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Bad Serine 170 - regulation and cellular effects Waissbluth, Ivan 2009-12-31

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BAD SER1NE 170- REGULATION AND CELLULAR EFFECTSbyIvan WaissbluthB.Sc. University of British Columbia, 2000A THESIS SUBMITTED IN PARTIAL FULFILLMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinThe Faculty of Graduate Studies(Experimental Medicine)THE UNIVERSITY OF BRITISH COLUMBIA(Vancouver)April 2009© Ivan Waissbluth 2009ABSTRACTThe balance between cell proliferation and cell death is imperative for homeostasis inmulticellular organisms. This homeostasis had long been thought to be the result oftwo separate processes, but there is recent evidence indicating that the processes ofproliferation and apoptosis are coupled. Here we demonstrate that Bad, a proapoptotic member of the Bcl-2 family of proteins that is thought to exert a death-promoting effect by heterodimerization with Bcl-xL, is able to interact directly withthe cell cycle machinery. Immunoprecipitation experiments indicate that Bad interactswith both Cdk2 and its late Gi cyclin partner, Cyclin E. This finding is relevant as wealso demonstrate that prior to the conversion of Bad into a death factor; Bad’sphosphorylation state in healthy mammalian cells, specifically at Ser- 170, is able toinfluence cell cycle progression. Here we show that transfection with Bad Si 70A, amutant form of Bad which mimics the unphosphorylated form, results in a prolongedS phase during cell cycle. We also show that the kinase activity towards Bad Ser- 170increases in S phase of the cell cycle. Together this suggests that Bad Ser-170 is aphosphorylation site which is targeted during S phase and is able to interfere with thenormal progression through S phase of the cell cycle. From this, it was of interest toelucidate the kinase responsible for phosphorylating Bad at Ser-170 as it may provideinsight into signal transduction pathways that converge in terms of controlling bothsurvival and cell proliferation, and ultimately cell expansion. Through a process ofcolumn purification, usage of chemical inhibitors, and gene knockdown, we showevidence that CaMKII-y mediates the phosphorylation of Bad Ser 170, thusestablishing a novel connection between CaMKII signaling and apoptosis inhematopoietic cells. We hypothesize that CaMKII-y plays a major role in controllingBad’s ability to induce apoptosis and to affect cell cycle progression, by controllingBad’s phosphorylation state at Ser-170.11TABLE OF CONTENTSAbstract iiTable of Contents iiiList of Tables viList of Figures viiList of Abbreviations ixAcknowledgements xiii1.0 CHAPTER 1-INTRODUCTION 11.1 APOPTOSIS 11.1.1 Apoptosis and the Bcl-2 Family 11.1.2 BcI-2 Family Members 21.1.3 Pro-Survival Bcl-2 Subfamily 41.1.4 Apoptotic Bcl-2 Subfamily 51.1.5 BH3-Only Subclass 61.1.6 Direct Activation Model 71.1.7 Indirect Activation Model 91.1.8 Embedding Together Model 111.2 BCL-2 FAMILY REGULATION 121.2.1 Regulatory mechanisms 121.2.2 BH3-only regulation 131.2.3 Bad regulation 141.3 CASPASES 161.3.1 Caspase activation 161.3.2 Apoptotic pathways leading to caspase activation 171.4 Ca27CALMODULIN-DEPENDENT PROTEIN KINASE II 211.4.1 CaMKII Expression 211.4.2 CaMKII Isoforms 211.4.3 CaMKII Holoenzymes 221.4.4 CaMKII Activation 231.4.5 CaMKII and Apoptosis 241.5 A LINK BETWEEN APOPTOSIS AND CELL CYCLE 251.5.1 Cell Cycle Summary 251111.5.2 Apoptotic and Proliferative SignallingPathways are Interconnected 271.5.3 Bcl-2 Family and Cell Cycle 281.5.4 Bad and Cell Cycle 311.6 BCL-2 FAMILY - A RATIONAL DRUG TARGET 321.6.1 Cancer Cells are Primed for Death 321.6.2 Targeting Bcl-2 331.6.3 Bad-DrugTemplate 331.6.4 Targeting the Apoptotic and ProliferativeSignaling Pathways 341.7 AIM OF STUDY 362.0 CHAPTER 2- METHODS AND MATERIALS 382.1 CELL LINES AND TISSUE CULTURE 382.2 REAGENTS 392.3 ANTIBODIES 392.4 PLASMIDS 412.4.1 Bacterial Transformation 412.5 PROTEIN ANALYSIS 412.5.1 Cell Treatment 412.5.2 Preparation of Total Cell Lysate 422.5.3 Preparation of Nuclear and Cytoplasmic Extracts 422.5.4 Co-Immunoprecipitation and Western Blotting 432.6 ASSAYS 432.6.1 Apoptosis Assay 432.6.2 Cell Cycle Assay 442.6.3 Cell Sorting Assay 442.7 RETROVIRAL INFECTION 442.8 COLUMN CHROMATOGRAPHY 452.9 KINASE ASSAY and siRNA 463.0 CHAPTER 3- A BAD CONNECTION BETWEENAPOPTOSIS AND CELL CYCLE 483.1 INTRODUCTION 48iv3.2 RESULTS.493.2.1 Phosphorylation at Serl7O Regulates Bad’sApoptotic Function 493.2.2 Phosphorylation of Bad Serl7O RegulatesNovel Cell Cycle Effect 523.2.3 Dephosphorylation at Bad Ser 170 StallsCell Cycle at S Phase 563.2.4 Bad Interacts with Cell Cycle Machinery 583.2.5 Bad’s Interaction with Cdk2 and Cyclin EOccurs in the Cytosol 613.2.6 Bad Interacts with Cdk2/CylinE in the cytosol 643.2.7 Bad’s Interactions and Cell Cycle Stage 683.2.8 Co-Immunoprecipitated Cdk2 shows kinase activityagainst Histone Hi protein 703.3 DISCUSSION 724.0 CHAPTER 4- CAMKII PHOSPHORYLATES BAD 774.1 INTRODUCTION 774.2 RESULTS 784.2.1 Identification of the Bad Serl7O Kinase 784.2.2 MonoQ Column Chromatography 794.2.3 Superdex S200 Column Chromatography 814.2.4 CaMKII Inhibitor Studies 834.2.5 kinase Activity Using Purified CaMKII Isoforms 864.2.6 siRNA Knockdown Experiment of CaMKII 884.3 DISCUSSION 905.0 CHAPTERS- BAD SER17O AND CELLULAR EFFECTS 925.1 INTRODUCTION 925.2 RESULTS 925.2.1 Cytokine Activation of CaMKII Activity 925.2.2 Inducing Apoptosis via Cytokine StarvationDoes Not Affect Bad/Cdk2 Interaction 975.2.3 Apoptosis Mediated by CaMKII Inhibitor isDependent Upon Bad S170 995.2.4 Kinase Activity Against Serl7O PeptideIncreases in S phase of Cell Cycle 1015.3 DISCUSSION 1046.0 CONCLUSION 107REFERENCES 113vLIST OF TABLESTable 2.1 Antibodies used.39viLIST OF FIGURESCHAPTER 1Figure 1.1 Pro-survival and pro-apoptotic Bcl-2 family members 3Figure 1.2 Direct and Indirect Activation Models 7Figure 1.3 Pro-apoptotic Bcl-2 family proteins neutralizespecific pro-survival proteins 9Figure 1.4 Schematic diagram of BH and transmembrane (TM)domains within Bcl-2 and Bad 14Figure 1.5 Apoptotic pathways overview 20Figure 1.6 CDKlCyclin Partners and the RegulatorsCell Cycle Progression 26Figure 1.7 Bad Ser 170 — regulation and cellular effects 35CHAPTER 3Figure 3.1.Figure 3.2Figure 3.3Figure 3.4Figure 3.5Figure 3.6Figure 3.7Figure 3.8Figure 3.9Figure 3.10Figure 3.11Figure 3.12Figure 3.13Stable infection of Bad S170 mutants 50S170 modulates Bad’s apoptotic function 51S170 Modulates Bad’s apoptotic function 53Mutation mimicking phosphorylation at Seri7O inhibitsBad’s apoptotic function 55Cells expressing Bad S17OA have a longer doubling time.. 57Bad S17OA Expression Stalls Cells in S Phase 59Increased levels of Bad S17OA or Bad S17OD results inincreased cell cycle effect 60Bad Co-Immunoprecipitates with Cdk2 and CyclinE 62Cdk2 and CyclinE Co-Immunoprecipitate with Bad 63Bad interacts with Cdk2/CyclinE in the cytosol 65Bcl-xL expression interferes with Bad/Cdk2 interaction 67Bad associates with cyclinE during GuS phase 69Bad’s Co-Immunoprecipitated complex shows kinaseactivity against histone Hi protein 71CHAPTER 4Figure 4.1 Mono-Q column fractionation and kinase Assay 80vuFigure 4.2 Superdex S200 fraction and kinase Assay 82Figure 4.3 Column activity and KN9383Figure 4.4 Superdex column activity and KN93 84Figure 4.5 Kinase activity of immunoprecipitated CaMKII 85Figure 4.6 Activity of purified CaMKIJ isoforms 87Figure 4.7 Superdex S200 fraction and kinase assay of 3T6 lysates 88Figure 4.8 CaMKII-y-Directed siRNA decreases kinase activityagainst Bad S170 89CHAPTER 5Figure 5.1 IL-3-stimulated activity against Bad S170 peptide 95Figure 5.2 Inducing apoptosis via IL-3 starvation does notappear to effect Bad’s ability to interact with Cdk2 98Figure 5.3 Induction of apoptosis by the CaMKII inhibitor, KN93.. . .100Figure 5.4 Cells synchronized in S phase show relative increase inCaMKII kinase activity 102Figure 5.5 CaMKII kinase activity Increase in S phase 103viiiLIST OF ABBREVIATIONa anti-I- knockoutAb antibodyADP adenosine diphosphateAIF apoptosis inducing factorAPAF-1 apoptosis protease activating factor 1Ask-i apoptosis signal regulating kinase-iATM ataxia telangiectasia mutatedATP adenosine triphosphateATR ataxia telangiectasia relatedBad Bcl-2 antagonist of cell death proteinBcl-2 B cell lymphomal leukemia-2BR Bcl-2 homologyBim Bcl-2-interacting mediator of cell deathBok Bcl-2 associated ovarian killerBrdU bromodeoxyuridineCaspase cysteine aspartate specific proteaseC. elegans Caenorhabditis elegansCAD caspase activated DNAaseCARD caspase recruitment domainCdk cyclin dependent kinaseCED cell death protein (C. elegans)Chkl/ Chk2 checkpoint kinase TI checkpoint kinaseIIC-terminal carboxy terminalDa daltonDD death domainDED death effector domainDIABLO direct lAP binding protein with Low pHDISC death-inducing signalling complexixDLC dynein light chainDNA deoxyribonucleic acidE. Coli Escherichia coilelF eukaryotic Initiation FactorELISA enzyme-linked immunosorbent assayER endoplasmic reticulumFACS fluorescence activated cell sorterFADD fas associated death domainFasL Fas ligandGFP green fluorescent proteinGMCSF granulocyte macrophage colony stimulatingfactorsGSK-3 glycogen synthase kinase-3h hourTAPs inhibitor of apoptosis proteinsICAD inhibitor of caspase activated DNAase1L3 interleukine-3IP immunoprecipitationMAC mitochondrial apoptosis-inducing channelMAPK-AP mitogen-activated protein kinase-activatedproteinMdl S short Mci-iMEK map kinase or erk kinaseMEKK1 MEK kinase 1Mito mitochondriaMMP mitochondrial membrane permeabilizationMOT multiplicity of infectionMOM mitochondrial outer membraneMPT mitochondrial permeability transitionMs mousemTOR mammalian target of RapamycinNF-AT nuclear factor of activated T-ceilsxNK cells natural killer cellsN-terminus amino-terminusP phosphoP.1. propidium iodidePARP poly ADP ribose polymerasePBS phosphate buffer salinePCNA proliferating cell nuclear antigenPCR polymerase chain reactionPDK-l phosphoinositide dependent kinase -lPDK-2 phosphoinositide dependent kinase -2PEST proline, glutamic acid, serine, threoninPH pleckstrin homologyP1 phosphatidylinositolP1-3 kinase phosphatidylinositol 3 -kinasePIKKs P1-3 kinase related kinasesPIP phosphatidylinositol phosphatePKB protein kinase BPKC protein kinase CPSR phosphatidylserine receptorPtdlns phosphoinositidePtdlns3,45P3 phosphatidylinositol 3 ,4,5-triphosphatePtdlns4,5P2 phosphatidylinositol 4,5-bisphosphatePTEN phosphatase and tensin homolog detected onchromosome tenPTP permeability transition porePUMA p53 up-regulated mediator of apoptosisRb rabbitrpm revolution per minuteSI T serine! threonineSDSIPAGE sodium dodecyl sulphate! polyacrylamide gelelectrophoresisSer serinexisFasL soluble fas ligandSH2 src homology-2SiRNA small interfering RNAsSMAC second mitochondria-derived activator ofcaspaseSTAT signal transducer and activator of transcriptiont(14:18) translocation (14:18)tBid truncated BidTCLs total cell lysatesThr threonineTNF tumor-necrosis factorTNF-R tumor necrosis factor receptorUV ultra violetVDAC voltage dependent anion channelWB western blotxiiACKNOWLEDGEMENTSI am deeply indebted to my supervisor Professor Dr. Vincent Duronio fromthe University of British Columbia whose kindness, patience and encouragementhelped me through the challenges of biological research and the writing of this thesis.I am very fortunate to have had the pleasure of working alongside the manyintelligent and kind laboratory colleagues at the Jack Bell Research Centre. Thewealth of support in the form ingenuity and deep knowledge of the field, fromResearch Associate Dr. Sarwat Jamil, played a major role in guiding my research andultimately completing this thesis. I want to also thank my dear friend and colleague,Dr. Sherry Wang, for all her patience and being the wonderful person that she is. Iwant to also express my sincere pleasure of working with Joseph Anthony, one of themost thoughtful and diligent people I know. A good portion of this thesis was onlypossible because of the contributing effort of Payman Hojabrpour. Payman was anexcellent person to work with. Beyond his expertise in a laboratory setting, Paymantaught me many things from negotiating prices with sales reps to maintaining ahealthy balance of work and family. I would also like to thank my wonderful and verybright girlfriend, Sarah Crome, who since we met has exemplified the spirit of hardwork and dedication. Her kind words and unwavering support propelled me tocomplete this degree.Especially, I would like to give my special and loving thank you to myMother and Father who gave me the courage to undertake this degree.xliiChapter 1INTRODUCTION1.1 APOPTOSIS1.1.1 Apoptosis and the Bcl-2 FamilyBy the 1960’s, several laboratories demonstrated that cell death was biologicallycontrolled (programmed), requiring the cell’s own proteins (1) and exhibited commonmorphological changes, including plasma membrane blebbing, cytoplasmicreorganization, chromatin condensation and DNA fragmentation (2). Thesemorphological changes and their underlying molecular mechanism are now commonlyreferred to as apoptosis, a term coined in 1972 by Kerr, Wyllie, and Currie (3).An early breakthrough in the study of apoptosis came with the observation that celldeath is usually accompanied by rapid activation of endonucleases (4) and subsequently,electrophoresis of cleaved DNA “fragments” (5) were specifically associated withapoptosis (6), though only until recently was the endonuclease responsible (DFF/CAD)identified (7, 8). The observation that phosphatidylserine, normally found on the innerside of the cell’s membrane, is exposed on the outer side of the cell membrance on dyingcells (9) provided not only another apoptosis marker, but also led to our currentunderstanding of how dead cells are recognized prior to their engulfment.The first evidence of a genetic program dedicated to physiological cell death camefrom developmental studies in C. elegans (10, 11) and the generation of the first ‘ced’(cell death abnormal) mutants in 1983 (12). Nobel-prize winning studies of Horvitz andcolleagues (11) had determined that the demise of the 131 somatic cells fated to dieduring worm development required two genes, CED-3 and CED-4, whereas another,CED-9, ensured the survival of all others (13, 14). However, the ‘modem’ era of celldeath research and the explosion of interest in the field resulted from the identification ofthe first component of the mammalian cell death machinery, the gene Bc12 (15), and theconvergence of two disparate fields brought about by studies showing that Bcl-2 was ableto prevent programmed cell death in C. elegans (16) and that CED-9 was Bcl-2’s1structural and functional counterpart (17).These landmark discoveries demonstratedthatthe programmed cell death observed inmammalian cells and in the nematodewas thesame highly conserved process (16).Though Bcl-2 was the first componentof the apoptotic system to be recognized,ithad been originally cloned notbecause it was an apoptotic gene,but because it was foundto be translocated in follicular lymphoma(B-cell CLL/Iymphoma, (18)).Investigatorsfirst thought that Bcl-2 may belike other oncogenes involved intranslocations, such asAbi and c-Myc which promote cellproliferation; however Bcl-2 wasnot shown tostimulate cell division, but rather preventedcell death when growth factor was removed(15). Several studies ensued providingearly evidence that one functionof p53 (19), thegene most commonly mutated in humancancers (20), is to cause apoptosiswhich can beblocked by Bcl-2 (21). Earlyexperiments using transgenic miceover-expressing Bcl-2also provided the first evidenceconnecting inhibition of cell deathwith autoimmunedisease (22). These studies, amongmany others, gave birth to the concept,now widelyembraced (23-26), that impaired apoptosisis a crucial step in tumorigenesisanddisregulated immune responses.1.1.2 Bcl-2 Family MembersOver the past decade it has becomeevident that Bcl-2 belongs toan extendedfamily of at least twenty five Bcl-2-relatedproteins in mammalian cells(27-30). The Bcl2 family of proteins function to control the“life/death switch” by integratingdiverse interand intracellular cues to determine whetheror not the signal shouldreach themitochondrion. Members ofthe Bcl-2 family are characterizedby the presence of distinctconserved sequence motifs knownas Bcl-2 homology (BH) domainsdesignated Bill,BH2, BH3 and BH4 (Fig. 1.1). The Bcl-2 family can be divided intotwo broad classes:those that inhibit apoptosis and those thatpromote apoptosis.2Multidomain—..— B...—LfFigure. 1.1 Pro-Survival and Pro-Apoptotic Bcl-2 family members. Bcl-2 familymembers share regions of homology termed BH domains (BH1, BH2,BH3, and BH4). Several family members also contain a domain (TM) thatmediates insertion into the outer membrane of the mitochondrion and/orendoplasmic reticulum. Pro-Survival family members contain Bill -4domains. Pro-Apoptotic family members are subdivided into multi Bildomain (Multidomain) or BH3 -only domain proteins. Adapted from (31).*The existence of a BH4 domain within Mci-i still remains controversial.BH4 BH3 Bill BH2 TM-Pro-SurvivalProApoptotic. •Wf1—Bc12BdxLMd-iAlBaxBakBokBadBidBirnNoxaPumaBH3-only-.3The pro-survival class has been divided into two subclasses based on the presenceof one or more BH domains; Bcl-2 and its closest relatives, Bcl-xL and Bcl-w, and themore divergent group consisting of Al and McI- 1.The pro-apoptotic class, mainly identified as Bcl-2-binding proteins, promote ratherthan antagonize apoptosis and fall into two distinct groups: Bax subclass and BH3-onlysubclass. The Bax subclass members (Bax, Bak, Bok) have sequences that are similar tothose in Bcl-2, especially in the BH1, BH2 and BH3 regions. The BH3-only subclass,exemplified by Bik, Bim and Bad, are largely unrelated in sequence to either Bcl-2 oreach other (32, 33), apart from the short BH3 motif (hence their name). These disparate‘BH3-only proteins’ cannot induce apoptosis in the absence of Bax and Bak (34, 35) andappear to function upstream, sensing death signals and intracellular damage; whereas theBax-like proteins act further downstream, probably in mitochondrial disruption (seebelow).1.1.3 Pro-Survival Bcl-2 SubfamilyThe abundance and type of Bcl-2 pro-survival members is thought to dictatewhether the cell will live when stimulated by physiological, pathogenic or cytotoxicstimuli. Bcl-2 and its closest homologues, Bcl-xL and Bcl-w, possess four BH regionsand a hydrophobic carboxy-terminal domain. The C-terminal hydrophobic sequencetargets or anchors these proteins to the cytoplasmic face of intracellular membranesincluding: the outer mitochondrial membrane, the endoplasmic reticulum (ER) and thenuclear envelope. The crucial interactions between pro-survival and pro-death Bcl-2family members appears to occur on these membranes and most members either normallyreside on these surfaces, or rapidly assemble there after an apoptotic signal (36). Thethree-dimensional structure of Bcl-xL (37), Bcl-2 (38) and Bcl-w (37) are remarkablysimilar and comprise a globular bundle of five amphipathic OL-helices that surround twocentral hydrophobic cL-helices. The resulting hydrophobic groove, formed by residuesfrom BR 1-3, can bind the BH3 (-24 residue x-helix) of an interacting BH3-only relative4(39); and this interaction interferes with the pro-survival protein’s ability to prevent Baxor Bak from perturbing the integrity of intracellular membranes.1.1.4 Apoptotic Bcl-2 subfamilyIt is widely agreed upon that BAX and BAK are critical components of the cellularapoptotic machinery (40-47). In response to cytotoxic signals, Bax translocates from itscytoplasmic location to intracellular membranes, and both Bax and Bak changeconformation and form membrane-associated homo-oligomers. The three-dimensionalstructure of monomeric Bax (48) closely resembles that of its pro-survival relatives. Baxhas a BH1/2/3 hydrophobic groove which is occluded by its hydrophobic carboxyterminal helix. In response to stress signals, Bax changes conformation and flips out itshydrophobic tail. Presumably the changes result in exposure of domains normally maskedin the inactivated proteins (48), targeting the protein to the mitochondrial membrane (49)and enabling homo-oligomerization, which is the active form of the proteins. Unlike Bax,Bak is an integral mitochondrial membrane protein, though it too changes conformationduring apoptosis (45, 47, 50-52). As the Bak BH3 domain is critical for Bak-mediatedapoptosis (53), it has been suggested that this motif not only allows restraint of Bak by itspro-survival counterparts but also for its homo-oligomerization, perhaps by allowing aBH3-exposed conformer (‘prime& Bak) to dimerize with an ‘unprimed’ receptor-like Bakconformer (53). Knockout studies suggest that both Bax and Bak are functionally similaras the loss of either gene has little effect in most cells and tissues. The absence of bothproteins, however, blocks apoptosis in many cell types (47, 54) and impairsdevelopmentally programmed attrition in several tissues, and results in perinatal death(55).Bax and Bak oligomers are considered pro-apoptotic since they are thought toprovoke or contribute to the permeabilization of the outer mitochondrial membrane,resulting in the release of apoptogenic proteins (28). The mechanism, however, remainscontroversial (56-5 8). One model, which is based on the structural resemblance of Bcl-2family members and diptheria toxin (37), is that Bax and Bak form channels. Consistent5with this hypothesis is the fact that Bax oligomers can form pores in liposomes (59) thatallow passage of cytochrome c (60, 61), and it has also been shown that mitochondriafrom apoptotic cells contain a novel channel (62). Alternatively, Bax and Bak