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Chk2 is negatively regulated by protein phosphatase 2a
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by Alyson Freeman.
[Tampa, Fla] :
b University of South Florida,
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Dissertation (Ph.D.)--University of South Florida, 2010.
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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'alpha 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'alpha 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'alpha and CHK2 proteins, however, it does not have an effect on the binding of B'alpha to CHK1. IR-induced dissociation is an early event and occurs in a dose-dependent manner. CHK2 and B'alpha 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'alpha in vitro; however, in vivo, irradiation has no effect on PP2A activity or localization. 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.
Advisor: Alvaro N. A. Monteiro, Ph.D.
DNA damage response
DNA double-strand break
x Cancer Biology
t USF Electronic Theses and Dissertations.
CHK2 is Negatively Regulated by Protein Phosphatase 2A by 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 r esponse phosphorylation, kinase, ionizing radiation, DNA double strand break Copyrig ht 2010, Alyson K. Freeman
DEDICATION To my husband Scott N. Freeman, my parents James P. and Carolyn C. Fay, and my sister Erin M. Fay.
ACKNOWLEDGEMENTS I fi rst and foremost would like to thank my me ntor, 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, Sylvi a 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 speci al 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.
i TABLE OF CONTENTS LIS T OF FIGURES iv LIST OF ABBREVIATIONS vi ABSTRACT viii INTRODUCTION 1 DNA Damage Response Pathway 1 Sensors Mre11 Rad50 Nbs1 and Rad9Rad1 Hus1 C omplexes 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 P hosphat ases 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 P 53 32 DNA Damage Response Proteins Whose Activities are Enhanced 35 ATM 35 ATR 36 DNAPK 36 BRCA1 37 P53 38
ii Summary and Rationale 38 MATERIALS AND METHODS 40 Cloning 40 Yeast Two Hybrid 40 Cell Cu ltu re, 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 B inds to the B' Subunit of PP 2A 46 CHK2 I nteracts with the B' Subunit of PP2A in Y east 46 CHK2 and B' Bind in Mammalian C ells 51 Binding B etween CHK2 and PP2A is Modulated by DNA D amage 54 B' and CHK2 Dissociate upon IR induced DNA D amage 54 CH K2 Binds to M any PP2A Subunits 58 B' and CHK2 are Dissociated A fter DNA Damage C aused by IR and D oxorubicin, but not B' and CHK1 61 Analysis of the IR induced D issociation of CHK2 and B' Dissociation is an Early Event and O ccur s in a D ose dependent M anner 63 Re A ssociation of CHK2 and B' does Not Correlate W ith R esolution of DNA D amage 67 The A ct ivity of ATM, but not PP2A, is N ecessary for the D issociation of CHK2 and B' Mutation of CHK2 Serines 33 and 35 Affects B inding to B' Analysis of the E ffects of CHK2 on PP2A 77 CHK2 can P hosphorylate B' in vitro 77 PP2A Localization is N ot I nfluenced by IR 79 PP2A Activ ity is Unaffected by IR and is N ot I nfluenced by CHK2 82 Analysis of the E ffects of PP2A on CHK2 87 Chan ges in CHK2 Phosphorylation D ue to IR 87 Changes in CHK2 Kinase A ctivity after IR 91 Mutation of CHK2 Serines 33 and 35 Affects Protein S tability 94
iii DISCUSSION 97 LIST OF REFERENCES 103 ABOUT THE AUTHOR END PAGE
iv LIST OF FIGURES Figu re 1. The DNA d amage response p athway 3 Figure 2. The s tructure of CHK2. 12 Figure 3. PP2A holoenzyme s tructure 20 Figure 4. Protein serine/threonine phosphatases in the DNA damage response pathway 24 Figure 5. B' binds to CHK2 amino acids 1107. 49 Figure 6. CHK2 binds to B' amino acids 89322. 50 Figure 7. CHK2 binds to FLAG B' in mammalian cells 52 Figure 8. Endogenous CHK2 and endogenous B' bind in mammalian cells 53 Figure 9. Binding between CHK2 and B' is dissociated after DNA damage. 56 Figure 1 0. Dissociation of CHK2 and B' is verified using another CHK2 antibody 57 Figure 1 1. CHK2 binds to many PP2A B' subunits and they are dissociated after IR 59 Figure 1 2. Endogenous PP2A C and CH K2 can bind and they are dissociated after IR 60 Figure 1 3. D issociation of CHK2 and B' is caused by DNA damage induced by IR and doxorubicin. 62 Figure 1 4. Dissociation of CHK2 and B' occurs in a dose dependent manner 65 Figure 15. Dissociation of CHK2 and B' is and early event after IR 66 Figure 16. CHK2 and B' are able to reassociate hours after 6 Gy IR 69 Figure 17. CHK2 and B' are able to reassociate hours after 50 Gy IR 70
v Figure 18. PP2A phosphatase activity is not required for CHK2 binding to or dissociating from B' 73 Figure 19. Inhibition of ATM and DNA PK kinase activity prevents the IR induced dissociation of B' and CHK2 74 Figure 20. Mutation of CHK2 serines 33 and 35 affects binding to B' 76 Figure 21. CHK2 can phosphorylate B' in vitro 78 Figure 22. B' 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' does not change after IR 85 Figure 26. Total PP2A activity does not differ in HCT116 wildtype and CHK2/ cells 86 Figure 27. Inhibition of PP2A activity increases CHK2 activity 89 Figure 28. Mut ation of CHK2 serines 33 and 35 affects CHK2 phosphorylation at other sites 90 Figure 29. B' overexpression negatively affects CHK2 kinase activity 92 Figure 30. Mutation of CHK2 serines 33 and 35 affects CHK2 kinase activity 93 Figure 31A Mutat ion 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
vi L IST OF ABBREVIATIONS 4NQO 4nitroquinoline N oxide 911 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 suscepti bility 1 CHK2 Checkpoint kinase 2 CHK1 Checkpoint kinase 1 CPT Camptothecin DDR DNA damage response DNAPK DNAdependent protein kinase DOX Doxorubicin DSB DNA double strand breaks ES Embryonic stem HR Homologous recombination HU Hydroxyurea IB Immunoblot IP Immunoprecipitation
vii IR Ionizing radiation MEF Mouse embryonic fibroblast MMC Mitomycin C MMS Methylmethane sulfonate MRN Mre11 Rad50Nbs 1 NBS Nijmegen Breakage Syndrome NCS N eocarzinostatin 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/th reonine phosphatase RDS Radioresistant DNA synthesis RFC Replication factor C RPA Replication protein A SSB Single strand break s VPR Viral protein R
viii CHK2 IS NEGATIVELY REGULATED BY PROTEIN PHOSPHATSE 2A Alyson K. Freeman ABSTRACT Checkpoint kinase 2 (CHK2) is a n 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' 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' binds to the SQ/TQ cluster domain of CHK2, which is a target of ATM phosphory lation. 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 br eaks by ionizing radiation (IR) as well as treatment with doxorubicin causes disso ciation of the B' and CHK2 proteins however, it does not have an effect on the binding of B' to CHK1 IR induced dissociation is an early event and occurs in a dose dependent manner. CHK2 and B' can reassociate hours after DNA damage and this is not dependent upon t he repair of the DNA. D issociation 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 dis sociation after IR CHK2 is able to phosphorylate B' in vitro ; however, in vivo irradiation has no effect on PP2A activity or localization.
ix 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.
