of PP4C caused a slower decrease in γ-H2AX foci after IR but PP2A knockdown did not affect foci. PP4C primarily dephosphorylated γ-H2AX associated with chromatin rather than in the

nucleoplasm, whereas PP2A did not. PP4C depleted cells delayed entry into mitosis, but did not have any problems with the initiation of the G2/M checkpoint (128).

In summary, the dephosphorylation of γ-H2AX is mediated through PP2A and

PP4. This is necessary for the recovery from the DSB-induced checkpoint.

P53

P53 is negatively regulated through direct dephosphorylation by PP1 and PP2A and through the regulation of its negative regulator, Mdm2. The phosphorylation of p53 at S37 is required for p53 transcriptional activity. PP2A binds to p53 following IR and dephosphorylates S37 (36). Inhibition of PP2A with OA or siRNA causes an increase in p53 phosphorylation at S15 and an increase in p53 activity (121).

By inhibiting PP1 and PP2A with 100nM OA and comparing to the inhibition of

only PP2A with 10nM OA, it was shown that PP1 inhibition induces apoptosis in rabbit

lens epithelial cells. This is accompanied by an induction of p53 and the p53 target Bax

(89). Inhibition of PP1, but not PP2A, with OA or calyculin A increased apoptosis in

human lens epithelial cells in a p53-dependent manner. Inhibition of PP1 only slightly increased p53 levels but dramatically increased p53 phosphorylation at S15 and S37 which enhance regulation of p53 targets Bcl2 and Bax. PP1 dephosphorylates p53 at S15

and S37 in vitro and in vivo and this decreases its transcriptional activity and attenuates apoptosis (90).

Wip1 is induced in response to IR in a p53-dependent manner as a part of a

negative feedback mechanism (26, 49). Wip1-/- MEFs exhibit a slight increase in p53

phosphorylation at S15 and an increase in the levels of p53 target p21 (26). In an in vitro

assay, Wip1 could dephosphorylate p53 on S15 but not S46. After IR, Wip1-/- MEFs had

an increase in phosphorylated S15. An increase in the protein levels of Wip1 decreased

p53 protein levels and S15 phosphorylation whereas knockdown of Wip1 with siRNA

resulted in increased p53 protein levels and S15 phosphorylation. In this system, Wip1

did not have an effect on ATM or ATR after IR or UV, respectively, therefore the effect

on p53 seems to be direct (106).

Wip1 plays an important role in the cell growth and cycle checkpoints in a p53-

dependent manner. Overexpression of Wip1 led to a decrease in colony formation (49).

Wip1-/- mice displayed a defect in T cell maturation due to sustained p53 activation (157).

-/- Wip1 MEFs have a more robust G1 arrest following IR, which may be due to increased

activity of p53 (26). Wip1 was required for recovery from arrest in G2 in a p53-

33 dependent manner (99). Overexpression of Wip1 decreased the S-phase and G2/M checkpoints whereas siRNA knockdown of Wip1 increased the intensity and length of the

S-phase and G2/M checkpoints after UV and IR (106). Interestingly, the Wip1 gene was found to be amplified in 11% of human breast tumors, most of which had wild-type p53

(15). These data collectively suggest that the negative regulation of p53 by Wip1 contributes to tumorigenesis.

Not only is p53 regulated by multiple PSPs directly, but it is also regulated indirectly through the regulation of Mdm2. Mdm2 was predicted to be a Wip1 substrate

(203). Indeed, Wip1, but not PP2A, dephosphorylates Mdm2 at S395, which is an ATM phosphorylation site (105, 114). Wip1 dephosphorylation at S395 increases the stability of Mdm2 and increases the Mdm2-p53 interaction. Wip1 inhibits Mdm2 auto- ubiquitination thereby stabilizing the protein (105).

The induction of p53 causes an induction of cyclin G and increased binding between cyclin G and PP2A B'γ3 (134). Cyclin G preferentially associates with B'γ3 in the active holoenzyme complex. It appears to act as a recruitment factor to bring PP2A to Mdm2 so PP2A can dephosphorylate Mdm2 at S216 and S166. This increases Mdm2 binding to p53, thereby negatively regulating it (135). Overexpression of PP2A B'γ3 and

B'γ2 increases the levels of cyclin G1 protein. Ectopic expression of B'γ3 counteracts the

DNA damage-induced destabilization of cyclin G1 (92).

In conclusion, the multiple modes of regulation of p53 and its activation of negative feedback loops signifies the importance of keeping p53 tightly regulated after

DNA damage. PSPs play a role in regulating p53 through direct dephosphorylation via

PP1 and PP2A as well as enhancement of Mdm2 negative regulation of p53 through

PP2A.

DNA Damage Response Proteins Whose Activities are Enhanced

In contrast to the many instances of negative regulation of DDR proteins by PSPs,

there are also specific cases in which certain phosphatases enhance the activity of

proteins in the pathway (Figure 4).

ATM

PP5 has been shown to play an important role in the activation of the DDR

through ATM. Cells with decreased PP5 protein or activity exhibited a decrease in ATM

autophosphorylation at S1981, a decrease in ATM kinase activity, and a phenotype of

RDS after DNA damage, which is consistent with the phenotype of cells lacking ATM

activity (4). PP5-deficient MEFs displayed a defect in the G2/M checkpoint after IR, a

decrease in ATM activity, and less phosphorylation of ATM targets CHK2 and Nbs1

after DNA damage (207). In fact, PP5 was found to bind to ATM, and this binding was

increased after treatment with NCS or IR (4).

Interestingly, ATM plays a role in activating PP1 and this is important for the

G2/M checkpoint. It has been shown that PP1 is activated following DNA damage

caused by IR (59). ATM phosphorylates the PP1 inhibitor I-2 at S43, causing dissociation of PP1 and I-2. This phosphorylation is necessary for the dephosphorylation of PP1 at T320 (183). These two actions cause an increase in PP1 activity (59, 183).

PP1 then inhibits Aurora-B kinase activity resulting in a G2/M checkpoint (183).

ATR

HIV-1 Vpr (viral protein R) induces a G2 checkpoint through ATR and the phosphorylation of Cdk1 at Y15 (43, 65, 145, 149). PP2A is necessary for this Vpr- induced checkpoint. Inhibition of PP2A with OA or knockdown of the PP2A A or C subunits resulted in a decrease in the amount of cells arrested at the Vpr-induced G2/M

checkpoint, a decrease in Vpr-induced Cdk1 Y15 phosphorylation, and a decrease in

phosphorylation of CHK1 S345 (43, 111). This was only in a Vpr-induced fashion, as

PP2A had no effect on pS345-CHK1 after UV or HU (91).

In addition to its role in activating ATM, PP5 was also shown to be important for

the activation of ATR. PP5 can bind to ATR after treatment with NCS, UV, and HU.

PP5 was necessary for the phosphorylation of CHK1 at S345 and the knockdown of PP5

inhibited the replication checkpoint after UV, inhibited the S-phase checkpoint after HU,

and decreased RPA phosphorylation and foci formation (211).

DNA-PK

PP1, PP2A, and PP6 have all been shown to positively influence DNA-PK

activity. When PP1 was added to DNA-PK complexes that were induced to autophosphorylate, the reduction in activity and disruption of the DNA-PK-DNA binding due to the autophosphorylation were reversed (120). PP2A dephosphorylates DNA-PKcs

as well as Ku70 and Ku80 leading to increased DNA-PK activity (39). This opposes the

autophosphorylation of DNA-PK that results in decreased activity of DNA-PK (20).

CPT-induced DSB increased the association between PP2A and Ku proteins. PP2A

dephosphorylation of DNA-PKcs and Ku increases the association between the DNA-PK

proteins and this promotes DNA repair (193).

PP6 and DNA-PK form a complex in cells and this binding increases after IR. IR

causes a translocation of DNA-PK and PP6 from the cytoplasm to the nucleus and it

appears that localization of the phosphatase is dependent on the kinase and vice versa.

Knockdown of PP6 almost completely abolished the increase in DNA-PK activity after

IR, caused a defect in DSB repair, and resulted in colongenic survival similar to DNA-PK

knockdown cells, which means they have a higher sensitivity to IR (123).

BRCA1

PP1 can dephosphorylate BRCA1 at the CHK2 site S988, the ATM site S1524,

and the ATR site S1423 (101, 208). The overexpression of PP1 can partially inhibit the

hyperphosphorylation of BRCA1 after IR (101). Since PP1 can dephosphorylate BRCA1

at sites that have been shown to be important for BRCA1 function, it was intriguing to

find that PP1 may actually act to enhance BRCA1 function. PP1 specifically binds to

BRCA1 amino acids 898-901 (KVTF) (197, 208). Mutation of this site negatively

affected HR and the localization of the HR factor Rad51 to the break site, which are

functions of BRCA1 (208). Interestingly, in human tissues, PP1 mRNA levels were

significantly higher in normal tissue as compared to sporadic breast tumors (197). This implicates PP1 in the activation of BRCA1.

P53

Although p53 is negatively regulated via multiple PSPs, its activity is also

enhanced by two phosphatases. PP2Cα overexpression increases p53 transcription

activity. The overexpression of PP2Cα inhibits cell proliferation, cell cycle progression,

and colony formation and induces apoptosis. These effects are partially p53-dependent

(133).

P53 phosphorylation at T55 leads to p53 degradation (94). PP2A containing the

B'γ1 or B'γ3 subunit dephosphorylates T55 in response to DNA damage which leads to the inhibition of cell proliferation and transformation. In addition, protein levels of B'γ subunits increase after IR (93).

In conclusion, recent literature has demonstrated that PSPs have important functions in both the activation of the DDR and its negative regulation. Negative regulation can mean keeping the protein in an inactive state or inactivating it following the repair of the DNA to allow for recovery from the cell cycle checkpoint.

Summary and Rationale

The DDR is extremely important for cells to prevent the propagation of mutations that can lead to cancer. A highly sophisticated network of cell signaling machinery coordinates cell cycle checkpoints, DNA repair, senescence, and apoptosis. CHK2 plays an integral role in the successful function of the DDR, especially in response to double-

strand breaks.

PSPs are increasingly being discovered to influence the activities of DDR proteins, both in positive and negative ways. We have decided to further examine the role of CHK2 in the DDR and its regulation through its interactions with other proteins.

We have identified the B'α subunit of PP2A as a CHK2 interacting protein and demonstrate that it negatively regulates CHK2 phosphorylation, activity, and protein stability. This work provides a novel mechanism for CHK2 regulation and may be important for checkpoint recovery.

MATERIALS AND METHODS

Cloning

CHEK2 was cloned into the pGBKT7 vector via PCR of a human mammary gland cDNA library (CLONTECH Laboratories). PCR was used to generate B'α fragments containing amino acid residues 1-486 (full length), 1-195, 1-322, 176-322,

176-486, 304-486, 44-322, 89-322, 133-322, 1-282, and 1-195 from the clone that was identified in the yeast-two hybrid screen to bind to CHK2. These fragments were all cloned into the pACT2 vector and the full-length was cloned into pCMV2-FLAG. PCR was also used to generate fragments of CHK2 containing amino acid residues 1-303, 1-

194, 1-107, 89-194, 143-390, 195-336, 217-543, and 304-543. These were cloned into

the pGBKT7 vector. Full length CHK2 was subcloned into the pCMV-Tag3B vector.

The D347A, S33/35A, and S33/35E mutant forms of CHK2 were constructed via site-

directed mutagenesis and cloned into the pCMV-Tag3B and pcDNA3 vectors.

Yeast Two-Hybrid

The yeast-two hybrid was performed via the MATCHMAKER Two-Hybrid

System 3 (CLONTECH Laboratories), briefly described here. First, full length CHEK2 in pGBKT7 was transformed into AH109 yeast and selection was made on SD -Trp media. Next, the human mammary gland library (CLONTECH Laboratories) in the pACT2 vector was transformed into the same yeast and selection was made on SD -Leu -

Trp -Ade -His minimal media to verify binding between CHK2 and any prey from the 40

library. For the deletion fragment analysis, the full length gene encoding B'α in pACT2

was co-transformed with each CHEK2 fragment in pGBKT7 or full length CHEK2 in

pGBKT7 was transformed with each B'α gene fragment in pACT2 into AH109 yeast and

selection was made on minimal media.

Cell Culture, Transfection, and Reagents

293T cells were grown in DMEM media (Sigma) supplemented with 7.5% Fetal

Bovine Serum (SAFC), 1% Penicillin Streptomycin (Gibco), and 0.5% Amphotericin-B

(Sigma). HCT15 cells were grown in RMPI media (Gibco; Sigma) supplemented with

10% Fetal Bovine Serum, 1% Penicillin Streptomycin, and 0.5% Amphotericin-B.

HCT116 wild type (WT) and HCT116 CHK2-/- cells were a gift from Bert Vogelstein and

they were maintained in McCoy's 5A media (Gibco) supplemented with 10% Fetal

Bovine Serum, 1% Penicillin Streptomycin, and 0.5% Amphotericin-B. 293T cells were

transfected using Fugene 6 (Roche) and the HCT15 and HCT116 CHK2-/- cells were transfected using Lipofectamine 2000 (Invitrogen). When indicated, cells were treated with wortmannin, okadaic acid, hydroxyurea, mitomycin C, or doxorubicin (all from

Sigma) at the indicated concentrations.

Immunoprecipitations

For endogenous co-immunoprecipitations, 293T cells were lysed with RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 5 mM EDTA pH 8.0, 1% Triton X-100, 0.1% sodium deoxycholate, 0.1% SDS) or NETN buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1

mM EDTA, 0.5% NP-40, 1X protease inhibitors (Sigma), 1 mM PMSF, 0.5 mM Sodium

Vanadate, 0.5 mM Sodium Molybdate) and incubated with an antibody to CHK2 and

Protein A/G PLUS-Agarose (Santa Cruz) in modified RIPA buffer (made without sodium

deoxycholate and SDS) or NETN. Samples were eluted via boiling in sample buffer and

analyzed by immunoblotting. For co-immunoprecipitations with overexpressed proteins,

293T cells were transfected with a pCEP-4HA plasmid containing B'α, B'β, B'δ, B'ε, or

B'γ3 (obtained from Addgene; originally deposited by D. Virshup), a pCMV2-FLAG

plasmid containing B'α, or co-transfected with pCMV2-FLAG-B'α and wild-type CHK2 or a mutated CHK2 construct in pcDNA3. For immunoprecipitations via the HA tag, cells were lysed with NETN buffer and incubated overnight with monoclonal anti-HA agarose conjugate (Clone HA-7; Sigma). Samples were eluted via boiling in sample buffer and analyzed by immunoblotting. For immunoprecipitations via the FLAG tag, lysates were incubated with anti-FLAG M2 Affinity Gel (Sigma). Samples were eluted with 150 ng/µL 3X FLAG peptide (Sigma) and analyzed by immunoblotting.

Immunoblotting

Cell lysis was performed with NETN buffer. Samples were incubated on ice for

15 min and then centrifuged for 20 min at 13,000 rpm at 4ºC. Lysates were separated by

SDS-PAGE gel and transferred to PVDF membranes. Membranes were immunoblotted using the following antibodies: CHK2 (Clone 7; Upstate), CHK2 (NT; ProSci, Inc.),

PP2A A (Clone H-300; Santa Cruz), PP2A C (Clone 1D6; Upstate), PP2A B' (Upstate),

CHK1 (Clone G4; Santa Cruz), CHK2 phospho-serine 19 (Cell Signaling), CHK2 phospho-serines 33/35 (Cell Signaling), CHK2 phospho-threonine 68 (Cell Signaling), 42

CHK1 phospho-serine 317 (Cell Signaling), Talin (Clone TA205; Upstate), PARP (BD

PharMingen), H2AX (Upstate), FLAG (M2, peroxidase conjugate; Sigma), β-actin

(Clone AC-74; Sigma), HA (Clone 3F10; Roche). Immobilon Western

Chemiluminescent HRP Substrate (Millipore) or Amersham ECL Plus Western Blotting

Detection Reagent (GE Healthcare) was used for detection.