have beenshown to influence the shape of the mitochondria possibly by affecting the fissionlfusionapparatus (63) and it has also been suggested that Bax and Bak interact with the existingpermeability transition pore to possibly create a larger channel (57, 58); however, severalstudies have found no evidence for such interactions (50, 56, 64).1.1.5 BH3-only SubclassBH3-only proteins, which include Bim, Bad, Bid, Bik, Bmf, Puma, Noxa and Hrk,act as sensors for distinct apoptotic pathways and are able to trigger apoptosis in responseto developmental cues or intracellular damage (32). The primary structure of BH3-onlyproteins share no domains homologous with each other and with other members of theBcl-2 family apart from the BH3 domain. Many studies have demonstrated that theassociation with pro-survival proteins is crucial for their pro-apoptotic effects and theseinteractions require a BH3 domain (65-73); however, how BH3-only proteins triggerapoptosis is still controversial. Two models, direct and indirect, have been put forwardthat attempt to account for the various experimental results. Both models conclude thatsome BH3 -only proteins are more effective killers than others but they base this ondifferent observations and reasoning. Another layer of complexity has been added bydistinguishing proteins or their states as being cytoplasmic or membrane bound. Mostnotably, Leber et al., have proposed the Embedding Together model which proposes thatboth pro- and anti-apoptotic Bcl-2 family proteins undergo similar protein-proteininteractions that are governed by membrane dependent conformational changes. Theseconformational changes are thought to culminate into either an aborted or executedpermeabilization of the membrane depending on the final oligomeric state of proapoptotic Bax and/or Bak (36).61.1.6 Direct Activation ModelThe direct activation model suggests that certain BH3-only proteins, termed‘activators’ namely Bim and the truncated form of Bid (tBid), can bind to Bax and Bakdirectly and promote their activation (42, 45, 74-77). Further studies have shown that ifBak (or Bax) is present, Bid is able to very rapidly trigger cytochrome c release andapoptosis (47, 78). Moreover, it has been suggested that tBid might act by inducing Baxand Bak to oligomerize and nucleate channel formation in the mitochondrial membrane(79). This hypothesis stems from Bid’s resemblance to the pore-forming subunit of somebacterial toxins (80). Bim and tBid can be bound and neutralized by Bcl-2 pro-survivalproteins and the role of other BH3 -only proteins, termed “sensitizers” in this model, is tobind to these pro-survival proteins and stop them from sequestering BimltBid. The“sensitizers” (eg. Bad, Bik, Noxa, Bmf) (42) exert their proapoptotic functions indirectlyby competing for the BH3 domain-binding cleft in anti-apoptotic proteins, displacing orpreventing the binding of activators (42, 45, 75, 81). Thus in this model “sensitizers” areconsidered inhibitors of the inhibitors of apoptosis and are different from direct“activators” (eg. Bid and Bim) (Figure 1.2).Direct Activation Model Indirect Activation ModelSensitizer Activator Selective PromiscuousBadBimBadBimtBidtBidBc12Bcl-2 Mci-iBclxLBclxL AlMci-iBaxBaxAl BakBakFigure 1.2 Direct and Indirect Activation Models. Adapted from (82).7The direct activation model was devised based on experiments in a cell-free systemusing synthetic BH3 peptides derived from representative BH3-only molecules. TheseBH3 peptides were applied to isolated mitochondria, and their ability to inducemitochondrial outer membrane permeabilization (MOMP) were investigated (45, 75). Itwas found that the BH3-peptides derived from Bim and Bid could induce cytochrome crelease in the presence of Bax (45); while BH3 peptides, including those of Bad, Bik andNoxa, cannot directly activate Bax and Bak nor induce MOMP by themselves (45). Thus,although direct binding of the proteins were not observed in these particular experiments,the detectable collaboration implies direct activation, presumably by binding. Howeverensuing studies have indicated that both Bid and Puma BH3-peptides are able to bind tothe N-terminus of Bax (41, 83); these results, however, should be interpreted carefully asit is uncertain whether whole BH3 -only proteins could reproduce this binding.Furthermore, systems used in many of these experiments are in some points removedfrom the physiological conditions and the induction of mitochondrial outer membranepermeabilization (MOMP) on isolated mitochondria by Bid BH3 peptides, for example,requires a 1,000-fold higher concentration of BH3 molecule than that required in theinduction of MOMP by the full-length activated Bid protein (45). It is also difficult toreconcile the direct binding model with recent data using gene-deficient mice. ActivatedT-cells die rapidly in culture; however this cell death is reduced in Bim-deficient T cells,slightly reduced in Puma-deficient T cells and almost completely prevented in BimlPumadouble-deficient T cells (84). These findings demonstrate that Bid is not sufficient for Tcell apoptosis (otherwise there would still be cell death in Bim/Puma-deficient T cells). Itfurther shows that Puma is able to induce apoptosis similarly to Bim, as the difference inapoptosis between Bim-deficient and Bim/Puma-deficient T cells is clearly due to Puma(77). Therefore, the model needs to be modified to the extent that Puma can act like adirect activator, although the experiments with BH3 -peptides did not suggest that (41,83).81.1.7 Indirect Activation ModelThe indirect activation model (also termed the displacement model) on the otherhand suggests that all the BH3-only proteins, through their BH3 domain(85), engageonly their pro-survival relatives and that the pro-survival proteins function mainlybyinhibiting Bax/Bak activation (53, 77, 86). In this model Bim, tBid and PUMA,forexample, are considered to be strong apoptotic inducers because they can engage allthepro-survival proteins (41, 42, 53, 77, 87, 88) while other BH3-only proteinsexhibitmarked selectivity and as result show weaker apoptotic properties. This model isbasedon the observation that BH3-peptides derived from the various BH3-only proteinshavevastly different affinities for Bcl-2-like proteins (42, 75, 82). Studies haverevealed, forinstance, Bad and Bmf bind only Bcl-2, Bcl-xL and Bcl-w, whereasNoxa binds onlyMcli and Al (82). Experiments stemming from these findings have suggestedthat thecomplementary binding profiles of Bad and Noxa represent an apoptosis requirement.More precisely, both classes of Bcl-2 pro-survival proteins, one comprising Bcl-2, Bcl-xLand Bcl-w and the other Mci-i and A- 1, are required to be neutralized in order to inducea strong apoptotic effect (Figure 1.3). This explains in part how Bad and Noxa, knowntobe weak killers, potently induce apoptosis when combined (82).CBirn)Bad__)(PumaDHNoxaFigure 1.3 Pro-apoptotic Bcl-2 family proteins neutralize specificpro-survivalproteins. Adapted from (82)9This model suggests that induction of apoptosis requires the activation of acombination of BH3-oniy proteins that cover and inactivate all Bcl-2 pro-survivalproteins in the cell. Therefore, based on a system where all Bcl-2 pro-survival proteinsare expressed, activation of Bim or Puma would be sufficient to induce apoptosis whereasBad on its own would require an additional stimulus that could be provided by Noxa.This model also suggests that Mci-i is probably unable to block Bad (as it does not bindwith high affinity), and Bcl-2 cannot inhibit Noxa or Bid efficiently. Functional studiessupporting this model have been reported recently (53): first, Bak was found by coimmunoprecipitation experiments to be bound to Bcl-xL and Mci-i but not other Bcl-2-like proteins and, secondly, neutralization of both Bcl-xL and Mci-i was required toactivate Bak and to induce apoptosis (77). Worth noting is that these experiments usedcells expressing rather high levels of BH3-oniy proteins, and it is still unclear whyneutralization of either Mci-i or Bci-xL on its own does not release sufficient Bak tohomo-dimerize and induce apoptosis. Additionally, co-expression of Noxa and Bad isable to induce apoptosis in cells lacking both Bim and tBid (77); which at the same timechallenges the direct activation model as tBid and Bim are described as direct activatorsof Bax and Bak and are required for the induction of apoptosis. Further support for theindirect activation model comes from studies (86, 89, 90) using BH3 mimetic drug,ABT-737 (9i). Despite its high affinity for Bcl-2, Bcl-x(L), and Bcl-w, many cell typesproved refractory to ABT-737 and this resistance is due to ABT-737’s inability to targetpro-survival relative, Mci-i (92). Downreguiation of Md-i resulted in an increase inapoptosis when treated with ABT-737, where on the other hand, increased Mci-iexpression conferred resistance (93). Interestingly cells overexpressing Bcl-2 remainedhighly sensitive to ABT-737 (92). Hence, ABT-737 may prove efficacious in tumors withlow Mel-i levels, or when combined with agents that inactivate Mci-i.The indirect activation model isn’t without its caveats. For instance, this particularmodel has difficulty explaining how cells tolerate relatively high levels of Bax and/orBak espression without these proteins being constitutively bound to anti-apoptoticmembers (34, 94-97), as several studies have indicated the isolation of endogenous Baxand/or Bak does not generally stoichiometrically co-purify with anti-apoptotic proteins10(94). This suggests that further changes in the effector molecules are required for theirinteraction or permeabilization capability. Moreover, findings presented in this document,combined with other published reports, show that Bcl-2 family proteins bind to non-Bcl-2family members. Some of these interactions have also been shown to be disrupted byonly a specific BH3 -only protein. For example, Puma has been shown to disrupt cytosolicp53 and Bcl-xL complex (98).1.1.8 Embedding Together ModelSeveral lines of evidence suggest that the interaction between BH3-only proteinsand anti-apoptotic members is dependent on a lipid environment. These observationshave lead to an emerging model termed Embedding Together (36) that combinesattributes of both the indirect- and direct- activation models, in addition to providing acellular context by also taking into account how the presence or absence of lipidmembranes affect the binding of Bcl-2 members. Most notably, this model proposes thattBid changes conformation when bound to membranes and as result increases its affinityfor Bax. The interaction between membrane bound tBid with peripherally-membrane-bound Bax triggers insertion of Bax into the membrane. Once integrated into themembrane, Bax can recruit additional Bax proteins and oligomerize to permeabilize theouter mitochondrial membrane. The model does not require that any one pro-apoptoticprotein performs all of the functions ascribed to tBid, but rather suggests that most Bcl-2family members will perform only a subset of them. Bak is proposed to have a similarfunction to Bax except that its insertion in the membrane will be controlled by differentmodulators (e.g. tBid, Bim) with different affinities. Since Bak’s inactive conformation isconstitutively membrane bound it is suggested that it will have a similar functional role ascompared to the peripheral membrane form of Bax. Bcl-xL pio-survival role is thought toarise from its ability to inhibit both tBid and Bax. Although it may bind to both themembrane bound and cytoplasmic forms of tBid, the affinity of Bcl-xL for membranebound tBid is higher. Binding of Bcl-xL to tBid triggers insertion of Bcl-xL intomembranes and once inserted recruits other Bcl-xL proteins to insert into membranes11and/or tBid dissociates. Bcl-xL/tBid complexes are thought to be neutral complexes thatneither prevent nor promote apoptosis. However, because the affinity of Bcl-xL for tBidis higher than that between Bax and tBid, interactions between tBid and Bcl-xL inhibitsBax recruitment to the membrane. Membrane bound Bcl-xL further prevents Baxinserting into the membrane by preventing the conformation change in Bax that occurs atthe membrane. This model suggests that other pro-survival Bcl-2 family membersfunction in a similar fashion by binding different pro-apoptotic proteins (e.g. tBid/Bax)with different affinities.1.2 BCL-2 FAMILY REGULATION1.2.1 Regulatory MechanismsThe expression and activity of pro-survival and pro-apoptotic Bcl-2 family proteinsinfluence whether or not cells undergo apoptosis, and thus can have a direct affect ontissue homeostasis. Overexpression of Bcl-2, for instance, provokes an abnormalaccumulation of non-cycling cells (22, 99, 100). On the other hand, the tissuedegeneration brought about by inadequate levels of these proteins can be prevented by areduction in their BH3-only antagonist (101, 102). The levels and activity of pro-survivalproteins are regulated by diverse mechanisms, including: transcriptional control, post-translational modification, and turnover. The levels of Bcl-xL, Al, Md-i, and Bcl-2 forexample, are closely coupled to the supply of cytokines, which affects both theirproduction and stability. Bcl-2 levels may also be controlled, in part, by micro-RNAs(103) and its activity is modulated in complex ways by phosphorylation (104). The Mel-iprotein is particularly labile and is rapidly lost by proteasomal degradation early inresponse to several cytotoxic signals (105, 106). Although Bax levels have been reportedto change during apoptosis (107, 108), in most, if not all cells the multi-domain proteinsare present as inactive forms and the activity of the proteins appears to be regulatedmainly at the post-translational level. This may explain why the levels of Bax and Bak donot seem to have great significance and the loss of single alleles of either, or even three oftheir (combined) four alleles, has little physiological impact (55). In contrast, levels of12BH3-only proteins can have profound effects on tissue homeostasis, exemplified by theloss of a single Bim allele which prevented renal failure provoked by loss of Bc12 geneand restored lymphocyte numbers (109).1.2.2 BH3-only RegulationRecent studies have shed light on the mechanisms by which BH3-only proteins arecontrolled. Depending on the stimulus, such as death signals or survival factors, they willbe activated or deactivated, respectively. To avoid unwanted cell death, BH3-onlyproteins are restrained by a wide range of mechanisms at the mRNA as well as at theprotein level, including post-translational modifications and sequestration in cellularstructures (33, 92, 110). The multiplicity of mammalian BH3-only proteins and theircomplex regulation is thought to allow more precise control over apoptosis (33, 92). Inhealthy cells, BH3 -only proteins remain either in an inactive state or are expressed at lowlevels, and modification or the induction of expression is usually required for theirapoptotic action (32, 33). In some cell types BH3-only proteins are expressed atsufficiently high levels to induce apoptosis (40, 111, 112); however, they remain inactiveand in many cases are sequestered away from the mitochondria, where they can interactwith multidomain members and exert their pro-apoptotic effects. For instance, Bad, Bimand Bmf require post-translational activating-modifications (33) to translocate to themitochondria and initiate apoptosis.On the other hand, both Puma and Noxa, which are transcriptionally induced by p53(68-70) in response to DNA damage, do not appear to be under post-translational controland are thought to translocate to the mitochondria unconditionally (33). Bim is anexample of a BH3-only protein which is subject to several types of post-translationalregulation. Bim’s two predominant alleles, which are regulated downstream of the Aktsignal pathway, are under transcriptional control of FOXO3a and Forkhead transcriptionfactors (113). The BimEL and BimL proteins are sequestered in the cytoplasm by theirassociation with dynein light chain (DLC) 1, a component of the microtubular dyneinmotor complex (114). Phosphorylation by c-Jun N-terminal kinase (iNK) enhances13Bim’s pro-apoptotic activity (115) by allowing it to detach from the cytoskeleton.Beyond localization control, Bim protein levels are regulated by phosphorylation by Erk,which triggers its degradation by the proteasome (116-118). Regulation of Bid reveals yetanother unique set of cellular controls. The engagement of cell surface death receptorsactivates caspase-8 (119, 120) which in turn cleaves the amino-terminal region of Bid.This gives rise to two fragments (p7/p 15) that remain non-covalently bound to each other.The complex is then myristoylated onp15(121). This process is thought to expose Bid’sburied BH3 domain (79, 80) and target tBid to the mitochondria. Furthermore, CaseinKinases I and II can phosphorylate Bid and inhibit caspase-8-mediated cleavage of Bid(121). Apart from the post-translational regulations mentioned, Bid is transcriptionallyinduced by p53 (122), indicating the contribution of Bid to apoptotic pathways other thandeath-receptor-mediated pathways.1.2.3 Bad RegulationBad, one of the highly regulated BH3-only proteins, is found constitutivelyexpressed at varying levels in all mammalian cells (123, 124). In healthy cells, Bad isgenerally maintained in a hyperphosphorylated state by several kinase pathways and issequestered in the cytosol by binding with 14-3-3 scaffold proteins (125-129). Deathsignals that result in dephosphorylation of Bad convert the Bad protein into a survivalantagonist, whereby Bad selectively binds to and neutralizes anti-apoptotic molecules,particularly Bcl-xL (65, 96, 130); thereby, permitting the oligomerization and activationof pro-apoptotic molecules, Bax and Bak (123). Bad’s pro-apoptotic function has beenshown to be inhibited by phosphorylation (96, 119). Through its multiplephosphorylation sites (see figure 1.1), Bad is able to reflect the availability of survivalfactors on the cell surface and hence act as a convergence point for numerous pathways.The function of the various phosphorylation sites, independent of their position, is torender Bad unable to induce apoptosis (112, 13 1-133). A total of six phosphorylationsites have been identified on BAD; Ser”2(134), Ser’36 (134), Ser’28 (77), Ser’55 (135,136), Ser’7°(137) and Thr20’(138).141 10 30 90 104 133 152 184 199 236BcI-2_____ _____I IBH4 BH3 BHI BH2 TM1 141 159 204mBad____-BH31inBad MGTPKQPSLA PAHALGLPKS DPGIRSLGSD AGGRRWRPAA QS43rnBad MFQIPEFEPS EQEDAS-ATD RGLGPSLTE DQPGPYL APGLLGSNIHhBad MFQIPEFEPS EQEDSSSA-E RGLGPSPAG DGPSGSGKHHRQ APGLLWDASH188112 136mBad QQGRAATNSH HGGAGANETR SRHSSYPAGT EEDEGNEEEL SPFRGRSRSAhBad QQEQPTSSSH IIGGAGAVEIR SRESSYPAGT EDDEGMGEEP SPFRGRSRSA51138 155 170mBad PPNLWAAQRY GRELRRMSDE FEGSF-KGLP RPKSAGTATQ NRQSAGWTRIhBad PPNLWAAORY GRELRRNSDE FVDSFKKGLP RPKSAGTATQ RQSSSWTRV101187mBad IQSWWDRNLG KGGSTPSQhBad FQSWWDRNLG RGSSAPSQ151Figure 1.4 Schematic diagram of BR and transmembrane (TM) domains withinBcl-2 and Bad. The sequence of murine and human Bad are aligned toshow conserved phosphorylation sites (red Si 12, S136, S155, 5170) andthe underlined BH3 domain.The kinases involved in phosphorylation of Bad are numerous: PKC (139), Rsk(140), PKA (141), PIKE (133), P13K (142), p7056K (143), CK2 (144), Raci (145), Jnk(13 8), Pak5 (146), Cdki (147), and Rafi (148); many of these act on more than one ofthe phosphorylation sites. There are also several phosphatases, PP1 (149), PP2A (150)and PP2B (151), that have been shown to control the phosphorylation status of Bad.Moreover, phosphatases have been shown to compete with 14-3-3 for Bad binding upon15apoptosis induction, therefore suggesting that phosphatases do not act indiscriminatelyand are specific positive regulators of Bad-mediated apoptosis (53).Though phosphorylating Bad at its various sites may have a common overallpurpose, the mechanisms by which phosphorylation neutralizes Bad’s apoptotic functionare different. Among the 4 main sites of phosphorylation, Sen 12 and Ser136 were shownto promote association of Bad with cytosolic 14-3-3 proteins (134), thus sequestering Badaway from Bcl-xL and the mitochondria. Neutralizing Bad’s apoptotic function is alsoexecuted by phosphorylation at Sen 55 which lies within the BH3 domain, thereforedisrupting the interaction of Bad with Bcl-xL (126, 135, 152). Phosphorylation of Bad atSerl7O also abrogates Bad’s apoptotic function; however, the precise mechanism is stillunclear (137). Phosphorylation of yet another site on Bad at Ser128 was reported topromote apoptosis (153). Similarly, Thr2Ol of murine Bad was reported to be a target ofphosphorylation, but the significance of this is unclear since the sequence of murine andhuman Bad are not conserved at that site (138).1.3 CASPASES1.3.1 Caspase ActivationApoptosis is executed by caspases, a family of intracellular cysteine proteases.More than a dozen different caspases are synthesized in mammalian cells and areresponsible for many of the morphogenic features of apoptotic cell death (154). Forexample, polynucleosomal DNA fragmentation is mediated by cleavage of ICAD(inhibitor of caspase-activated DNase), the inhibitor of the endonuclease CAD (caspaseactivated DNase) that cleaves DNA into the characteristic oligomeric fragments (155).Likewise, proteolysis of several cytoskeletal proteins such as actin or fodrin leads to lossof overall cell shape, whereas degradation of lamin results in nuclear shrinking (154).Caspases are present in healthy cells as catalytically dormant pro-enzymes(zymogens), which are activated in response to developmentally programmed cues andstress stimuli that trigger apoptosis. There are two types of caspases