1 INTRODUCTION DNA Damage Resp onse P athway 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 d amage 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 th e DNA damage response (DDR) is to induce cell cycle arrest, repair the incurred DNA damage, or, in cas es where the extent of damage is beyond repair, signal for the initiation of apoptosis or cellular senescence (214) The different types of DNA da mage as well as each repair mechanism were extensively reviewed recently (153) Here we will focus primarily on the DNA damag e 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 nonhomologous 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 re paired. Distinct cell cycle checkpoints include G1/S or G1, intra S phase and
2 G2/M or G2. In the case that the damage cannot be repair ed 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 )
3 Figure 1. The DNA damage response pat hway. DNA breaks and replication stress signal through the sensors (MRN and 911), 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.
4 Sensors Mre11 Rad50 Nbs 1 and Rad9Rad1Hus1 C omplex es DNA damage is recognized by sensor proteins that initiate the activation of the DDR. These sensor s include the Mre11 Rad50Nbs 1 (MRN) complex and the Rad9Rad1 Hus1 (911) compl ex 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 11 complex res embles the proliferating cell nuclear antigen (PCNA) sliding clamp that is loaded onto DNA at points of replication (139) The Rad17replication factor C (RFC) complex acts as the 9 11 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 Nbs 1, DNA, and Rad50 and possesses DNA exonuclease, endonuclease, and unwinding activities (196) Rad50 may function to keep the broken ends of the DNA together. Nbs 1 functions to recruit signal transducing kinases to the break site and mediate s the DDR signal (215) Signal T ransducing K inases ATM and ATR The localization of the MRN and 911 complexes to the sites of DNA damage signals to the signal transducing kinases to become active. These kinases are Atax ia 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) P rimarily in response to DSB, ATM is activated by autophosphorylation on S 1981 c ausing inactive dimers to dissociate into active monomers (7). Interestingly the ATM protein was first identified in patients with ataxia telangiectasia (AT) (156) AT
5 c auses genomic instability, a predisposition to cancer, immunodeficiency, and several neurological impairments (33) Mutation of Mre11 and Nbs 1 in humans causes the similar disorders AT like disorder (ATLD) and Nijmegen Breakage Syndrome (NBS) respectively (172, 188) Nbs 1 seemingly acts both upstream and downstream of ATM as A TM is known to phosphorylate Nbs 1 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 i s dependent upon the 91 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 phosphoryla ted 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 ta rgeting subunit, the Ku70Ku80 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 K inases CHK1 and CHK2 The signal transducing kinases ATM and ATR signal t hrough the effector kinases CHK1 and CHK2 (checkpoint kinas e 1 and checkpoint kinase 2) (8). CHK1 and CHK2 work alongside ATM and ATR to perpetuate the DDR signal. CHK2 is activated
6 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, t here is also crosstalk between the se two pathways. The processing of DSB in an ATM and Mre11dependent manner leads to RPA coating the single strand ed 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 Nbs 1 in addition to ATR (53) The importance o f 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.
7 Mediators H2AX, BRCA1, MDC1, and 53BP1 The mediator proteins H2AX, BRCA1, MDC1, and 53BP1 work to coordinate the localizatio n of various factors in the DDR, promote their activation, and regulate substrate accessibility (176) The histone variant H2 AX comprises 1015% of total cellular histone H2A as revealed through mouse studies (19) H2AX is phosphorylated by ATM, ATR, and DNAPK on S139 and is commonly referred to as H2AX (148) 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) H2AX is required for the foci formation of Nbs 1 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) Phosphor ylated BRCA1 forms distinct nuclear foci that co localize with 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 H2AX and co localizes with 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
8 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 check points 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 p hosphorylate Cdc25C on S216 which allows 1433 binding and subsequent sequestration of Cdc25C in the cytoplasm, away from substrates. Cdc25B1 and Cdc25B2 are phosphorylated on S309 and S 323, respectively leading to inactivation through the same 1433 m ediated mechanism (38, 210) The transcription factor p 53 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 multiorgan cancer s yndrome, Li F raumeni 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,
9 depending upon the cellular stress (63) Mdm2 negat ively regulates p53 through ubiquitinmediated 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, DNAPK, CHK1 and CHK2 stabilize p53 by direct phosphoryl ati on on S15 and S20 which prevent p53 binding to Mdm2 (5, 160, 161) ATM and ATR inhibit Mdm2 by phosphoryl ating 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 1433 binding and degradation of Mdm X (24, 82) Ther e fore, 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 the se cellular effects have not yet been determined.
10 Checkpoint K inase 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 A ctivation CHK2 conta ins three major domains ( Figure 2): an SQ/TQ domain (amino acid residues 1969), an FHA domain (amino acid residues 112175), and a serine/threonine kinase domain (amino acid residues 220486), all of which play a role in the activation of CHK2. The SQ/TQ domain contains seven serine or t hreonine 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 T 68 in the SQ/TQ domain (3, 113, 119) The FHA domain of one molecule of CHK2 binds to the phosphorylated T 68 of another, allowing autophosphorylation of T 383 and T 387 within the kinase domain (83, 200) CHK2 then dissociates into active monomers (2). CHK2 is also phosphorylated at ser ines 19, 33, and 35 in the SQ/TQ domain by ATM and autophosphorylated at S516 and these ev ents enhance CHK2 activity (16, 79, 124, 198) Phosphorylation of CHK2 at S456 regulates CHK2 stability and autophosphorylation at S379 leads to CHK2
11 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 dom ain 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)
12 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.