Kinase Activity Assay

293T cells, 293T cells transiently transfected with pCMV2-FLAG empty vector or one containing FLAG-B'α, or HCT15 cells transiently transfected with a pCMV-

Tag3B empty vector or one containing myc-tagged CHK2 WT, D347A, S33/35A, or

S33/35E were irradiated with 20 Gy IR or mock treated. Cells were lysed with NETN buffer and immunoprecipitated using an antibody to CHK2 or the c-Myc tag (Clone

9E10; Santa Cruz) and Protein A/G PLUS-Agarose (Santa Cruz). Immunoprecipitates were washed with NETN followed by kinase assay buffer (50 mM Tris-HCl pH7.5, 10 mM MgCl2, 2 mM dithiothreitol, 1X protease inhibitors (Sigma), 1 mM PMSF, 0.5 mM

sodium vanadate, 0.5 mM sodium molybdate). Kinase reactions were carried out at 30ºC

for 30 min in kinase assay buffer containing 25 µM ATP and 10 µCi [γ-32P]ATP with

either 2 µg GST-B'α or 1 µg of GST-Cdc25C (amino acids 200-256) as a substrate using

equivalent amounts of GST as a negative control. Reactions were stopped by addition of

sample buffer, boiled, and separated by SDS-PAGE gels which were then immunoblotted

for CHK2 and autoradiographed. The plasmid containing GST-Cdc25C (amino acids

200-256) was a gift from Larry Karnitz (154).

Subcellular Fractionation

Cells were fractionated into cytoplasmic, nucleoplasmic, and chromatin fractions

using a modified EMSA protocol (146). Buffer A (20 mM Tris pH 7.4, 10% glycerol, 10

mM KCl, 0.2% NP-40, 1 mM EDTA, 1X protease inhibitors (Sigma), 1 mM PMSF, 0.5

mM Sodium Vanadate, 0.5 mM Sodium Molybdate, 0.6 mM β-mercaptoethanol) was

added to cells, mixed by flicking, and left on ice for 15 min. Samples were spun down

for 2 min at 14,000 rpm at 4ºC and the supernatant was saved as the cytoplasmic fraction.

The pellet was washed once with Buffer A, then Buffer B (20 mM Tris pH 7.4, 20%

glycerol, 10 mM KCl, 400 mM NaCl, 1 mM EDTA, 1X protease inhibitors (Sigma), 1

mM PMSF, 0.5 mM Sodium Vanadate, 0.5 mM Sodium Molybdate, 0.6 mM β- mercaptoethanol) was added and mixed by pipeting. Samples were incubated on a rocking platform at 4ºC for 20 min then centrifuged at 14,000 rpm for 15 min at 4ºC.

The supernatant was saved as the nuclear fraction. Acid extraction buffer (0.5 M HCl,

10% glycerol, 100 mM β-mercaptoethanol) was added to the remaining pellet, vortexed, incubated for 2 min, and centrifuged at 14,000 rpm for 5 min. Neutralization buffer (40 mM Tris pH 7.4, protease inhibitors) was added and the samples were adjusted to a pH of

7.4 and saved as the chromatin fraction. Immunoblot analysis was performed.

Phosphatase Activity Assay

Phosphatase activity was measured using the PP2A Immunoprecipitation

Phosphatase Assay Kit (Upstate) following the manufacturer's instructions. Briefly, cells were lysed with NETN buffer and immunoprecipitated with an antibody to PP2A C (for total PP2A activity) or FLAG (for activity of PP2A containing FLAG-B'α).

Immunoprecipitates were incubated with a phosphopeptide for 10 min at 30ºC and free

phosphate was measured by addition of Malachite Green Detection Solution and the

absorbance levels were read at 650 nm.

Protein Stability

HCT15 cells were transiently transfected with a pcDNA3 plasmid containing

CHK2 WT, S33/35A, or S33/35E. 24 h later, 100 µg/mL cycloheximide was added to all

plates to inhibit new protein synthesis and the cells were mock-irradiated or irradiated

with 20 Gy IR and collected every two hours. The 0 h time point was mock-irradiated

only. Cells were lysed with NETN buffer and analyzed by immunoblotting.

AlphaEaseFC software (Alpha Innotech) was used for densitometry analysis of the

immunoblot bands. This data was plotted on a graph and the best-fit line was generated to allow for comparison of the slopes of each line, which corresponds with the rate at which the protein was being degraded.

RESULTS

CHK2 Binds to the B'α Subunit of PP2A

While much is known about the signaling networks that function in the DDR, not

much is known about the negative regulation of the proteins involved in the absence of

damage as well as during recovery from the checkpoint. To this end, we decided to

identify novel interacting partners of CHK2 using the yeast two-hybrid system, identify

the binding domains, and verify the interaction in mammalian cells.

CHK2 Interacts with the B'α Subunit of PP2A in Yeast.

In order to identify novel interacting proteins of CHK2, we utilized the yeast two-

hybrid system. The full length CHEK2 gene was cloned into the pGBKT7 vector, which

expresses proteins fused to the GAL4 DNA binding domain and allows growth on media

lacking Tryptophan. We used this construct to screen a human mammary gland cDNA

library in the pACT2 vector, which expresses proteins fused to the GAL4 Activation

domain and allows growth on media lacking Leucine. When there is an interaction

between the protein fused to the DNA binding domain and the protein fused to the

activation domain, this results in the expression of genes which allow growth on media

lacking Adenine and Histidine. Therefore, interacting partners of CHK2 were isolated based on their ability, when co-expressed with wild-type CHK2, to allow yeast to grow on media lacking Adenine, Histidine, Leucine, and Tryptophan.

In this screening, we identified two independent clones of the B'α regulatory

subunit of PP2A (gene name: PPP2R5A). As proof that our CHK2 construct was

properly folded, we also identified two interacting proteins of CHK2 that have been

previously described: KPNA2 and Mus81. Karyopherin KPNA2 has been shown to bind

to a NLS of CHK2 and this interaction is important for proper CHK2 localization in the

nucleus (209). Mus81, a damage tolerance protein, was shown to bind to the FHA

domain of the S. pombe homologue of CHK2, Cds1, and appears to be important in the

function of the DNA damage response cell cycle checkpoints in response to UV and

replication stress (13).

To further investigate the interaction between B'α and CHK2, we mapped the B'α

interacting domain in CHK2. Multiple deletion constructs of CHEK2 were constructed

via PCR and cloned into the pGBKT7 vector. Each was co-transformed with the pACT2

vector containing the full length B'α. Binding was determined using the yeast-two hybrid system and was defined as growth of colonies on minimal media (-Ade, -His, -Leu, -Trp)

as in the original yeast two-hybrid screening. The minimal CHK2 fragment that bound to

full length B'α was comprised of amino acid residues 1-107. This region contains the entire SQ/TQ region (Figure 5), where ATM phosphorylates CHK2 at S19, S33, S35 and

T68, leading to the full activation of CHK2.

To determine the CHK2 binding region in B'α, multiple deletion constructs were constructed of B'α in pACT2 and each was co-expressed with full length CHK2 in pGBKT7. Binding was determined using the yeast-two hybrid system, again defined as growth in minimal media. The minimal binding region in B'α was found to contain amino acid residues 89-322 (Figure 6). Although this region spans almost half of the 47 protein, the crystal structure of the PP2A holoenzyme containing B'γ1, which is closely related to B'α, reveals unique features of this enzyme (25, 201). B'γ1 contains eighteen α helices which form eight HEAT-like repeats and the interaction to the A and C subunits is mediated by many residues scattered throughout the B'γ1 protein (201), as noted in

Figure 6. Therefore, it is probable that there are select residues within this span of amino acid residues 89-322 that are particularly important for the interaction to CHK2.

Figure 5. B'α binds to CHK2 amino acids 1-107. The full length CHK2 protein is shown with phosphorylation sites and domains highlighted. The SQ/TQ domain is displayed in light gray, the FHA domain in dark gray, and the kinase domain in black.

Using the yeast two-hybrid system, deletion mutants of CHK2 were co-transformed into

yeast with full-length B'α. 49

Figure 6. CHK2 binds to B'α amino acids 89-322. Full length B'α is shown with the residues responsible for binding to the PP2A A subunit in light gray and the C subunit in dark gray (201). Using the yeast two-hybrid system, deletion mutants of B'α were co- transformed with full-length CHK2. 50

CHK2 and B'α Bind in Mammalian Cells

We next wanted to verify the binding of B'α and CHK2 in a mammalian system.

293T cells were transiently transfected to express FLAG-B'α. Whole cell extracts were

immunoprecipitated using a FLAG antibody and eluted using an excess of FLAG peptide. This prevents the appearance of the heavy and light chains of the immunoglobulin on the Western blot and significantly reduces background. The immunoprecipitate was immunoblotted for endogenous CHK2, demonstrating that B'α

and CHK2 interact in vivo (Figure 7). Immunoblotting also detected the endogenous

PP2A A and C subunits, indicating that FLAG-B’α is able to function as a member of the

potentially active heterotrimeric PP2A complex.

To ensure that the binding of CHK2 with B'α occurs without overexpression of

the B'α protein, immunoprecipitation of the endogenous proteins was utilized. Whole

cell lysates of 293T cells were immunoprecipitated using a CHK2 antibody and were

then analyzed via Western blot using an antibody to B'α. As shown in Figure 8, B'α was

able to bind to CHK2, verifying that this interaction occurs with the endogenous levels of

both proteins.

Figure 7. CHK2 binds to FLAG-B'α in mammalian cells. Whole cell lysates of 293T cells expressing FLAG-B'α were immunoprecipitated (IP) using the FLAG antibody and immunoblotted (IB) for the indicated proteins. Cell lysates that were not expressing

FLAG-B'α were used as a negative control.

Figure 8. Endogenous CHK2 and endogenous B'α bind in mammalian cells. Whole cell lysates of 293T cells were immunoprecipitated using an antibody to CHK2 and immunoblotted for B'α. Normal mouse IgG was used as a negative control for the immunoprecipitation. 53

Binding Between CHK2 and PP2A is Modulated by DNA Damage

It is well characterized that CHK2 is activated upon the induction of DNA

damage and this involves the phosphorylation of CHK2 at multiple sites, including the region that binds to B'α. Therefore, we determined whether the binding between CHK2 and B'α is modified under these conditions. We also determined the extent to which

CHK2 could bind other members of the B' family and the PP2A C catalytic subunit.

B'α and CHK2 Dissociate upon IR-Induced DNA Damage.

Since CHK2 is activated upon DNA damage, we examined the binding between

CHK2 and B'α for any changes upon the induction of DNA DSB by exposing cells to IR.

Whole cell extracts of 293T cells transiently expressing FLAG-B'α were

immunoprecipitated using a FLAG antibody and immunoblotted for endogenous CHK2,

PP2A A and PP2A C (Figure 9). Interestingly, when cells were exposed to 20 Gy IR and incubated for 1 h, this markedly reduced the binding of CHK2 to B'α without affecting the binding of B'α to the PP2A A and C subunits.

Phosphorylation of CHK2 serine 33 and 35 is recognized by the same antibody and therefore we will refer to them as S33/35. Immunoblotting of the lysate input verifies that IR causes phosphorylation of CHK2 at S19, S33/35, and T68 as previously described

(3, 16, 113, 119), verifying the activation of CHK2 after IR (Figure 9).

To ensure that the dissociation seen between CHK2 and B'α in the previous experiment was not an artifact of the antibody that was used, we repeated the experiment but immunoblotted with a different CHK2 antibody from another company. Again,

CHK2 was seen to bind to B'α under normal conditions and after exposure to IR, CHK2 54 and B'α were dissociated (Figure 10). This confirms that CHK2 and B'α are dissociated after IR and this is not due to specific reagents that were used.

Figure 9. Binding between CHK2 and B'α is disrupted after DNA damage. 293T

cells transiently expressing FLAG-B'α or empty vector (FLAG) were irradiated with 20

Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated using a FLAG antibody and immunoblotted for the indicated proteins. The CHK2 antibody used is anti-

CHK2 (clone 7) from Upstate Cell Signaling Solutions. 56

Figure 10. Dissociation of CHK2 and B'α is verified using another CHK2 antibody.

293T cells transiently expressing FLAG-B'α were irradiated with 20 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated using a FLAG antibody and immunoblotted for the indicated proteins. The CHK2 antibody used is anti-CHK2 (NT), from Pro Sci, Incorporated.

CHK2 Binds to Many PP2A Subunits.

We have determined that CHK2 binds to one member of the PP2A B' family, namely B'α. The B' family of regulatory subunits is comprised of at least nine members encoded from five genes. To determine the specificity of CHK2 for additional B' proteins, HA-tagged B'α, B'β, B'δ, B'ε, and B'γ3 proteins were transiently expressed in

293T cells. Whole cell lysates were immunoprecipitated via an antibody to the HA tag.

Endogenous CHK2 was efficiently immunoprecipitated by all of the B’ proteins tested

(Figure 11), in agreement with a previous study that demonstrated CHK2's ability to bind to many B' proteins using an in vitro binding assay (40). In our study, CHK2 had the strongest interaction to B'γ3 and the weakest to B'β, with binding to B'α, B'δ, and B'ε residing within this range. Additionally, the CHK2-B' complexes were dissociated after the cells were exposed to IR. It should be noted that the ability of the B'β protein to bind to the PP2A A and C subunits was compromised compared to the other B' proteins, suggesting there may be a problem with the conformation of this overexpressed B'β.

In order to determine whether CHK2 can bind to an active PP2A complex, we immunoprecipitated CHK2 from the whole cell lysates of 293T cells and immunoblotted for the PP2A C catalytic subunit (Figure 12). Indeed, endogenous CHK2 was able to bind to endogenous PP2A C, suggesting that CHK2 can associate with an active PP2A complex through one of the B' regulatory subunits. After the cells were exposed to IR to induce DNA damage, the binding between CHK2 and PP2A C is severely diminished, as was seen with the B' subunits.

Figure 11. CHK2 binds to many PP2A B' subunits and they are dissociated after

IR. 293T cells transiently transfected with HA-tagged B'α, B'β, B'δ, B'ε or B'γ3 were irradiated with 20 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated with the HA antibody and immunoblotted for the indicated proteins. CHK2 was able to bind to all B' proteins tested and all were at least partially dissociated by exposure to IR. 59

Figure 12. Endogenous PP2A C and CHK2 can bind and they are dissociated after

IR. 293T cells were irradiated with 20 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated with an antibody to CHK2 and immunoblotted for PP2A C. 60

B'α and CHK2 are Dissociated After DNA Damage Caused by IR and Doxorubicin, but

not B'α and CHK1

CHK2 can be activated by other types of DNA damage in addition to DSB caused

by IR. Therefore, other DNA damaging agents were also tested for any effects on the

binding between CHK2 and B'α. 293T cells transiently expressing FLAG-B'α were

exposed to IR, UV, HU, mitomycin C (MMC), or doxorubicin (Dox). FLAG-B'α was immunoprecipitated and immunoblotted for endogenous CHK2, PP2A A and PP2A C.