which aredifferentiated by their activation mechanism. One form of activation, exemplified by16caspases 1, 2, 4, 5, 8 — 12, have long N-terminal pro-domains that undergo homotypicinteraction with specific adaptor proteins, such as FADD/MORT1 or Apaf-1. Thoughthese zymogens have little proteolytic activity, it is thought that the close interactionsbetween these molecules is sufficient to bring about autocatalytic processing resulting inthe generation of polypeptides (..20 kDa and 10 kDa) that assemble into the fully activecaspases (156). The second type of caspases, for example caspases 3, 6, and 7, have shortpro-domains and are activated predominantly through proteolysis by already activecaspases (or by granzymes: aspartate-specific serine proteases). Thus, it is thought thatthe adaptor protein-regulated (long pro-domain) caspases ignite the death effectormachinery and cause a self-amplifying (short pro-domain) caspase cascade thatultimately performs most of the proteolysis of vital substrate (157).Caspase activity in the cell can be controlled at two levels. First, caspase activity isinduced by upstream apoptosis signaling pathways that are highly regulated via the Bcl-2family (discussed below). Second, certain caspases can be antagonized by the inhibitor ofapoptosis proteins (lAPs). These TAPs bind to the active site of caspases, acting ascompetitive inhibitors. The anti-apoptotic effect of TAPs is neutralized upon release ofcertain mitochondrial proteins, such as Smac/DIABLO and HTRA2, which can sequesterthe lAPs (158).1.3.2 Apoptotic Pathways Leading to Caspase ActivationTwo principal apoptotic pathways, termed extrinsic and intrinsic, activate specificcaspases involved in apoptosis. The evolutionarily conserved intrinsic pathway, alsoknown as the ‘mitochondrial’ pathway, is primarily regulated by the Bcl-2 family and istriggered by developmental cues and diverse intracellular stresses. The intrinsic pathwayleads to the perturbation of the mitochondrial membrane and the release of apoptogenicfactors such as cytochrome c, apoptosis-inducing factor (AIF), Smac (secondmitochondria-derived activator of caspase)/DIABLO (direct inhibitor of apoptosis protein(IAP)-binding protein with low P1), Omi/HtrA2 or endonuclease G from themitochondrial intermembrane space (159-161). Cytochrome c, released from damaged17mitochondria, binds Apaf-l/caspase-9 to form what is known as the ‘apoptosome’complex resulting in caspase-9 activation which then activates the effector caspase-3.Other proteins released from the mitochondria, Smac/DIABLO and Omi/HtrA2, promotecaspase activation by neutralizing the inhibitory effects of TAPs (161). The triggering ofcaspase-3 activation leads to a caspase activation cascade that leads to complete cellulardestruction as hundreds of cellular proteins are degraded (Fig. 1.5)The extrinsic pathway begins outside a cell, when conditions in the extracellularenvironment determine that a cell must die. The extrinsic pathway, also known as the‘death receptor’ pathway, involves at least five transmembrane receptors belonging to theTNF (tumor necrosis factor)/NGF (neuronal growth factor)-receptor superfamily or byperform and granzyme B released from activated, cytotoxic lymphocytes. Cytotoxiclymphocytes express FasL and release granules containing granzyme B and perform.Binding of Fas ligand to the death receptor CD95 (Fas) results in clustering of receptorsand initiates the extrinsic pathway. The cytoplasmic portion of Fas contains a “deathdomain”, which plays a crucial role in transmitting the death signal from the cell’s surfaceto intracellular pathways (162). Unlike the intracellular regions of other transmembranereceptors involved in signal transduction, the death domain does not (162) possessenzymatic activity, but mediates signaling through protein—protein interactions.Stimulation of Fas by FasL results in receptor aggregation (152) and recruitment of theadaptor molecule Fas-associated death domain-containing protein (FADD) (163) throughinteraction between its own death domain and the clustered receptor death domains.FADD also contains a death-effector-domain (DED) that binds to an analogous domainrepeated in tandem within the zymogen form of caspase-8 (164). Thus, activation of Fasresults in receptor aggregation and formation of the so-called death-inducing-signalingcomplex (DISC) (165), containing trimerized Fas, FADD and procaspase-8. Procaspase-8oligomerization results in its autocatalysis and subsequent activation which results in thecleavage and activation of pro-effector caspases (caspase-3, 6, and 7) leading to apoptosis(149). In the case of cytotoxic lymphocytes, which express FasL and release granulescontaining granzyme B, granzyme B can enter target cells and directly activateprocaspases such as caspase-3.18The two pathways are largely independent, as overexpressed Bcl-2 does not protectlymphocytes from apoptosis induced by death receptor ligands (166, 167). However incertain cell types (e.g. hepatocytes), the extrinsic pathway engages the intrinsic pathwayvia a cleaved form of BH3-only protein Bid (tBid) (168).19oz(691)woJpogpoy81.UOAOrn1.o1.dod1ua11.suioppuiosiojaioouionpoiAosoonput(1.uonbosqnsP!H8 -osdsAqpiuwoidAJUO-Hfl04JO1A10JOSOAJOAUIiupuOqoOpwoiodoooisjoijwojupuSEosids13o-oJdo1.1AT1.oi(J1.oalTpoiojqq‘sogoojduuCjo!xololAo(qpoonawsjpoW41.osiooipujsTso1.dod1o1.upiof‘EosidsiooidS0WAfl0‘u1n1u‘!qI6osidsiooidjouoiWATp1oipsoowoidpuiS11.TO0J‘j-jdJoUOTWZU0WOijO04S0ZAJ1?0001UOJpO1iC300UTOIqoO1.(0JOoqoonpUiAoq1.0104M‘uupUoqoowu044ojjosoiAo044wagowoojsrnui.01.sioquiouiAjruuj-p3i4o4dod1-oJdsoonputS501451T1°3-£iiniimo4dodvo!su!JX1siso4dod1ouTpioj‘0513ds130OWAT1.o13O6osidsioum&ojp‘6osidsiopossoooidipisaVIJouoTwToossoquidmsip&qarog‘sJoqornpuiqAqsosidsonouonTqlqui044SOAOTIOJsiso4dodL?JouoT4onpUTuodn1napuo1po4TuJ044wagpos1oJaivw!uIoPUIsosixTsiojoJO41A1401jrn.ipuoipoiwpuooosTso4dod104u!p1301‘EosidsioodS0WAI1.0‘uamrn‘ii6osidsioodjoUOT413AT40041.SO4OUTOJdpuiS1.Tn.Iipiq‘j-JdJoU0T1.1ZuowoiJo044SO4OUJOJdouoaipoAjoowonpo(ojoosopi044oOnpUii(0440104A‘lnlpUOqoOwu041.04JC)50JJ(3041.W0Jj01.10OJSU1?J1.01.soqwowXJRUIJ-poi4o4dodg-ojdsoonpui550148‘i’’iio-Aiip.j0!JOJdOdV3!SH!BU1AeMqeILILT1111JdvUCUpUCLClIpIUOJJ&€IJiodrdod9UJGJO1Aiqjnd0!l0ld0dVci.—odcvD’s-wJI‘Isgcse..IIii‘Ip,.•s_I___••.JILnii82OJaavduiqwtuWSRId6GDJJJLi_itTt.ttatIlnwisDloIdoth11.4 Ca2/CALMODULIN - DEPENDENT PROTEIN KINASE II1.4.1 CaMKII ExpressionSince we show evidence thatCa217Calmodulin (CaM)-dependent protein kinase II(CaMKII) phosphorylates the BH3-only protein, Bad, a short description of CaMKII’sregulation and role in apoptosis is described below.Ca2/Calmodu1in (CaM)-dependent protein kinases (CaMK5) - CaMKI, CaMKII,eEF2K (previously called CaMKIII), and CaMKIV - are Ser/Tbr protein kinases that actas effectors by translating Ca2 signaling into the appropriate cellular responses (170).These protein kinases, which are all linked to Ca2 via the ubiquitous intracellular Ca2receptor CaM, have common as well as unique features with respect to their structure,regulation and activation.CaMKII is found in most tissues, is responsive to numerous signal transductionpathways and has broad substrate specificity. Substrates phosphorylated by CaMKII areimplicated in many aspects of cellular function, including the regulation of metabolism(glycogen synthetase and pyruvate kinase), membrane current (Ca2, C1, and Kchannels), neurotransmitter synthesis (tyrosine hydroxylase and tryptophan hydroxylase)and release (synapsin I), transcription (C/EBP and CRE-binding protein), cytoskeletalorganization (microtubule-associated protein 2), intracellular calcium homeostasis (1P3receptor and Ca2/ATPase), long-term potentiation and neuronal memory (AMPAreceptor) and as will be presented in this document, apoptosis (Bad).1.4.2 CaMKII IsoformsCaMKII comprises a family of isoforms derived from four closely related genes(ct, f3,‘y,and),all of which, are highly conserved among mammalian species. Forexample, the coding region of rat CaMKIIcL mRNA is 93% identical to the humanCaMKIIa mRNA and at the protein level, the human and rat a isoforms are identical inamino acid composition. Four different isoforms of CaMKII (a, f3, ‘ and)are encodedby four distinct genes: a (171-174),13(175-178),‘ (179-181), and ö (182-184); however,21all the isoforms from all four genes have a conserved core structure and share 89%—93%sequence homology in their catalytic and regulatory domains (180). The molecularweight of these kinase isoforms range from 54 kDa (a subunit) to 58 - 72 kDa(13,y, ösubunits).CaMKII isoforms are expressed at varying levels in different tissues and differ intheir cellular and subcellular localization. In the brain, for example, the a subunit is thepredominant form in forebrain, whereas the13subunit is the dominant form in thecerebellum (185). The y and isoforms are broadly distributed but at much lower levelsthan the a and f3 isoforms (186, 187). CaMKII isoforms also show distinct cellularlocalization; for example, as shown by immunohistochemistry, the distribution of the aand f3 subunits can differ even within the same neuronal cell (188).1.4.3 CaMKII HoloenzymesInterestingly, CaMKII isoforms expressed and localized in similar cellularlocations are thought to assemble into large holoenzymes. Using measurements of theradii and sedimentation coefficients, holoenzymes are thought to be composed of 8-12subunits (189-191). Co-expression and electron microscopy studies indicate that there islittle or no selectivity in the assembly (192, 193) and since cells often express multipleisoforms, the possibility exists for the formation of heteromultimers. The formation ofheteromultimers and their composition represents an additional means of modulatingCaMKII activity, as the localization, sensitivity of activation and autophosphorylation(194) are altered depending on the number and type of isoforms involved in theheteromultimer (189, 195, 196). High resolution images of forebrain and cerebellarholoenzymes (193) depicts a central ring with a hole in the center. The ring appears to becomposed of 8—10 smaller particles that appeared to be tethered by spokes that radiatedfrom the central core (193). The N-terminal region of the kinase (catalytic/regulatorydomain) is thought to be part of the small particles on the perimeter since calciumtracking experiments showed that Ca2/CaM appeared to gather at these particles. It wasalso noted that the rat forebrain kinase, which is predominantly a-CaMKII, appeared to22have 10 particles, whereas the rat cerebellar kinase, which is predominantly3-CaMKII,appeared to have only 8; indicating that the sizeof the holoenzyme also influencesCaMKII’s function and activity.1.4.4 CaMKII ActivationThe multimeric CaMKII is phosphorylatedby an intra-holoenzymeautophosphorylation reaction that is directed at eitherthe autoregulatory domain or theCaM-binding domain, producing diverse effects in its autoregulationand sensitivity toCa2/CaM. Prior to CaMKJI being activated, CaMKII is maintainedin an inhibited basalstate by an autoregulatory domain that acts as a pseudosubstrate preventingsubstratesfrom binding. The Ca2/CaM binding domain overlaps withthe autoregulatory domain.The isoforms share autophosphorylation sites within the autoregulatoryregions and basedon the numbering of the ct. isoform, the sites are Thr286 (Thr287for f3,y, )in the coreregulatory domain and Thr305/Thr306 (Thr306/Thr307 forf3,‘y,ö) in the CaM bindingdomain (197, 198). By far the best explored and understood is theCa2/CaM-stimu1atedautophosphorylation of Thr286. This phosphorylationinvolves a kinase cascade of sorts,with each subunit of the holoenzyme acting as both a kinase anda kinase of a CaMK.The resulting autophosphorylation of Thr286 producesa state of CaMKII, known asCa2/CaM-independent activity, where it becomesautonomous of its normal stimulus,Ca2/CaM, without affecting its maximal Ca2/CaM-stimulatedactivity. This is aconsequence of phosphorylation of Thr286 disablingthe autoinhibitory domain. CaMKII,when autophosphorylated at Thr286 also undergoes a 1000-foldincrease in its affinity forCa2/CaM, known as CaM trapping; however,autophosphorylation within the CaM-binding domain following CaM dissociation of activated autophosphorylatedenzymerestricts or prevents CaM from rebil)ding (CaM capping).The mechanisms andconsequences of autophosphorylation are centralto CaMKII’s intricate autoregulation,potentially underlying its ability to become differentially activatedin response to thelength and frequency of calcium spikes. This ability to detectspike frequency allowsCaMKII to act as a ‘molecular switch’ in learning andmemory, as a readout of synapticactivity. All of these characteristics are possible dueto the functional properties of23CaMKII, and its unique multimeric structure, autoregulationlactivation andautophosphorylation.It is worth noting that even though CaMKII isoforms share some homology withCaMKI, CaMKIII, and CaMKIV, CaMKII differs in terms of monomeric structure andmode of regulation (199, 200). Though each of these kinases, including CaMKII, requireCa2/CaM for activation and are activated by an upstream kinase that itself is a CaMkinase, CaMKI and CaMKIV also require phosphorylation of their activation ioop formaximal enzymatic activity (200).1.4.5 CaMKII and ApoptosisOnly recently has there been interest in CaMKII’ s role in apoptosis; however, a majorityof these studies have been limited to neuronal and myocyte cells (20 1-206). Moreover,the few studies regarding the specific role of CaMKII in apoptosis have been inconsistent(207-2 11). For instance, CaMKII expression which was reportedly involved in IAP-2regulation, prevented apoptosis in response to LPS and TNFCL (212). However an earlierstudy suggested that TNFL-induced apoptosis in U937 cells was a result of Ca2+-independent CaMKII activation and blocking CaMKII activation reduced the levels ofapoptosis (213). The reasons for the variable reports regarding CaMKII function may berelated to the differing expression levels of the 4 isoforms (and several reported splicevariants) in different cell types. Since many of the studies mentioned use of a CaMKIIinhibitor, which is not isoform specific, it’s not surprising that the results will varydepending on the expression and the current activation level of each of the isoforms. Forinstance, many reports have indicated that increased activation of CaMKII results inincreased survival in neuronal systems. Conversely, a majority of the myocyte studies,arising from cardiomyopathy and heart attack models, have indicated that CaMKIIactivation results in increased apoptosis and increased atrophy (201-204).241.5 A LINK BETWEEN APOPTOSIS AND CELL CYCLE1.5.1 Cell Cycle SummaryHere we describe a brief overview of the cell cycle with emphasis on the G 1 to Sphase transition control since several findings presented in this thesis relate to cell cycleregulation. The cell cycle is a term that refers to a series of events that take place in aeukaryotic cell leading to its replication. These events can be divided into two periods: i)interphase, during which the cell grows, accumulating nutrients needed for mitosis andduplicating its DNA and ii) the mitotic (M) phase, during which two distinct cells form.M phase is itself composed of two tightly coupled processes: a) mitosis, in which thecell’s chromosomes are divided between the two daughter cells, and b) cytokinesis, inwhich the cell’s cytoplasm divides forming distinct cells.Gi phase, S phase, and G2 phase are known collectively as interphase. Activationof each phase is dependent on the proper progression and completion of the previous one.Cells that have temporarily or reversibly stopped dividing are said to have entered a stateof quiescence called G0 phase.The complex macromolecular events of the eukaryotic cell cycle are regulated bya small number of heterodimeric protein kinases called cyclin dependent kinases (Cdks).The catalytic subunit of Cdks is only active as a protein kinase when bound to aregulatory cyclin protein. Whereas the levels of Cdks do not change significantly duringthe cell cycle, cyclin abundance is modulated during the cell cycle through programmedsynthesis and degradation.Because proper regulation of cell cycle phase transition is critical for anorganism’s survival, these protein kinases are exquisitely regulated at differentmechanistic levels and in response to a large variety of intrinsic and extrinsic signals(214, 215). The most prominent Cdks that function at the GuS phase transition are Cdk2, 4, and 6 (214-216). Cdk4 and its close relative, Cdk6, are controlled by D-type cyclins(Dl, D2, and D3) and act primarily by phosphorylating Retinoblastoma (Rb) and relatedproteins (214, 217, 218). Gi progression and S phase initiation, depends on the sustainedexpression of D-type cyclins, which, in turn, depends on continuous mitogenic25stimulation. This suggests that D-type cyclins may act as a link between mitogensignaling and the cell-cycle machinery. In support of this, D-type cyclin levels have beenshown to be regulated at the translational level through the phosphatidylinositol 3-kinase(PT 3-kinase) pathway (218-224) via the initiation factor eIF-4E (225).Following Cdk4/6 activation, Cyclin E binds and activates Cdk2 resulting in thephosphorylation of nucleophosmin, which is necessary for centrosome duplication.Phosphorylation ofNuclear Protein Ataxia-Telangiectasia (NPAT) then facilitates histonesynthesis and establishment of pre-replication complexes (jre-RC) at origins of DNAreplication (226). This entire process is thought to be necessary for proper S phase entry.Cyclin E expression appears to be more periodic than that of D-type cyclins. Cyclin EmRNA and protein begin to accumulate in late Gi, peak at the GuS transition and aredownregulated during S phase (215). Cyclin E transcription is activated when pRb ishyperphosphorylated and no longer exerts repression via E2F/DP, the transcription factorcomplex that targets pRb-mediated repression (227). In addition to expression control,cyclin levels are controlled via degradation through the ubiquitinlproteasome pathway(228). The importance and the fidelity of cyclin regulation is highlighted in studies whereoverexpression of D-type cyclins or cyclin E during early Gi leads to untimely Cdkactivation and premature S phase entry (218, 226).Rb Dephos’nCyclen BCdc2CDKIICycin AoCD<2______Rb phos’nFigure 1.6 CDK/Cyclin Partners and the Regulators Cell Cycle ProgressionCyclin ECDK226In addition to Cyclin levels, Cdk activation is also regulated by two classes ofCdk inhibitors (CKI5). The two classes are the Cip/Kip family and the INK4 family. TheCip/Kip family is composed of three members:21WAF1/CIP1 27K1P1and p572.TheINK4 family is composed of four members: p15, p16, p18 and p19 (229, 230). Thisclassification is based on their structure as well as their Cdk affinity. The Cip/Kip CKIshave Cdk-inhibitory domains that are thought to function by anchoring the inhibitorypolypeptide to the cyclin and occupying the substrate-binding site (231). Cip/Kipinhibitors are considered broad spectrum CKIs in that they can bind to and inhibit bothcyclin-D—Cdk4/6 kinases, as well as cyclin-E/A—Cdk2, although it has been reported thatthe efficiency of Cdk4/6 inhibition may vary for the different Cip/Kip inhibitors (232).The INK4 inhibitors share a common structural motif and mechanism ofinhibition and are narrow-spectrum CKIs as they only bind to and inhibit Cdk4 and Cdk6.INK4 inhibitors consist of repeating structural units known as ankyrin repeats (231).Ankyrin repeats form a concave structure, which in the case of 1NK4 inhibitors bindacross the back (non-catalytic) side of the target Cdk forcing a conformation that cannotsupport catalysis (231, 233). In addition, INK4-bound Cdks cannot bind to cyclin andthus are isolated as Cdk—LNK4 heterodimers (233).In addition to CKI’s inhibition of Cdk activity, phosphorylation of Cdksrepresents another form of direct regulation. For instance, Cdk2 is inhibited ifphosphorylated on tyrosine 15 (234). Conversely, Cdc25 phosphatase family membersact at discrete times during the cell cycle to remove the inhibitory phosphates (235, 236).Experiments involving the ectopic expression of CDC25A show an accelerated GuSphase transition. This suggests Cdc25 phosphatase activity may function to accumulateand maintain cyclin-E—Cdk2 complexes in a pre-active state (237). Recent studies havealso shown that full activation of Cdk2, for example, requires phosphorylation ofthreonine 160 by Cdk-activating-kinases (CAK) or more precisely by homologs of thewee-i kinase (238-242). All of which illustrates a complex mechanism aimed atcontrolling Cdk activity and ultimately cell cycle progression.271.5.2 Apoptotic and Proliferative Signalling Pathways are InterconnectedThe balance between cell proliferation and cell death is imperative forhomeostasis in multicellular organisms. This homeostasis had long been thought to be theresult of two separate processes, but there is evidence indicating that the processes ofproliferation and apoptosis are coupled. For instance, evidence exists suggesting theinvolvement of cdks in the process of apoptosis. Studies have shown that cdks areactivated in apoptosis arising in factor-deprived neurons, and during induction ofapoptosis in lymphocytes by Granzyme B, tumor necrosis factor (TNF) or humanimmunodeficiency virus Tat protein (243-247). Conversely, it has been shown thatseveral apoptosis regulatory proteins themselves can impinge on the cell cycle machinery(248-252). The concept of both survival and replication being coupled is furthersupported by observations that oncogenes sensitize cells to a wide range ofmechanistically different triggers of apoptosis including DNA