13 CHK2 in C ancer CHK2 is considered a tumor suppressor a nd m utations 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 LiFraumeni 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 T P53 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 Je wish 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 th e UACC812 breast cancer cell line (195) In addition, m ultiple 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 vitr o (199) The T908A mutation also causes an early stop codon at L303 (195) R145W has reduced kinase activity in vitro is not
14 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 activa tion (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 base d 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 g ene 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 i dentified in LiFraumeni families, primary
15 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 P henotypes Two CHK2/ mouse m odels 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 CHK1null 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, t hymocytes 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 conti nued to replicate DNA after DSB instead of activat ing 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 p53mediated DDR response Knockdown of CHK2 in HCT116 cells inhibited Cdc25C phosphorylation at
16 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 phosphorylati on at S20 in response to IR intact G1/S and G2/M checkpoint s following IR and an appropriate apoptotic response to 5fluorouracil when compared to the HCT116 wildtype cells. Gene targeti ng 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 de monstrate 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, t he function of CHK2 in cells is often measur ed by RDS, apoptosis, and the G1/S and G2/M checkpoints
17 Protein Serine/Threonine Phosphatases The re are two major families of protein serine/threonine phosphat a ses (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 a n ankyrin repeat subunit (123) PP2B, also known as PP3 and calc i neurin, and PP7 are stimulated by Ca2+. PP2B consists of an A catalytic subunit and a B subunit that is related to calmo dulin. PP7 contains EF hand domains (127) PP5 is unique in the fact that it has three TRP domains which mediate proteinprotein interactions, and a peptidylpro l yl cis trans isomeraselike 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. PP2C is also known as Wip1 or PMDD1. Wip1, PP2C PP 2C and PP2C 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 for ms of the A subunit, and that have 87% identity (66). T he re are also two forms of the C
18 subunit and 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 A C and B' 1 was solved, revealing the active enzyme structure as well as binding sites between the three subunits (25, 201) The A subunit contains 15 HEAT repeats of antiparallel helices and forms a horseshoe shape. The C subunit forms a compact ellipsoidal structure. The B' 1 subunit forms a shape similar to the A subunit and is comprised of 18 helices and 8 pseudoHEAT repeats. The full B' family was originally cloned in 1995 and is comprised of five genes two of which are differentially splic ed to form three isoforms each (116) The names of all of the B' family members are as follows: B' B' B' B' 1, B' 2, B' 3, B' 1, B' 2, and B' 3. Although this nomenclature i s 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, t he literature of the B' family can often be confusing. I n 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,
19 but not B' or B'' proteins, which likely affects PP2A substrate specificity (102) The serine/threonine kinase PKR phosphorylates B' and this increases PP2A activity in vitro (202) Therefore, in addition to s ubstrate specificity based on the B subunit in the holoenzyme, post translational modifications can affect PP2A activity.
20 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.
21 PP2A F unction 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 b e 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 C results in embryoni c lethality (56) Transgenic mice overexpressing B' 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 cardiomyopat hy 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 overexpressi on of c Myc has been found in many human tumors (31) PP2A containing B' binds to c Myc and dephosphorylates it at S62, resulting in decreased c Myc protein levels and decreased cMyc transcriptional activity (6). PP2A has a negative effect o n the G2/M transition through the nega tive 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 1433. PP2A co ntaining B counteracts this by dephosphorylating T138 in Cdc25C Chk1 phosphorylates PP2A B at S37, enhancing B' binding to the A and C subunits. Therefore, Chk1 keeps the cell
22 from progressing into mitosis by enhancing dephosphorylation of T138, keeping 1433 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' subunit and ceramide promotes translocation of B' to the mitochondrial membrane (151) PP2A in C ancer Several independent lines of evidence suggest a role for the PP2A hol oenzyme 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 SV40mediated 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 A 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 A mutations E64D in a lung carcinoma, E64G in a breast cancer, R418W in a melanoma cell line, and a frameshift mutation at amino acid F 170 in a breast cancer were rece ntly identified (18) These m utations 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)
23 Protein Serine/Threonine Phosphatases in the DNA Damage R esponse 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 w ill review the recent literature implicating the importance of phosphatases in the DDR alongside kinases. DNA D amage Response Proteins that are N egatively 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 event s Indeed, there are many proteins that are negatively regulated in this manner ( Figure 4 )
24 Figure 4. Protein serine/threonine phos phatases 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.
25 ATM and ATR The first evidence that PP2A might play a role in the ATM dep endent 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 inhi bition of PP2A with OA or expres sion 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 t he ATM and ATR phosphorylation of a 32kD protein and TIP prevented this function of PP2A (115) In X. laevis nucleoplasmi c extracts, DSB inhibit replication by inhibiting the loading of Cdc45, RPA, and polymerase 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 show n a role for Wip1 in negatively regulatin g ATM. E1A+Ras expressing Wip1null 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) Spl enocytes from Wip1+/ and Wip1/ mice in an E myc background displayed an increase in ATM phosphorylation and p53 S18 phosphorylation (164) Wip1+/ and Wip1/ mice were more resistant to
26 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 depend ent 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 dephosphoryla ted 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 r egulates CHK1. Cells lacking Dis2 have a prolonged G2 arrest following treatment with the double strand break inducer MMS ( m ethylmethane sulfonate) or the UV mimetic 4NQO ( 4nitroquinoline N oxide ) but
27 DNA repair is not affected. The overexpression of D is2 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 i n vitro PP2A was able to directly dephosphorylate CHK1 (88) In X. laevis egg extracts, the addition of PP2A C reversed CHK1 phosphorylation at S344 (huma n 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, wherea s 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/svC57BL6 background showed a greater increase in phosphorylation at S345 in C HK1 after IR than wild type MEFs whereas Wip1+/+ and Wip1/ splenocytes expressing E 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)
28 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 e ndonuclease 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 th en adaptation to the chec kpoint 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. Pph3Psy2 binds to Rad53 and dephosphorylates it directly. The Pph3Psy2 complex is necessary for the dephosphorylation of Rad53 during
29 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 M MS 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 fol lowing 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, CH K2 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 coimmunoprecipitation, CHK2 was found to bind to PP2A A, C, and many B' subunits. CHK2 was able to phosphorylate B' 1 and B' 3 in vitro and this increased PP2A activity in vitro The overexpression of B' 3 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)
30 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 expressi ng 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 I R, 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.