Interestingly, treatment with UV, HU, or MMC did not have any effect on the ability of

CHK2 to bind to FLAG-B'α (Figure 13). However, treatment with Dox did decrease the

binding between the two proteins, but not to the extent that IR did. By examining the

phosphorylation of CHK2 in the lysate input via immunoblotting, it can be seen that the

pattern of CHK2 phosphorylation was different after each treatment. After IR and Dox,

S19, S33/35, and T68 were all phosphorylated; after UV and MMC, T68 was primarily

phosphorylated and S19 was weakly phosphorylated; and after HU, S19 and T68 were

phosphorylated. The dissociation between CHK2 and B'α occurs after IR and Dox: the

only conditions that resulted in S33/35 phosphorylation at the time points studied.

Since CHK1 is also phosphorylated and activated in response to DNA damage in

a similar manner to CHK2, we also tested whether CHK1 could bind to PP2A B'α and

whether this binding was disrupted by DNA damage (Figure 13). Interestingly, CHK1

was able to bind to B'α, but this binding was not disrupted by treatment with IR, UV,

HU, MMC, or Dox. CHK1 was highly phosphorylated at S317 after IR, UV, and HU,

which is in agreement with previous studies (53, 213). This demonstrates that the

modulation of the interaction by DNA damage is specific to the CHK2-B'α complex. 61

Figure 13. Dissociation of CHK2 and B'α is caused by DNA damage induced by IR

and doxorubicin. 293T cells transiently transfected with FLAG-B'α were exposed to

either 20 Gy IR and incubated for 1 h, 50 J/m2 UV and incubated for 2 h, 2 mM

hydroxyurea (HU) for 24 h, 0.5 µg/mL mitomycin C (MMC) for 24 h, or 1 mM doxorubicin (DOX) for 2 h. Whole cell lysates were immunoprecipitated using the

FLAG antibody and immunoblotted for the indicated proteins.

Analysis of the IR-Induced Dissociation of CHK2 and B'α

The previous experiments have demonstrated that CHK2 can bind to many B'

family members as well as the C subunit and they are dissociated after specific types of

DNA damage. Since the dissociation of CHK2 and B'α occurs the most after treatment

with IR, we decided to further characterize this dissociation by examining any dose

dependence, the timing of dissociation, and whether they could re-associate at later points

in time. We also examined the role of phosphorylation in the binding and dissociation of

CHK2 and B'α since the dissociation correlated with the phosphorylation of CHK2 at

certain sites.

Dissociation is an Early Event and Occurs in a Dose-Dependent Manner

Since we have observed that IR causes the most dissociation of B'α and CHK2 of all of the DNA damaging agents that we studied, we next ascertained which doses of IR provide maximal dissociation. 293T cells transiently expressing FLAG-B'α were exposed to 2, 5, 10, 20, or 50 Gy, and FLAG-B'α was immunoprecipitated and immunoblotted (Figure 14). Maximal dissociation of endogenous CHK2 and B'α reached a peak after exposure to 10 Gy IR. By examining the phosphorylation of CHK2 in the lysate input, it can be seen that S19 phosphorylation increased with the increasing doses of IR. T68 phosphorylation was maximal after 2 and 5 Gy, and then decreased with the increasing IR doses. However, we observed the phosphorylation of S33/35 to be maximal after 10 Gy of IR. This corresponds to the IR dose that induces maximal dissociation between CHK2 and B'α.

In order to determine the precise timing of B'α and CHK2 dissociation after IR,

293T cells transiently expressing FLAG-B'α were irradiated with 20 Gy IR and collected every half an hour from 0.5 h to 2.5 hours following IR. FLAG-B'α was immunoprecipitated and it was found that CHK2 binding was minimal 0.5 h after IR, and the binding was slightly restored over the remainder of the time course (Figure 15).

Significantly, the dissociation at 0.5 h correlated with maximal phosphorylation of CHK2 at S33/35, but did not correlate with phosphorylation of S19 or T68.

Figure 14. Dissociation of CHK2 and B'α occurs in a dose-dependent manner.

293T cells were transiently transfected with FLAG-B'α and irradiated with 2, 5, 10, 20, or 50 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated with the

FLAG antibody and immunoblotted for the indicated proteins.

Figure 15. Dissociation of CHK2 and B'α is an early event after IR. 293T cells were transiently transfected with FLAG-B'α and irradiated with 20 Gy and incubated for 0.5,

1.0, 1.5, 2.0, or 2.5 h. Whole cell lysates were immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins.

Re-Association of CHK2 and B'α does Not Correlate With Resolution of DNA Damage

CHK2 plays an important role in activating cell cycle checkpoints after DNA damage (8). Since we have established that PP2A B'α and CHK2 dissociate after IR, we investigated whether re-association at a later time was correlated to the resolution of

DNA damage. We used two different doses of IR for comparison. First, we exposed cells to a sub-lethal dose of 6 Gy, the lowest dose after which we could detect a significant dissociation between CHK2 and B'α and which would also allow for recovery. We also exposed the cells to 50 Gy, which is considered lethal. 293T cells expressing FLAG-B'α

were collected 1 h, 4 h, 8 h, and 12 h after exposure to both doses of IR (Figure 16 and

Figure 17). Interestingly, the kinetics of dissociation after 50 Gy were similar to that

after 6 Gy. CHK2 was dissociated from B'α after 1 h and began to re-associate

approximately 4 h after IR with a subsequent increase through the remaining time points.

After both doses, the phosphorylation of S19, S33/35, and T68 followed similar patterns.

S33/35 had maximal phosphorylation after 1 h, again correlating with the most

dissociation between CHK2 and B'α. There does seem to be some difference in CHK2

phosphorylation between the two doses, since after 6 Gy IR, the CHK2 that was bound to

B'α after 4-12 h existed as a doublet, suggesting the existence of hyper and

hypophosphorylated forms (Figure 16).

In order to assess how dissociation and re-association correlated with the

resolution of DNA damage, we determined the levels of histone H2AX phosphorylated at

S139, also known as γ-H2AX, which is a well characterized marker of DSB (148). Thus,

higher levels of γ-H2AX denote the presence of DSB. When the breaks are repaired, γ-

H2AX levels return to levels found before DNA damage (in some cases it can no longer 67

be detected by Western blot or immunofluorescence). In our experiments, after 6 Gy IR,

the maximal amount of γ-H2AX appeared after 4 h, returning to pre-damage levels by 12

h (Figure 16). On the other hand, after 50 Gy IR, maximal levels of γ-H2AX were

detected after 1 h and did not decline throughout the remainder of the time points (Figure

17). Since B'α and CHK2 were shown to re-associate throughout the time course, and

DNA DSB were present during these times as show by the presence of γ-H2AX, the re- association does not correlate with the resolution of the DSB.

In summary, dissociation of CHK2 and B'α is induced in a dose-dependent manner and is an early event after IR. CHK2 and B'α re-association after DNA damage does not require resolution of the DNA damage. Moreover, dissociation and re- association correlate with phosphorylation of S33/35 of CHK2.

Figure 16. CHK2 and B'α are able to re-associate hours after 6 Gy IR. 293T cells

were transiently transfected with FLAG-B'α and irradiated with 6 Gy and incubated for

1, 4, 8, or 12 h. Whole cell lysates were immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins. 69

Figure 17. CHK2 and B'α are able to re-associate hours after 50 Gy IR. 293T cells

were transiently transfected with FLAG-B'α and irradiated with 50 Gy and incubated for

1, 4, 8, or 12 h. Whole cell lysates were immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins. 70

The Activity of ATM, but not PP2A, is Necessary for the Dissociation of CHK2 and B'α.

We next investigated the importance of PP2A activity on the binding and dissociation of CHK2 and B'α. OA is a phosphatase inhibitor that, when used at 0.5 µM, blocks the PP2A catalytic subunit's activity without affecting PP1 (55). 293T cells transiently expressing FLAG-B'α were treated with OA prior to IR exposure. FLAG-B'α was immunoprecipitated using an antibody to the FLAG tag and immunoblot analysis was performed (Figure 18). In the mock treated cells, CHK2 was able to bind to FLAG-

B'α, and this binding was unaffected by OA treatment alone. After IR, CHK2 and B'α dissociated and the presence of OA did not affect the dissociation. The inhibition of

PP2A by OA also had no effect on the ability of FLAG-B'α to bind to the PP2A A and C subunits, indicating that it can still form the holoenzyme complex. Therefore, PP2A activity is not required for B'α binding to, or dissociation from, CHK2.

As we have seen thus far, the most pronounced dissociation between CHK2 and

B'α occurs when CHK2 is phosphorylated on S33/35, which are phosphorylated in an

ATM-dependent manner (16, 79, 124). ATM belongs to the PIKK family along with

ATR and DNA-PK. To determine whether the CHK2-B'α dissociation is a PIKK- dependent event as is the phosphorylation of CHK2 S33/35, the PIKK inhibitor wortmannin was utilized at a concentration that inhibits ATM and DNA-PK, but not ATR activity in vivo (155). 293T cells transiently expressing FLAG-B'α were incubated with wortmannin prior to exposure to IR. FLAG-B'α was immunoprecipitated using a FLAG antibody and immunoblot analysis was performed (Figure 19). Pre-incubation with wortmannin prevented the dissociation of CHK2 from FLAG-B'α after IR. This

71 treatment also inhibited the phosphorylation of S19 and S33/35, indicating that the phosphorylation of CHK2 is required for the dissociation of CHK2 and B'α.

Figure 18. PP2A phosphatase activity is not required for CHK2 binding to or dissociating from B'α. 293T cells were transiently transfected with FLAG-B'α, treated with 0.5 µM OA for 1 h and then irradiated with 20 Gy IR and incubated for 1 h. Whole cell lysates were immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins. 73

Figure 19. Inhibition of ATM and DNA-PK kinase activity prevents the IR-induced dissociation of B'α and CHK2. 293T cells were transiently transfected with FLAG-B'α,

treated with 20 µM wortmannin for 1 h then irradiated with 10 Gy IR and incubated for 1

h. Whole cell lysates were immunoprecipitated with the FLAG antibody and

immunoblotted for the indicated proteins. 74

Mutation of CHK2 Serines 33 and 35 Affects Binding to B'α

To further investigate the importance of the phosphorylation of CHK2 S33/35 on

the binding and dissociation between CHK2 and B'α, we made two CHK2 mutants. The

substituted alanines for serines 33 and 35 to prevent phosphorylation (S33/35A) and the

substituted glutamic acids to mimic phosphorylation (S33/35E). Wild-type CHK2

(CHK2 WT), S33/35A and S33/35E were co-transfected along with FLAG-B'α into

HCT116 CHK2-/- cells, which were then mock-irradiated or irradiated with 20 Gy.

FLAG-B'α was immunoprecipitated and immunoblot analysis was performed (Figure

20). In mock treated cells, we saw an increase in S33/35A binding to B'α and a decrease in S33/35E binding as compared to CHK2 WT. Alternatively, after irradiation, CHK2

WT and S33/35A bound at the same levels as mock treated CHK2 WT, however,

S33/35E binding is completely abrogated.

Figure 20. Mutation of CHK2 serines 33 and 35 affects binding to B'α. HCT116

CHK2-/- cells were transfected with FLAG-B'α and either wild-type CHK2 or the

S33/35A or S33/35E mutants. Whole cell lysates were immunoprecipitated with the

FLAG antibody and immunoblotted for CHK2.

Analysis of the Effects of CHK2 on PP2A

Thus far, we have documented the binding between CHK2 and many PP2A subunits. This is an interesting case in which a kinase and a phosphatase bind under certain circumstances. As described above in detail, CHK2 phosphorylates a number of substrates which can positively and negatively regulate them. PSPs play important roles in both positively and negatively regulating DDR proteins as well. Therefore, it is possible that CHK2 kinase activity has an effect on PP2A activity, and vice versa. In this next section, we will investigate whether PP2A B'α can be used as a substrate by CHK2 and the extent to which CHK2 or DNA damage could affect PP2A localization or activity.

CHK2 can Phosphorylate B'α in vitro.

Since CHK2 is a kinase that is activated after irradiation, we determined whether

B'α could be used as a substrate for CHK2 in an in vitro kinase assay. 293T cells were irradiated to activate CHK2 which was then immunoprecipitated and incubated in a kinase reaction with GST-B'α (Figure 21). Indeed, phosphorylated B'α was detected, indicating that in an in vitro system, CHK2 can phosphorylate B'α.

Figure 21. CHK2 can phosphorylate B'α in vitro. Whole cell lysates from irradiated

293T cells were immunoprecipitated using an antibody to CHK2. CHK2 kinase activity towards GST-B'α was measured using an in vitro kinase reaction. 78

PP2A Localization is Not Influenced by IR

Since CHK2 was able to phosphorylate B'α in vitro, we next determined if this

had an effect on PP2A localization. 293T cells transiently expressing FLAG-B'α were

mock-irradiated or irradiated with 20 Gy, separated into the cytoplasmic, nucleoplasmic

and chromatin fractions, and subjected to immunoblot analysis (Figure 22). CHK2 was

located in the cytoplasm and nucleoplasm. The slower migrating form of CHK2 after IR

is indicative of its hyperphosphorylated active status. The PP2A A and C subunits were

also found in the cytoplasm and nucleoplasm, whereas FLAG-B'α was located in all three cellular compartments. The localization in the chromatin fraction may be an effect of its overexpression, as the endogenous A and C subunits could not be detected. Exposure of the cells to IR did not change the localization of any proteins examined, indicating that

PP2A localization is not affected by DNA DSB caused by IR.

CHK2 was also found to bind to other B' family members in addition to B'α.

Therefore, using an antibody that recognizes many B' proteins, we also examined the

subcellular localization of the endogenous B' family. As seen in Figure 23, there are

endogenous B' proteins that are located in the cytoplasm and nucleoplasm, and the

localization of each protein is not affected by exposure of the cells to IR.

Figure 22. B'α localization is not changed by IR. 293T cells transiently expressing

FLAG-B'α were mock-irradiated or irradiated with 20 Gy and incubated for 1 h. Using a subcellular fractionation method, cells were separated into the cytoplasmic, nucleoplasmic, and chromatin fractions, and immunoblot analysis for the indicated proteins was performed. Talin was used as a marker for the cytoplasm, PARP for the nucleoplasm, and H2AX for the chromatin. 80

Figure 23. Localization of endogenous PP2A proteins do not change after IR. 293T cells were mock-irradiated or irradiated with 20 Gy and incubated for 1 h. Using a subcellular fractionation method, cells were separated into the cytoplasmic, nucleoplasmic, and chromatin fractions, and immunoblot analysis for the indicated proteins was performed. Talin was used as a marker for the cytoplasm, PARP for the nucleoplasm, and H2AX for the chromatin. 81

PP2A Activity is Unaffected by IR and is Not Influenced by CHK2

If CHK2 is indeed able to phosphorylate B'α in vivo, it may have an effect on

PP2A activity. We first examined the total PP2A activity in the cell. The C subunit is

responsible for all of the PP2A catalytic activity in the cell, regardless of which

regulatory subunit is bound to the complex. Therefore, 293T cells were either exposed to

IR or mock treated and immunoprecipitated using an antibody to the C subunit to assess

total PP2A activity. Phosphatase activity of the immunoprecipitates was measured using

an in vitro phosphatase assay (Figure 24). The levels of PP2A activity were not changed

by exposure to IR.

Next, the activity of the PP2A complexes specifically containing FLAG-B'α was determined. 293T cells transiently expressing FLAG-B'α were either exposed to IR or mock treated and immunoprecipitated using a FLAG antibody to measure only the

activity of PP2A complexes containing B'α. Phosphatase activity was measured using an

in vitro phosphatase assay and was unaffected by exposure to IR (Figure 25). Since the assay measures activity against a small peptide, this may not accurately reflect changes in phosphatase activity toward specific substrates, however, it is a measure of the total activity of PP2A containing the B'α subunit.