damage, nutrientdeprivation, interferon, protein synthesis inhibitors, hypoxia, TNF, and CD95 (253-259),many of which exert no obvious direct effect on cell proliferation. Thus it appears as ifthe activation of cell proliferation primes the cellular apoptotic program and unlesscounteracted by appropriate survival signals, automatically removes the cell. Conversely,the process of undergoing apoptosis requires the engaging of the cell cycle machinery.1.5.3 Bcl-2 Family and Cell CycleThe Bcl-2 family of proteins, as mentioned previously, are recognized as criticalregulators of apoptosis. However, accumulating evidence suggests Bcl-2 family membersmay also play an important role in cell cycle progression. The connection between cellcycle and cell death is supported by numerous observations describing cycling cells asmore susceptible to cell death as compared to quiescent cells. The morphologicalappearance of apoptotic cells and its similarities to cells undergoing mitosis has also beennoted. From this, suggestions have been made that the mechanisms that regulate mitosismay play a role in apoptosis (260). Conversely, cells undergoing programmed cell death28often exhibit activation of cell cycleevents, such as cdk activation andabortive cell cycleprogression (261, 262).Bcl-21spro-survival function wasfirst observed to affect cellcycle progression inIL-3-dependent FDC-Pl cells over-expressingBcl-2. It was observed that growthfactorwithdrawal resulted in the reductionof cell size as compared to unstarvedcells and cellswere mostly arrested in G0/Gl (15).From this, it was thought Bcl-2 arrestedcells in G0 tomaintain viability when deprivedof growth factor. A similar observationof Bcl-2’seffect on proliferation was thatbone marrow-derived IL-3-dependentBAF3 cellsexpressing Bcl-2 promoted survivalupon IL-3 removal and arrestedcells in G0/Gl (15).It was also noted that these cellsdid not undergo cell cycle re-entryupon IL-3 restimulation. From these early experiments,a series of papers ensued, includingseveralstudies examining the effect ofBcl-2 not only on G0/Gl arrest butalso on cell cycleprogression. Many of the earlystudies regarding the physiologicaleffects of Bcl-2’s cellcycle inhibitory role were demonstratedby Stan KorsmeyerTslaboratory, by studyingandcomparing T-cells from modelsderived using bcl2-deficient, bcl2-heterozygous,wild-type, and bcl2 transgenic mice(250). Studying cell cycleentry in response to T-cellactivation, it was shown that increasingexpression of Bcl-2 correlatedwith a higherG0/Gl fraction and lower S-phase fraction(252, 263, 264). Several experimentsinvolving activation of quiescent Tand B cells and serum stimulationexperiments usingarrested NIH3T3 cells showedthat Bcl-2 expression delayedthe onset of S phase,indicating inhibition of G0 to S progression(264). Another early study whichused HL6Ocells (promyelocytic leukemiacell line) demonstrated that whencells overexpressingBcl-2 were treated with DMSO,a differentiative stimulus that did notinvolve cell death,cells decreased R11A content morequickly than control cells. Thissuggested that Bcl-2expression facilitated exit toG0 (265) and this effect was separate fromits anti-apoptoticfunction. Parallel studies usingBcl-2 homologs (Bcl-xL, BCL-w,and E1BI9K) alsoshowed retarded progressionto S phase; demonstrating Bcl-2’s abilityto affect cell cycleprogression is found in otherantiapoptotic molecules within theBcl-2 family, and is notcell type restricted (15, 266).Based on the above studies, Bcl-2was thought to begenerally growth inhibitory, however,growth rate measurements in conventionaland29continuous chemostat cultures later revealed that in cycling cells, Bcl-2 does notsignificantly affect growth rates under normal growth conditions, but prolongs Gi insuboptimal conditions (250, 251, 263, 267-269). Furthermore, cells sorted for the samesize, regardless of Bcl-2 or Bcl-xL expression level, entered cell cycle with similarkinetics, indicating that the main function of Bcl-2 and Bcl-xL is to drive cells into G0(270).The notion that Bcl-2 may somehow be connected directly or indirectly to thecell cycle machinery prompted several more studies investigating the molecularmechanism by which Bcl-2 is able to inhibit cell cycle progression. One study, usingmurine IL-3—dependent NSF/Ni .H7 cells, aimed at studying the effect of Bcl-2’sphosphorylation state on cell cycle progression, observed that Bcl-2 mono-(Ser7O) ormulti-site (Thr69, Ser7O, and Ser87) phosphorylation in its flexible loop domain wasshown to regulate intracellular reactive oxygen species levels; and subsequently inhibitcell cycle progression by delaying the G1/S transition (271). Another study, using Bcl-2transgenic mice, showed that Bcl-2 is able to delay cell cycle entry by delaying theaccumulation of E2F 1, a critical inducer of cell cycle entry (272). Various groups showedthat negative cell cycle regulators p27 and p130, which binds E2F4 during G0 to inhibitE2F-i expression, were elevated significantly more than usual in Bcl-2 cells during arrest(249, 250, 272, 273). Further studies showed that activation of cyclinE/cdk2 andcyclinD/cdk4, which is required for normal Gi to S progression, was reduced in Bcl-2and Bcl-xL cells; and this decrease in cdk activity was due to high p27 in the cyclinlcdkcomplexes (272, 274). Further evidence supporting p27 as an important mediator of Bcl-2and Bcl-xL’s cell cycle effect was shown by the inability of Bcl-2 and Bcl-xLoverexpression to delay proliferation in cells from p27 transgenic mice (272, 274). Itwas found that the protein levels associated with the induction of G 1 entry, including cFos, c-Jun, Myc, cyclin D expression, were not affected by Bcl-2 and Bcl-xL (250, 274),indicating the early signaling events initiating G0 to Gi transition are intact. However, thecritical activation of cdk2/4, which is required for transition into S phase, is delayed.301.5.4 Bad and Cell CycleInterestingly Bcl-xL and Bcl-2 antagonist, BH3-only protein Bad, has beenimplicated in several studies with cell cycle regulation. Bad has been shown to bephosphorylated at S128 by Cdki, a G2/M regulator in neuronal cells and Bad expressionalters Cyclin D expression in breast cancer cells (275). However, neither of these findinghave been supported in other cell types. Moreover, strong evidence involving Bad andcell cycle regulation arise from the finding that Bcl-xL can prolong the G0 phase andinduce cell cycle arrest, both of which can be blocked through Bad expression (270).Furthermore, it was demonstrated that a BH3 mutant form of Bad (Li 51 A), which isunable to bind Bcl-xL and Bcl-2, had no effect on the ability of Bcl-xL to delay the onsetof S phase. This demonstrated that Bad’s ability to modulate Bcl-xL’s cell cycle functionis dependent on its interaction with Bcl-xL. Therefore, these experiments demonstratedthat a molecule known to inhibit the anti-apoptotic function of Bcl-xL also inhibited itscell cycle activities. This further supports the suggestion that the cell cycle effectsobserved are a result of Bcl-xL expression. Furthermore, it was shown thatoverexpression of Bad not only prevented cell cycle arrest, but also showed increasedactivation of Cdk2 specifically during G1-S transition (248, 270). The precise timing ofCdk2 activation, caused by Bad expression, is worth noting as Cdk2 activation has beenassociated with Bax-induced cell death (276, 277). Interestingly, experiments using Bclx‘ and Bcl2 cells demonstrated that the cell cycle activity of Bad is not simply the resultof inactivating Bcl-xL as cells lacking Bcl-xL arrest normally. Furthermore, it has beenshown that BaJ’ cells arrest normally and as such supports the hypothesis that neitherBad nor Bcl-xL is required for G0/Gi arrest, though Bad/Bcl-xL heterodimerization canovercome the G0/Gl checkpoint. Thus, the increase in Cdk2 activationand the ability ofBad/Bcl-xL heterodimers to push cells into S phase, demonstrates that the function ofBad may not only.be to inactivate Bcl-xL or Bcl-2, but that Bad may be actively involvedin cell cycle control. This is further supported by studies showing that phosphorylation ofBad at Ser-170 can promote cell cycle progression, an effect that appears to be separatefrom its pro-survival function (137).311.6 BCL-2 FAMILY PROTEINS AS RATIONALDRUG TARGETS1.6.1 Cancer Cells are Primed for DeathThe process whereby a normal cell becomes cancerousrequires a minimum oftwo alterations: deregulation of proliferation andsuppression of apoptosis. Deregulationof proliferation, as seen in many cancers, is a consequenceof overproduction and/orincreased activation of specific proteinsthat promote cell duplication. Cells, however,have a fail-safe mechanism that is able to detect extremeproliferative signals and activatethe apoptotic machinery. For this reason even thoughoncogenes can stimulate cellproliferation and tissue growth, in manycases a net loss of cells results over time (278-280). Furthermore, studies have shown thatcells with deregulated proliferationmechanism die more readily than normal cells inconditions where nutrients are scarce(281). For instance, up-regulation of Myc inducesproliferation, but at the same timeengages the apoptotic machine and as a result thecell becomes more vulnerable to deathsignals. Therefore in order to become cancerous, cellsmust undergo further cellularalterations that permit uncontrolled proliferationwithout promoting apoptosis. Onecommon death-defying strategy is to enhance the activitiesof apoptosis-inhibitingproteins; therefore, blocking death signals from reaching themitochondria (282) . It is forthis reason that transformed cells where oncogenes increasethe rate of proliferation, inmany cases, have also been shown to have up-regulatedexpression of pro-survivalproteins such Bcl-2 or Bcl-xL (253, 258). It is noteworthy thatwhile cancer cellsinactivate elements of the apoptotic pathway, theynever disable the entire signalingcascade. For instance, cells that have been transformedby Bcl-2:Myc synergy, haveturned on the apoptotic machine, however thecell’s demise is prevented by Bcl-2’sability to neutralize Bax-like proteins (15, 23, 253). Asa result, even though the apoptoticmachine is ‘running,’ Bcl-2 has disengagedit from being able to perturb themitochondrion and trigger apoptosis. Nevertheless, theaggressive proliferative effect ofMyc is thought to prime the cell for apoptosis andas a result, the prospect of re-engagingthe apoptotic machinery to the mitochondrion makes Bcl-2 andits pro-survival relatives(Bcl-xL and Bcl-w) rational therapeutic targets. Hence, directlyinhibiting Bcl-2 is likely32to restore a functional apoptotic system and thus engage the over-activated deathmachinery in cancerous cells while also increasing the sensitivity towards chemotherapy.1.6.2 Targeting BcI-2Several proof-of-concept studies have been performed based on earlier work byStrasser et al. which showed that mice overexpressing both Bcl-2 and Myc develop animmature lymphoblastic leukemia, as seen by excessive amounts of white blood cells andenlarged spleen (283). Breakthrough studies by Letai et al. using transgenic mice thatconditionally express Bcl-2 and Myc showed that reducing Bcl-2 results in a dramaticdecrease in the number of white blood cells and the enlarged spleen returned to normalsize within 2 weeks (284). Furthermore, mice that had Bcl-2 levels reduced lived onaverage 145 days as compared to mice with high levels of Bcl-2 which lived 82 days(284). This demonstrates that the elimination of Bcl-2 expression clearly results inremission of the leukemia and prolonged survival of the mice. Furthermore, normal cellshave been shown to tolerate reduced Bcl-2 levels, and mice that have lowered expressionof Bcl-2 are completely healthy (102). This further strengthens the rationale of Bcl-2 as atherapeutic target since the inhibition of the target protein must have little effect on cellsother than those that are cancerous. These results combined with our increasedunderstanding of the functional significance of specific Bcl-2 family members has fueledthe pharmacological pursuit for therapeutic peptides and peptidomimetics that inhibitspecific pro-survival proteins such as Bcl- Bad - A drug templateBad has been used as a template for peptidomimetic design since Bad has beenshown to have high affinity for Bcl-2 and functional studies have demonstrated Bad’sability to antagonize Bcl-2’s pro-survival effect (101, 285, 286). In addition, Bad ‘ micehave been shown to develop lymphomas with increased age, and in response to sublethalradiation (287, 288). Therefore, the lack of Bad clearly plays an important role in the33development of cancer and as such the introduction of a molecule with Bad-like-bindingproperties may be able to re-sensitize transformed cells to apoptosis signaling pathways.Further support comes from knock-in mice expressing Bad with Ser mutated to Ala at112, 136 and 155 sites (Bad 3SA). Bad 3SA knock-in cells showed increased growthfactor responsiveness and lowered threshold for mitochondrial disruption, as compared toBad knock-in and wild type cells (287). These studies among the many others describingBad’s pro-apoptotic regulation, via phosphorylation, will undoubtedly lead to a moreaccurate template from which to design an effective therapeutic peptide. As such, ourwork investigating the cellular and molecular effects of Bad’s phosphorylation state atSer-170 will contribute to the overall understanding of Bad’s regulation and possiblyform the basis of a more potent and specific inducer of apoptosis.1.6.4 Targeting the apoptotic and proliferative signaling pathwaysThe potential role of Bad in cell cycle regulation, as presented in this thesis, alsorepresents a new direction in the study of this protein as well as a new potential fortherapeutic development. The data presented (see Chapter 3) indicating Bad directlyinteracts with the proliferative pathway raises the possibility that Bad, when notphosphorylated, may harbor the potential of modulating both the apoptotic andproliferative pathways that are hyperactive in cancer cells, and redirect them towards theactivation or amplification of the cell death machinery. Other data presented in Chapters4 and 5, show that cells expressing Bad preferentially undergo apoptosis when treatedwith CaMKII inhibitor (KN93). This finding is relevant as we also show that CaMKII-phosphorylates Bad at Ser-170, a site known to modulate Bad’s apoptotic ability. Thustargeting CaMKII activity may be another means of engaging the apoptotic machinery.Moreover, well recognized genes involved in cancers, such as p53, which is akey regulator of the cell cycle and apoptosis (289, 290), have been the pharmacologicaltarget of many potential and current anti-cancer drugs (291-294). This highlights theimportance of trying to understand the mechanism of Bad’s regulation of cell cycleprogression. Similar potential has attracted increased attention to cyclins and their34respective kinase partners, cdks, since both have been recently shown to play a role inapoptosis in addition to their well established role in cell cycle regulation (185, 277, 295-297). At least theoretically it should be possible to selectively harness their pro-apoptoticpotential and direct it towards selective activation of programmed cell death in cancercells. Thus, understanding the roles of these proteins will certainly pave a way towardsmore scientific advancement in the field of cancer biology and will provide solidfoundations for the discovery of novel drug targets that would kill, or at least controlcancer progression. Furthermore, the sophisticated dual role of the Bcl-2 family membersin the intricate regulation of apoptosis and the cell cycle makes them ideal therapeutictargets in the numerous diseases characterized by deregulated cell cycle and apoptoticpathways.aFigure 1.7 Bad Ser 170 — regulation and cellular effects11-3 stimulation was shown to increase the activity of levels of CaMKII andsubsequently increase the level of phosphorylation of its activatingphosphorylation site (Thr287). CaMKII-y was shown to be capable ofphosphorylating Bad at Ser 170. Bad’s apoptotic ability was shown to be reducedwhen phosphorylated at Ser 170. Dephosphorylation of Bad at Ser 170 was shownto increase the time required for cells to transit from G1 to S phase of the cellcycle. Bad was also shown to interact with key GuS cell cycle regulartors, Cdk2and Cyclin E.•I..Apoptosis351.7 AIMS OF STUDYThe studies in this thesis are aimed at furthering our understanding of Bad’sregulation specifically with respect to its phosphorylation at the Ser 170 site. We wereinterested in knowing whether the dephosphorylation of Ser 170 site could contribute tothe potency of the already apoptotic mutant protein, Bad Serll2,136,155Ala (Bad 3SA).Conversely, we were also interested in determining if phosphorylation of Ser 170 couldreduce the apoptotic effect of Bad 3SA. Beyond studying the effects of Ser 170 on Bad’sability to induce apoptosis, we also describe a novel cell cycle effect that appears to becontrolled at the level of phosphorylation at Ser 170. The increased replication time thatoccurs when cells overexpress Bad mutant form, mimicking dephosphorylation at Ser170 (Bad Si 70A), was an intriguing observation. From this, we were interested intracking the DNA content of cells expressing Bad Si 70A to see whether there was aspecific stage in the cell cycle that was hindered or whether the entire cell cycle wasslowed. From these studies, we show that cells expressing Bad Si 70A have markedincrease in the percentage of cells in S phase which indicates a slowing of the cell cyclespecifically at S phase. Based on this finding, we were interested in studying how Badmay be effecting cell cycle progression. Similar studies ongoing in our lab at the time,involving Mci-i and its association with Cdkl (298), prompted us to investigate thepotential association of Bad with cell cycle regulatory proteins. Endogenous Bad andflag-tagged Bad were immunoprecipitated and these IPs were probed for the presence ofa number of cell cycle regulators. Association of Bad with both cyclin E and Cdk2 wasdetected. Interestingly, both cyclin E and Cdk2 are late Gi and S phase regulators, and assuch we were interested in determining whether this novel interaction was responsible forthe cell cycle effects described above. Unfortunately, we were unable to determine whatregulates the interaction between Bad and Cdk2/cyclin E. We do show that the interactionoccurs primarily in the cytosol and the overexpression of Bcl-xL appears to negativelyaffect the level of interaction of Bad and Cdk2/CyclinE. However, we did not observe asignificant change in Bad’s ability to interact with Bcl-xL or Cdk2 when Bad is mutatedat Ser 170 to mimic phosphorylation (Bad S17OD) or dephosphorylation (Bad Si7OA).The second major experimental effort was aimed at identifying the kinaseresponsible for phosphorylating Bad at Ser-170. From these studies, we identified36CaMKII--y as the kinase that phosphorylates Bad at Sen 70. Parallel studies, investigatingpathways involved in regulating Bad’s phosphorylation at Ser 170 revealed that bothcytokine stimulation and cell cycle stage affect the level of kinase activity against the Ser170 site. Follow up studies involving the inhibition of CaMKII using small moleculeinhibitor, KN93, demonstrated that FDC-P1 cells underwent apoptosis in conditionswhere CaMKII was inhibited. Both the apoptosis and kinase activity studies support theour newly formed hypothesis: CaMKII-y is regulated by both proliferative and survivalsignaling pathways, and increased CaMKII-y activity promotes survival, at least in part,by phosphorylating Bad at Ser 170.37Chapter 2MATERIALS AND METHODS2.1 CELL LINES AND TISSUE CULTUREMC/9 and FDC-Pl cell lines were culturedin RPMI 1640 medium supplementedwith 10% fetal bovine serum, 2mML-glutamine, 1mM sodium pyruvate,50 nM13-mercaptoethanol. For MC/9 and FDC-P1 cells,the above medium was supplementedwith 2.5% WEHI-3-conditioned mediumas a source of mouse IL-3. MC/9 cells over-expressing Bcl-xL and Bcl-2 (MC/9-Bci-xL), or Bcl-2(MC/9-Bci-2), and FDC-Pl cellsover-expressing Bcl-xL (FDC-Pl/BcI-xL)were cultured in RPMI 1640 mediumsupplemented with 10% fetal bovine serum,2mM L-glutamine, 1mM sodium pyruvate,50 nM 3-mercaptoethanol, and 2.5%WEHI-3-conditioned medium. The MC/9-Bcl-2 celllines were maintained in the presenceof (200 ig/m1) hygromycin. The MC/9-Bci-xL, andthe FDC-P1/Bcl-xL were maintainedin the presence of(0.5 mg/mi) G418. MC/9-Bcl-xLand FDC-P1/BcI-xL expressing flag-taggedBad WT and flag-tagged Bad mutants(S17OA, S17OD, 3SA, 4SA, 3SAI7OD) were maintainedin the presence of (2.5mg/mi)puromycin in addition to (0.5mg/ml) G41 8. 3T6 cells were grown in DMEM mediumsupplemented with 10% fetal bovine serum, 2mM L-glutamine, and 1 mM sodiumpyruvate. Cells were maintained at 37°Cand 5% CO2 in a humidified incubator. TheMC/9-Bcl-2, MC/9-Bcl-xL and FDC-P1/Bcl-xLwere constructed using retroviralinfection. MC/9-Bcl-xL, and FDCP-1/Bcl-xLcells persisting after 7 days of continuousselection were used for transfection of BAD-pMXpuroconstructs.Bone marrow-derived mast cells(BMMCs) were derived by aspiratingfrom thefemurs and tibias of 6-8 weeks old femaleCD1 mice as described (299). Non-adherentcells BM cells were plated at 1x106cells/ml inIMDM containing 3Ong/ml of WEHI-3.Cell cultures were maintained between2 x and 8 x cells per ml.382.2 REAGENTSRecombinant IL-3, was purchased from R&D Systems(Minneapolis, MN). Theinhibitors LY-294002, wortmannin and rapamycinwere from Calbiochem (EMD).Propidium iodide (P.1.) was from Sigma-Aldrich (Saint Louis,MO). The BP and LRclonase enzymes, Zeocin and Blastocidin werefrom Invitrogen. The Effectene andLipofectamine 200 transfection reagents were from GIBCOBRL and Oligofectinetransfection reagent, from Invitrogen (Burlington,ON) was used for siRNA knockdownexperiments.2.3 ANTIBODIESTable 2.1: Antibodies usedAntibody Type Dilution ProcedurerSourcei-Bad Rb 1:1000 W, IP BD Biosciences,, 610392t-Bad Ms 1:1000 W SantaCruz:Biotechnology,SC-943ti-Flag Ms 1 :25 00W, IP Sigma Chemical,F-3 165c,.