31 H2AX PP1, PP2A and PP4 have been implicated in negatively regul ating H2AX. In yeast, d eletion of any of the PP4 like complex HTP C genes (Pph3, Psy2, and Ybl046w) increases the amount of cellular H2AX. HTP C can dephosphorylate H2AX in vitro Although Pph3 deficient cells had similar rates of DSB repair and loss of H2AX foci as wild type cells, even though H2A X remained phosphorylated, Rad9 (human 53BP1) and Rad53 (human CHK2) remained active longer and this correlated with maintenance of the G2/M checkpoint (76). In huma n cells, t he inhibition of PP1 partially inhibited the elimination of H2AX after DNA damage induced by IR (129) The inhibition of PP2A or knockdown with siRNA increased the amount of H2AX, increased the amoun t 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 H2AX in nucleosomes in vitro PP2A is at least 25 times more active toward H2AX in monomeric form or when incorporated into nucleosomes (27, 129) Indeed, PP2A C colocalizes with and binds to H2AX after CPT treatment and the binding increases with increasing DNA damage (27) Thus far, PP2A and PP4 have been implicated in negatively regulating H2AX. In cells with both PP4C and PP2A C knocked down, 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
32 dephosphorylate H2AX from mononuclosomes in vitro (28) Both PP2A and PP4, but not PP1, can dephosphorylate H2AX in vitro from acid extracted histones. Knockdown of PP4C and PP2A C caused an increase in the total levels of H2AX in untreated and irradiated cells (128) Further examination of the specific roles of PP2A and PP4 i n the regulation of H2AX has found distinct roles for each. PP4C silenced cells, but not PP2A silenced cells, showed an increase in H2AX even before DNA damage (28) PP2A C sile nced cells were slightly weaken ed 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 H2AX foci after IR but PP2A knockdown did not affect foci. PP4C primarily dephosphorylated H2AX associated with chrom atin 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 H2AX is mediated through PP2A and PP4. This is necessary for the recovery from the DSB induced checkpoint. P 53 P53 is negatively regulated through direct dephosphorylation by PP1 and PP2A and through the regulation of its negative regulator, Mdm2. Th e p hosphorylation 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 incr ease in p53 phosphorylation at S15 and an increase in p53 activity (121)
33 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 induce s 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 it s 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 de creased 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 p53dependent 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-
34 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 checkp oints after UV and IR (106) Interestingly, the Wip1 gene was found to be amplified in 11% of human breast tumors, most of which had wildtype p53 (15) These data collectively suggest that the negative regulation of p53 by Wip1 contributes to tumorigene si s. Not only is p53 regulated by multiple PSPs directly, but it is also regulated indi rectly t hrough the regulation of Mdm2. Mdm2 was predicted to be a Wip1 substrate (203) Indeed, Wip1, but not PP2A, dephosphorylates M dm2 at S395, which is an ATM phosphorylation site (105, 114) Wip1 dephosphorylation at S395 increases the stability of Mdm2 and increases the Mdm2p53 interaction. Wip1 inhibits Mdm2 autoubiquitination thereby stabilizing the protein (105) The induction of p53 causes an induction of cyclin G and increased binding between cyclin G and PP2A B' 3 (134) Cyclin G prefer entially associates with B' 3 in the active holoenzyme complex. It appears to act as a recruitment factor to bring PP2A to Mdm2 so PP2A can dephos phorylate Mdm2 at S216 and S166. This increases Mdm2 binding to p53, thereby negatively regulating it (135) Overexpression of PP2A B' 3 and B' 2 increas e s the levels of cyclin G1 protein. Ectopic expression of B' 3 counteracts the DNA damage induced destabilization of cyclin G1 (92) In conclusion, the multiple modes of regulation of p53 and its activation of ne gative feedback loops signifies the importance of keeping p53 tightly regulated after DNA damage. PSPs play a role in regulating p53 through direct dephosphorylation via
35 PP1 and PP2A as well as enhancement of Mdm2 negative regulation of p53 through PP2A. DNA Damage Response Proteins W hose Activities are Enhanced In contrast to the many instance s 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 ( Figu re 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 Nbs 1 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 kin ase activity resulting in a G2/M checkpoint (183)
36 ATR HIV 1 Vpr (viral protein R) induces a G2 checkpoint through ATR and the phosphorylation of Cdk1 at Y 15 (43, 65, 145, 149) PP2A is necessary for this Vpr induced checkpoi nt. 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 Y 15 phosphorylation, and a decrease in phosphorylation of CHK 1 S345 (43, 111) This was only in a Vpr induced fashion, as PP2A had no effect on pS345CHK1 after UV or HU (91) I n 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 inhibite d the replication checkpoint after UV, inhibited the S phase checkpoint after HU, and decreased RPA phosphorylation and foci formation (211) DNAPK PP1, PP2A, and PP6 have all been shown to positively influence DNAPK 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
37 dephosphorylation of DNA PKcs and Ku increases the associati on between the DNA PK proteins and this promotes DNA repair (193) PP6 and DNAPK form a complex in cells and this bindi ng increases after IR IR causes a t ranslocation 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 tissu e as compared to sporadic breast tumors (197) This implicates PP1 in the activation of BRCA1.
38 P 53 Although p53 is negatively regulate d via multiple PSPs, its activity is also enhanced by two phosphatases. PP2C overexpression increases p53 transcription activity The overexpression of PP2C inhibits cell proliferation, cell cycle progression, and colony formati on and induces apoptosis. These effects are partially p53 dependent (133) P 53 phosphorylation at T55 leads to p53 degradation (94) PP2A containing the B' 1 or B' 3 subunit dephosphorylates T55 in response to DNA damage which leads to the inhibition of cell proliferation and transformation. In addition, protein levels of B' 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 inac tivating it following the repair of the DNA to allow for recovery from the cell cycle checkpoint. Summary and R ationale The DDR is extremely important for cells to prevent the propagation of mutations that can lead to cancer. A highly sophisticated ne twork 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.
39 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' 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.