In order to determine whether CHK2 has an effect on PP2A activity, the HCT116 wild-type (WT) cells and the isogenic HCT116 CHK2-/- cell lines were utilized. Both

cell lines were either exposed to IR or mock treated and immunoprecipitated using an

antibody for the PP2A C subunit to measure total PP2A activity (Figure 26). The in

vitro phosphatase assay revealed no difference in PP2A activity in the mock treated or

irradiated samples in either cell line. Also, there was no difference observed between the 82

HCT116 WT or CHK2-/- cell lines, suggesting that CHK2 has no effect on PP2A activity under these conditions. Taken together, this data indicates that PP2A activity is not affected by IR exposure or by CHK2.

Figure 24. Total PP2A phosphatase activity does not change after IR. 293T cells were irradiated with 20 Gy or mock treated and incubated for 1 h. Whole cell extracts were immunoprecipitated with an antibody to the PP2A C subunit. Phosphatase activity of the immunoprecipitates was determined using an in vitro phosphatase assay. The activity of the mock sample was defined as 100%. 84

Figure 25. Phosphatase activity of PP2A containing B'α does not change after IR.

293T cells transiently transfected with FLAG-B'α were irradiated with 20 Gy or mock treated and incubated for 1 h. Whole cell extracts were immunoprecipitated using the

FLAG tag. Phosphatase activity was determined and the activity of the mock treated sample expressing FLAG-B'α was defined as 100%. 85

Figure 26. Total PP2A activity does not differ in HCT116 wild-type and CHK2-/-

cells. HCT116 wild-type and HCT116 CHK2-/- cells were irradiated with 20 Gy and incubated for 1 h. Whole cell extracts were immunoprecipitated using an antibody to the

PP2A C subunit. Phosphatase activity was determined and the mock sample from the

HCT116 wild-type cells was defined as 100%.

Analysis of the Effects of PP2A on CHK2

We have shown that CHK2 does not seem to have any effect on PP2A activity or

localization, even though in vitro CHK2 can use B'α as a substrate. We next wanted to determine the extent to which PP2A can affect CHK2 phosphorylation, activity, and protein stability.

Changes in CHK2 Phosphorylation due to IR

In order to determine the extent to which PP2A has an effect on the phosphorylation of CHK2, the PP2A inhibitor OA was utilized. 293T cells were treated with OA in the presence and absence of irradiation, and the amount of phosphorylation of

CHK2 at specific sites was measured via immunoblotting (Figure 27). Pre-treatment with OA increased the phosphorylation of CHK2 after IR, primarily at S19, although there were also slight increases in the phosphorylation of S33/35 and T68, demonstrating that PP2A can dephosphorylate CHK2 at multiple sites.

Since we have shown that mutation of S33/35 can influence the binding between

B'α and CHK2, this led us to investigate any effect phosphorylation at these sites has on

CHK2 function. Therefore, we examined the ability of the S33/35A and S33/35E mutants to be phosphorylated at other CHK2 sites. HCT15 cells (which lack functional

CHK2) transiently expressing myc-tagged CHK2 wild-type (WT), kinase dead (D347A),

S33/35A, or S33/35E were either exposed to IR or mock treated. The phosphorylation status of each CHK2 protein was determined via immunoblotting (Figure 28). In the mock treated samples, CHK2 WT and S33/35A showed a very faint phosphorylation of

S19. However, S33/35E displayed a much stronger phosphorylation, even in the absence

of DNA damage. After exposure to IR, CHK2 WT was phosphorylated on S19, with levels comparable to the mock treated S33/35E. The phosphorylation of S33/35A at S19 was slightly compromised. T68 phosphorylation was slightly higher in the S33/35A and

S33/35E in both conditions as compared to CHK2 WT. Therefore, the S33/35 mutations primarily affect S19 phosphorylation, the same site that was affected most by PP2A inhibition.

Figure 27. Inhibition of PP2A activity increases CHK2 phosphorylation. 293T cells were incubated with 0.5 µM OA for 1 h then irradiated with 20 Gy and incubated for 1 h.

Whole cell extracts were examined for phosphorylation levels of CHK2 via immunoblot. 89

Figure 28. Mutation of CHK2 serines 33 and 35 affects CHK2 phosphorylation at other sites. HCT15 cells transiently transfected with pCMV-Tag3B empty vector

(vector) or myc-tagged CHK2 WT, D347A, S33/35A or S33/35E were exposed to 20 Gy

IR or mock treated and incubated for 1 h. Whole cell extracts were examined for CHK2 phosphorylation via immunoblot. 90

Changes in CHK2 Kinase Activity after IR

To directly measure the affect of PP2A on CHK2 activity, we transfected 293T

cells with FLAG-B'α and measured CHK2 kinase activity using an in vitro kinase assay

with GST-Cdc25C (amino acid residues 200-256) as a substrate (Figure 29). Cells were

either mock treated or irradiated, CHK2 was immunoprecipitated, and the kinase activity

was measured. In the mock treated samples, B'α did not have any effect on CHK2 kinase activity. However, after irradiation, overexpression of B'α prevented the full activation

of CHK2.

Since mutation of S33/35 to glutamic acid positively affects CHK2

phosphorylation primarily on S19, the effect on CHK2 kinase activity was also examined

since phosphorylation of this site has been shown to enhance CHK2 activity (16).

HCT15 cells expressing myc-tagged CHK2 WT, D347A, S33/35A, or S33/35E were

exposed to IR or mock treated. CHK2 activity was determined using an in vitro kinase

assay with GST-Cdc25C (amino acid residues 200-256) as a substrate (Figure 30). In the mock treated samples, S33/35E had higher kinase activity than CHK2 WT, whereas

S33/35A had lower kinase activity. After IR, S33/35E had slightly higher kinase activity than CHK2 WT, while the S33/35A mutant had significantly less. These data suggest that phosphorylation of S33/35 is important for CHK2 activity and that mimicking this phosphorylation can increase CHK2 activity even in the absence of DNA damage.

Figure 29. B'α overexpression negatively affects CHK2 kinase activity. 293T cells

expressing FLAG-B'α or FLAG (as a negative control) were irradiated with 20 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated using a CHK2 antibody and incubated in an in vitro kinase assay using GST-Cdc25C (amino acids 200-256) as a substrate. Autoradiographs (top panel) were normalized against the levels of CHK2 protein per sample and represented by graphs (bottom panel). 100% activity was defined by the amount in the negative control sample after IR. 92

Figure 30. Mutation of CHK2 serines 33 and 35 affects CHK2 kinase activity.

HCT15 cells were transiently transfected with empty vector (vector), CHK2 WT,

S33/35A, or S33/35E and mock-irradiated or exposed to 20 Gy IR and incubated for 1 h.

Cell lysates were immunoprecipitated and incubated in an in vitro kinase assay using

GST-Cdc25C (amino acids 200-256) as a substrate (top panel). Five experiments were plotted on a single graph (bottom panel). Activity of 1 was defined as the activity in the

CHK2 WT mock-treated sample. The median of each set of data points is shown with a black bar. 93

Mutation of CHK2 Serines 33 and 35 Affects Protein Stability

Another mode of CHK2 regulation is via its protein stability. CHK2 that is

expressed in HCT15 cells is phosphorylated at S456 after irradiation, stabilizing the

protein (75). Since we have shown that phosphorylation of S33/35 affects CHK2 kinase

activity, we also investigated the role it may play in protein stability. HCT15 cells

transiently expressing CHK2 WT, D347A, S33/35A, or S33/35E were incubated with

cycloheximide to prevent the synthesis of new proteins. Cells were then either exposed

to IR or mock treated and collected every two hours (Figure 31A). By plotting the best- fit line for each data set, the slope of the line could be examined to measure how fast the protein is being degraded (Figure 31B). In mock treated cells, the S33/35E protein was substantially more stable than WT. S33/35A had a slightly stabilizing effect as well.

After exposure to IR, the WT protein became more stable, whereas the stability of the

S33/35A and S33/35E proteins remained similar to the mock treated samples. These results denote the importance of phosphorylations of S33/35 in the increase of CHK2 stability after IR.

Figure 31A. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability.

HCT15 cells were transiently transfected with CHK2 wild-type (WT), S33/35A, or

S33/35E in the pcDNA3 vector. 100 µg/mL cycloheximide was added to all cells which were then either mock treated or exposed to 20 Gy IR and collected at the indicated time points. Levels of each protein were analyzed by immunoblotting. 95

Figure 31B. Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability.

Levels of CHK2 proteins from Figure 31A were normalized against the 0 h sample for each condition and the best fit line was plotted for each data set. The slope of the mock treated CHK2 WT line is -0.1250, S33/35A is -0.0831, and S33/35E is -0.0443. The slope of the IR CHK2 WT line is -0.0733, S33/35A is -0.0738, and S33/35E is -0.0457. 96

DISCUSSION

After DNA damage, the cell must activate cell cycle checkpoints to allow time to repair the breaks, or if the damage is too extensive, trigger apoptosis signaling (214).

ATM phosphorylates CHK2 on multiple sites in the SQ/TQ domain, leading to full

CHK2 activation (3, 16, 79, 83, 113, 119, 124, 200). Here, we propose a novel mechanism for CHK2 regulation in which ATM phosphorylation causes a dissociation of

PP2A from CHK2, thus allowing CHK2 activation. PP2A can then bind to CHK2 hours later, providing a mechanism for CHK2 down-regulation after DNA damage (Figure 32).

We have identified the B'α subunit of PP2A as a CHK2-interacting protein via a yeast two-hybrid screen and have confirmed the interaction in mammalian cells. Full length CHK2 was found to bind to B'α amino acids 89-322. Our inability to further narrow down the interacting region suggests that residues that mediate the interaction are scattered within this region. Interestingly, the structure of the PP2A holoenzyme indicates that the majority of interactions between the B' subunit and the A and C subunits are mediated by amino acid residues located at intervening loops between the 18 α-helices

(25, 201). Thus, it is likely that this may also be the case for the interaction with CHK2.

Identification of these residues could reveal the basis for substrate specificity of PP2A enzymes containing different B' subunits. This large portion of B'α that binds to CHK2 is a highly conserved region between the B' family members (42). Indeed, when we tested the specificity of CHK2 for the B' family, we found that CHK2 could bind to B'β,

B'δ, B'ε, and B'γ3 as well and all of the interactions were dissociated upon exposure to irradiation.

In mammalian cells, binding between B'α and CHK2 is markedly decreased by exposure to IR. However, other types of damage caused by UV, HU, or MMC did not disrupt binding. Doxorubicin had an effect, but not to the extent to which IR did, although comparisons of agents with different mechanisms of action are challenging. B'α can also bind CHK1, though the interaction is not disrupted by any of the DNA damage- inducing agents tested. This demonstrates a unique aspect of the relationship between

CHK2 and B'α: there is binding under normal conditions, but this is disrupted only after

DSB induced by IR, when serines 33 and 35 are phosphorylated. CHK2 and B'α are able to re-associate hours after IR, and this correlates with the dephosphorylation of S33/35, but not with repair of the bulk of damaged DNA as measured by phosphorylation of histone H2AX.

We have found that maximal dissociation between CHK2 and B'α correlates with maximal phosphorylation of CHK2 S33/35, which are phosphorylated in an ATM- dependent manner (16). When we inhibited ATM with wortmannin, the phosphorylation of S33/35 was prevented as well as the dissociation between CHK2 and B'α. The region in CHK2 that binds to B'α is comprised of amino acids 1-107, which contains the entire

SQ/TQ domain. Serines 33 and 35 are located in this domain, suggesting that the phosphorylation of these sites could directly contribute to the dissociation of the two proteins. We therefore mutated CHK2 S33/35 to alanine to prevent phosphorylation

(S33/35A) or to glutamic acid to mimic phosphorylation (S33/35E) and tested the effects on binding to B'α. Our data indicates that phosphorylation at S33/35 is necessary for the 98

dissociation of B'α and CHK2, but it is not sufficient on its own; other phosphorylation

sites are probably involved as well. Interestingly, wild-type CHK2 did not dissociate

from B'α after IR when they were both overexpressed. This may be because when CHK2

is overexpressed, it can become activated without DNA damage, allowing for

dissociation in the absence of DNA damage (3, 200).

In mock treated cells, S33/35E had a level of phosphorylation at S19 that was

comparable to CHK2 WT after IR. Pharmacologic PP2A inhibition with OA also

increases phosphorylation at S19. Alternatively, the S33/35A mutant had slightly less

phosphorylation after IR compared to CHK2 WT. The S33/35E mutant also had

increased kinase activity in mock treated conditions compared to CHK2 WT.

Conversely, the S33/35A mutant had decreased kinase activity after IR, in agreement

with a previous study (16). CHK2 kinase activity is decreased when B'α is

overexpressed as well.

Mutations at S33/35 also altered CHK2 protein stability. The S33/35E mutant was more stable as compared to the CHK2 WT under mock treated conditions. After exposure to IR, the stability of the S33/35A and S33/35E proteins did not change; however, the CHK2 WT protein stability increased after IR, in agreement with a previous study that demonstrated CHK2 expressed in HCT15 cells is more stable after IR (75).

Our work demonstrates the importance of phosphorylation of S33/35 in this process.

PP2A has already been demonstrated to negatively regulate other DNA damage

response proteins under various conditions, including ATM, ATR, RPA, H2AX, CHK1,

and p53, signifying the importance of PP2A in the DNA damage response pathway (27,

28, 36, 48, 55, 88, 115, 121, 122, 128, 129, 142). In addition, two previous papers have

implicated a role for PP2A in regulating CHK2. It was demonstrated that CHK2 can

phosphorylate B'γ in vitro, increasing PP2A activity towards CHK2 in vitro (40). In an in

vitro kinase assay, we found that CHK2 could phosphorylate B'α, however, this did not

have an effect on PP2A activity or localization. It has also been revealed that treating

cells with okadaic acid or incubating CHK2 protein with PP2A affected CHK2

phosphorylation at T68, in agreement with our results (96).

Another mode of CHK2 regulation in humans has been characterized. The Wip1

phosphatase has been shown to dephosphorylate T68 of CHK2 which leads to a decrease

in its kinase activity and ability to induce apoptosis (50, 136). Interestingly, Wip1

binding to CHK2 is enhanced by DNA damage, in opposition to the relationship between

PP2A and CHK2, which are dissociated after IR. Interestingly, both Wip1 and PP2A B'α

bind to the SQ/TQ domain of CHK2 (136, 206).

Mutations in CHEK2, the gene encoding the protein CHK2, have been associated with various cancers. Since the main role of CHK2 in the cell is to prevent the propagation of mutations via activation of DNA damage response cell cycle checkpoints, any mutation that negatively affects CHK2 function might increase the likelihood that a cell becomes cancerous. In fact, CHK2 mutations that cause decreased or eliminated kinase activity, an inability to bind to substrates, inefficient phosphorylation on T68, and/or destabilization of the protein have been identified (46, 85, 158, 169, 199). These mutations are all located in the FHA domain, the kinase domain, or the region between

these two domains.

In the context of the interaction between CHK2 and PP2A, mutations at S33

and/or S35 that prevent the dissociation of PP2A and CHK2 would also prevent the full

100

activation and stabilization of CHK2. To date, no mutations of S33/35 have been found

to be associated with cancer, even though they would negatively affect CHK2 function.

However, it cannot be assumed that because no mutations have been found specifically at

S33/35 that phosphorylation at these sites is not affected by mutations located elsewhere in the protein. In fact, the 1100delC and E161del mutations have been shown to exhibit inefficient phosphorylation of T68 in response to DNA damage (169). It will be necessary to examine these and other cancer-associated CHK2 mutations for the affect they have on the ability of CHK2 to be phosphorylated on S33/35 as well.