-Bc12 Rb 1:1000W, IP Santa CruzBiotechnology-_____________________SC-23960ci-BclxL Rb 1:10001W, IP BD Biosciences,556361ti-Cyclin A Rb 1:500 W, IPSanta CruzBiotechnologySC-3 1084IL-Cyclin D Rb 1:500 W, IP Santa CruzBiotechnology.____________________ SC-450ti-Cyclin E Rb 1:500 W, IP Santa CruzBiotechnologySC-25303ti-Cdk2 Rb 1:2000 W, IPSanta CruzBiotechnologySC-7082939Antibody Type Dilution [Procedure Sourcec-Cdk4 Rb 1:2000 W, IP Santa CruzBiotechnologySC-70832cL-Cdk6 Rb 1:2000 W, IP Santa CruzBiotechnologySC-53638a-Chkl Rb 1:2000 W, IP Santa CruzBiotechnology,SC-7898a-CaMKII alpha Ms 1:500 W, IP Santa CruzBiotechnology,SC-70492ci-CaMKII beta Ms 1:500 W, IP Santa CruzBiotechnology,SC-1540cL-CaMKII gamma Rb 1:500 W, IP Santa CruzBiotechnology,SC-1541z-Gsk3 Rb 1 :25 00 W Santa CruzBiotechnology,SC-8 1463i-Histone Hi Ms 1:2500 W Santa CruzBiotechnology,SC-8030cL-P Thr 286 CaMKII P 1:500 W Milipore,AB3827cL-Vinculm Ms 1:5000 W Santa CruzBiotechnology,SC-73614x=anti, P=phospho, IPimmunoprecipitation, W=westem blot, Ab=antibody, Rb=rabbit,Ms=mouse,402.4 PLASMIDSPlasmids CTV83 expressing human Bcl-xLand CTV87 expressing human Bcl-2were a gift from Dr. Rob Kay. pMXpuro plasmidsexpressing Bad and Bad mutants Bad(SI7OA, S17OD, 3SA) were constructedby Shaynoor Dramsi. The Bad 4SA constructwas prepared commercially by Seqwright Inc.PCR based site directed mutagenesis was performedusing pMXpuro plasmidexpressing mutated Bad 4SA tocreate Bad 3SA17OD. Specifically thecytosine atposition 977 was replaced with an adenine which resultedin the alanine (GCC) becomingaspartic acid (GAC). Primer used:5’-GGACTTCCTCGCCCAAAGGACGCAGGCACTGCAACACAG-3’.2.4.1 Bacterial TransformationConstructs generated were propagated by transformingthe DHG competent cells.Briefly, one microliter each of BP orLR reaction mix was added to 50 il of DH5competent cells. The reactions were incubatedon ice for 30 minutes followed by heatshock at 42°C for 30 seconds. The cells were thenput on ice for 1-2 minutes followed byaddition of 450 p1 of LB medium and incubation at37°C for 1 hour. The cells were thenspread on LB plates containing 50 jig/ml kanamycinand 100 jig/ml ampicillin. Thekanamycin or ampicillin resistantcolonies were selected and the plasmids werepropagated and purified using plasmid midi-prep kit(Qiagen).2.5 PROTEIN ANALYSIS2.5.1 Cell TreatmentsFor analysis of proteins by Westernblotting, cytokine-dependent cells werestarved of cytokine by overnight incubationleaving 10% of the original IL-3 containingmedium, or alternatively, cells were washed threetimes with PBS and incubated incytokine free medium for the indicatedtime. For experiments involving cytokinestimulation, cells were stimulated with10 jig/mi synthetic IL-3 in conditions previously41shown to induce maximal serine phosphorylation (Duronio Ct al., 1992; Weiham et a!.,1994; Ettinger et al., 1997). In experiments involving KN93 (CaMKII inhibitor) or itsinactive analog, KN92, cells were treated in the presence of cytokine for 24 hours. Finalconcentrations used were 10 ig/ml and 50 jig/mI KN93 or KN92.2.5.2 Preparation of Total Cell Lysate (TCLs)Total cell lysates were obtained by lysing cells in ice-cold solubilization buffer[50 mM Tris/HC1, pH 7.7, 1% Triton X-100, 10% (v/v) glycerol, 100 mM NaC1, 2.5 mMEDTA, 10 mM NaF, 40 j.ig/ml phenylmethylsuifonyl fluoride, 1 mM pepstatin, 0.5mg/mi ieupeptin, 10 mg/mi soybean trypsin inhibitor, 0.2 mM Na3VO4,1 mM Na3 MoO4and 1 mg/mi microcystin-L for 5 mm followed by centrifugation at 13000 rpm for 10mm. The supernatants containing the total cell proteins were boiled for 3 minutes in SDSsample buffer containing 1% ç3-mercaptoethanol and subsequently used for Westernblotting.2.5.3 Preparation of Nuclear and Cytoplasmic ExtractsFor extraction of cytosolic and nuclear proteins, cells were lysed in buffer Acontaining 10 mM HEPES (pH 7.9), 10 mM KC1, 1.5 mM MgCI2,0.34 M sucrose, 10%glycerol plus 1 mM DTT, 0.1% Triton X- 100, 0.2 mM Na3VO4,1 mM Na3 MoO4,andthe protease inhibitor cocktail for 5 minutes followed by centrifugation at 4000 rpm(2000 g) for 5 minutes to pellet primarily the nuclei. The supernatants were furthercentrifuged at 13000 rpm (15000 g) for 10 minutes and subsequently stored as cytosolicfractions and pellets were used for extraction of nuclear proteins. The pellets werewashed twice with buffer A and the nuclei were resuspended in buffer B containing 0.2mM EGTA (pH 8), 3 mM EDTA (pH 8), plus 1 mM DTT, RNaseA, DNAaseA, 0.2 mMNa3VO4,1mM Na3 MoO4,and the protease inhibitor cocktail for 30 minutes followed bysonication using a Sonic Dismembrator 550 (Fisher Scientific, Nepean, ON, Canada) atsetting 3 for 10 seconds. The extracted proteins were centrifuged at 4,000 rpm (2,000 g)for 5 minutes. The supernatants contained the nuclear proteins. The nuclear and cytosolicextracts were then boiled in SDS sample buffer containing 1% -Mercaptoethanol for 5minutes.422.5.4 Co-Immunoprecipitations (IPs) and Western BlottingTypically, 4-6 mg of protein extracts was used for immunoprecipitations.Extracted proteins were incubated overnight at 4C with the antibodies listed in section2.3 at the indicated concentrations. Immuno-complexes were captured with 4Ojil ofprotein G-Sepharose beads slurry at 4°C for 1 h. Beads were washed 3 times with coldlysis buffer and boiled in SDS sample buffer containing 2% p-Mercaptoethanol for 5minutes.TCLs and IPs were separated by SDS/PAGE followed by Western blot analysis.Transfers were made by semi-dry blotting on to nitrocellulose membranes. Themembranes were blocked for 1 h in 5% (w/v) low-fat dry milk in Tris-buffered salinewith 0.05% Tween 20 followed by overnight incubation at 4°C with the antibodies listedin section 2.3 at the indicated concentrations. Anti-rabbit, anti-mouse or anti-goatantibodies conjugated to horseradish peroxidase were used to detect theimmunocomplexes by enhanced chemiluminescence (Amersham International, Oakville,ON, Canada)2.6 ASSAYS2.6.1 Apoptosis assaysApoptosis was assayed by annexin V and propidium iodide (P1) staining. Sampleswere read using flow cytometry (BD FACSCant0) and analyzed with FCS Express v.2(DeNovo software). The cells were cultured until reaching a density of no more than 5 xcells per ml, followed by washing 3 times with complete RPMI 1640 mediumwithout WEHI-3. Cells were harvested after various times of IL-3 starvation, washedwith PBS twice and centrifuged at 200 x g for 4 mm. The pellet (5 x i05 cells) wasresuspended in 500 jtL of binding buffer (PBS containing 0.1% glucose, 100 jig/mlRNAse A). The staining solution [Annexin V-fluorescein labeling reagent: PT = 1:2 (v/v)]was incubated for 15 mm at room temperature in the dark. Percentage of cells undergoingapoptosis was determined from both Annexin V positive cells together with cells thatwere both Annexin V and P1 positive. Analysis of cells at various time points verified43that at early times, Annexin V positive cells were observed and at later times, these cellsbecame double positive.2.6.2 Cell Cycle AssayCells were synchronized in late G1 phase of the cell cycle by treating cells withHydroxyurea (100 mM) for 18 h. Cells were released from Gi by washing cells threetimes with PBS and re-culturing in normal growth media plus WEHI-3. Cells wereharvested at various time points after release and fixed in ice cold 70% ethanol for 15minutes. The cells were then pelleted and incubated in PBS containing 0.1% glucose, 100jig/mi RNAse A and 50 jig/ml propidium iodide for 30 mm in the dark. The sampleswere analyzed by flow cytometry. By analyzing cell size and DNA content, cells werecategorized as being in Gi, S and G2 phases of the cell cycle.2.6.3 Cell Sorting AssayCells growing in normal growth media with WEHI-3 were used for cell sortingexperiments. Cells were washed with PBS just prior to being sorted using the BD FACSVantage SE Turbo sort cell sorter. Cells were sorted based on cell size and DNA contentand are here referred to as cells in either: Gi, S or G2 phases of the cell cycle. Sortedcells were lysed instantly as described in section RETROVIRAL INFECTIONThe retroviral infection was done to over-express human Bcl-xL into cytokinedependent FDC-P1 and MC/9 cell lines. Briefly, Plat-E (300) packaging cells were platedin 6 well plates. Once cells were 50% confluent, cells were transfected with Effectene(Qiagen) according to the manufacturer’s protocol. Approximately 1 jig of either CTV83-Bcl-xL (gift from Dr. Rob Kay) or CTV87-Bcl-2 retroviral vectors were used to transfectthe cells using the Effectene transfection kit (Qiagen). Twenty-four hours followingtransfection, the medium throughout was replaced with fresh media and 72 hours later theviral supernatant was collected and centrifuged at high speed to remove all the cell44debris. The viral supernatant was subsequently filtered through a 0.45 jim filterand thesupernatant was then used to infect FDC-Pl and MC/9 cells. The cell mediawas replacedwith the viral supernatant and 48 hours after infection the cells were selectedwith eitherG4l8 (0.5 mg/mi) or Hygromycin (200 jig/mi) for cells infected with CTV83-Bcl-xLretrovirus or CTV-Bcl-2 retrovirus, respectively. MC/9-Bcl-xL, and FDC-Pl/Bcl-xLcellspersisting after 7 days of continuous selection were used for transfectionof BADpMXpuro constructs. BAD-pMXpuro constructs include: Bad Serl7OAla (170A),BadSerl7OAsp (170D), Bad- Serll2, 136, l55Ala (3SA), Bad-Serll2, 136,155, l7OAla(4SA) and Bad-Sen 12, 136, l55Ala, l7OAsp (3SA17OD). Stable FDC-Pl/Bcl-xLandMC/9-Bcl-XL transfectants expressing various FLAG-tagged Bad constructs in the pMXpuro vector were selected in complete medium containing 2.5 jig/mL puromycin,andstable clones were isolated by serial dilution in 96 well plates.2.8 COLUMN CHROMATOGRAPHYCells were pelleted at 200 x g for 4 minutes and lysed with ice-cold solubilizationbuffer. Lysates were spun down at 13,000 rpm (15,000g) for 10 minutes at 4°C toremove the nuclei and insoluble material. The supernatant(500 g of protein) was thenloaded into Mono-Q HR 5/5 column from Pharmacia. Mono-Q-buffer-A contained50mM of Tris-HC1 pH 8.8, 2.5 mM of EDTA pH 8.0, 1 mM of sodium vanadateand 0.01%Triton X-100. Mono-Q-buffer-B is the same as Buffer A except that it alsocontains afinal concentration of 1 M NaC1 and a linear gradient of 0 to 1 M NaCl was usedto elutebound proteins. Samples of 0.5 ml were collected at flow rate of 0.25mi/mm.For Superdex S200 column fractionation, the cell extracts (500g of protein)were loaded and the column washed with buffercontaining 50 mM Tris 7.7 pH, 0.05%Triton X- 100. Fractions of 0.5 ml were collected with a flow rate of 0.25 ml/min.452.9 KINASE ASSAYA peptide encoding the Seri7O site of Bad was synthetically prepared at theBiomedical Research Centre, UBC. The synthetic peptide contained the followingsequence: RRGGPRPKSAGVA (PRPKSAG corresponds to residues 166-172 of murineBad or 130-136 of human Bad). The substrate for CaMKII kinase activity was the peptidePLSTRLSVSS from Santa Cruz Biotechnology. For Chkl kinase activity, the substratewas ChkTide (Sigma). For Cdk2 kinase activity, the substrate was purified histone Hi(Gibco-BRL). Kinase assays were performed by adding 5ig of peptide, 10 jiL of thekinase (fraction collected from column or IP’d kinase) and 10ilof the ,-[32Pj-ATPprepared in assay dilution buffer which contained 25 mM of f3-glycerophosphate, 20 mMMOPS, pH 7.2, 5 mlvi EGTA, 2 mM EDTA, 20mM MgC12,250 iM DTT and 5 jiM ofmethyl aspartic acid. The final reaction volume was 30 jil and reaction was incubated at30° C for 15 mm. Subsequently, 15iilof the assay was spotted on Whatman p81chromatography filter paper. The spotted filter papers were washed in 1% 0-PhosphoricAcid for 40 minutes with multiple changes to remove unbound 32P. The activity of theeach sample was obtained by scintillation counting of the filter papers. In someexperiments, KN-93, a selective inhibitor of CaMKII was incubated with the kinase at 10jiM final concentration, for 20 minutes at 3 7°C, prior to assay. In assays of commercialCaMKII, the CaM Kinase II Assay Kit (Upstate, 17-135) was used and Ca2 andcalmodulin were added for full activity of the enzyme. However, for immunoprecipitatedCaMKII from cells, the presence or absence of Ca2 was found to have no significanteffect on the kinase activity.463.0 s1RNAGene Silencing by siRNA — 3T6 cells were plated at a density of 2 x 1 O cells/cm2in 6-well plates. Twenty four hours after plating, cells were transfected using Oligofectamine(Invitrogen) as recommended by the manufacturer. Cells were harvested 48 h later. Threedifferent CaMKII-f3 and ‘ siRNA variants where used together to knockdown expression.The CaMKII-’ siRNA sequences are as follows:5’-ACCAAGAAGUUGUCCGCCCGAGAU-3’5’ -AUCUCGGGCGGACCACUUCUUGGUA-3’5’ -UGAGAACUUGCUGCUGGCGAGUAAA-3’The three CaMKII- siRNA sequences are as follows:5 ‘-CCAGUGGACGGGAUUAAGGAAUCUU-3’5 ‘-AAGAUUCCUUAAUCCCGUCCACUGG-3’5 ‘-CCAAAGCCCGGAAGCAGGAAAUCAU-3’47Chapter 3Bad: Cross Talk between Cell Cycle and Apoptosis3.1 INTRODUCTIONAll mammalian cells have an intricate network of signaling molecules that areable to sense and interpret internal and external signals. Specific combinations of growthfactors act as biological commands that direct cells to proliferate, grow, and differentiatewhile the absence of growth factors is a signal to either enter a state of quiescence, or toundergo apoptosis. Hemopoietic cells serve as an excellent model system to studyapoptosis since their survival is dependent upon specific cytokines, which can stimulatecell growth as well as maintain cell viability by inhibiting apoptosis (301-304). The deathof cytokine-deprived hemopoietic cells proceeds via the mitochondrial pathway,involving regulation of various Bcl-2 family proteins (113, 123, 305).Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought toexert a death-promoting effect by heterodimerization with Bcl-xL and disrupting its antiapoptotic activity. Based on this feature, Bad has been labeled as a death effector;however, there have been several recent studies demonstrating its importance in healthycells and implicating Bad in cell cycle regulation (270, 275).Our laboratory reported that Bad is phosphorylated at a novel site, Ser 170 (137).The functional relevance of this phosphorylation was assessed by way of site directedmutagenesis. These studies revealed that transfection with the Bad Si 70A mutant, whichmimics constitutive dephosphorylation at this site, enhanced the ability of this protein toinduce apoptosis; whereas, Bad S17OD, a mutant which mimics constitutivephosphorylation at Si 70, results in a protein that is virtually unable to promote apoptosisand causes at least a doubling in cell number over several days. Thus, it was hypothesizedthat Ser 170 represents a regulatory site able to inhibit Bad’s apoptotic function andpromote cell cycle progression.48In this chapter, results are presented that have further analyzed the function of theSerl7O residue in controlling Bad’s apoptotic function. Here, we also present datasupporting a novel cell cycle effect associated with the phosphorylation state of Bad Ser170 along with a direct interaction between Bad and proteins involved in cell cycleregulation. All immunoblots shown in the following sections, are representative of aminimum of three replicates.3.2 RESULTS3.2.1 Phosphorylation at Serl7O Regulates Bad’s Apoptotic FunctionThe BH3-only proteins, including Bad, can have a profound effect on apoptosisregulation by virtue of their interactions with other Bcl-2 family proteins. Bad isphosphorylated at several Ser residues, which control its interaction with Bcl-xL and/or14-3-3 proteins. To further characterize the functional role of phosphorylation at the Ser170 site on Bad, which was previously reported in cytokine dependent MC/9 mast cells(137), Flag-tagged Bad (WT) and the corresponding mutants at the Serl7O site (S17OAand Si 70D) were expressed via retroviral transfer in another cytokine-dependenthemopoietic progenitor cell line, FDC-P 1, in which Bcl-xL was also over-expressed.FDC-P 1 cells were used for apoptosis studies since they where more sensitive to IL-3withdrawal than MC/9 cells, in terms of increased levels of apoptosis within 24 hours ofstarvation. In our hands, as well in other laboratories (Dr. Schrader and Dr. McNagnypersonal communication), MC/9 cells became IL-3 independent and for this reason theywere not as useful for apoptosis studies. Thus, while studies monitoring the proliferationand cell cycle status used MC/9 cells, we first wanted to confirm and extend the effect ofchanges to Bad at Serl7O in another cytokine-dependent cell line. Immunoblot analysisof stably infected cells demonstrated expression of the Flag-tagged mutant forms of Badproteins (Bad S17OA and Bad S17OD), as well as wild type Bad, and showed that levelsof endogenous Bad were unaffected as compared to control cells expressing only Bcl-xL(Fig. 3.1).49c’ C - a a.? r-. r-.) L -U) U) Cl) U)BcIxLI I 1F-BADVinculin—— I L——’—p85BADVincuHnFigure 3.1. Stable infection of Bad S170 mutantsFDC-P 1 cells expressing Bcl-xL (BclxL) were retrovirally infected with Flag-tagged Bad-Serl7OAla (S17OA) or Bad-Serl7OAsp (S17OD) or Bad wild type(WT). Cell extracts were separated by SDS-PAGE and immunoblot analysis wasperformed using anti-Bad (BAD), anti-Flag (F-BAD) and anti-Bcl-xL (BCLxL)antibodies. F-BAD represents the amount of flag-tagged Bad; and BADrepresents endogenous levels of Bad protein.The IL-3 dependent FDC-P 1 cells and the transfected variants were measured forsigns of apoptosis after 24 hrs of cytokine starvation. Apoptosis was measured using flowcytometry analysis for binding of Annexin-V and incorporation of propidiumiodide(P.1.). Annexin-V staining is regarded as an indicator of early stage apoptosis, while P.1.staining occurs at later stages of apoptosis. As shown in Figure3.2, FDC-Pi cellsexpressing Bcl-xL were much more resistant to apoptosis following cytokine withdrawalwhen compared to parental cells. Expression of wild type Bad blocked the pro-survivaleffect of Bcl-xL as expected, and thus these cells regained cytokine-dependent survival.Supportive of previously published findings, cells expressingBad-S 1 70D underwentsignificantly less apoptosis following cytokine starvation than thesame cells expressingBad-S17OA (t-test, p<O.05; n3). When measuring Annexin V staining, cells expresingS170D were not significantly (t-test, p>O.05) different from cells without the addedBadprotein, suggesting that mutation of the Si 70 site to a phospho-mimetic residue is able to0-Ja a2 i—. I—C) - —c Cl) U)50prolong the onset of apoptosis. Conversely, cells expressing Bad-Si 70A showed similarlevels of apoptosis as compared to cells expressing wild type Bad (Fig. 3.2).P1 Stain*U)>aParentalBcIxLU)JBadWTU)I17OD0•170A*t-test p<O.05Annexin V StainU)>fl Parentalj BclxLJBadWTIi170D•170A0*t-test p<O.05** t-test p>O.05P1 and Annexin V Stain60*40 —ParentalBclxL— QBadWT20—liii 170D•170A0— *t-test p<O.05Figure 3.2 S170 modulates Bad’s apoptotic functionFDC-Pi cells expressing Bcl-xL (BclxL) and Bad-Serl7OAla(S17OA) or BadSer 1 7OAsp (Si 70D) or Bad wild type (WT) where grown in normal conditions(Control) or where starved of cytokine for 24 hours (Starved). Using FACS,apoptosis was calculated as the percentage of cells showing either Annexin V orP.1. positive staining or double positive staining.Control Starved**ControlStarved1*Control Starved513.2.2 Phosphorylation at Serl7O mutes Bad’s pro-apoptotic abilityBased on previous work in our lab showing that phosphorylation of Bad at Ser170 could overcome the apoptotic effect of dephosphorylation of Ser 112; we wereinterested in testing whether phosphorylation at the Ser 170 site could overcome ordiminish the apoptotic effect of Bad mutant 1 12A/136A/155A (3SA). Using FDC-P1-Bcl-xL cells (Fig 3.3A), apoptotic analysis studies comprising of Annexin V and P.1.staining revealed that cells expressing 1 12A/136A!155A1170D (3SA170D) hadsignificantly lower levels of apoptosis during cytokine starvation, as compared to cellsexpressing Bad-3SA or Bad mutant 1 12AJ136A/155AJ170A (4SA) (Fig. 3.3B). Fromthese results, it appears that phosphorylation at Ser 170 is not only able to reduce theapoptotic effect of Bad, but can at least partially overcome the potent pro-apoptotic effectof dephosphorylation at the three other key residues of Bad. Furthermore, thedephosphorylation of this site is able to transform Bad into a more potent death effector.52AFDCP1-BclxL3SA 4SA 3SADF-BAD—.Vin____CU0CUCU________________CU•5 4UC)00FDCP13SA 4SA 3SADCIBI ‘CU00CCUCU ‘tvCU200CU00U).000CUU)00UWT•3SAQ4SAD3SAD__________*t-test p<O.05•WT•3SAQ4SAD3SAD*t-test p<0.05Figure 3.3 S170 modulates Bad’s apoptotic function(A) FDC-Pl parental cells and FDC-Pl cells expressing Bcl-xL, were retrovirallyinfected to express Flag-tag-Bad-Si 12AIS136AIS155A (3SA), BadSi12A/S136AJS155AIS17OA (4SA) or S112A/S136AJSi55AIS17OD (3SAD).Lysates of cells expressing Bad mutants 3SA, 4SA and 3SAD were separated bySDS page and immunoblotted using anti-Flag antibody (F-Bad). Anti-Vinculinantibody (Vin) was used to ensure equal loading.