40 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' fragments containing amino acid residues 1486 (full length), 1195, 1322, 176322, 176486, 304486, 44322, 89322, 133322, 1282, and 1195 from the clone that was identified in the yeasttwo 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 wa s also used to generate fragments of CHK2 containing amino acid residues 1303, 1194, 1107, 89194, 143390, 195336, 217543, and 304543. 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 H ybrid The yeast two hybrid was performed via the MATCHMAKER Two Hybrid System 3 (CLONTECH Laboratori es), 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 yeas t and selection was made on SD Leu Trp Ade His minimal media to verify binding between CHK2 and any prey from the
41 library. For the deletion fragment analysis, the full length gene encoding B' in pACT2 was co transformed with each CHEK2 fragment in pG BKT7 or full length CHEK2 in pGBKT7 was transformed with each B' gene fragment in pACT2 into AH109 yeast and selection was made on minimal media. Cell Culture, Transfection, and R eagents 293T cells were grown in DMEM media (Sigma) supplemented with 7.5% Fetal Bovine Serum (SAFC), 1% Penicillin Streptomycin (Gibco), and 0.5% AmphotericinB (Sigma). HCT15 cells were grown in RMPI media (Gibco; Sigma) supplemented with 10% Fetal Bovine Serum, 1% Penicillin Streptomycin, and 0.5% AmphotericinB. HCT116 wil d 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% AmphotericinB. 293T cells were transfected using Fug ene 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 conce ntrations. Immunoprecipitations For endogenous coimmunoprecipitations, 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
42 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 deoxy cholate and SDS) or NETN Samples were eluted via boiling in sample buffer and analyzed by immunoblotting. For coimmunoprecipitations with overexpressed proteins, 293T cells were transfected with a pCEP 4HA plasmid containing B' B' B' B' or B' 3 (obtained from Addgene; originally deposited by D. Virshup), a pCMV2FLAG plasmid containing B' or cotransfected with pCMV2 FLAG B' and wildtype 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, lys ates were incubated with anti FLAG M2 Affinity Gel (Sigma). Samples were eluted with 150 ng/ L 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 4C. Lysates were separated by SDS PAGE gel a nd 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 phosphoserine 19 (Cell Signaling), CHK2 phosphoserines 33/35 (Cell Signaling), CHK2 phosphothreonine 68 (Cell Signaling),
43 CHK1 phosphoserine 317 (Cell Signaling), Talin (Clone TA205; Upstate), PARP (BD PharMingen), H2AX (Upstate), FLAG (M2, perox idase conjugate; Sigma), 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 A ctivity As say 293T cells, 293T cells transiently transfected with pCMV2FLAG empty vector or one containing FLAG B' 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 irradia ted 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 b y 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 30C for 30 min in kinase assay buffer containing 25 M ATP and 10 Ci [ -32P]ATP with either 2 g GST B' or 1 g of GST Cdc25C (amino acids 200256) as a substrate using equivalent amounts of GST as a negative control. Reactions were stopped by addition of sample buffer, boiled, and separated by SD S PAGE gels which were then immunoblotted for CHK2 and autoradiographed. The plasmid containing GST Cdc25C (amino acids 200256) was a gift from Larry Karnitz (154)
44 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% glycer ol, 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 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 4C 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 mercaptoethanol) was added and mixed by pipeting. Samples were incubated on a rocking platform at 4C for 20 min then centrifuged at 14,000 rpm for 15 min at 4C. The supernatant was saved as the nuclear fraction. Acid extraction buffer (0.5 M HCl, 10% glycerol, 100 mM mercaptoethanol) was added to the remaining pellet, vortexed, incu bated 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. P hosp hatase Activity A ssay 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 PP 2A C (for total PP2A activity) or FLAG (for activity of PP2A containing FLAG B' ).
45 Immunoprecipitates were incubated with a phosphopeptide for 10 min at 30C and free phosphate was measured by addition of Malachite Green Detection Solution and the absorba nce 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 g/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 mockirradiated 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.
46 RESULTS CH K2 B inds to the B' S ubunit of PP 2A 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 twohybrid system, identify the binding domains, and verify the interaction in mammalian cells. CHK2 I nteracts with the B' Subunit of PP2A in Y east 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 Tr y ptophan. 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 Leu cine. 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 Ade nine and His tidine Therefore, interacting partners of CHK2 were isolated based on their ability, when coexpressed with wild type CHK2, to allow yeast to grow on media lacking Ade nine His tidine Leu cine, and Tr y ptophan.
47 In this screening, w e identified two independent clones of the B' 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 M us 81. 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 t he 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 interact ion between B' and CHK2, we mapped the B' interacting domain in CHK2. Multiple deletion constructs of CH E K2 were constructed via PCR and cloned into the pGBKT7 vector. E ach was co transformed with the pACT2 vector containing the full length B' Binding was determined using the yeast two hybrid system and was defined as growth of colonies on minimal media ( A de, His L eu T rp ) as in the original yeast two hybrid screening The minimal CHK2 fragment that bound to full length B' was comprised of amino acid residu es 1 107. This region contains the entire SQ/TQ region ( Fig ure 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' multiple deletion constructs were constructed of B' 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' was found to contain amino acid residues 89 3 22 ( Fig ure 6 ). Although this region spans almost half of the
48 protein, the crystal structure of the PP2A holoenzyme containing B' 1, which is closely related to B' reveals unique features of this enzyme (25, 201) B' 1 contains eighteen 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' 1 protein (201) as noted in Fig ure 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.
49 Figure 5 B' 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 hy brid system, deletion mutants of CHK2 were cotransformed into yeast with fulllength B'
50 Figure 6 CHK2 binds to B' amino acids 89322. Full length B' is shown with the residues responsible for binding to the PP2A A subunit in light gray a nd the C subunit in dark gray (201) Using the yeast two hybrid system, deletion mutants of B' were co transformed with full length CHK2.
51 CHK2 and B' Bind in Mammalian C ells We next wanted to verify the binding of B' and CHK2 in a mammalian system. 293T cells were transiently transfected to express FLAG B' Whole cell extracts were immuno precipitated 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 immunoblot ted for endogenous CHK2, demonstrating that B' and CHK2 interact in vivo ( Figure 7 ). Immunoblotting also detected the endogenous PP2A A and C subunits, indicating that FLAG B is able to function as a member of the potentially active heterotrimeric PP2A complex. To ensure that the binding of CHK 2 with B' occurs without overexpression of the B' 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' As shown in Figure 8 B' was able to bind to CHK2, verifying that this interaction occurs with the endogenous levels of both proteins.
52 Figure 7 CHK2 binds to FLAG B' in mammalian cells. Whole cell lysates o f 293T cells expressing FLAG B' were immunoprecipitated (IP) using the FLAG antibody and immunoblotted (IB) for the indicated proteins. Cell lysates that were not expressing FLAG B' were used as a negative control.
53 Figu re 8 Endogenous CHK2 and endogenous B' bind in mammalian cells. Whole cell lysates of 293T cells were immunoprecipitated using an antibody to CHK2 and immunoblotted for B' Normal mouse IgG was used as a negative control for the immunoprecipitation.
54 Binding B etween CHK2 and PP2A is M odulated 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' Therefore, we determined whether the binding between CHK2 and B' 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' and CHK2 Dissociate upon IR Induced DN A D amage. Since CHK2 is activated upon DNA damage, we examined the binding between CHK2 and B' for any changes upon the induction of DNA DSB by exposing cells to IR. Whole cell extracts of 293T cells transiently expressing FLAG B' were immunoprecipitated using a FLAG antibody and immunoblotted for endogenous C HK2, 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' without affecting the binding of B' t o 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 S 33/35. Immunoblotting of the lysate input verifies that IR causes phosphorylation of CHK2 at S 19, S 33/35, and T 68 as previously described (3, 16, 113, 119) verifying the activ ation of CHK2 after IR ( Figure 9 ). To ensure that the dissociation seen between CHK2 and B' 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' under normal conditions and after exposur e to IR, CHK2
55 and B' were dissociated ( Figure 10). This confirms that CHK2 and B' are dissociated after IR and this is not due to specific reagents that were used.