Not only have no mutations been found at S33/35, but no cancer-associated mutations have been found in the entire SQ/TQ domain. It is not known how a mutation in the SQ/TQ domain would affect the structure of the protein or the function of the enzyme since this domain is unstructured and has not been crystallized (17). It is possible that mutations in the SQ/TQ domain may prevent the binding of PP2A to CHK2 under normal circumstances, leading to an inappropriate activation of CHK2 that could inhibit cell proliferation even in the absence of DNA damage.

Proper regulation of the DDR is important for cell function. Activation of the

DDR is necessary to prevent the propagation of mutations that could lead to diseases such as cancer. Negative regulation of the DDR is important for the cell to prevent inappropriate checkpoint signaling as well as returning the cell to the cell cycle after the damage is repaired. Indeed, the multiple modes of CHK2 regulation, by two phosphatases and by regulation of kinase activity and protein stability, may highlight this importance.

101

Figure 32. Model of PP2A regulation of CHK2 through binding and dissociation.

CHK2 binds to PP2A through the B'α subunit under normal conditions. IR-induced DSB causes dissociation through ATM phosphorylation of CHK2 S33/35. When these are dephosphorylated, PP2A can bind back to CHK2 and dephosphorylate it. This leads to decreased CHK2 activity and protein stability. 102

LIST OF REFERENCES

1. Ahn, J., M. Urist, and C. Prives. 2003. Questioning the role of checkpoint

kinase 2 in the p53 DNA damage response. J Biol Chem 278:20480-9.

2. Ahn, J. Y., X. Li, H. L. Davis, and C. E. Canman. 2002. Phosphorylation of

threonine 68 promotes oligomerization and autophosphorylation of the Chk2

protein kinase via the forkhead-associated domain. J Biol Chem 277:19389-95.

3. Ahn, J. Y., J. K. Schwarz, H. Piwnica-Worms, and C. E. Canman. 2000.

Threonine 68 phosphorylation by ataxia telangiectasia mutated is required for

efficient activation of Chk2 in response to ionizing radiation. Cancer Res

60:5934-6.

4. Ali, A., J. Zhang, S. Bao, I. Liu, D. Otterness, N. M. Dean, R. T. Abraham,

and X. F. Wang. 2004. Requirement of protein phosphatase 5 in DNA-damage-

induced ATM activation. Genes Dev 18:249-54.

5. Appella, E., and C. W. Anderson. 2001. Post-translational modifications and

activation of p53 by genotoxic stresses. Eur J Biochem 268:2764-72.

6. Arnold, H. K., and R. C. Sears. 2006. Protein phosphatase 2A regulatory

subunit B56alpha associates with c-myc and negatively regulates c-myc

accumulation. Mol Cell Biol 26:2832-44.

7. Bakkenist, C. J., and M. B. Kastan. 2003. DNA damage activates ATM through

intermolecular autophosphorylation and dimer dissociation. Nature 421:499-506.

103

8. Bartek, J., and J. Lukas. 2003. Chk1 and Chk2 kinases in checkpoint control

and cancer. Cancer Cell 3:421-9.

9. Bassing, C. H., K. F. Chua, J. Sekiguchi, H. Suh, S. R. Whitlow, J. C.

Fleming, B. C. Monroe, D. N. Ciccone, C. Yan, K. Vlasakova, D. M.

Livingston, D. O. Ferguson, R. Scully, and F. W. Alt. 2002. Increased ionizing

radiation sensitivity and genomic instability in the absence of histone H2AX. Proc

Natl Acad Sci U S A 99:8173-8.

10. Bell, D. W., J. M. Varley, T. E. Szydlo, D. H. Kang, D. C. Wahrer, K. E.

Shannon, M. Lubratovich, S. J. Verselis, K. J. Isselbacher, J. F. Fraumeni, J.

M. Birch, F. P. Li, J. E. Garber, and D. A. Haber. 1999. Heterozygous germ

line hCHK2 mutations in Li-Fraumeni syndrome. Science 286:2528-31.

11. Bermudez, V. P., L. A. Lindsey-Boltz, A. J. Cesare, Y. Maniwa, J. D. Griffith,

J. Hurwitz, and A. Sancar. 2003. Loading of the human 9-1-1 checkpoint

complex onto DNA by the checkpoint clamp loader hRad17-replication factor C

complex in vitro. Proc Natl Acad Sci U S A 100:1633-8.

12. Binz, S. K., A. M. Sheehan, and M. S. Wold. 2004. Replication protein A

phosphorylation and the cellular response to DNA damage. DNA Repair (Amst)

3:1015-24.

13. Boddy, M. N., A. Lopez-Girona, P. Shanahan, H. Interthal, W. D. Heyer, and

P. Russell. 2000. Damage tolerance protein Mus81 associates with the FHA1

domain of checkpoint kinase Cds1. Mol Cell Biol 20:8758-66.

14. Brewis, N., K. Ohst, K. Fields, A. Rapacciuolo, D. Chou, C. Bloor, W.

Dillmann, H. Rockman, and G. Walter. 2000. Dilated cardiomyopathy in

104

transgenic mice expressing a mutant A subunit of protein phosphatase 2A. Am J

Physiol Heart Circ Physiol 279:H1307-18.

15. Bulavin, D. V., O. N. Demidov, S. Saito, P. Kauraniemi, C. Phillips, S. A.

Amundson, C. Ambrosino, G. Sauter, A. R. Nebreda, C. W. Anderson, A.

Kallioniemi, A. J. Fornace, Jr., and E. Appella. 2002. Amplification of PPM1D

in human tumors abrogates p53 tumor-suppressor activity. Nat Genet 31:210-5.

16. Buscemi, G., L. Carlessi, L. Zannini, S. Lisanti, E. Fontanella, S. Canevari,

and D. Delia. 2006. DNA damage-induced cell cycle regulation and function of

novel Chk2 phosphoresidues. Mol Cell Biol 26:7832-45.

17. Cai, Z., N. H. Chehab, and N. P. Pavletich. 2009. Structure and activation

mechanism of the CHK2 DNA damage checkpoint kinase. Mol Cell 35:818-29.

18. Calin, G. A., M. G. di Iasio, E. Caprini, I. Vorechovsky, P. G. Natali, G.

Sozzi, C. M. Croce, G. Barbanti-Brodano, G. Russo, and M. Negrini. 2000.

Low frequency of alterations of the alpha (PPP2R1A) and beta (PPP2R1B)

isoforms of the subunit A of the serine-threonine phosphatase 2A in human

neoplasms. Oncogene 19:1191-5.

19. Celeste, A., S. Petersen, P. J. Romanienko, O. Fernandez-Capetillo, H. T.

Chen, O. A. Sedelnikova, B. Reina-San-Martin, V. Coppola, E. Meffre, M. J.

Difilippantonio, C. Redon, D. R. Pilch, A. Olaru, M. Eckhaus, R. D.

Camerini-Otero, L. Tessarollo, F. Livak, K. Manova, W. M. Bonner, M. C.

Nussenzweig, and A. Nussenzweig. 2002. Genomic instability in mice lacking

histone H2AX. Science 296:922-7.

105

20. Chan, D. W., and S. P. Lees-Miller. 1996. The DNA-dependent protein kinase is

inactivated by autophosphorylation of the catalytic subunit. J Biol Chem

271:8936-41.

21. Chaturvedi, P., W. K. Eng, Y. Zhu, M. R. Mattern, R. Mishra, M. R. Hurle,

X. Zhang, R. S. Annan, Q. Lu, L. F. Faucette, G. F. Scott, X. Li, S. A. Carr,

R. K. Johnson, J. D. Winkler, and B. B. Zhou. 1999. Mammalian Chk2 is a

downstream effector of the ATM-dependent DNA damage checkpoint pathway.

Oncogene 18:4047-54.

22. Chehab, N. H., A. Malikzay, M. Appel, and T. D. Halazonetis. 2000.

Chk2/hCds1 functions as a DNA damage checkpoint in G(1) by stabilizing p53.

Genes Dev 14:278-88.

23. Chen, J., B. L. Martin, and D. L. Brautigan. 1992. Regulation of protein

serine-threonine phosphatase type-2A by tyrosine phosphorylation. Science

257:1261-4.

24. Chen, L., D. M. Gilkes, Y. Pan, W. S. Lane, and J. Chen. 2005. ATM and

Chk2-dependent phosphorylation of MDMX contribute to p53 activation after

DNA damage. Embo J 24:3411-22.

25. Cho, U. S., and W. Xu. 2007. Crystal structure of a protein phosphatase 2A

heterotrimeric holoenzyme. Nature 445:53-7.

26. Choi, J., B. Nannenga, O. N. Demidov, D. V. Bulavin, A. Cooney, C. Brayton,

Y. Zhang, I. N. Mbawuike, A. Bradley, E. Appella, and L. A. Donehower.

2002. Mice deficient for the wild-type p53-induced phosphatase gene (Wip1)

106

exhibit defects in reproductive organs, immune function, and cell cycle control.

Mol Cell Biol 22:1094-105.

27. Chowdhury, D., M. C. Keogh, H. Ishii, C. L. Peterson, S. Buratowski, and J.

Lieberman. 2005. gamma-H2AX dephosphorylation by protein phosphatase 2A

facilitates DNA double-strand break repair. Mol Cell 20:801-9.

28. Chowdhury, D., X. Xu, X. Zhong, F. Ahmed, J. Zhong, J. Liao, D. M.

Dykxhoorn, D. M. Weinstock, G. P. Pfeifer, and J. Lieberman. 2008. A PP4-

phosphatase complex dephosphorylates gamma-H2AX generated during DNA

replication. Mol Cell 31:33-46.

29. Cimprich, K. A., and D. Cortez. 2008. ATR: an essential regulator of genome

integrity. Nat Rev Mol Cell Biol 9:616-27.

30. Clarke, P. R., I. Hoffmann, G. Draetta, and E. Karsenti. 1993.

Dephosphorylation of cdc25-C by a type-2A protein phosphatase: specific

regulation during the cell cycle in Xenopus egg extracts. Mol Biol Cell 4:397-

411.

31. Cole, M. D. 1986. The myc oncogene: its role in transformation and

differentiation. Annu Rev Genet 20:361-84.

32. Cortez, D., Y. Wang, J. Qin, and S. J. Elledge. 1999. Requirement of ATM-

dependent phosphorylation of brca1 in the DNA damage response to double-

strand breaks. Science 286:1162-6.

33. Crawford, T. O. 1998. Ataxia telangiectasia. Semin Pediatr Neurol 5:287-94.

34. de Bock, G. H., M. Schutte, E. M. Krol-Warmerdam, C. Seynaeve, J. Blom,

C. T. Brekelmans, H. Meijers-Heijboer, C. J. van Asperen, C. J. Cornelisse,

107

P. Devilee, R. A. Tollenaar, and J. G. Klijn. 2004. Tumour characteristics and

prognosis of breast cancer patients carrying the germline CHEK2*1100delC

variant. J Med Genet 41:731-5.

35. den Elzen, N. R., and M. J. O'Connell. 2004. Recovery from DNA damage

checkpoint arrest by PP1-mediated inhibition of Chk1. Embo J 23:908-18.

36. Dohoney, K. M., C. Guillerm, C. Whiteford, C. Elbi, P. F. Lambert, G. L.

Hager, and J. N. Brady. 2004. Phosphorylation of p53 at serine 37 is important

for transcriptional activity and regulation in response to DNA damage. Oncogene

23:49-57.

37. Dong, X., L. Wang, K. Taniguchi, X. Wang, J. M. Cunningham, S. K.

McDonnell, C. Qian, A. F. Marks, S. L. Slager, B. J. Peterson, D. I. Smith, J.

C. Cheville, M. L. Blute, S. J. Jacobsen, D. J. Schaid, D. J. Tindall, S. N.

Thibodeau, and W. Liu. 2003. Mutations in CHEK2 associated with prostate

cancer risk. Am J Hum Genet 72:270-80.

38. Donzelli, M., and G. F. Draetta. 2003. Regulating mammalian checkpoints

through Cdc25 inactivation. EMBO Rep 4:671-7.

39. Douglas, P., G. B. Moorhead, R. Ye, and S. P. Lees-Miller. 2001. Protein

phosphatases regulate DNA-dependent protein kinase activity. J Biol Chem

276:18992-8.

40. Dozier, C., M. Bonyadi, L. Baricault, L. Tonasso, and J. M. Darbon. 2004.

Regulation of Chk2 phosphorylation by interaction with protein phosphatase 2A

via its B' regulatory subunit. Biol Cell 96:509-17.

108

41. Durocher, D., J. Henckel, A. R. Fersht, and S. P. Jackson. 1999. The FHA

domain is a modular phosphopeptide recognition motif. Mol Cell 4:387-94.

42. Eichhorn, P. J., M. P. Creyghton, and R. Bernards. 2009. Protein phosphatase

2A regulatory subunits and cancer. Biochim Biophys Acta 1795:1-15.

43. Elder, R. T., M. Yu, M. Chen, X. Zhu, M. Yanagida, and Y. Zhao. 2001. HIV-

1 Vpr induces cell cycle G2 arrest in fission yeast (Schizosaccharomyces pombe)

through a pathway involving regulatory and catalytic subunits of PP2A and acting

on both Wee1 and Cdc25. Virology 287:359-70.

44. Esplin, E. D., P. Ramos, B. Martinez, G. E. Tomlinson, M. C. Mumby, and G.

A. Evans. 2006. The glycine 90 to aspartate alteration in the Abeta subunit of

PP2A (PPP2R1B) associates with breast cancer and causes a deficit in protein

function. Genes Chromosomes Cancer 45:182-90.

45. Everett, A. D., C. Kamibayashi, and D. L. Brautigan. 2002. Transgenic

expression of protein phosphatase 2A regulatory subunit B56gamma disrupts

distal lung differentiation. Am J Physiol Lung Cell Mol Physiol 282:L1266-71.

46. Falck, J., C. Lukas, M. Protopopova, J. Lukas, G. Selivanova, and J. Bartek.

2001. Functional impact of concomitant versus alternative defects in the Chk2-

p53 tumour suppressor pathway. Oncogene 20:5503-10.

47. Falck, J., N. Mailand, R. G. Syljuasen, J. Bartek, and J. Lukas. 2001. The

ATM-Chk2-Cdc25A checkpoint pathway guards against radioresistant DNA

synthesis. Nature 410:842-7.

48. Feng, J., T. Wakeman, S. Yong, X. Wu, S. Kornbluth, and X. F. Wang. 2009.

Protein phosphatase 2A-dependent dephosphorylation of replication protein A is

109

required for the repair of DNA breaks induced by replication stress. Mol Cell Biol

29:5696-709.

49. Fiscella, M., H. Zhang, S. Fan, K. Sakaguchi, S. Shen, W. E. Mercer, G. F.

Vande Woude, P. M. O'Connor, and E. Appella. 1997. Wip1, a novel human

protein phosphatase that is induced in response to ionizing radiation in a p53-

dependent manner. Proc Natl Acad Sci U S A 94:6048-53.

50. Fujimoto, H., N. Onishi, N. Kato, M. Takekawa, X. Z. Xu, A. Kosugi, T.

Kondo, M. Imamura, I. Oishi, A. Yoda, and Y. Minami. 2006. Regulation of

the antioncogenic Chk2 kinase by the oncogenic Wip1 phosphatase. Cell Death

Differ 13:1170-80.