(B) Using FACS, apoptosis was calculated as the percentage of cells showing eitherAnnexin V or P.1. positive staining or double positive staining. Apoptosis wasassayed using FDC-Pi-BelxL cells expressing Flag—tagged Bad (WT), BadS112AIS136A/S155A (3SA) or Bad-S112AIS136AISi55A/S17OA (4SA) andBad-Si 12A/S136AISi55AIS17OD (3 SAD) under normal growth conditions(control) or after 24 hour cytokine starvation (Starved).B *P1 Stainri40Control StarvedWT•3SAQ 4SAO 3SAD*t-test p<O.05Annexin V StainTi]rControl StarvedP1 and Annexin V Stain *E__rr0 —Control Starved53Further experiments examining the effect of phosphorylation of Ser 170 wereperformed using parental FDC-P1 cells (not overexpressing Bcl-xL). From this we wereable to examine whether the observed increase in survival of cells expressing 3SA17ODversus 3SA and 4SA required the co-expression of pro-survival protein, Bcl-xL. FDC-P1cells were retrovirally infected with Bad constructs: 3SA, 4SA and 3SA17OD. Cells wereplaced under selection for 48 h at which point cells were washed in order to remove deadnon-transfected cells and debris. Following the initial selection, apoptosis was measuredfor 96 hours following infection under normal growth conditions. The high level ofapoptosis observed when cells express Bad mutant 3SA or 4SA is consistent with ourearlier findings as well as those of others (287). As shown in Figure 3.4, we did notobserve a significant increase (t-test, p>O.O5) in levels of apoptosis in cell expressing Badmutant 4SA as compared to 3SA; nevertheless, expression of either 3SA or 4SA showeda consistently high level of apoptosis. Interestingly, cells expressing Bad mutant3SA17OD showed a marked and significant decrease (t-test, p<O.O5) in the level ofapoptosis as compared to 3SA and 4SA at 72 and 96 hours. In fact, FDC-Pl cellsexpressing 3SA17OD continued to grow for weeks under constant selection without coexpression of Bcl-xL.54P1 Stain90980a70.9 60• 504030‘S 20100Figure 3.4 Mutation mimicking phosphorylation at Serl7O inhibits Bad’sapoptotic function without overexpression of BcI-xLUsing FACS, apoptosis was calculated as the percentage of cells showing eitherAnnexin V or P.1. positive staining or double positive staining. Apoptosis wasassayed using FDC-P1 cells transfected with Flag—tagged BadS112A!S136A/S155A (3SA), Bad-S112AIS136A/S155A/S17OA (4SA) or BadS112A/S136A/S155A/S17OD (3SAD) at 36, 48, 72 and 96 hours posttransfection.>0)0C,,.90)a)C-,Ca)>C,)C.9C,)a>‘-C036haurO48 hours•72 houra96hour•36 hourso48 hours•72 hours96 hours•36 hoursO48hourE•72 hours96hourP1 and Annexin V Stain3SA 4SA 3SAD553.2.3 Phosphorylation of Bad Serl7O RegulatesNovel Cell Cycle EffectAs mentioned, previous studies performed by Dramsi et al. (137) showedthat aSi 70A mutation enhanced the pro-apoptotic activityof Bad, while the SI 70D Badmutant showed very little apoptotic activity. Beyondthe apoptotic effect, there was alsothe unexpected finding that the Bad Si 70D not only rendered Badless apoptotic, but italso promoted cell proliferation.We followed up the earlier studies, using MC/9 cells,to confirm that Bad’s abilityto affect cell cycle progression could be observed by comparing MC/9-BclxL-Si 70A andMC/9-BclxL-S 1 70D cells (Fig. 3 .5A). Cells expressingBad Si 70A were found to requirefewer passages to maintain appropriate cell numbers.In other words, these cells appearedto have a slower doubling time. From this we were interested in knowingif the increasein doubling time was due to a higher percentage ofcells undergoing apoptosis or whetherthe cells have a slower rate of cell cycle. Using FACSanalysis, double positive (P.1. andAnnexin V) staining was assessed to determineapoptosis levels of MC/9-Bcl-xL cellsexpressing Bad Si7OA, Bad SI7OD andBad WT (Fig. 3.5B). We observed nearly equaland very low level of apoptosis when cells weremaintained in normal growth conditions(t-test p>O.05; comparing BclxL to other cell types).These results were consistent withprevious results (i37) produced in our lab, usingthese MC/9 cells, and suggest thatphosphorylation of Bad at Si 70 may be affectingcell cycle progression.We examined the growth rate of MC/9 cells expressingBad Seri7O mutants, byseeding cultures with the same number of cells andrecorded cell numbers over 5 days(Fig. 3.5C). By the third day, there werea significantly lower (t- test, p<O.O5) number ofcells expressing Bad-Si 70A as compared to cells expressing Bad-Si70D and BclxLalone. However, the cells expressing Bad 5170A had the longest doubling time, but inthese anaylsis, the rate was not significantlydifferent from cells expressing Bad WT (ttest, p>0.05). Since apoptosis levels were nearly identical forall cells regardless of Badmutant expressed, we were increasingly confident thatthe difference in cell numbers wasdue largely to the effects of mutations of Bad Si70 affectingthe cell cycle rate.563‘:iiliBCLxL WT SI7OA S1700Cell Line Rate Doubling TimeMc9BclxL 1.823 26.3Mc9-BdxL4ad 1.619 29.6Mc9-BdxLBadS170A 1.522 31.6Mc9-BclxL-BadSl7OD 1.812 26.5* ttest, p<0.05Figure 3.5 Cells Expressing Bad S17OA Have a Longer Doubling Time(A) Flag-tagged Bad and the corresponding mutants at the Sen 70 site (Si 70A andSi 70D) were expressed via retroviral transfer in MC/9 cells in which Bcl-xL wasalso overexpressed (BCLxL). Immunoblot analysis of stably infected cellsdemonstrated expression of the Flag-tagged Bad (WT) and mutant forms of Badproteins.(B) MC/9-BclxL cells expressing Bad-wt (WT), Bad-S 170A (S17OA) or Bad-S 170D(Si7OD) were stained with P1 and Annexin-V. Using FACS,apoptosis wascalculated as the percentage of cells showing Annexin and P1 positive stainingExperiment repeated three times (n = 3).(C) MC/9-Bcl-xL cells expressing Bad WT, Bad Si 70A, or Bad Si 70D were seededat a concentration 25,000 cells/mL and cell numbers were counted every 24 hoursfor 5 days. The Rate is defined as the multiple of times a cell doubles everytwenty four hours. The Doubling Time is define the amount of time require forcells to double in number. Experiment repeated three times (n = 3).Annexin V and P1 SlainA B<-JF-BADp85C30 100025xZ20151000ICFz0-+- Mc9-BclxL-.- Mc9-Bdxl-Bad-k- Mc,9-Bdxl-SI7OA-a-- Mc9-BdxL.-SI700A.123Days45 12 34 5Days573.2.4 Dephosphorylation at Bad Ser 170 Stalls Cell Cycle at S PhaseCareful examination at Bad’s ability to affect cell cycle was accomplished bymeasuring the number of cells in Gi, S and G2 stages of the cell cycle (Fig. 3.6A).Through this cell cycle analysis we observed that cells expressing Bad Si 70A mutantshowed a consistent increase in the number of cells in S phase as compared to cellsexpressing Bad wild type and Bad S17OD (Fig. 3.6A). The increase in the number of cellsin S phase is indicative of an increase in the time required to transit through S phase; andas such we hypothesized that expression of Bad Si 70A prolongs the time during whichcells are in S phase. In order to examine transit time through S phase, cells were firstsynchronized in G 1 stage of the cell cycle using Hydroxyurea. Cells were released fromcell cycle block and analyzed via FACS at various time points after release (Fig. 3.6B).The percentage of cells in either G 1, S or G2 stage of the cell cycle was determined.From this we observed that expression of either Bad Si 70A or Si 70D had little to noaffect on the the time require to enter S phase; however, we did note a digression in thetime required to transit S phase. A majority of cells expressing Bad Si 70A are in S phaseat 8 hours after release where cells expressing Bad S17OD, Bad wt, or BclxL alone wereshown to have entered G2 phase by that time. None of the mutants caused any differencein the exit of cells from Gi, while the cells expressing BadS170A took longer to appearin G2. These results reaffirm our hypothesis that expression of Bad Si 70A slowsprogression through S phase of the cell cycle, suggesting that phosphorylation of Bad at5cr 170 may control both Bad’s ability to induce apoptosis and Bad’s novel cell cycleeffect.The results in Fig. 3.6 were obtained using mixed populations of infected celllines. Thus, to further confirm Bad mutant S17OA’s effect on cell cycle, we wereinterested in studying the effect of expression levels of respective Bad Serl 70 mutants oncell cycle progression. To do so, we created several clonal cell lines expressing varyinglevels of either Bad wt, Bad S17OA or Bad S17OD (Fig 3.7A). This enabled a moreprecise analysis as cells with known expression levels of respective forms of Bad.58A100. 80D MC9-BcIxL60 FThU MC98ad• MC9-S1 70A2 40MC9SI7ODB20GiII100-—.- MC9-S17OA60• MC9S17OD40 --MC9BadMC9-BcIxL2:Oh lb 2h 4h Gb80S160 _-—------ MC9-S17OAa - ,--f..- MC9-S17ODMC9-Bad20- MC9 BclxL0Oh lb 2h 4h Sb 8h lOb100G2804— -- MC9-S17OAa60. MC9-S1700, 40 _—-- MC9-Bad20MC9 BdxL0•4lb 2h 4h Sb Sb lObTimeFigure 3.6 Bad S17OA Expression Stalls Cells in S Phase(A) MC/9-Bcl-xL and MC/9-Bcl-xL Flag-Bad WT, Si 70A and Si 70D were fixed andstained with PT and analyzed using FACS. Cell cycle analysis was performed bymeasuring DNA content and cell size. Cells were classified as being in Gi, S orG2 phase.(B) MC/9-Bcl-xL and MC/9-Bcl-xL Flag-Bad WT, S17OA and S17OD weresynchronized at Gi and released. Cells were fixed and stained with P1 andanalyzed using FACS. The percentage of cells in Gi, S or G2 was determinedover a 0 to 10 hour time course after release.59Cells expressing higher or lower levels of Bad mutant (Bad Si 70A, Bad Si 70D)and wild type proteins were directly compared (Fig. 3.7B). This experiment supportedinitial findings and showed once again that cells expressing Bad Si 70A appeared to takelonger to transit through S phase as compared to cells expressing Bad S17OD (Fig 3.6).Interestingly, the cell line showing high level of expression of Bad Si 70D showed adecrease in the percentage of cells in S phase. Conversely, increase expression of BadSi 70A resulted in an even greater increase in the number of cells in S phase. Thereforethe effects of Bad Si 70A and Si 70D expression appears to be dose dependent, whichfurther validates our initial findings indicating that phosphorylation of Sen 70, or lack of,is responsible for the observed cell cycle effect.ABad SI7OD clones Bad S17QA clones%j;:à:::46IB___.1:___ ___READ_____ _____[•I r•.S17(’A S170A S17OD S170[) BclxL#6 #3 #1 #4Figure 3.7 Increased levels of Bad S17OA or Bad S17OD results in increased cellcycle effect.(A) Lysates from clonal populations of MC/9-BclxL cells (BclxL) expressing BadSi 70A and Bad Si 70D, were immunoprecipitated using anti-Flag antibody andwestern blotted using anti-Flag antibody.(B) Cell cycle analysis was performed using clonal populations of MC/9-BclxL cells(BclxL) expressing Bad Si 70A and Bad Si 70D. Cell lysates of respective clonalpopulations were separated by SDS-PAGE and immunoblotted using anti-Flagantibody.603.2.5 Bad Interacts with Cell Cycle MachineryBased on the above findings demonstrating that Bad, depending on itsphosphorylation state at Ser 170, can influence the time required for cells to transitionthrough S phase; we were interested in examining the molecular mechanism by whichBad is able to affect cell cycle progression. We first examined the possibility that Badinteracts with cell cycle regulatory proteins. This was assessed by means ofimmunoprecipitation experiments. Endogenous Bad was immunoprecipitated and boundproteins were separated using SDS PAGE. Immuno-detection was performed using aseries of antibodies against a wide range of cell cycle regulatory proteins (for exampleCdkl, Cdk2, Cdk4, Cdk6, Cyclin B, Cyclin D, p27, and p21); however we were only ableto consistently detect Cyclin-Dependent Kinase 2 (Cdk2) within the immuno-complex.This Bad/Cdk2 interaction was observed using both MC/9 cells and primary mast cells(mouse) (Fig. 3.8A) while immunoprecipitating endogenous Bad.Based on the above results, we were interested in examining if the observedBad/Cdk2 interaction is part of a greater complex of proteins. More specifically, we wereinterested in knowing if Cdk2’s GuS cyclin partners, Cyclin A and E, also formed part ofthis interaction. Performing similar experiments as described above, we observed aconsistent detection of Cyclin E; however, we were unable to detect any Cyclin A withinthe complex (Fig. 3 .8A). To further strengthen the specificity argument of theseinteractions, cells expressing Flag-tagged Bad were used. When Flag-Bad wasimmunoprecipitated using anti-Flag antibody, association with both cyclin E and Cdk2were also observed (Fig. 3.8B), showing that this was not a result of non-specific coimmunoprecipitation.61AIP:BAD TLAb MC9 MC9Cdk2*________________BadIP:BAD TLAb MC9 MC9______________CycEIBadIP:BAD TLMC9 MC9 Ab— CycAi 4IIBadCdk2*BadCdk2*CycEIP:Bad TLAb Mast Mast—Figure 3.8 Bad Co-Immunoprecipitates with Cdk2 and CyclinE(A) MC/9 (MC9) and bone-marrow derived mast (Mast) cell extracts wereimmunoprecipitated (IP) using anti-Bad antibody and the immuno-complex wasseparated by SDS-PAGE and immunoblotted with anti- Bad (Bad), Cdk2(CDK2), Cyclin E (CycE) and Cyclin A (CycA) antibodies.(B) MC/9-BclxL-Flag-tagged Bad (FBad) cell extracts were immunoprecipitated (IP)using anti-Flag antibody and separated by SDS-PAGE and immunoblotted withanti-Bad (BAD), Cdk2 (CDK2) and Cyclin E (CycE) antibodies.*Denotes Ig L*Denotes Ig LBIP: FLAGFBad Abtarn’* —Bad*Denotes Ig L62We also performed immunoprecipitation experiments using Cdk2 and Cyclin Eantibodies and immuno-blotted using Bad antibody (Fig. 3.9A,B). In both cases, Bad wasdetected within the immunoprecipitated complex. Together, these results suggest thatBad, Cdk2, and Cyclin E form a complex or are part of a larger complex of proteins.A*BadCycE IPCycEr —‘:*1iCdk2 IPAb IP TL1.1m1LnrzFigure 3.9 Cdk2 and CyclinE Co-Immunoprecipitate with Bad(A) MC/9 cell extracts were inimunoprecipitated (IP) using anti-CyclinE antibody(CycE IP) and the immuno-complex was separated by SDS-PAGE andimmunoblotted with anti- Bad (BAD) and Cyclin E (CycE) antibodies.(B) MC/9 cell extracts were immunoprecipitated (1P) using anti-Cdk2 antibody andseparated by SDS-PAGE and immunoblotted with anti-Bad (Bad), Cdk2 (CDK2)antibodies.Ab IP TL-B*BadCdk2**Denotes Ig L633.2.6 Bad’s Interaction with Cdk2 and Cyclin E occurs in the cytosolSince both Cyclin E and its catalytic subunit, Cdk2, translocate to the nucleuswhere they regulate the phosphorylation of many proteins including retinoblastoma, itwas of interest to detennine if the observed Bad/Cdk2 and Bad/CyclinE interactionsoccur specifically in the nucleus. Figure 3.1OA is meant to demonstrate the efficacy of thenuclear preparations. This was performed by immunoblot analysis of both the cytosolicand nuclear fractions using GSK-3 and Histone Hi antibodies. GSK-3 is known tolocalize to the cytosol and Histone Hi is found within the nuclear fraction. The detectionof both these proteins enabled us to demonstrate that our nuclear preparations hadminimal contamination of cytosolic proteins (Fig. 3.1 OA). Nuclear and cytosolic lysatefractions were immunoprecipitated using anti-Bad antibody and immuno-detection ofbound proteins was performed using anti-Cdk2 and Cyclin E antibodies. We did notdetect any interaction between Bad and Cdk2 or Cyclin E when using nuclear fractions;though, we did observe Bad/Cdk2/CyclinE interactions when using cytosolic fractions(Fig. 3.1OB,C). Since there is little evidence in the literature that Bad is present in thenucleus, in addition to the great majority of Cdk2 and Cyclin E expression being incytosolic fraction, it was not surprising that no association of the proteins in the nucleuswas detected.64A___________C NMC9 BdxL 8d2 MC9 BdxL Bd2tP:BAD TLjir --—— r —____*Denotes Iq LCCycEBadTL IPAb C N C N. IFigure 3.10 Bad interacts with Cdk2/CyclinE in the cytosol(A) Cytosolic and nuclear fractions of MC/9 (MC9); MC/9- BclxL (BclxL); andMC/9-Bc12 (Bcl2) cell lysates were separated by SDS-PAGE and immunoblottedusing anti-Gsk3 (GSK3) and Histone Hi (Hill) antibodies.(B) Nuclear (N) and cytosolic (C) fractions of MC/9 cells expressing either BclxL(BclxL) or Bcl-2 (Bcl2) were used to perform immunoprecipitation experimentsusing anti-Bad antibody (IP:BAD), followed by separation by SDS-PAGE andimmunoblotting with anti- Bad (BAD)and Cdk2 (CDK2),(C) Nuclear (N) and cytosolic (C) fractions of MC/9 cell extracts were used toperform immunoprecipitation experiments using anti-Bad antibody (IP), followedby separation by SDS-PAGE and immunoblotting with anti-Cyclin E (CycE) andBad (Bad) antibodies.C NMC9 8dxL 8c12 MC9 BcIxL Bc12GSK3 —BCdk2BadN CAb BcxL BcL2 8cIxL Bc12_______HHIN C -BclxL Bc12 BclxL Bc1265It has been recently reported that BadJBcl-xL complex affectscell cycleprogression (248); therefore, we investigated the effects of overexpressingBad’s pro-survival binding partners, Bcl-xL and Bcl-2, on the formation of the Bad!Cdk2andBadlCyclin E complexes. Using parental MC/9 and MC/9 cells overexpressingeitherBcl-xL or Bcl-2 (Fig. 3.11 A), immunoprecipitation experiments using anti-Badantibodyshowed that overexpression of BclxL, as compared to the overexpressionof Bcl-2 andendogenous levels of Bcl-xL, negatively affected the amount of Cdk2associated withBad (Fig 3.1 1B); however, we did not observe a significant change in theamount ofassociated Cyclin E (Fig 3.11 C).Together these findings indicate Bad, a pro-death Bcl-2 member, isable to hindercell cycle progression, specifically at the Gl-S transition; and Bad is ableto interactdirectly with established GuS cell cycle regulators Cdk2and Cyclin E in the cytosol; andfurthennore, expression of Bad’s survival antagonist,Bcl-xL, appears to effect Bad’sability to associate with Cdk2.66ABcIxL MC9 Bc12BCLXLBADII——IBc12 BcIxL MC9BCL2ICycEBIP:BADAb MC9 BcIxL BcI-2Cdk2 —* —‘----— — —BADCCycEIP:BAD IP:CycEAb BcIxL Bc12 BcIxL Bc12 Ab—_— lit’CycEFigure 3.11 Bcl-xL expression interferes with Bad/Cdk2 interaction(A) MC/9 (MC9); MC/9- BclxL (BclxL); and MC/9-Bc12 (Bc12) cells were assessedfor Bcl-xL (BCLxL), Bcl-2 (BCL2), Bad (BAD), and Cdk2 (CDK2) expression.Cyclin E expression levels were assessed via immunoprecipitation of Cyclin Eusing anti-Cyclin E antibody (CycE).(B) MC/9 (MC9); MC/9- BclxL (BclxL); and MC/9-Bc12 (Bc12) cell lysates wereinimunoprecipitated using anti-Bad antibody. Proteins were separated by SDSPAGE and immunoblotted using anti-Cdk2 (CDK2), Bad (BAD) and(C) anti-Cyclin E (CycE) antibody.IP: CycEMC9 BcIxL Bc12 Ab673.2.7 Bad’s Interactions and Cell Cycle StageSince overexpression of Bad S17OA and Bad S17OD showeda cell cycle effect atS phase transition, we were interested in determining whether Bad’sinteraction withCdk2 and Cyclin E was altered at various stages ofthe cell cycle. Using a Gi cell cycleinhibitor, hydroxyurea, which arrests cells by blockingDNA synthesis, we were able tosynchronize cells (Fig 3.1 2A). Over a specified timecourse we analyzed the expressionof Bad, Cdk2, and Cyclin E (Fig. 3.12B,C) and overthe same specified time course wedetermined if the observed Bad/Cdk2/CyclinE interactions changethroughout the cellcycle. These experiments revealed that Cyclin E expression was highestat 0 h anddecreased over the observed time points (0, 2, 4, 6,8 h) after hydroxyurea release,whereas Cdk2 levels remained relatively constant.Bad IP experiments over a similartime course showed that Bad/Cdk2 interaction remained unchangedafter release (Fig3.1 2B), while the amount of interaction betweenBad and Cyclin E changed over thecourse of the cell cycle. More precisely, we observeda greater amount of Cyclin Ecomplexed with Bad at GuS and early S phase of thecell cycle (Fig 3.12C). At this pointwe cannot be sure whether the observed changesin Bad/Cyclin E interactions are simplythe result of increased expression of Cyclin E at lateG 1 and early S phase (Fig 3.1 2C) orthis interaction is truly indicative of a molecular mechanism, involvingBad and Cyclin E,that acts to regulate cell cycle progression. However,since the level of Cdk2 associatedwith Bad appears to remain constant, the increase co-immuniprecipitationof Cyclin Emay result from its association with Cdk2.68ABIP: BADAb 0 hr 1 hr 2 hr 4 hr 6 hr 8 hr 20 hrCdk2 —.— —CBadTL0 hr 1 hr 2 hr 4 hr 6 hr 8 hr 20 hrI — — 1Ohr 2hr 4hr 6hr 8hrCycEI —BADFigure 3.12 Bad associates with CyclinE during GuS phase(A) MC/9 cells were synchronized at late GuS. Times refer to hours after release.Cells were fixed and stained with P1 and were analyzed using FACS.(B) Cell extracts, from corresponding time points, were immunoprecipitated usinganti-Bad antibody (IP:BAD). Immunoprecipated proteins and total lysates (TL)from corresponding extracts were separated by SDS PAGE and immunoblottedwith anti- Cdk2, and Bad antibodies.