56 Figure 9 Binding between CHK2 and B' is disrupted after DNA da mage. 293T cells transiently expressing FLAG B' 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 us ed is anti CHK2 (clone 7) from Upstate Cell Signaling Solutions.
57 Figure 10 Dissociation of CHK2 and B' is verified using another CHK2 antibody 293T cells transiently expressing FLAG B' 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.
58 CHK2 B inds to Many PP2A S ubunits We have determined that CHK2 binds to one member of the PP2A B' family, namely B' 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' B' B' B' and B' 3 proteins were transiently expressed in 293T cell s. 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 m any B' proteins using an in vitro binding assay (40). In our study, CHK2 had the strongest interaction to B' 3 and the weakest to B' with binding to B' B' and B' residing within this range. Additionally, the CHK2B' complexes were dissociated after the cells were exposed to IR. It should be noted that the ability of the B' 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' 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 PP 2A 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' regula tory 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.
59 Figure 11 CHK2 binds to many PP2A B' subunits and they are diss ociated after IR 293T cells transiently transfected with HA tagged B' B' B' B' or B' 3 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.
60 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.
61 B' and CHK2 are Dissociated A fter DNA Damage Caused by IR and D oxorubicin, but not B' 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 t he binding between CHK2 and B' 293T cells transiently expressing FLAG B' were exposed to IR, UV, HU mitomycin C (MMC), or doxorubicin (Dox). FLAG B' was immunoprecipitated and immunoblotted for endogenous CHK2, PP2A A and PP2A C. Interestingly, t re atment with UV, HU, or MMC did not have any effect on the ability of CHK2 to bind to FLAG B' ( Fig ure 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, S 19, S 33/35, and T 68 were all phosphorylated; after UV and MMC, T 68 was primarily phosphorylated and S 19 was weakly phosphorylated; and after HU, S 19 and T6 8 were phosphorylated. The dissociation between CHK2 and B' occurs after IR and Dox: the only conditions that resulted in S 33/35 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' and whether this binding was disrupted by DNA damage ( Fig ure 13 ). Interestingly, CHK1 was able to bind to B' but this binding was not disrupted by treatment with IR, UV, HU, MMC, or Dox. CHK1 was highly phosphorylated at S 317 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' complex.
62 Figure 13 D issociation of CH K2 and B' is caused by DNA damage induced by IR and doxorubicin 293T cells transiently transfected with FLAG B' 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 g/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.
63 Analysis of the IR Induced D issociation of CHK2 and B' 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 t he dissociation of CHK2 and B' 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' since the dissoc iation correlated with the phosphorylation of CHK2 at certain sites. Dissociation is an Early E vent and Occurs in a Dose Dependent Manner Since we have observed that IR causes the most dissociation of B' 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' were exposed to 2, 5, 10, 20, or 50 Gy, and FLAG B' was immunoprecipitated and immunoblotted ( Fig ure 14 ). Maximal dissociation of endogenous CHK2 and B' reached a peak after exposure to 10 Gy IR. By examining the phosphorylation of CHK2 in the lysate input, it can be seen that S 19 phosphorylation increased with the increasing doses of IR. T 68 phosphorylation was maximal after 2 and 5 Gy, and then decreased with the increasing IR doses. However, we observed the phosphorylation of S 33/35 to be maximal after 10 Gy of IR. This corresponds to the IR dose that induces maximal dissociation between CHK2 and B'
64 In order to determine the precise timing of B' and CHK2 dissociation after IR 293T cells transiently expressing FLAG B' were irradiated with 20 Gy IR and collected every half an hour from 0.5 h to 2.5 hours following IR FLAG B' 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 ( Fig ure 15 ). Significantly, the dissociation at 0.5 h correlated with maximal phosphorylation of CHK2 at S 33/35, but did not correlate wit h phosphorylation of S 19 or T 68.
65 Figure 14 Dissociation of CHK2 and B' occurs in a dosedependent manner 293T cells were transiently transfected with FLAG B' 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 indic ated proteins.
66 Figure 15 Dissociation of CHK2 and B' is an early event after IR 293T cells were transiently transfected with FLAG B' and irradiated with 20 Gy and incubated for 0.5, 1.0, 1.5, 2.0, or 2.5 h. Whole cell lysates w ere immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins.
67 Re A ssociation of CHK2 and B' does N ot Correlate W ith 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' and CHK2 dissociate after IR, we investigated whether reassociation 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 sublethal dose of 6 Gy, the lowest dose after which we could detect a significant dissociation between CHK2 and B' and which would also allow for recovery. We also exposed the cells to 50 Gy, which is considered lethal. 293T cells expressing FLAG B' were collected 1 h, 4 h, 8 h, and 12 h aft er exposure to both doses of IR ( Fig ure 16 and Figure 17 ). Interestingly, the kinetics of dissociation after 50 Gy w ere similar to that after 6 Gy. CHK2 was dissociated from B' 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 S 19, S 33/35, and T 68 followed similar patterns. S 33/35 had maximal phosphorylation after 1 h, again correlating with the most dissociation between CHK2 and B' Th ere 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' after 4 12 h existed as a doublet, suggesting the existence of hyper and hypophosphorylated forms ( Fig ure 1 6 ). 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 S 139, also known as H2AX, which is a w ell characterized marker of DSB (148) Thus, higher levels of H2AX denote the presence of DSB When the breaks are repaired, H2AX levels return to levels found before DNA damage (in some cases it can no longer
68 be detected by Western blot or immunofluorescence). In our experiments, a fter 6 Gy IR, the maximal amo unt of H2AX appeared after 4 h, returning to pre damage levels by 12 h ( Fig ure 16 ). On the other hand, after 50 Gy IR, maximal levels of H2AX were detected after 1 h and did not decline throughout the remainder of the time points ( Fig ure 17). Since B 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 H2AX, the re association does not correlate with the resolution of the DSB In summary, dissociation of CHK2 and B' is induced in a dose dependent manner and is an early event after IR. CHK2 and B' re association after DNA damage does not require resolution of the DNA damage. Moreover, dissociation and re association correlate with phosphorylation of S 33/35 of CHK2.
69 Figure 16. CHK2 and B' are able to re associate hours after 6 Gy IR 293T cells were transiently transfected with FLAG B' and irradiated with 6 Gy and incubated for 1, 4, 8, or 12 h. Whole cell lysates were immunoprecipitated wit h the FLAG antibody and immunoblotted for the indicated proteins.