51. Fuku, T., S. Semba, H. Yutori, and H. Yokozaki. 2007. Increased wild-type

p53-induced phosphatase 1 (Wip1 or PPM1D) expression correlated with

downregulation of checkpoint kinase 2 in human gastric carcinoma. Pathol Int

57:566-71.

52. Gatei, M., S. P. Scott, I. Filippovitch, N. Soronika, M. F. Lavin, B. Weber,

and K. K. Khanna. 2000. Role for ATM in DNA damage-induced

phosphorylation of BRCA1. Cancer Res 60:3299-304.

53. Gatei, M., K. Sloper, C. Sorensen, R. Syljuasen, J. Falck, K. Hobson, K.

Savage, J. Lukas, B. B. Zhou, J. Bartek, and K. K. Khanna. 2003. Ataxia-

telangiectasia-mutated (ATM) and NBS1-dependent phosphorylation of Chk1 on

Ser-317 in response to ionizing radiation. J Biol Chem 278:14806-11.

54. Gatei, M., B. B. Zhou, K. Hobson, S. Scott, D. Young, and K. K. Khanna.

2001. Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related

110

kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo

assessment using phospho-specific antibodies. J Biol Chem 276:17276-80.

55. Goodarzi, A. A., J. C. Jonnalagadda, P. Douglas, D. Young, R. Ye, G. B.

Moorhead, S. P. Lees-Miller, and K. K. Khanna. 2004. Autophosphorylation of

ataxia-telangiectasia mutated is regulated by protein phosphatase 2A. Embo J

23:4451-61.

56. Gotz, J., A. Probst, E. Ehler, B. Hemmings, and W. Kues. 1998. Delayed

embryonic lethality in mice lacking protein phosphatase 2A catalytic subunit

Calpha. Proc Natl Acad Sci U S A 95:12370-5.

57. Guillemain, G., E. Ma, S. Mauger, S. Miron, R. Thai, R. Guerois, F.

Ochsenbein, and M. C. Marsolier-Kergoat. 2007. Mechanisms of checkpoint

kinase Rad53 inactivation after a double-strand break in Saccharomyces

cerevisiae. Mol Cell Biol 27:3378-89.

58. Guo, C. Y., D. L. Brautigan, and J. M. Larner. 2002. ATM-dependent

dissociation of B55 regulatory subunit from nuclear PP2A in response to ionizing

radiation. J Biol Chem 277:4839-44.

59. Guo, C. Y., D. L. Brautigan, and J. M. Larner. 2002. Ionizing radiation

activates nuclear protein phosphatase-1 by ATM-dependent dephosphorylation. J

Biol Chem 277:41756-61.

60. Haber, J. E. 2000. Recombination: a frank view of exchanges and vice versa.

Curr Opin Cell Biol 12:286-92.

61. Hahn, W. C., S. K. Dessain, M. W. Brooks, J. E. King, B. Elenbaas, D. M.

Sabatini, J. A. DeCaprio, and R. A. Weinberg. 2002. Enumeration of the

111

simian virus 40 early region elements necessary for human cell transformation.

Mol Cell Biol 22:2111-23.

62. Hainaut, P., and M. Hollstein. 2000. p53 and human cancer: the first ten

thousand mutations. Adv Cancer Res 77:81-137.

63. Harris, S. L., and A. J. Levine. 2005. The p53 pathway: positive and negative

feedback loops. Oncogene 24:2899-908.

64. Haupt, Y., R. Maya, A. Kazaz, and M. Oren. 1997. Mdm2 promotes the rapid

degradation of p53. Nature 387:296-9.

65. He, J., S. Choe, R. Walker, P. Di Marzio, D. O. Morgan, and N. R. Landau.

1995. Human immunodeficiency virus type 1 viral protein R (Vpr) arrests cells in

the G2 phase of the cell cycle by inhibiting p34cdc2 activity. J Virol 69:6705-11.

66. Hemmings, B. A., C. Adams-Pearson, F. Maurer, P. Muller, J. Goris, W.

Merlevede, J. Hofsteenge, and S. R. Stone. 1990. alpha- and beta-forms of the

65-kDa subunit of protein phosphatase 2A have a similar 39 amino acid repeating

structure. Biochemistry 29:3166-73.

67. Hinds, T. D., Jr., and E. R. Sanchez. 2008. Protein phosphatase 5. Int J

Biochem Cell Biol 40:2358-62.

68. Hirao, A., A. Cheung, G. Duncan, P. M. Girard, A. J. Elia, A. Wakeham, H.

Okada, T. Sarkissian, J. A. Wong, T. Sakai, E. De Stanchina, R. G. Bristow,

T. Suda, S. W. Lowe, P. A. Jeggo, S. J. Elledge, and T. W. Mak. 2002. Chk2 is

a tumor suppressor that regulates apoptosis in both an ataxia telangiectasia

mutated (ATM)-dependent and an ATM-independent manner. Mol Cell Biol

22:6521-32.

112

69. Honda, R., H. Tanaka, and H. Yasuda. 1997. Oncoprotein MDM2 is a

ubiquitin ligase E3 for tumor suppressor p53. FEBS Lett 420:25-7.

70. Iwabuchi, K., P. L. Bartel, B. Li, R. Marraccino, and S. Fields. 1994. Two

cellular proteins that bind to wild-type but not mutant p53. Proc Natl Acad Sci U

S A 91:6098-102.

71. Jallepalli, P. V., C. Lengauer, B. Vogelstein, and F. Bunz. 2003. The Chk2

tumor suppressor is not required for p53 responses in human cancer cells. J Biol

Chem 278:20475-9.

72. Janssens, V., and J. Goris. 2001. Protein phosphatase 2A: a highly regulated

family of serine/threonine phosphatases implicated in cell growth and signalling.

Biochem J 353:417-39.

73. Jazayeri, A., J. Falck, C. Lukas, J. Bartek, G. C. Smith, J. Lukas, and S. P.

Jackson. 2006. ATM- and cell cycle-dependent regulation of ATR in response to

DNA double-strand breaks. Nat Cell Biol 8:37-45.

74. Jin, J., T. Shirogane, L. Xu, G. Nalepa, J. Qin, S. J. Elledge, and J. W.

Harper. 2003. SCFbeta-TRCP links Chk1 signaling to degradation of the

Cdc25A protein phosphatase. Genes Dev 17:3062-74.

75. Kass, E. M., J. Ahn, T. Tanaka, W. A. Freed-Pastor, S. Keezer, and C.

Prives. 2007. Stability of checkpoint kinase 2 is regulated via phosphorylation at

serine 456. J Biol Chem 282:30311-21.

76. Keogh, M. C., J. A. Kim, M. Downey, J. Fillingham, D. Chowdhury, J. C.

Harrison, M. Onishi, N. Datta, S. Galicia, A. Emili, J. Lieberman, X. Shen, S.

Buratowski, J. E. Haber, D. Durocher, J. F. Greenblatt, and N. J. Krogan.

113

2006. A phosphatase complex that dephosphorylates gammaH2AX regulates

DNA damage checkpoint recovery. Nature 439:497-501.

77. Kim, S. T., D. S. Lim, C. E. Canman, and M. B. Kastan. 1999. Substrate

specificities and identification of putative substrates of ATM kinase family

members. J Biol Chem 274:37538-43.

78. Kubbutat, M. H., S. N. Jones, and K. H. Vousden. 1997. Regulation of p53

stability by Mdm2. Nature 387:299-303.

79. Kurz, E. U., P. Douglas, and S. P. Lees-Miller. 2004. Doxorubicin activates

ATM-dependent phosphorylation of multiple downstream targets in part through

the generation of reactive oxygen species. J Biol Chem 279:53272-81.

80. Laitman, Y., B. Kaufman, E. L. Lahad, M. Z. Papa, and E. Friedman. 2007.

Germline CHEK2 mutations in Jewish Ashkenazi women at high risk for breast

cancer. Isr Med Assoc J 9:791-6.

81. Lavin, M. F. 2004. The Mre11 complex and ATM: a two-way functional

interaction in recognising and signaling DNA double strand breaks. DNA Repair

(Amst) 3:1515-20.

82. LeBron, C., L. Chen, D. M. Gilkes, and J. Chen. 2006. Regulation of MDMX

nuclear import and degradation by Chk2 and 14-3-3. Embo J 25:1196-206.

83. Lee, C. H., and J. H. Chung. 2001. The hCds1 (Chk2)-FHA domain is essential

for a chain of phosphorylation events on hCds1 that is induced by ionizing

radiation. J Biol Chem 276:30537-41.

84. Lee, J. H., and T. T. Paull. 2007. Activation and regulation of ATM kinase

activity in response to DNA double-strand breaks. Oncogene 26:7741-8.

114

85. Lee, S. B., S. H. Kim, D. W. Bell, D. C. Wahrer, T. A. Schiripo, M. M.

Jorczak, D. C. Sgroi, J. E. Garber, F. P. Li, K. E. Nichols, J. M. Varley, A. K.

Godwin, K. M. Shannon, E. Harlow, and D. A. Haber. 2001. Destabilization of

CHK2 by a missense mutation associated with Li-Fraumeni Syndrome. Cancer

Res 61:8062-7.

86. Lee, T. H., C. Turck, and M. W. Kirschner. 1994. Inhibition of cdc2 activation

by INH/PP2A. Mol Biol Cell 5:323-38.

87. Leroy, C., S. E. Lee, M. B. Vaze, F. Ochsenbien, R. Guerois, J. E. Haber, and

M. C. Marsolier-Kergoat. 2003. PP2C phosphatases Ptc2 and Ptc3 are required

for DNA checkpoint inactivation after a double-strand break. Mol Cell 11:827-35.

88. Leung-Pineda, V., C. E. Ryan, and H. Piwnica-Worms. 2006. Phosphorylation

of Chk1 by ATR is antagonized by a Chk1-regulated protein phosphatase 2A

circuit. Mol Cell Biol 26:7529-38.

89. Li, D. W., U. Fass, I. Huizar, and A. Spector. 1998. Okadaic acid-induced lens

epithelial cell apoptosis requires inhibition of phosphatase-1 and is associated

with induction of gene expression including p53 and bax. Eur J Biochem

257:351-61.

90. Li, D. W., J. P. Liu, P. C. Schmid, R. Schlosser, H. Feng, W. B. Liu, Q. Yan,

L. Gong, S. M. Sun, M. Deng, and Y. Liu. 2006. Protein serine/threonine

phosphatase-1 dephosphorylates p53 at Ser-15 and Ser-37 to modulate its

transcriptional and apoptotic activities. Oncogene 25:3006-22.

115

91. Li, G., R. T. Elder, K. Qin, H. U. Park, D. Liang, and R. Y. Zhao. 2007.

Phosphatase type 2A-dependent and -independent pathways for ATR

phosphorylation of Chk1. J Biol Chem 282:7287-98.

92. Li, H., K. Okamoto, M. J. Peart, and C. Prives. 2009. Lysine-independent

turnover of cyclin G1 can be stabilized by B'alpha subunits of protein phosphatase

2A. Mol Cell Biol 29:919-28.

93. Li, H. H., X. Cai, G. P. Shouse, L. G. Piluso, and X. Liu. 2007. A specific

PP2A regulatory subunit, B56gamma, mediates DNA damage-induced

dephosphorylation of p53 at Thr55. Embo J 26:402-11.

94. Li, H. H., A. G. Li, H. M. Sheppard, and X. Liu. 2004. Phosphorylation on

Thr-55 by TAF1 mediates degradation of p53: a role for TAF1 in cell G1

progression. Mol Cell 13:867-78.

95. Li, J., B. L. Williams, L. F. Haire, M. Goldberg, E. Wilker, D. Durocher, M.

B. Yaffe, S. P. Jackson, and S. J. Smerdon. 2002. Structural and functional

versatility of the FHA domain in DNA-damage signaling by the tumor suppressor

kinase Chk2. Mol Cell 9:1045-54.

96. Liang, X., E. Reed, and J. J. Yu. 2006. Protein phosphatase 2A interacts with

Chk2 and regulates phosphorylation at Thr-68 after cisplatin treatment of human

ovarian cancer cells. Int J Mol Med 17:703-8.

97. Lin, W. C., F. T. Lin, and J. R. Nevins. 2001. Selective induction of E2F1 in

response to DNA damage, mediated by ATM-dependent phosphorylation. Genes

Dev 15:1833-44.

116

98. Linares, L. K., A. Hengstermann, A. Ciechanover, S. Muller, and M.

Scheffner. 2003. HdmX stimulates Hdm2-mediated ubiquitination and

degradation of p53. Proc Natl Acad Sci U S A 100:12009-14.

99. Lindqvist, A., M. de Bruijn, L. Macurek, A. Bras, A. Mensinga, W.

Bruinsma, O. Voets, O. Kranenburg, and R. H. Medema. 2009. Wip1 confers

G2 checkpoint recovery competence by counteracting p53-dependent

transcriptional repression. Embo J 28:3196-206.

100. Liu, Q., S. Guntuku, X. S. Cui, S. Matsuoka, D. Cortez, K. Tamai, G. Luo, S.

Carattini-Rivera, F. DeMayo, A. Bradley, L. A. Donehower, and S. J.

Elledge. 2000. Chk1 is an essential kinase that is regulated by Atr and required

for the G(2)/M DNA damage checkpoint. Genes Dev 14:1448-59.

101. Liu, Y., D. M. Virshup, R. L. White, and L. C. Hsu. 2002. Regulation of

BRCA1 phosphorylation by interaction with protein phosphatase 1alpha. Cancer

Res 62:6357-61.

102. Longin, S., K. Zwaenepoel, J. V. Louis, S. Dilworth, J. Goris, and V.

Janssens. 2007. Selection of protein phosphatase 2A regulatory subunits is

mediated by the C-terminus of the catalytic subunit. J Biol Chem.

103. Lou, Z., K. Minter-Dykhouse, X. Wu, and J. Chen. 2003. MDC1 is coupled to

activated CHK2 in mammalian DNA damage response pathways. Nature

421:957-61.

104. Lovly, C. M., L. Yan, C. E. Ryan, S. Takada, and H. Piwnica-Worms. 2008.

Regulation of Chk2 ubiquitination and signaling through autophosphorylation of

serine 379. Mol Cell Biol 28:5874-85.

117

105. Lu, X., O. Ma, T. A. Nguyen, S. N. Jones, M. Oren, and L. A. Donehower.

2007. The Wip1 Phosphatase acts as a gatekeeper in the p53-Mdm2

autoregulatory loop. Cancer Cell 12:342-54.

106. Lu, X., B. Nannenga, and L. A. Donehower. 2005. PPM1D dephosphorylates

Chk1 and p53 and abrogates cell cycle checkpoints. Genes Dev 19:1162-74.

107. Lu, X., T. A. Nguyen, S. H. Moon, Y. Darlington, M. Sommer, and L. A.

Donehower. 2008. The type 2C phosphatase Wip1: an oncogenic regulator of

tumor suppressor and DNA damage response pathways. Cancer Metastasis Rev

27:123-35.

108. Lukas, J., C. Lukas, and J. Bartek. 2004. Mammalian cell cycle checkpoints:

signalling pathways and their organization in space and time. DNA Repair (Amst)

3:997-1007.

109. Malkin, D., F. P. Li, L. C. Strong, J. F. Fraumeni, Jr., C. E. Nelson, D. H.

Kim, J. Kassel, M. A. Gryka, F. Z. Bischoff, M. A. Tainsky, and et al. 1990.

Germ line p53 mutations in a familial syndrome of breast cancer, sarcomas, and

other neoplasms. Science 250:1233-8.