(C) Time point extracts were immunoprecipitated using anti-Bad antibody (IP:BAD).Immunoprecipitated proteins and total lysates (TL) were separated by SDS PAGEand immunoblotted with anti- Cyclin E and Bad antibodies.A. LA.L.G1S•G2Ohr 2hr 4 hrA.6 hr 8hr lOhrIP: BADAb Ohr 2hr 4hr 6hrCycElø ‘ —TLBADII693.2.8 Co-Immunoprecipitated Cdk2 shows kinase activity against Histone HiproteinSince we have shown that Bad co-interacts with Cdk2 and CyclinE, we wereinterested in determining whether the co-immunoprecipitated Cdk2 was active. HistoneHi peptide substrate, an established Cdk2 subtstrate (306, 307), was used as a Cdk2substrate while performing kinase assays. Using a Gi chemical cell cycle inhibitor,hydroxyurea, we synchronized MC/9 cells at late Gi phase of the cell cycle. Cell lysatesfrom designated time points after release were used to immunoprecipitate Bad. Weevaluated whether Bad’s co-immunoprecipitated complex showed kinase activity againstHistone Hi and whether the activity changed depending on what stage of the cell cyclethe inununoprecipitate was assessed. These experiments reveal that the anti- Badimmunoprecipitated complex has kinase activity against Histone Hi (Fig. 3.13). Thisresult suggests that Cdk2 may be active, though it is difficult to say with certainty that theobserved activity is not a consequence of another kinase that forms part of the coimmunoprecipitated complex that is also able to phosphorylate Histone Hi. We didhowever observe a consistent and significant (t-test, comparing the 3 hour time point withother time points) increase in activity in immunoprecipitated fractions at the 3 hour timepoint after release compared to all other time points except for 0 hour. This findingcorresponds with published data (307, 308) showing peak Cdk2 activity during GuSphase transition and our data showing a majority of cells entering S phase within 4 hoursof Hydroxyurea release (see Fig 3.6 and Fig 3.12). Thus, the increase in activity is likelydue to the overall increase in Cdk2 activity rather than an increase in Cdk2/Badinteraction since we have shown that the level of Cdk2/Bad interaction is not affected bycell cycle stage (see Fig. 3.6). Moreover, we can safely suggest that the increased Cdk2activity in the anti-Bad ip’ s may be directly correlated to the increased level of cyclin Eassociated with Cdk2/Bad complex..7030Time after release* t-test p<0.05n=3Figure 3.13 Bad’s co-immunoprecipitatedcomplex shows kinase activity againstHistone Hi proteinMC/9 cells were synchronized using Hydroxyureaand then released. Lysatescorresponding to various time points afterrelease were used to immunoprecipitateBad using Bad antibody. Controlrepresents non-synchronized cells. Kinaseassays were performed against HistoneHi peptide. The consistently observedpeak in activity at 3 hours after release wasstatistically(*)different from all othertime points measured except 0 hour. Theresults presented are representativeofthree replicate experiments (n3).EC-)c?z>:>ciCuCuI::T2520151050-IF4.*1Control Oh 1.5h 3h4.5h SnI I I713.3 DISCUSSIONRegulation of apoptosis and cell cycle control are both important to oncogenesisand vital to normal homeostasis. Disruption of cell cycle regulation can result inapoptosis and demise of the organism (309-3 11). Conversely, failure to execute apoptosiscorrectly can lead to a cancerous phenotype with abnormal cell cycle kinetics (312, 313).This homeostasis had long been thought as a result of two separate processes, but there isgrowing evidence indicating that the processes of proliferation and apoptosis are coupled.The first portion of this chapter presents data supporting that Bad is highlyregulated by numerous phosphorylation events, at multiple Serine residues (35, 75, 314,315), for which Bad thus serves as a cell ‘sensor.’ We demonstrate that phosphorylationof Bad at Serl7O, as shown indirectly by expression of a mutant form of Bad in which theSerl7O site is mutated to the phospho-mimetic residue aspartate, can reduce its apoptoticeffect. This was shown in experiments comparing the effects of Bad mutants: 3 SA versus3 SA 1 70D and 1 70A versus 1 70D. The apoptotic ability of Bad, depending on the level ofdephosphorylation, strongly supports Bad’s role as an integral sensor of apoptotic andsurvival signals. Furthermore, we show that the neutralizing effect of phosphorylation ofSer 170 may act independently of Bcl-xL expression. Thus, Bad’s ability to promoteapoptosis may not be limited to a mechanism in which Bad antagonizes Bcl-xL. It isimportant to note that since cells studied are overexpressing proteins, one should view theobserved cellular effects with caution. The various fold increase in Bad expression mayresult in cells becoming far more sensitive to cytokine withdrawal regardless of thephosphorylation state of Bad. The high expression of Bad may result in the inability ofthe cell to contain or control Bad’s activity, which may result in Bad protein beinglocated in cellular compartments or interacting with various proteins that in normalconditions it would not.The Bcl-2 family, an established class of regulators of apoptosis, has morerecently been associated with other physiological roles such as cell cycle regulation (250-252, 266, 272, 273, 277). For instance, Bcl-2 and Bcl-xL have been shown to delay cell72cycle re-entry from the resting G0 state, and BAX can accelerate entry into S phase (249,316). Bid’ knockout mice have revealed an intriguing role in both cell proliferation (317)and DNA damage response via ATM (318, 319); and more recently, our group has shownthat Md- 1, a Bcl-2 family member known to suppress cell growth when overexpressed,interacts with inactive Cdki in the nucleus (298), as well as playing a role in checkpointresponse via Chkl kinase (320).Other related studies have implicated Bad, a member of the ‘BH3-only’ sub-groupof the Bcl-2 family of proteins (32, 87, 314), in cell cycle regulation (137, 248, 321). Arecent study has shown that Bad can cause continued cell cycle progression in serumstarvation or contact inhibition conditions and this ability to overcome cell cycle arrest isdirectly affected by the heterodimerization of Bad and Bcl-xL (248). More specifically, ithas been suggested that the ability of BadlBcl-xL heterodimers to push cells into S phasewithout causing significant apoptosis is an indication that the function of Bad may notmerely be to inactivate Bcl-xL or Bc12, but that Bad may be actively involved in cellcycle control. In 2001, Dramsi et al. (137) reported that Bad, when phosphorylated at Ser170, loses much of its apoptotic potency and promotes cell cycle progression. This cellcycle phenomenon, along with parallel studies involving Mci-i and G2/M cell cycleregulators being performed in our lab, initiated many of the studies presented involvingBad and G i/S cell cycle regulatory proteins. In this chapter we have presented evidencethat dephosphorylation of Ser 170 not only primes the cell for death, but also hinders cellcycle progression. Specifically, cells expressing Bad S17OA, a mutation mimickingdephosphorylation, had a higher percentage of cells in the S phase of the cell cycle;indicating the cells were taking longer to duplicate their DNA as compared to cellsexpressing a mutant form mimicking phosphorylation (Bad Si 70D) and wild type Bad(Bad WT). This was further supported when examining individual mutant clones withvarying levels of expression. If Bad’s phosphorylation state at Ser 170 was affecting cellcycle progression, one would expect that increasing the expression level of either mutantshould magnify the effects, and this is precisely what we observed. In addition, it isimportant to note that the observed cell cycle effect was unrelated to apoptosis, sincethese cells, regardless of which Bad Ser 170 mutant was expressed, underwent similarlow levels of apoptosis when maintained in the appropriate growth conditions. These73observations together suggest that phosphorylation of Bad at Sen 70 can control a uniquefunction of this protein. Hence, Bad may be playing a role in the coupling of twoprocesses involved in cell expansion: cell survival and cell proliferation.Even though several studies have implicated Bcl-2 family members with cellcycle regulation, very few studies have revealed any direct connection between the cellcycle and apoptotic machines. Here we have presented evidence that Bad was able toassociate with the cyclin dependent kinase, Cdk-2, and its 01/S cyclin partner, Cyclin E.This association was tested using antibodies against Bad, Flag-Bad, Cdk2 and Cylin E. Inall cases, independent of which antibody is used for immunoprecipitation, we were ableto observe Bad’s association with Cdk2 and Cyclin E.The GuS cell cycle checkpoint represents a critical period for cells to commit togrowth arrest or proliferation. This stage also represents a period where the cells areresponsive to cytokines; therefore, once cells are committed to enter S phase, additionalstimulation by growth factors is surplus to the cell’s proliferation requirements (214).That is, once the Rb is activated by phosphorylation by Cyclin D/Cdk-4, or -6 complexesin early Gi and in late 01 by Cyclin E/Cdk2 complexes; and Rb is able to dissociatefrom its repressor, E2FI, the cell has committed to entering the cell cycle process. SinceCdk2/Cyclin E phosphorylates Rb in the nucleus, we were interested in examiningwhether the observed association between Bad, Cdk2 and Cyclin E was altereddepending on its cellular localization. From these studies we observed no interaction inthe nucleus and all the observed interaction was shown to occur in the cytosol. Todetermine whether Bad’s association with Cdk2 and Cyclin is altered depending on thestage of the cell cycle, we synchronized the cells and immunoprecipitated Bad from celllysates from 01 through late S phase. Bad’s association with Cdk2 did not alterthroughout the cell cycle, however Bad showed increased association with Cyclin Eduring late 01 and early S phase. The association decreased substantially by mid S phase.This observation is complicated by the fact that Cyclin E showed its greatest levels ofexpression during the same period of the cell cycle; therefore it is difficult to say withcertainty that the increase in association between Bad and Cyclin E is not a reflection ofthe increased Cyclin E expression as opposed to an insight into the mechanism by which74Bad may function to regulate cell cycle progression. We also tested whether theimmunoprecipitated complex show kinase activity against Histone Hi, a known Cdk2kinase target. We showed that the immuno-complex has kinase activity against HistoneHi and this activity is highest when cells are in S phase. Together this, supports ourfinding that Bad interacts with Cdk2, however it has not yet been shown with certaintythat Cdk2 is responsible for the observed kinase activity.Further attempts to understand which factors may be affecting Bad’s associationwith the above cell cycle regulators lead us to test whether expression of Bad’s apoptosisantagonists, Bcl-xL and Bcl-2, had an effect. We did not observe an effect on Bad’sassociation with cyclin E and Cdk2 when expressing Bcl-2; however we did observe adecrease in the levels of association between Bad and Cyclin E in cells expressing highlevels of Bcl-xL. Since both Bcl-xL and Bcl-2 are known Bad heterodimer partners, thedifference observed may be due to Bad’s higher affinity for Bcl-xL as compared to BcI-2.On the other hand, the observed difference may in fact be a hint into how Bad’sassociation with Cdk2 and Cyclin E is controlled.We performed further studies examining the effect of phosphorylation of Ser 170and Bad’s association with Cdk2, Cyclin E and Bcl-xL. In support of previouslypublished findings, we did not observe a relationship in the levels of Bad and Bcl-xLassociation and the phosphorylation state of Bad Ser 170. When examining the levels ofCdk2 (Cyclin E exp. in progress) associated with Bad, we failed to observe anysignificant changes regardless of whether Bad was mutated to mimic phosphorylation ordephosphorylation at Ser 170. These findings indicate that even though phosphorylationof Bad at Ser 170 appears to have a specific effect on late Gi and early S phase, theinteraction of Bad and Cdk2 or Bad and Cyclin E may be an indirect relationshipcontrolled by means other than Bad’s phosphorylation state at Ser-170. Thus, themolecular target of Bad when dephosphorylated at Ser 170 remains to be elucidated. Thecell cycle mechanism of the Bcl-2 family may involve complex regulation of bothinhibitors and activators of key cell cycle mediators. Bad may be playing an active role incell cycle regulation or it is possible that Bad may be acting similarly to its proposedsensory function. Therefore, the association may be a result of a mechanism allowing the75apoptotic machinery to sense the proper operation of the cell cycle. Furthermore, studiesdemonstrating cell cycle activities of Bc12, Bcl-xL, and Bad, together with their knownmitochondrial membrane localization, also raises the possibility that the mitochondriaand their energy producing function may have a role in the observed cell cycle effect.Together, the results in this chapter strengthen the notion that Bad’s apoptoticeffect is modulated by its numerous phosphorylation sites and that phosphorylation ofBad at the Serl7O site plays an important role in the regulation of its pro-apoptoticfunction. Moreover, the neutralizing effect of the phospho-mimetic mutation at Ser 170does not appear to require the expression of Bcl-xL. This observation is in agreementwith previous findings demonstrating that the phosphorylation state of Ser 170 does noteffect Bad’s ability to heterodimerize Bcl-xL. Moreover, the observations that Bad,depending on the phosphorylation state of Sen 70, can influence cell cycle progression,combined with the novel interaction with cell cycle regulators Cdk2 and Cyclin E, revealsa novel convergence between two major events controlling total cellular content in anycomplex organism: apoptosis and cell division.76Chapter 4CAMKII- MEDIATES PHOSPHORYLATION OF BAD AT SER17O4.1 INTRODUCTIONOne of the highly regulated BH3-only proteinsis Bad, whose pro-apoptoticfunction was shown to be inhibited by phosphorylation at multiplesites (123, 134). Asdiscussed earlier, Bad and its multiple phosphorylation sites (Ser-1 12, 128, 136, 155,and170 in the murine protein) is able to converge signals from numerous pathways aswell assensitize cells to apoptotic signals. These apoptotic signals are manifestedin the theblocking of upstream kinases that are able to phosphorylate Bad.Bad is constitutively expressed at varying levelsin all healthy mammalian cellsand is generally maintained in a hyperphosphorylated state by severalkinase pathwaysincluding PKA (135, 136), p90rsk (322) and PKB/AKT (131, 133).The latter, which isthe most widely reported, is thought to play an importantrole in the well known survivaleffects of the P13-kinase pathway. However, we and others have foundthat this pathwaymay have no role in Bad phosphorylation in some cell types (112, 323). Nevertheless,death signals that result in dephosphorylation of Bad convertthe Bad protein into asurvival antagonist. As mentioned earlier, Bad selectively bindsand neutralizes antiapoptotic molecules, particularly Bcl-xL (65, 96, 130); therebypermitting activation ofthe multi-domain pro-apoptotic molecules, Bax and Bak.Studies analyzing phosphorylation of Bad, performedin our laboratory,discovered a site near the carboxy terminus,Serl7O, as an additional site ofphosphorylation involved in regulation of Bad (137).As presented in chapter 3, thedegree of phosphorylation of Serl7O site appears to affect notonly Bad’s ability topromote apoptosis, but also cell cycle progression. In this chapterwe describe the studiesand results that lead to the identification of the kinase that regulates phosphorylationofSerl7O on murine Bad (corresponding to Ser134 of human Bad),and thus demonstrate adirect role of this kinase in cell survival and proliferation.774.2 RESULTS4.2.1 Identification of the Bad Serl7OkinaseWe sought the identity of the kinasethat phosphorylates Bad at Ser 170 byassaying kinase activity against a peptide based onthe sequence corresponding to the Ser170 in murine Bad (PRPKSAG). Aswill be shown below, kinase activitythatphosphorylates this site could be readilyassayed using extracts from MC/9 cells,whichwere the cell type first used to identifythis phosphorylation, as well as from othercelltypes, including FDC-P1. We first tested a seriesof known kinase inhibitors: LY-294002,wortmannin, U0126, rapamycin, SB203580and Ro-31-8220 to obtain clues regarding theidentity of the kinase; however, littleor no effect was observed at concentrations knownto selectively block various protein kinases (data notshown). Work done in our lab hadpreviously shown, through the use ofstandard in vitro kinase assays, that thefollowingimmunoprecipitation of kinases: PKB/akt, erki!2,Cdk-2, Cdk-4, Cdk-6, and GSK-3showed no significant activity againstthe peptide compared to their activity againststandard substrates (data not shown).The search for alternative potential kinasesthat may phosphorylate the Si70 siteof Bad was carried out using ScanProsite(http://expasy.org/tools/scanprosite/) againsttheSwissProt database. The short sequencePRPKS was found to be surprisinglyrare in thedatabase, with only 37 hits found (3of which were Bad — human, mouse andrat). A motifscan under high stringency was perfonnedon all 37 proteins to analyze potentialserine/threonine phosphorylation sites.Residue S846 within the protein MSulf-l, whichcorresponded to the serine of the PRPKSsequence, was the only protein foundto have apredicted kinase that phosphorylatedthe site. In this case, Calmodulin-dependentKinaseII was predicted to be a possible kinasethat phosphorylated this site. It was alsoreportedrecently that the checkpoint kinase, Chkl,was able to phosphorylate the Sen 70site inBad (324). As will be described below,these two kinases were further investigatedtodetermine their potential role in phosphorylatingthe Sen 70 site of Bad.784.2.2 MonoQ Column ChromatographyThe separation of Bad-S 170 kinase activity by column chromatography wasperformed. Cell lysates of both MC/9 and FDC-P1 cells were each separated by MonoQand Superdex S200 fractionation. MonoQ fractionation of either MC/9 or FDC-P1resulted in a single peak of kinase activity that co-migrated when fractions were assayedusing the peptide corresponding to Bad-S170, or the peptide: PLSTRLSVSS, a reportedCaMKII substrate (Fig. 4.1A). The peak activity eluted between 0.4 and 0.5 M NaCl.When the same column fractions were assayed using a peptide that is commonly used todetect Chkl activity, there were two major peaks of activity that eluted at earlier points inthe salt gradient (Fig. 4.1 B). This result suggested that Chkl was unlikely to be the kinaseresponsible for S170 phosphorylation.791 2 3 4 5 6 7 8 9 10111213141516. NaCiConcentrattonMonoQ Fractions • CaMKII substrate• Sl7OpeptideB• Chkl substrate_________________1 2 3 4 5 6 7 8 9 1011 12131415 16MonoQ FractionsFigure 4.1 Mono-Q column fractionation and kinase Assay(A) Mono-Q fractionation of MC/9 cell extracts assayed for kinase activity. Thefirst 5 fractions represent column flow-through, at which point the 0-1.0 MNaC1 gradient was begun. In (A), red squares represent activity againstBad-S 170 peptide and blue circles represent activity against a CaMKIIsubstrate, PLSTRLSVSS. The green circles represent the NaC1concentration.(B) Data represents activity against the Chkl substrate in identical fractions tothose shown in panel (A).804.2.3 Superdex S200 Column ChromatographySuperdex S200 fractionation of MC/9 lysatealso showed a single major peak ofkinase activity that was capable of phosphorylatingthe Bad-S 170 peptide. This peak wascentered at an approximate molecular weightof 400-5 00 kDa and overlapped with a peakof activity measured against the CaMKII substrate(Fig. 4.2A). As shown in Fig. 4.2B,the same fractions assayed againsta peptide used to detect Chkl activity showedanoverlapping profile, but the major peaksof activity were clearly distinct and theChklactivity migrated with a much smallermolecular weight. It is important to note thatthemigration of the CaMKII activity withan apparent size of greater than 440 kDaisexpected for the multimeric formsof this kinase family.In order to further purify the fractioncontaining the Bad S170 kinase,weperformed a double column purification; wherethe peak Superdex S200 fraction wasrunon the MonoQ column. Unfortunately, despitenumerous attempts, we found that mostofthe kinase activity was lost following any combinationof these two column purificationsteps and thus further purification by conventionalchromatography was not pursued.To verify that CaMKII and Chkl proteinare present in peak fractions predictedfrom the kinase assays, SuperdexS200 column fractions were precipitatedwith coldacetone, then run on SDS-PAGE and immunoblottedto detect CaMKII or Chkl. Asshown in Figure 2C, the antibody to CaMKIIdetected bands correspondingto theisoforms of the enzyme, which are knownto migrate at approximately 60-65 kDa. Thesebands appeared in Superdex S200 fractions9-12, which correspond to thepeak of kinaseactivity that could phosphorylate theSen 70-containing peptide and the CaMKIIsubstrate. Immunoblotting forthe Chkl enzyme was performed todetermine where itmigrated on the same SuperdexS200 column. The band at approximately50 kDacorresponding to Chkl was detectedin fractions 17-19, which correspondedto the peakof activity against the ChkTide substrate.Together, our data appears to rule out Chklasthe relevant kinase that is responsiblefor phosphorylation of Bad at Serl7O,andsubsequent studies focused on the activityof CaMKII.81A70-440K 230K 67K60S504030/1 2 3 4 5 b7 8 910 11 1213 141516171819Superdex Fractions. Si 70 peptideB • CaMKII substrate12040K 230K 67K•Chkl Substrate1 2 3 4 5 6 7 8 91011 12 13141516 171819Superctex FractionsC9 10 11 12 13 14—— CaMKII15 16 17 18 19 2050 KDa— ChklFigure 4.2 Superdex S200 fraction and kinase Assay(A) Superdex S200 fractionation of MC/9 cell extracts. The blue dots representkinase activity against Bad-S 170 peptide and black dots represent activityagainst the CaMKII substrate.(B) The activity against the Chkl substrate was monitored in the same fractionsas in panel (A). Migration of three molecular weight standards is indicatedin panel (A) and (B).