70 Figure 17. CHK2 and B' are able to re associate hours after 50 Gy IR 293T cells were transiently transfected with FLAG B' 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.
71 The A ctivity of ATM, but n ot PP2A, is Necessary for the D issociation of CHK2 and B'. We next investigated the importance of PP2A activity on the binding and dissociation of CHK2 and B' OA is a phosphatase inhibitor that, when used at 0.5 M blocks the PP2A catalytic subunit's activity without affecting PP1 (55) 293T cells transiently expressing FLAG B' were treated with OA prior to IR exposure. FLAG B' was immunoprecipitated using an antibody to the FLAG tag and immunoblot analysis was performed ( Fig ure 18 ). In the mock treated cells, CHK2 was able to bind to FLAG B' and this binding was unaffecte d by OA treatment alone. After IR, CHK2 and B' 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' to bind to the PP2A A and C subunits, indicating that it can s till form the holoenzyme complex. Therefore, PP2A activity is not required for B' binding to, or dissociation from, CHK2. As we have seen thus far, the most pronounced dissociation between CHK2 and B' occurs when CHK2 is phosphorylated on S 33/35, which are phosphorylated in an ATM dependent manner (16, 79, 124) ATM belongs to the PIKK family along with ATR and DNAPK. To determine whether the CHK2 B' dissociation is a PI KKdependent event as is the phosphory lation of CHK2 S 33/35, the PI KK 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' were incubated with wortmannin prior to exposure to IR. FLAG B' was immunoprecipitated using a FLAG antibody and immunoblot analysis was performed ( Fig ure 19 ). Pre incubation with wortmannin prevented the dissociation of CHK2 from FLAG B' after IR. This
72 treatment also inhibited the phosphorylation of S 19 and S 33/35, indicating that the phosphorylation of CHK2 is required for the dissociation of CHK2 and B'
73 Figure 18. PP2A phosphatase activity is not required for CHK2 binding to or dissociat ing from B' 293T cells were transiently transfected with FLAG B' treated with 0.5 M 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 indic ated proteins.
74 Figure 19. Inhibition of ATM and DNAPK kinase activity prevents the IR induced dissociation of B' and CHK2 293T cells were transiently transfected with FLAG B' treated with 20 M wortmannin for 1 h then irradiated w ith 10 Gy IR and incubated for 1 h. Whole cell lysates were immunoprecipitated with the FLAG antibody and immunoblotted for the indicated proteins.
75 Mutation of CHK2 Serines 33 and 35 Affects B inding to B' To further investigate the importance of the phosphorylation of CHK2 S 33/35 on the binding and dissociation between CHK2 and B' 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 (S3 3/35E). Wildtype CHK2 (CHK2 WT), S33/35A and S33/35E were co transfected along with FLAG B' into HCT116 CHK2/ cells, which were then mock irradiated or irradiated with 20 Gy. FLAG B' was immunoprecipitated and immunoblot analysis was performed ( Fig u re 20). In mock treated cells, we saw an increase in S33/35A binding to B' 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.
76 Figure 20. Mutation of CHK2 serines 33 and 35 affects binding to B' HCT116 CHK2/ cells were transfected with FLAG B' 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.
77 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 a nd 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 prot eins as well. Therefore, i t 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' 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 P hosphorylate B' in vitro Since CHK2 is a kinase that is activated after irradiation, we determined whether B' could be used as a substrate for CHK2 in an in vitro kinase assay. 293T cells were irradi ated to activate CHK2 which was then immunoprecipitated and incubated in a kinase reaction with GST B' ( Fig ure 21 ). Indeed, phosphorylated B' was detected, indicating that in an in vitro system, CHK2 can phosphorylate B'
78 Figure 21. CHK2 can phosphorylate B' in vitro Whole cell lysates from irradiated 293T cells were immunoprecipitated using an antibody to CHK2. CHK2 kinase activity towards GST B' was measured using an in vitro kinase reaction.
79 PP2A L ocali z ation is N ot I nfluenced by IR Since CHK2 was able to phosphorylate B' in vitro we next determined if this had an effect on PP2A localization. 293T cells transiently expressing FLAG B' were mock irradiated or irradiated with 20 Gy, separated into the cytopl asm ic nucleoplasm ic and chromatin fractions, and subjected t o 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 PP2 A A and C subunits were also found in the cytoplasm and nucleoplasm, whereas FLAG B' 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 localiza tion is not affected by DNA DSB caused by IR. CHK2 was also found to bind to other B' family members in addition to B' Therefore, using an antibody that recognizes many B' proteins, we also examined t he 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.
80 Figure 22. B' localization is not changed by IR 293T cells transiently expressing FLAG B' 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 c hromatin 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 Figure 23. Localization of endogenous PP2A protei ns 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 analysi s for the indicated proteins was performed. Talin was used as a marker for the cytoplasm, PARP for the nucleoplasm, and H2AX for the chromatin.
82 PP2A Activity is U naffected by IR and is N ot I nfluenced by CHK2 If CHK2 is indeed able to phosphorylate B' 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. Theref ore, 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 ( Fig ure 24). The levels of PP2A activity were not changed by exposure to IR. Next, the activity of the PP2A complexes specifically containing FLAG B' was determined. 293T cells transiently expressing FLAG B' were either exposed to IR or mock treated and immunopre cipitated using a FLAG antibody to measure only the activity of PP2A complexes containing B' Phosphatase activity was measured using an in vitro phosphatase assay and was unaffected by exposure to IR ( Fig ure 25 ). Since the assay measures activity again st 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' subunit. In order to determine whether CHK2 has an effect on PP2A act ivity, 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 ( Fig u re 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
83 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.
84 Figure 24. Total PP2A phosphatase activity does not change after IR 293T cel ls 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. T he activity of the mock sample was defined as 100%.
85 Figure 25. Phosphatase activity of PP2A containing B' does not change after IR 293T cells transiently transfected with FLAG B' 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' was defined as 100%.
86 Figure 26. Total PP 2A activity does not differ in HCT116 wild type and CHK2/ cells HCT116 wildtype 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. Phosphata se activity was determined and the mock sample from the HCT116 wildtype cells was defined as 100%.