110. Margolis, S. S., J. A. Perry, C. M. Forester, L. K. Nutt, Y. Guo, M. J. Jardim,

M. J. Thomenius, C. D. Freel, R. Darbandi, J. H. Ahn, J. D. Arroyo, X. F.

Wang, S. Shenolikar, A. C. Nairn, W. G. Dunphy, W. C. Hahn, D. M.

Virshup, and S. Kornbluth. 2006. Role for the PP2A/B56delta phosphatase in

regulating 14-3-3 release from Cdc25 to control mitosis. Cell 127:759-73.

111. Masuda, M., Y. Nagai, N. Oshima, K. Tanaka, H. Murakami, H. Igarashi,

and H. Okayama. 2000. Genetic studies with the fission yeast

118

Schizosaccharomyces pombe suggest involvement of wee1, ppa2, and rad24 in

induction of cell cycle arrest by human immunodeficiency virus type 1 Vpr. J

Virol 74:2636-46.

112. Matsuoka, S., M. Huang, and S. J. Elledge. 1998. Linkage of ATM to cell cycle

regulation by the Chk2 protein kinase. Science 282:1893-7.

113. Matsuoka, S., G. Rotman, A. Ogawa, Y. Shiloh, K. Tamai, and S. J. Elledge.

2000. Ataxia telangiectasia-mutated phosphorylates Chk2 in vivo and in vitro.

Proc Natl Acad Sci U S A 97:10389-94.

114. Maya, R., M. Balass, S. T. Kim, D. Shkedy, J. F. Leal, O. Shifman, M. Moas,

T. Buschmann, Z. Ronai, Y. Shiloh, M. B. Kastan, E. Katzir, and M. Oren.

2001. ATM-dependent phosphorylation of Mdm2 on serine 395: role in p53

activation by DNA damage. Genes Dev 15:1067-77.

115. McConnell, J. L., R. J. Gomez, L. R. McCorvey, B. K. Law, and B. E.

Wadzinski. 2007. Identification of a PP2A-interacting protein that functions as a

negative regulator of phosphatase activity in the ATM/ATR signaling pathway.

Oncogene 26:6021-30.

116. McCright, B., A. M. Rivers, S. Audlin, and D. M. Virshup. 1996. The B56

family of protein phosphatase 2A (PP2A) regulatory subunits encodes

differentiation-induced phosphoproteins that target PP2A to both nucleus and

cytoplasm. J Biol Chem 271:22081-9.

117. Meek, K., V. Dang, and S. P. Lees-Miller. 2008. DNA-PK: the means to justify

the ends? Adv Immunol 99:33-58.

119

118. Meijers-Heijboer, H., A. van den Ouweland, J. Klijn, M. Wasielewski, A. de

Snoo, R. Oldenburg, A. Hollestelle, M. Houben, E. Crepin, M. van Veghel-

Plandsoen, F. Elstrodt, C. van Duijn, C. Bartels, C. Meijers, M. Schutte, L.

McGuffog, D. Thompson, D. Easton, N. Sodha, S. Seal, R. Barfoot, J.

Mangion, J. Chang-Claude, D. Eccles, R. Eeles, D. G. Evans, R. Houlston, V.

Murday, S. Narod, T. Peretz, J. Peto, C. Phelan, H. X. Zhang, C. Szabo, P.

Devilee, D. Goldgar, P. A. Futreal, K. L. Nathanson, B. Weber, N. Rahman,

and M. R. Stratton. 2002. Low-penetrance susceptibility to breast cancer due to

CHEK2(*)1100delC in noncarriers of BRCA1 or BRCA2 mutations. Nat Genet

31:55-9.

119. Melchionna, R., X. B. Chen, A. Blasina, and C. H. McGowan. 2000.

Threonine 68 is required for radiation-induced phosphorylation and activation of

Cds1. Nat Cell Biol 2:762-5.

120. Merkle, D., P. Douglas, G. B. Moorhead, Z. Leonenko, Y. Yu, D. Cramb, D.

P. Bazett-Jones, and S. P. Lees-Miller. 2002. The DNA-dependent protein

kinase interacts with DNA to form a protein-DNA complex that is disrupted by

phosphorylation. Biochemistry 41:12706-14.

121. Messner, D. J., C. Romeo, A. Boynton, and S. Rossie. 2006. Inhibition of

PP2A, but not PP5, mediates p53 activation by low levels of okadaic acid in rat

liver epithelial cells. J Cell Biochem 99:241-55.

122. Mi, J., E. Bolesta, D. L. Brautigan, and J. M. Larner. 2009. PP2A regulates

ionizing radiation-induced apoptosis through Ser46 phosphorylation of p53. Mol

Cancer Ther 8:135-40.

120

123. Mi, J., J. Dziegielewski, E. Bolesta, D. L. Brautigan, and J. M. Larner. 2009.

Activation of DNA-PK by ionizing radiation is mediated by protein phosphatase

6. PLoS ONE 4:e4395.

124. Mochan, T. A., M. Venere, R. A. DiTullio, Jr., and T. D. Halazonetis. 2003.

53BP1 and NFBD1/MDC1-Nbs1 function in parallel interacting pathways

activating ataxia-telangiectasia mutated (ATM) in response to DNA damage.

Cancer Res 63:8586-91.

125. Mochan, T. A., M. Venere, R. A. DiTullio, Jr., and T. D. Halazonetis. 2004.

53BP1, an activator of ATM in response to DNA damage. DNA Repair (Amst)

3:945-52.

126. Mohammad, D. H., and M. B. Yaffe. 2009. 14-3-3 proteins, FHA domains and

BRCT domains in the DNA damage response. DNA Repair (Amst) 8:1009-17.

127. Moorhead, G. B., L. Trinkle-Mulcahy, and A. Ulke-Lemee. 2007. Emerging

roles of nuclear protein phosphatases. Nat Rev Mol Cell Biol 8:234-44.

128. Nakada, S., G. I. Chen, A. C. Gingras, and D. Durocher. 2008. PP4 is a

gamma H2AX phosphatase required for recovery from the DNA damage

checkpoint. EMBO Rep 9:1019-26.

129. Nazarov, I. B., A. N. Smirnova, R. I. Krutilina, M. P. Svetlova, L. V.

Solovjeva, A. A. Nikiforov, S. L. Oei, I. A. Zalenskaya, P. M. Yau, E. M.

Bradbury, and N. V. Tomilin. 2003. Dephosphorylation of histone gamma-

H2AX during repair of DNA double-strand breaks in mammalian cells and its

inhibition by calyculin A. Radiat Res 160:309-17.

121

130. Norbury, C. J., and B. Zhivotovsky. 2004. DNA damage-induced apoptosis.

Oncogene 23:2797-808.

131. O'Neill, B. M., S. J. Szyjka, E. T. Lis, A. O. Bailey, J. R. Yates, 3rd, O. M.

Aparicio, and F. E. Romesberg. 2007. Pph3-Psy2 is a phosphatase complex

required for Rad53 dephosphorylation and replication fork restart during recovery

from DNA damage. Proc Natl Acad Sci U S A 104:9290-5.

132. O'Neill, T., A. J. Dwyer, Y. Ziv, D. W. Chan, S. P. Lees-Miller, R. H.

Abraham, J. H. Lai, D. Hill, Y. Shiloh, L. C. Cantley, and G. A. Rathbun.

2000. Utilization of oriented peptide libraries to identify substrate motifs selected

by ATM. J Biol Chem 275:22719-27.

133. Ofek, P., D. Ben-Meir, Z. Kariv-Inbal, M. Oren, and S. Lavi. 2003. Cell cycle

regulation and p53 activation by protein phosphatase 2C alpha. J Biol Chem

278:14299-305.

134. Okamoto, K., C. Kamibayashi, M. Serrano, C. Prives, M. C. Mumby, and D.

Beach. 1996. p53-dependent association between cyclin G and the B' subunit of

protein phosphatase 2A. Mol Cell Biol 16:6593-602.

135. Okamoto, K., H. Li, M. R. Jensen, T. Zhang, Y. Taya, S. S. Thorgeirsson,

and C. Prives. 2002. Cyclin G recruits PP2A to dephosphorylate Mdm2. Mol

Cell 9:761-71.

136. Oliva-Trastoy, M., V. Berthonaud, A. Chevalier, C. Ducrot, M. C. Marsolier-

Kergoat, C. Mann, and F. Leteurtre. 2007. The Wip1 phosphatase (PPM1D)

antagonizes activation of the Chk2 tumour suppressor kinase. Oncogene 26:1449-

58.

122

137. Oliver, A. W., A. Paul, K. J. Boxall, S. E. Barrie, G. W. Aherne, M. D.

Garrett, S. Mittnacht, and L. H. Pearl. 2006. Trans-activation of the DNA-

damage signalling protein kinase Chk2 by T-loop exchange. Embo J 25:3179-90.

138. Pallas, D. C., V. Cherington, W. Morgan, J. DeAnda, D. Kaplan, B.

Schaffhausen, and T. M. Roberts. 1988. Cellular proteins that associate with the

middle and small T antigens of polyomavirus. J Virol 62:3934-40.

139. Parrilla-Castellar, E. R., S. J. Arlander, and L. Karnitz. 2004. Dial 9-1-1 for

DNA damage: the Rad9-Hus1-Rad1 (9-1-1) clamp complex. DNA Repair (Amst)

3:1009-14.

140. Paull, T. T., E. P. Rogakou, V. Yamazaki, C. U. Kirchgessner, M. Gellert,

and W. M. Bonner. 2000. A critical role for histone H2AX in recruitment of

repair factors to nuclear foci after DNA damage. Curr Biol 10:886-95.

141. Pereg, Y., D. Shkedy, P. de Graaf, E. Meulmeester, M. Edelson-Averbukh,

M. Salek, S. Biton, A. F. Teunisse, W. D. Lehmann, A. G. Jochemsen, and Y.

Shiloh. 2005. Phosphorylation of Hdmx mediates its Hdm2- and ATM-dependent

degradation in response to DNA damage. Proc Natl Acad Sci U S A 102:5056-61.

142. Petersen, P., D. M. Chou, Z. You, T. Hunter, J. C. Walter, and G. Walter.

2006. Protein phosphatase 2A antagonizes ATM and ATR in a Cdk2- and Cdc7-

independent DNA damage checkpoint. Mol Cell Biol 26:1997-2011.

143. Poyurovsky, M. V., C. Priest, A. Kentsis, K. L. Borden, Z. Q. Pan, N.

Pavletich, and C. Prives. 2007. The Mdm2 RING domain C-terminus is required

for supramolecular assembly and ubiquitin ligase activity. Embo J 26:90-101.

123

144. Rainey, M. D., E. J. Black, G. Zachos, and D. A. Gillespie. 2008. Chk2 is

required for optimal mitotic delay in response to irradiation-induced DNA

damage incurred in G2 phase. Oncogene 27:896-906.

145. Re, F., D. Braaten, E. K. Franke, and J. Luban. 1995. Human

immunodeficiency virus type 1 Vpr arrests the cell cycle in G2 by inhibiting the

activation of p34cdc2-cyclin B. J Virol 69:6859-64.

146. Rios-Doria, J., A. Velkova, V. Dapic, J. M. Galan-Caridad, V. Dapic, M. A.

Carvalho, J. Melendez, and A. N. Monteiro. 2009. Ectopic expression of

histone H2AX mutants reveals a role for its post-translational modifications.

Cancer Biol Ther 8:422-34.

147. Rogakou, E. P., C. Boon, C. Redon, and W. M. Bonner. 1999. Megabase

chromatin domains involved in DNA double-strand breaks in vivo. J Cell Biol

146:905-16.

148. Rogakou, E. P., D. R. Pilch, A. H. Orr, V. S. Ivanova, and W. M. Bonner.

1998. DNA double-stranded breaks induce histone H2AX phosphorylation on

serine 139. J Biol Chem 273:5858-68.

149. Roshal, M., B. Kim, Y. Zhu, P. Nghiem, and V. Planelles. 2003. Activation of

the ATR-mediated DNA damage response by the HIV-1 viral protein R. J Biol

Chem 278:25879-86.

150. Ruediger, R., H. T. Pham, and G. Walter. 2001. Disruption of protein

phosphatase 2A subunit interaction in human cancers with mutations in the A

alpha subunit gene. Oncogene 20:10-5.

124

151. Ruvolo, P. P., W. Clark, M. Mumby, F. Gao, and W. S. May. 2002. A

functional role for the B56 alpha-subunit of protein phosphatase 2A in ceramide-

mediated regulation of Bcl2 phosphorylation status and function. J Biol Chem

277:22847-52.

152. Ruvolo, P. P., X. Deng, T. Ito, B. K. Carr, and W. S. May. 1999. Ceramide

induces Bcl2 dephosphorylation via a mechanism involving mitochondrial PP2A.

J Biol Chem 274:20296-300.

153. Sancar, A., L. A. Lindsey-Boltz, K. Unsal-Kacmaz, and S. Linn. 2004.

Molecular mechanisms of mammalian DNA repair and the DNA damage

checkpoints. Annu Rev Biochem 73:39-85.

154. Sarkaria, J. N., E. C. Busby, R. S. Tibbetts, P. Roos, Y. Taya, L. M. Karnitz,

and R. T. Abraham. 1999. Inhibition of ATM and ATR kinase activities by the

radiosensitizing agent, caffeine. Cancer Res 59:4375-82.

155. Sarkaria, J. N., R. S. Tibbetts, E. C. Busby, A. P. Kennedy, D. E. Hill, and R.

T. Abraham. 1998. Inhibition of phosphoinositide 3-kinase related kinases by the

radiosensitizing agent wortmannin. Cancer Res 58:4375-82.

156. Savitsky, K., A. Bar-Shira, S. Gilad, G. Rotman, Y. Ziv, L. Vanagaite, D. A.

Tagle, S. Smith, T. Uziel, S. Sfez, M. Ashkenazi, I. Pecker, M. Frydman, R.

Harnik, S. R. Patanjali, A. Simmons, G. A. Clines, A. Sartiel, R. A. Gatti, L.

Chessa, O. Sanal, M. F. Lavin, N. G. Jaspers, A. M. Taylor, C. F. Arlett, T.

Miki, S. M. Weissman, M. Lovett, F. S. Collins, and Y. Shiloh. 1995. A single

ataxia telangiectasia gene with a product similar to PI-3 kinase. Science

268:1749-53.

125

157. Schito, M. L., O. N. Demidov, S. Saito, J. D. Ashwell, and E. Appella. 2006.

Wip1 phosphatase-deficient mice exhibit defective T cell maturation due to

sustained p53 activation. J Immunol 176:4818-25.

158. Schwarz, J. K., C. M. Lovly, and H. Piwnica-Worms. 2003. Regulation of the

Chk2 protein kinase by oligomerization-mediated cis- and trans-phosphorylation.

Mol Cancer Res 1:598-609.

159. Shaag, A., T. Walsh, P. Renbaum, T. Kirchhoff, K. Nafa, S. Shiovitz, J. B.

Mandell, P. Welcsh, M. K. Lee, N. Ellis, K. Offit, E. Levy-Lahad, and M. C.

King. 2005. Functional and genomic approaches reveal an ancient CHEK2 allele

associated with breast cancer in the Ashkenazi Jewish population. Hum Mol

Genet 14:555-63.

160. Shieh, S. Y., J. Ahn, K. Tamai, Y. Taya, and C. Prives. 2000. The human

homologs of checkpoint kinases Chk1 and Cds1 (Chk2) phosphorylate p53 at

multiple DNA damage-inducible sites. Genes Dev 14:289-300.

161. Shieh, S. Y., M. Ikeda, Y. Taya, and C. Prives. 1997. DNA damage-induced

phosphorylation of p53 alleviates inhibition by MDM2. Cell 91:325-34.