87 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' as a substrate. We next wanted to determine the extent to which PP2A can affect CHK2 phosphorylation, activity and protein stability. Changes in CHK2 P hosphorylation 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 ( Fig ure 27 ). Pre tr eatment with OA increased the phosphorylation of CHK2 after IR, primarily at S 19, although there were also slight increases in the phosphorylation of S 33/35 and T 68, demonstrating that PP2A can dephosphorylate CHK2 at multiple sites. Since we have shown t hat mutation of S 33/35 can influence the binding between B' 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 wildtype (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 det ermined via immunoblotting ( Fig ure 28 ). In the mock treated samples, CHK2 WT and S33/35A showed a very faint phosphorylation of S 19. However, S33/35E displayed a much stronger phosphorylation, even in the absence
88 of DNA damage. After exposure to IR, CHK 2 WT was phosphorylated on S 19, with levels comparable to the mock treated S33/35E. The phosphorylation of S33/35A at S 19 was slightly compromised. T 68 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 S 19 phosphorylation, the same site that was affected most by PP2A inhibition.
89 Figure 27. Inhibition of PP2A activity increases CHK2 phosphorylation 293T cells were in cubated with 0.5 M 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.
90 Figure 28. Mutation of CHK2 serines 33 and 35 affects CHK2 phosph orylation 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 CHK 2 phosphorylation via immunoblot.
91 Changes in CHK2 K inase A ctivity after IR To directly measure the affect of PP2A on CHK2 activity, we transfected 293T cells with FLAG B' and measured CHK2 kinase activity using an in vitro kinase assay with GST Cdc25C (amino acid residues 200 256) as a substrate ( Fig ure 29 ). Cells were either mock treated or irradiated, CHK2 was immunoprecipitated, and the kinase activity was measured. In the mock treated samples, B' did not have any effect on CHK2 kinase activity. However, after irradiation, overexpression of B' prevented the full activation of CHK2. Since mutation of S 33/35 to glutamic acid positively affects CHK2 phosphorylation primarily on S 19, 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 myctagged 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 ( Fig ure 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 S 33/35 is important for CHK2 activity and that m imicking this phosphorylation can increase CHK2 activity even in the absence of DNA damage.
92 Figure 29. B' overexpression negatively affects CHK2 kinase activity. 293T cells expressing FLAG B' or FLAG (as a negativ e control) were irradiated with 20 Gy and incubated for 1 h. Whole cell lysates were immunoprecipitated using a CHK2 antibody and incubated in a n in vitro kinase assay using GST Cdc25C (amino acids 200256) 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.
93 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 mockirradiated or exposed t o 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 200256) 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.
94 Mutation of CHK2 Serines 33 and 35 Affects Protein Stability Another mode of CHK2 regulation is via its protein stabilit y. CHK2 that is expressed in HCT15 cells is phosphorylated at S 456 after irradiation, stabilizing the protein (75) Since we have shown that phosphorylation of S 33/ 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 ( Fi gure 31 A ). 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 treate d 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 S 33/35 in the increase of CHK2 stability after IR.
95 Figure 31 A Mutation of CHK2 serines 33 and 35 affects CHK2 protein stability. HCT15 cells w ere transiently transfected with CHK2 wild type (WT), S33/35A, or S33/35E in the pcDNA3 vector. 100 g/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.
96 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 bes t 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.
97 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 ac tivation (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 t hen bind to CHK2 hours later, providing a mechanism for CHK2 downregulation after DNA damage ( Figure 32 ) We have identified the B' subunit of PP2A as a CHK2interacting protein via a yeast two hybrid screen and have confirmed the interaction in mammalian cells. Full length CHK2 was found to bind to B' 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 aci d residues located at interve ning loops between the 18 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 d ifferent B' subunits. This large portion of B' 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'
98 B' B' and B' 3 as well and all of the interactions were dissociated upon exposure to irradiation. In mammalian cells, binding between B' and CHK2 is markedly decreased by exposure to IR. However, other types of damage caused by UV, HU, or MM C d id 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' can also bind CHK1, though the interaction is not disrupted by any of the DN A damage inducing agents tested. This demonstrates a unique aspect of the relationship between CHK2 and B' : 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' are able to re associate hours after IR, and this correlates with the dephosphorylation of S 33/35, 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 a nd B' correlates with maximal phosphorylation of CHK2 S 33/35, which are phosphorylated in an ATM dependent manner (16) When we inhibited ATM with wortmannin, the phosphorylation of S 33/35 was prevented as well a s the dissociation between CHK2 and B' The region in CHK2 that binds to B' is comprised of amino acids 1107, which contains the entire SQ/TQ domain. Serines 33 and 35 are located in this domain, suggesting that the phosphorylation of these sites coul d directly contribute to the dissociation of the two proteins. We therefore mutated CHK2 S 33/35 to alanine to prevent phosphorylation (S33/35A) or to glutamic acid to mimic phosphorylation (S33/35E) and tested the effects on binding to B' Our data indi cates that phosphorylation at S 33/35 is necessary for the
99 dissociation of B' 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' 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, S3 3/35E had a level of phosphorylation at S 19 that was comparable to CHK2 WT after IR. Pharmacologic PP2A inhibition with OA also increases phosphorylation at S 19. Alternatively, the S33/35A mutant had slightly less phosphorylation after IR compared to CHK 2 WT. T he 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' is overexpressed as well. Mutations at S 33/35 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 S 33/35 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
100 implicated a role for PP2A in regulating CHK2. It was demonstrated that CHK2 can phosphorylate B' in vitro increasing PP2A activity towards CHK2 in vitro (40) In an in vitro kinase assay, we found that CHK2 could phosphorylate B' 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 T 68, in agreement with our results (96) Another mode of CHK2 regulation in humans has been characterized. The Wip1 phosphatase has been shown to dephosphorylate T 68 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' 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 propaga tion 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 kina se 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 be tween 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
101 activation and stabilization of CHK2. To date, no mutations of S33/35 have been found to be associated with cancer, even though they would ne gatively affect CHK2 function. However, i t cannot be assumed that because no mutations have been found specifically at S33/35 that phosphorylation at these sites is not affected by mu tations located elsewhere in the protein. In fact, t he 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 mu tations for the affect they have on the ability of CHK2 to be phosphorylate d 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 m utations in the SQ/TQ domain may prevent the binding of PP2A to CHK2 under normal circumstances leading to an inappropriate activation of CHK2 that c ould 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 prev ent 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.
102 Figure 32. Model of PP2A regulation of CHK2 through binding and dissociation. C HK2 binds to PP2A through the B' 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.
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ABOUT THE AUTHOR Alyson K. Freeman (maiden name Alyson K. Fay) received a Bachelor of Science degree in Bio chemistry and Molecular Biology from the University of Massachusetts in 2002. Afterwards, s he 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 Biolog y Ph.D. program at the University of South Florida and conducted resea rch in the laboratory of Dr. Al var o N. A. Monteiro at the H. Lee Moffitt Cancer Center and Research Institute. Three years of her graduate research was funded through a predoctoral awar d 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.