162. Shinozaki, T., A. Nota, Y. Taya, and K. Okamoto. 2003. Functional role of

Mdm2 phosphorylation by ATR in attenuation of p53 nuclear export. Oncogene

22:8870-80.

163. Shreeram, S., O. N. Demidov, W. K. Hee, H. Yamaguchi, N. Onishi, C. Kek,

O. N. Timofeev, C. Dudgeon, A. J. Fornace, C. W. Anderson, Y. Minami, E.

Appella, and D. V. Bulavin. 2006. Wip1 phosphatase modulates ATM-

dependent signaling pathways. Mol Cell 23:757-64.

126

164. Shreeram, S., W. K. Hee, O. N. Demidov, C. Kek, H. Yamaguchi, A. J.

Fornace, Jr., C. W. Anderson, E. Appella, and D. V. Bulavin. 2006.

Regulation of ATM/p53-dependent suppression of myc-induced lymphomas by

Wip1 phosphatase. J Exp Med 203:2793-9.

165. Smith, G. C., and S. P. Jackson. 1999. The DNA-dependent protein kinase.

Genes Dev 13:916-34.

166. Snouwaert, J. N., L. C. Gowen, A. M. Latour, A. R. Mohn, A. Xiao, L.

DiBiase, and B. H. Koller. 1999. BRCA1 deficient embryonic stem cells display

a decreased homologous recombination frequency and an increased frequency of

non-homologous recombination that is corrected by expression of a brca1

transgene. Oncogene 18:7900-7.

167. Sodha, N., S. Bullock, R. Taylor, G. Mitchell, B. Guertl-Lackner, R. D.

Williams, S. Bevan, K. Bishop, S. McGuire, R. S. Houlston, and R. A. Eeles.

2002. CHEK2 variants in susceptibility to breast cancer and evidence of retention

of the wild type allele in tumours. Br J Cancer 87:1445-8.

168. Sodha, N., R. S. Houlston, S. Bullock, M. A. Yuille, C. Chu, G. Turner, and

R. A. Eeles. 2002. Increasing evidence that germline mutations in CHEK2 do not

cause Li-Fraumeni syndrome. Hum Mutat 20:460-2.

169. Sodha, N., T. S. Mantoni, S. V. Tavtigian, R. Eeles, and M. D. Garrett. 2006.

Rare germ line CHEK2 variants identified in breast cancer families encode

proteins that show impaired activation. Cancer Res 66:8966-70.

127

170. Srivastava, S., Z. Q. Zou, K. Pirollo, W. Blattner, and E. H. Chang. 1990.

Germ-line transmission of a mutated p53 gene in a cancer-prone family with Li-

Fraumeni syndrome. Nature 348:747-9.

171. Stevens, C., L. Smith, and N. B. La Thangue. 2003. Chk2 activates E2F-1 in

response to DNA damage. Nat Cell Biol 5:401-9.

172. Stewart, G. S., R. S. Maser, T. Stankovic, D. A. Bressan, M. I. Kaplan, N. G.

Jaspers, A. Raams, P. J. Byrd, J. H. Petrini, and A. M. Taylor. 1999. The

DNA double-strand break repair gene hMRE11 is mutated in individuals with an

ataxia-telangiectasia-like disorder. Cell 99:577-87.

173. Stewart, G. S., B. Wang, C. R. Bignell, A. M. Taylor, and S. J. Elledge. 2003.

MDC1 is a mediator of the mammalian DNA damage checkpoint. Nature

421:961-6.

174. Stiff, T., S. A. Walker, K. Cerosaletti, A. A. Goodarzi, E. Petermann, P.

Concannon, M. O'Driscoll, and P. A. Jeggo. 2006. ATR-dependent

phosphorylation and activation of ATM in response to UV treatment or

replication fork stalling. Embo J 25:5775-82.

175. Stone, S. R., J. Hofsteenge, and B. A. Hemmings. 1987. Molecular cloning of

cDNAs encoding two isoforms of the catalytic subunit of protein phosphatase 2A.

Biochemistry 26:7215-20.

176. Stracker, T. H., T. Usui, and J. H. Petrini. 2009. Taking the time to make

important decisions: the checkpoint effector kinases Chk1 and Chk2 and the DNA

damage response. DNA Repair (Amst) 8:1047-54.

128

177. Stucki, M., J. A. Clapperton, D. Mohammad, M. B. Yaffe, S. J. Smerdon, and

S. P. Jackson. 2005. MDC1 directly binds phosphorylated histone H2AX to

regulate cellular responses to DNA double-strand breaks. Cell 123:1213-26.

178. Stucki, M., and S. P. Jackson. 2006. gammaH2AX and MDC1: anchoring the

DNA-damage-response machinery to broken chromosomes. DNA Repair (Amst)

5:534-43.

179. Suganuma, M., H. Fujiki, H. Suguri, S. Yoshizawa, M. Hirota, M. Nakayasu,

M. Ojika, K. Wakamatsu, K. Yamada, and T. Sugimura. 1988. Okadaic acid:

an additional non-phorbol-12-tetradecanoate-13-acetate-type tumor promoter.

Proc Natl Acad Sci U S A 85:1768-71.

180. Takagi, Y., M. Futamura, K. Yamaguchi, S. Aoki, T. Takahashi, and S. Saji.

2000. Alterations of the PPP2R1B gene located at 11q23 in human colorectal

cancers. Gut 47:268-71.

181. Takai, H., K. Naka, Y. Okada, M. Watanabe, N. Harada, S. Saito, C. W.

Anderson, E. Appella, M. Nakanishi, H. Suzuki, K. Nagashima, H. Sawa, K.

Ikeda, and N. Motoyama. 2002. Chk2-deficient mice exhibit radioresistance and

defective p53-mediated transcription. Embo J 21:5195-205.

182. Takai, H., K. Tominaga, N. Motoyama, Y. A. Minamishima, H. Nagahama,

T. Tsukiyama, K. Ikeda, K. Nakayama, M. Nakanishi, and K. Nakayama.

2000. Aberrant cell cycle checkpoint function and early embryonic death in

Chk1(-/-) mice. Genes Dev 14:1439-47.

129

183. Tang, X., Z. G. Hui, X. L. Cui, R. Garg, M. B. Kastan, and B. Xu. 2008. A

novel ATM-dependent pathway regulates protein phosphatase 1 in response to

DNA damage. Mol Cell Biol 28:2559-66.

184. Travesa, A., A. Duch, and D. G. Quintana. 2008. Distinct phosphatases mediate

the deactivation of the DNA damage checkpoint kinase Rad53. J Biol Chem

283:17123-30.

185. Uldrijan, S., W. J. Pannekoek, and K. H. Vousden. 2007. An essential function

of the extreme C-terminus of MDM2 can be provided by MDMX. Embo J

26:102-12.

186. Uziel, T., Y. Lerenthal, L. Moyal, Y. Andegeko, L. Mittelman, and Y. Shiloh.

2003. Requirement of the MRN complex for ATM activation by DNA damage.

Embo J 22:5612-21.

187. Vahteristo, P., J. Bartkova, H. Eerola, K. Syrjakoski, S. Ojala, O. Kilpivaara,

A. Tamminen, J. Kononen, K. Aittomaki, P. Heikkila, K. Holli, C. Blomqvist,

J. Bartek, O. P. Kallioniemi, and H. Nevanlinna. 2002. A CHEK2 genetic

variant contributing to a substantial fraction of familial breast cancer. Am J Hum

Genet 71:432-8.

188. Varon, R., C. Vissinga, M. Platzer, K. M. Cerosaletti, K. H. Chrzanowska, K.

Saar, G. Beckmann, E. Seemanova, P. R. Cooper, N. J. Nowak, M. Stumm,

C. M. Weemaes, R. A. Gatti, R. K. Wilson, M. Digweed, A. Rosenthal, K.

Sperling, P. Concannon, and A. Reis. 1998. Nibrin, a novel DNA double-strand

break repair protein, is mutated in Nijmegen breakage syndrome. Cell 93:467-76.

130

189. Virshup, D. M., and S. Shenolikar. 2009. From promiscuity to precision:

protein phosphatases get a makeover. Mol Cell 33:537-45.

190. Wang, B., S. Matsuoka, P. B. Carpenter, and S. J. Elledge. 2002. 53BP1, a

mediator of the DNA damage checkpoint. Science 298:1435-8.

191. Wang, H., S. Wang, L. Shen, Y. Chen, X. Zhang, Z. Wang, C. Hu, and W.

Yue. 2009. Chk2 down-regulation by promoter hypermethylation in human bulk

gliomas. Life Sci.

192. Wang, H. C., W. C. Chou, S. Y. Shieh, and C. Y. Shen. 2006. Ataxia

telangiectasia mutated and checkpoint kinase 2 regulate BRCA1 to promote the

fidelity of DNA end-joining. Cancer Res 66:1391-400.

193. Wang, Q., F. Gao, T. Wang, T. Flagg, and X. Deng. 2009. A nonhomologous

end-joining pathway is required for protein phosphatase 2A promotion of DNA

double-strand break repair. Neoplasia 11:1012-21.

194. Wang, S. S., E. D. Esplin, J. L. Li, L. Huang, A. Gazdar, J. Minna, and G. A.

Evans. 1998. Alterations of the PPP2R1B gene in human lung and colon cancer.

Science 282:284-7.

195. Wasielewski, M., P. Hanifi-Moghaddam, A. Hollestelle, S. D. Merajver, A.

van den Ouweland, J. G. Klijn, S. P. Ethier, and M. Schutte. 2009.

Deleterious CHEK2 1100delC and L303X mutants identified among 38 human

breast cancer cell lines. Breast Cancer Res Treat 113:285-91.

196. Williams, R. S., J. S. Williams, and J. A. Tainer. 2007. Mre11-Rad50-Nbs1 is a

keystone complex connecting DNA repair machinery, double-strand break

signaling, and the chromatin template. Biochem Cell Biol 85:509-20.

131

197. Winter, S. L., L. Bosnoyan-Collins, D. Pinnaduwage, and I. L. Andrulis.

2007. The interaction of PP1 with BRCA1 and analysis of their expression in

breast tumors. BMC Cancer 7:85.

198. Wu, X., and J. Chen. 2003. Autophosphorylation of checkpoint kinase 2 at

serine 516 is required for radiation-induced apoptosis. J Biol Chem 278:36163-8.

199. Wu, X., S. R. Webster, and J. Chen. 2001. Characterization of tumor-associated

Chk2 mutations. J Biol Chem 276:2971-4.

200. Xu, X., L. M. Tsvetkov, and D. F. Stern. 2002. Chk2 activation and

phosphorylation-dependent oligomerization. Mol Cell Biol 22:4419-32.

201. Xu, Y., Y. Xing, Y. Chen, Y. Chao, Z. Lin, E. Fan, J. W. Yu, S. Strack, P. D.

Jeffrey, and Y. Shi. 2006. Structure of the protein phosphatase 2A holoenzyme.

Cell 127:1239-51.

202. Xu, Z., and B. R. Williams. 2000. The B56alpha regulatory subunit of protein

phosphatase 2A is a target for regulation by double-stranded RNA-dependent

protein kinase PKR. Mol Cell Biol 20:5285-99.

203. Yamaguchi, H., S. R. Durell, D. K. Chatterjee, C. W. Anderson, and E.

Appella. 2007. The Wip1 phosphatase PPM1D dephosphorylates SQ/TQ motifs

in checkpoint substrates phosphorylated by PI3K-like kinases. Biochemistry

46:12594-603.

204. Yang, S. I., R. L. Lickteig, R. Estes, K. Rundell, G. Walter, and M. C.

Mumby. 1991. Control of protein phosphatase 2A by simian virus 40 small-t

antigen. Mol Cell Biol 11:1988-95.

132

205. Yoda, A., K. Toyoshima, Y. Watanabe, N. Onishi, Y. Hazaka, Y. Tsukuda, J.

Tsukada, T. Kondo, Y. Tanaka, and Y. Minami. 2008. Arsenic trioxide

augments Chk2/p53-mediated apoptosis by inhibiting oncogenic Wip1

phosphatase. J Biol Chem 283:18969-79.

206. Yoda, A., X. Z. Xu, N. Onishi, K. Toyoshima, H. Fujimoto, N. Kato, I. Oishi,

T. Kondo, and Y. Minami. 2006. Intrinsic kinase activity and SQ/TQ domain of

Chk2 kinase as well as N-terminal domain of Wip1 phosphatase are required for

regulation of Chk2 by Wip1. J Biol Chem 281:24847-62.

207. Yong, W., S. Bao, H. Chen, D. Li, E. R. Sanchez, and W. Shou. 2007. Mice

lacking protein phosphatase 5 are defective in ataxia telangiectasia mutated

(ATM)-mediated cell cycle arrest. J Biol Chem 282:14690-4.

208. Yu, Y. M., S. M. Pace, S. R. Allen, C. X. Deng, and L. C. Hsu. 2008. A PP1-

binding motif present in BRCA1 plays a role in its DNA repair function. Int J Biol

Sci 4:352-61.

209. Zannini, L., D. Lecis, S. Lisanti, R. Benetti, G. Buscemi, C. Schneider, and D.

Delia. 2003. Karyopherin-alpha2 protein interacts with Chk2 and contributes to

its nuclear import. J Biol Chem 278:42346-51.

210. Zeng, Y., and H. Piwnica-Worms. 1999. DNA damage and replication

checkpoints in fission yeast require nuclear exclusion of the Cdc25 phosphatase

via 14-3-3 binding. Mol Cell Biol 19:7410-9.

211. Zhang, J., S. Bao, R. Furumai, K. S. Kucera, A. Ali, N. M. Dean, and X. F.

Wang. 2005. Protein phosphatase 5 is required for ATR-mediated checkpoint

activation. Mol Cell Biol 25:9910-9.

133

212. Zhang, P., J. Wang, W. Gao, B. Z. Yuan, J. Rogers, and E. Reed. 2004. CHK2

kinase expression is down-regulated due to promoter methylation in non-small

cell lung cancer. Mol Cancer 3:14.

213. Zhao, H., and H. Piwnica-Worms. 2001. ATR-mediated checkpoint pathways

regulate phosphorylation and activation of human Chk1. Mol Cell Biol 21:4129-

39.

214. Zhou, B. B., and S. J. Elledge. 2000. The DNA damage response: putting

checkpoints in perspective. Nature 408:433-9.

215. Zhou, J., C. U. Lim, J. J. Li, L. Cai, and Y. Zhang. 2006. The role of NBS1 in

the modulation of PIKK family proteins ATM and ATR in the cellular response to

DNA damage. Cancer Lett.

216. Zhou, J., H. T. Pham, R. Ruediger, and G. Walter. 2003. Characterization of

the Aalpha and Abeta subunit isoforms of protein phosphatase 2A: differences in

expression, subunit interaction, and evolution. Biochem J 369:387-98.

134

ABOUT THE AUTHOR

Alyson K. Freeman (maiden name Alyson K. Fay) received a Bachelor of Science degree in Biochemistry and Molecular Biology from the University of Massachusetts in

2002. Afterwards, she worked as a research technician in the laboratory of Dr. Junona

Moroianu at Boston College until 2004. She then pursued her graduate degree in the

Cancer Biology Ph.D. program at the University of South Florida and conducted research in the laboratory of Dr. Alvaro N. A. Monteiro at the H. Lee Moffitt Cancer Center and

Research Institute. Three years of her graduate research was funded through a predoctoral award from the Florida Breast Cancer Coalition Research Foundation. She will continue to conduct cancer research as a postdoctoral fellow in the laboratory of Dr.

Deborah Morrison at the National Cancer Institute.