(C) Column fractions numbers correspond to those in Fig. 2 (A) and (B). Peakfractions of Serl7O and ACT III kinase activity (9-11) correspond tofractions in which CaMKII isoforms of 60-65 kDa are detected. Fractions18 and 19, which show a peak of activity against the Chkl substrate, have aband below 55kDa, which is the size expected for Chkl.824.2.4 CaMKII inhibitor studiesThe column fractionation experiments and immunoblot analysis providedgoodevidence that CaMKII co-migrated with the peak fractions from eitherthe MonoQ orSuperdex S200 columns. In order to test whether CaMKII was responsible fortheobserved activity against both the Bad Si 70 and commerciallyavailable CaMKIIsubstrate (AutoCamTide II peptide), we performed a series of experiments usingaCaMKII inhibitor, KN-93 (lOuM final concentration). KN93 is widelyused and has beendescribed as a selective inhibitor of CaMKII kinase activity (325).Our first set ofexperiments involved verifying KN93’s ability to inhibit CaMKIIactivity (Fig. 4.3A).Using purified CaMKII protein, we assessed the activity against AutoCamTideII peptide(ACTII) and tested whether the activity was specific. We observed very littlephosphorylation of Histone Hi and H2B; and the high levels of phosphorylationagainstACTII were reduced by nearly 70% in the presenceof KN93. These confirmatoryexperiments led to further inhibitor studies wherewe examined whether the fractionsshowing peak activity against both the CaMKII substrate andBad S 170-containingpeptide could be inhibited using KN93. Based on these studieswe observed that peakfractions from either the MonoQ (Fig. 4.3B) or Superdex S200A B- 16010• Nohbftorb 120• 5170peptideI I::____CaMKII substrateCaMK Hi H2 Peak KN93subFigure 4.3 Column activity and KN93(A) The kinase activity of purified CaMKII was assayed against commerciallyavailable CaMKII substrate, Histone Hi, HistoneH2B; in the presence andabsence of CaMKII inhibitor, KN93 (10 jig/ml). Thisfigure is arepresentative of 3 separate experiments.(B) The kinase activity was determined for MonoQ columnpeak fraction(corresponding to fraction 14 in Fig. 4.1A) with and withoutKN93.83(Fig. 4.4A) are greatly inhibited by KN93. Inhibition by KN93 reduced activity of peakfractions from either column by nearly 80%. Furthennore, the inactive analog (KN92)had no inhibitory effect (Fig. 4.4B).E&*IFigure 4.4 Superdex column activity and KN93(A) Superdex S200 column peak fraction (corresponding to fraction 10 in Fig.2A) was assayed for kinase activity against either the Bad S 170-containingpeptide, or the AutoCamTide II peptide (ACT III); in the presence orabsence of the CaMKII inhibitor, KN-93 (10 jig/mi). Results are averagesof triplicate determinations +1- standard deviation from a single experiment.Similar results were obtained in at least 3 independent experiments.(B) Superdex S200 column peak fraction (corresponding to fraction 10 in Fig.4.2A) was assayed for kinase activity against Bad S 170-containing peptide;in the presence or absence of the CaMKII inhibitor, KN-93 (10 jig/mi) andit’s inactive analog KN92. Similar results were obtained in at least 3independent experiments.A Ba 5170 pptidci CaMKH subtratPeak KN93a si 70 pptidepeak KN9 KN9284Further data supporting CaMKII as a Bad Serl7O kinase, was derived fromimmunoprecipitation experiments. Using MC/9 cell lysates, CaMKII wasimmunoprecipitated, using anti-CaMKII antibody, and subsequently tested to determinewhether the immunoprecipitate showed kinase activity against Bad Ser 170 peptide. Asshown in figure 4.5, immunoprecipitated CaMKII showed activity against both acommercially available substrate and the Bad Sen 70 peptide. Moreover, KN93 inhibitedthe observed activity, hence eliminating the possibility that unknown proteins forming acomplex with CaMKII may be responsible for the observed activity.Therefore, fractionation of cellular proteins by size or by charge results in singlefractions, shown to contain CaMKII protein, which is able to phosphorylate both CaMKIIsubstrate peptide and the Si 70 peptide; and the activity against both substrates are greatlyreduced by the selective CaMKII inhibitor KN93. This marked decrease in activityagainst the Si 70 peptide in the presence of CaMKII inhibitor, suggests that CaMKII isable and is responsible for the majority of the observed phosphorylation of Bad at Ser170.4030 • NoKN93KN93Bad S170 ACTIIIpeptideFigure 4.5 Kinase activity of immunoprecipitated CaMKIICaMKII was immunoprecipitated using anti-CaMKII antibody and kinaseactivity of the immuno-complex was assayed against both the ACTIII andS170 containing peptide; with and without KN93 (10 ig/m1). Similarresults were obtained in at least 3 independent experiments.854.2.5 Kinase activity using purified CaMKII isoformsThe column purification experiments along with theCaMKII inhibitor studiesprovided strong evidence that CaMKII is ableto phosphorylate Bad S 170-containingpeptide. Therefore, we were interested in determining whichof the four major isoformsof CaMKII (a, J3, y, and ö) were present in the peak fractionsof both the MonoQ andSuperdex S200 column purification. The peaks fromthe column fractionation containingkinase activity were separated using SDS PAGE and we probed immunoblotsfor thepresence of CaMKII isoforms using antibodies against CaMKII-a, f3, y, and ö.Unfortunately, the antibodies available to CaMKII isoforms, particularlyanti-CaMKII- y,and -ö, were not found to be useful in immunoblots to distinguish between them.As wewere unable to detect which protein isoforms were expressedin cell types of interest, wedid however obtain commercial sources of purified isoforms ofCaMKII- a, f3,y,andwhich allowed us to test for their ability to phosphorylatethe peptide corresponding toBad S 170 or the CaMKII substrate peptide. As shownin Figure 4.6, all of these kinasesphosphorylated the CaMKII substrate, with some differencesin their relative level ofactivity that might be explained by specific activities ofthe enzyme preparations.However, when assayed using the peptide correspondingto Bad S 170, there was astriking difference in the activities, with only the gamma isoformof CaMKII havingrobust activity, while the others had little to no activity. As expected,the KN-93 inhibitorwas able to block the activity of each of the isoforms, in assaysagainst both substrates,although in our hands it may have been slightly less effective against CaMKII-a.86700EQ.C.)0x>>ci)Cl)(‘3Figure 4.6 Activity of purified CaMKII isoformsRecombinant CaMKII- ct, f3, y, and 6 were used in kinase assays againsteither the Bad S 170-containing peptide (Bad S 170) or AutoCamTide III(ACTIII), in the presence or absence of KN-93 (10 igJm1).6005004003002001000ITITTIlTBad S170 Bad S170+ KN93U CaMKII- pB CaMKII-y• CaMKII- 6• •CaMKII-ctIACTIII ACTIII+ KN93874.2.6 siRNA knockdown experiment of CaMKIITo confirm that CaMKII-’ was the cellular kinase responsible for the Bad S 170kinase activity, we used 3T6 cells treated with siRNA to knock down expression ofCaMKII-’y. 3T6 cells were used since siRNA knockdown, based on past experience,cannot be successfully done in either MC/9 or FDC-P1 cells. To ensure 3T6 cells hadsimilar kinase activity profiles, as compared to MC/9 and FDC-P 1, we performedSuperdex S200 fractionation of 3T6 lysates (Fig 4.7A). The fractions were tested foractivity against Bad Ser- 170 peptide. The profile was nearly identical to that seen whenusing MC/9 and FDC-P1 cells (see Fig. 4.2). Fraction #10 showed the greatest amount ofkinase activity against the Bad S 170 peptide and this activity was greatly reduced whentreated with CaMKII inhibitor KN93 (Fig 4.7B).ABE0x0< 20800604020#10 #10+KN93Figure 4.7 Superdex S200 fraction and kinase assay of 3T6 lysates(A) Superdex S200 fractionation of 3T6 cell extracts. The black dots representkinase activity of respective fractions against Bad-S 170 peptide.(B) Kinase activity of superdex S200 peak fraction (#10) with and withoutCaMKII inhibitor (KN93)6040440K 230K 67K1 2 3 4 5 6 7 8 9 10 11 12 13 1415 16 17 1819Fractions88We chose siRNA specificfor CaMKII-3 as a negativecontrol, since we hadalready demonstrated thatthe purified form of this isoform wasunable to phosphorylateBad S 170 peptide. In supportof this result we didnot observe any decrease in kinaseactivity against Si 70 peptidewhen cells were treatedwith siRNA, specific for CaMKII3. However, when cells were treatedwith siRNA specific for CaMKII-y,the activity ofcell lysates against both substrateswas significantly reduced(Fig.4.8). Knockdown ofCaMKII-’’ had a greater effecton the kinase activity againstBad Sen 70 peptide thanagainst the CaMKII substratepeptide. The appearance ofa differential inhibition wassomewhat expected as the activity ofthe remaining isoforms is thoughtto contribute tothe total CaMKII activityobserved. We were eager tofollow up these studies withwestern blot analysis in orderto verify that CaMKII-y proteinlevels had in fact beenknocked down; but at thetime these experiments wereperformed, there were nocommercially availableCaMKII-y antibodies.Figure 4.8 CaMIUI--directedsiRNA decreases kinase activityagainst Bad S170Represent studies performedthree times. 3T6 cellswere untreated(Control), or incubated withsiRNA directed against CaMKII-3or—y,asindicated. Cell lysates wereused in a kinase assay usingBad Si70-containing peptide(S 170) or AutoCamTide III (ACTIII) as indicated.Results are averages of triplicatedeterminations +7- standarddeviationfrom a single experiment.Similar results were obtainedin at least 3independent experiments.90jg606040t5$ 20C100O SI 70 peptideR ACTIIIGamma sIRNA Beta sIRNAControl894.3 DISCUSSIONMuch work has been conducted to understand the mechanismby which Bcl-2family members control mitochondrial mediated cell death. The BH3-only subfamily ofproteins have been described as “sensitizers” since they influence the apoptotic responsebut are unable to disrupt the mitochondrial membrane as can be doneby the “effector”type Bcl-2 family proteins, Bax and Bak. Bad is one of the BH3-only proteins, and it hasa unique role as a sensor of multiple upstream kinases that target its phosphorylation atmultiple sites. Phosphorylation of Bad at Serll2 and Ser136 can allow association withcytosolic 14-3-3 proteins, and phosphorylation at Ser155, which is in the BH3 domain,serves to disrupt associations with Bcl-2 and Bcl-xL. Inearlier sections, we showevidence that phosphorylation of Bad at Serl7O plays a role in regulation of Bad’s proapoptotic activity and a novel cell cycle regulatory function. Even thoughthe Sen 70 sitewas first discovered in 2000, no studies to date had characterizedthe kinase responsiblefor phosphorylation of Bad at Serl7O, which became the focusof the studies in thischapter.When the Serl7O phosphorylation site was first described, it was shown that itsphosphorylation was not dependent upon either PI3K!PKB or MEK!erk dependentpathways (137, 326). Other relevant studies reported thata peptide based on thisphosphorylation site could be phosphorylated by the checkpoint kinase, Chkl (324). Amore recent study describes Pim3 as being able to phosphorylate Badat multiple serineresidues including Sen 70 (327), though the cellular effect of Bad being phosphorylatedat Serl7O is not pursued.Our initial studies established conditions under which we could readily detectkinase activity in specific cell extracts (MC/9 and FDC-P1). Using Bad Ser 170containing peptide, we then proceeded to examine a series of rational kinase candidatesby testing their respective inhibitors. We were unsuccessful in our initial attempts, thoughpredictive software did eventually help us identify a promisinglead candidate.In a series of in vitro assays of kinase activity, we have presented convincingevidence that the primary activity that is responsible forphosphorylation of a peptide90encoding the Si 70 of Bad, corresponds toCaMKII. The use of a selective inhibitorofCaMKII, KN-93, reinforces the identityof the S170 kinase as CaMKII. The analog,KN92, which has no effect on CaMKII,also does not inhibit Si 70 activity.Furthermore,kinase activity of immunoprecipitatedCaMKII also showed activity against the S170 site.In further characterization of the differentCaMKII isoforms that might beresponsible forphosphorylation of Bad-S 170, we weresurprised to find a striking difference intheability of the four enzymes to phosphorylatethis site. While each of the purifiedenzymescould phosphorylate the CaMKJI substrate,and the activity was inhibitable by KN-93,only CaMKII-y phosphorylated the peptideencoding S 170.Another intriguing aspect of the studiesis the specificity of the CaMKII-yenzyme. Comparison of the fourisoforms of CaMKII shows that they arehighlyhomologous, particularly in the kinasedomain. The Seri7O site inBad is a non-conventional site of phosphorylationthat is unlike any other known kinase substrate.Therefore, it will be interesting to investigatethe features of the CaMKII1’enzyme thatresults in its unique ability to phosphorylateBad at Serl7O. Finally, the role of CaMKII-yin phosphorylating Bad combined withthe data indicating the effects ofBad Ser 170phosphorylation on promoting cell survivaland cell cycle survival may lead to furthernovel studies, most likely havinga greater impact in the field of neuroscienceas both Badand CaMKII have already been establishedas key proteins in the function and survivalofneuronal cells.91Chapter 5Cellular Effects of CaMKII Activation and Bad Phosphorylation at Serl7O5.1 INTRODUCTIONCaMKII is a ubiquitous serine/threonine protein kinase that is activated bycalcium and CaM and is known to phosphorylate diverse substrates involved in multiplecellular functions including metabolism, neurotransmitter release, membrane fusion, cellcycle control and apoptosis (328-33 1). As shown in chapter 4, we have providedevidence that CaMKII, and more specifically, CaMKII-y, is able to directlyphosphorylate Bad at Sen 70, indicating CaMKII-y can act as a signaling intermediaterelaying survival signals by neutralizing Bad’s apoptotic ability as well as affecting Bad’scell cycle effect.Bad is a pro-apoptotic member of the Bcl-2 family of proteins that is thought toexert a death-promoting effect by heterodimerizing with Bcl-xL and disrupting its antiapoptotic activity. Bad’s apoptotic function is modulated by its phosphorylation atmultiple serine sites including Ser-170. The Ser-170 site was first discovered as a sitethat was hyperphosphorylated when MC/9 cells were stimulated with IL-3. Moreover,phosphorylation at Ser-170 was found to inhibit Bad’s apoptotic functions and thusprovides a probable means by which IL-3 promotes survival. However, the kinaseresponsible for relaying the IL-3 survival signal and phosphorylating Bad at Serl7O hadremained elusive until now. As such, this chapter aims to describe the effects of variousstimulations on CaMKII activation and whether the inhibition of CaMKII effects cellsurvival.5.2 RESULTS5.2.1 Cytokine activation of CaMKII activityThe ability of cytokines to activate CaMKII activity has never been described andtherefore we investigated whether the CaMKII activity in FDC-P 1 cells was modulated92by cytokine deprivation and re-addition. We utilized the peak kinase activity fromSuperdex S200 fractionation which we had previously shown to contain CaMKII. WhenFDC-P 1 cells were deprived of cytokine for 4 hours, which we have shown previouslywas able to almost completely suppress activity of other kinases such as PKB or erk 1/2(332), the kinase activity within the peak fraction was reduced by approximately 30%when assayed against Bad S170 peptide (5.1A). However, when the kinase activity wasassayed against CaMKII substrate (5.1 B), the reduction in activity was not as pronounced(15%). These results suggest that IL-3 signaling increases CaMKII activity against bothBad Serl7O and CaMKII substrate. The difference in activity levels may be aconsequence of CaMKII isoforms being differentially activated by IL-3 stimulatiOn.Since all the isoforms have been shown to phosphorylate the CaMKII substrate, thechange in one or more isoforms activity levels would be difficult to capture in this type ofexperiment. Whereas we have shown that CaMKII-y is likely the only isoform thatphosphorylates Bad Serl7O in FDC-P1 cells and as such we can expect a morepronounced effect against the Bad S170 peptide if IL-3 stimulation activates CaMKII.Moreover, the fact that cytokine starvation did not lead to complete suppression ofCaMKII activity is consistent with our previous results showing that when cells werestarved of cytokine, there was always a basal level of Bad phosphorylation specifically atthe Serl7O site, and the level of phosphorylation was increased by cytokine treatment ofcells (peptide 2 in (326)).Re-addition of IL-3 to the starved cells for 20 mm resulted in an increase inactivity back to the pre-starvation levels, when assayed against either the Bad S 170peptide or the CaMKII substrate peptide. The results shown in figure 5.1A and B arerepresentative of several replicate experiments. The data is presented in this manner sincethe experiments were performed with different age radioisotope and as such theradioactivity levels cannot be compared directly.Similar results were obtained when CaMKII activity was assayed followingimmunoprecipitation from cytokine-starved cells and from starved cells treated with IL-3(Figure 5.1 C). At the time these experiments were performed, we did not have antibodiesselective for each of the isoforms, thus we were unable to determine which of the93CaMKII isoforms were affectedby IL-3 stimulation. In morerecent studies done byP.Hojabrpour, immunoprecipitationof CaMKII-y usinga specific antibody hasdemonstrated that stimulationof cytokine-starved FDC-P1 cells with IL-3 resultsin arobust stimulation of kinase activity.The level of activity of the CaMKIIgroup of enzymes canbe closely correlatedwith the level of phosphorylationat a highly conserved autophosphorylationsite, Thr286(numbering from CaMKII-alpha),found in all isozymesof CaMK-II. This has beenshown in studies with specificinhibitors, as well asspecific substrates of the kinase(333,334). Phosphorylation atthis “autonomy site” enablesCaMK-II to remain active aftertheinteraction with Ca+2/CaMhas ended (33 3-335). Consistentwith our previous findingsusing Superdex S200 peakfractions, stimulation of cytokine-starvedFDC-Pl cells withIL-3 resulted in an increase in phosphorylationof Thr286 on CaMKII (Figure5.ID).Related studies just recentlyperformed by P. Hojabrpour,have shown that a similarincrease in autophosphorylationcan be observed in the immunoprecipitatedCaMKII-isoform. Together, these findingsindicate that Bad Ser-170phosphorylation and itsassociated anti-apoptotic effectare controlled by IL-3 signallingvia CaMKII-y activation.94B5040Note Repsentatve of 3 experimentsCortro 4h Starved IL-3 TrtedIL-3 Stimulation10070550 mm 2 mm 5 mm 10 mm%- Phospho-CaMKAE 80&6040200 ‘ITControl 4h 5Thrvd IL-3lreatedNte Rprroentative of 6 xpetmeInsC60E&50403020100DNote Rpresentatlve of 3 expimentL-3 Treated 4h StarvedVinculin95Figure 5.1 IL-3-stimulates kinase activityagainst Bad S170 peptide(A) FDC-Pl cells were harvested after growthunder normal conditions (Control), orafter starvation of cytokine for 4 hours (4hStarvation), or cells were starved for 4hours and re-stimulated with IL-3 for 20 minutes(IL-3 treated). Cell extracts wereseparated by Superdex S200 and kinaseactivity against the peptide correspondingto Bad-S170 was assayed. This isa representative result showing average ofduplicate samples from a single experiment thatwas repeated 6 times with similarresults.(B) Same experimental setup as inA, except Superdex S200 fractions were assayedfor kinase activity against the CaMKIIsubstrate peptide This is a representativeresult showing average of duplicatesamples from a single experiment that wasrepeated 3 times with similar results.(C) FDC-Pl cells were starved of cytokine orre-stimulated with IL-3 as in A. Lysateswere immunoprecipitated with anti-CaMKIIantibody and the immunoprecipitateswere used in a kinase assay withthe Bad-S 170 peptide as substrate. Resultsshown are from a single experiment, but similarresults were obtained in at least 4independent experiments.(D) IL-3 stimulates phosphorylation of CaMKIIat Thr286. FDC-P1 cells were starvedfor 4 hours, followed by stimulation withIL-3 for the time indicated. Cell lysateswere separated by electrophoresis andimmunoblotted using an antibody directedagainst the phosphorylated T286 of CaMKII (phospho-CaMKII).Numbers at leftindicate relative molecular weight(in kDa) of molecular weight standards. Thesame blot was blotted with anti-Vinculin antibody.965.2.2 Inducing apoptosis via cytokine starvation does not affect Bad/Cdk2interactionStemming from experiments analyzing CaMKII’ s kinase activity against Bad Ser170 when cells are starved of cytokine (Fig 5.1), we chose to also test whether cytokinestarvation affects Bad’s ability to interact with Cdk2. As described previously, Bad’sphosphorylation status is altered when starved of cytokine and Bad’s Ser-170 site hadbeen shown to become hyper-phosphorylated when stimulated with cytokine.Admittedly, without understanding the mechanism or the cellular consequences ofBadJCdk2 interaction, we thought to interrogate the effects of cytokine withdrawal on theformation of the BadICdk2 complex. We show in Figure 5.2A that 6 hours of cytokinestarvation has very little effect on Bad’s ability to interact with Cdk2 as compared tounstarved cells. As to avoid the complications associated with comparing studies whencells are undergoing apoptosis, we were limited to a narrow window of starvation timepoints. It was shown that by 8 hours there was a sharp increase in the percentage of cellsshowing initial signs of apoptosis (Fig. 5.2B). The lack of any detectable change in BadCdk2 association following cytokine starvation again gave us no conclusive resultsregarding this event and the phosphorylation of Sen 70.97ABad IPAb Oh 6h TLCdk2 • .• •— -‘:Densitornetry 2974 28772Bad__________________BAnnexin V StainOOLsPh8LStarvation timeFigure 5.2 Inducing apoptosis via IL-3 starvation does not appear to effect Bad’sability to interact with Cdk2(A) MC/9 cells were starved of cytokine for 0 hours (Oh) and 24 hours (24h). Cellextracts were immunoprecipitated using anti-Bad antibody (Bad IP) and theimmuno-complex was separated by SDS-PAGE and immunoblotted with antiCdk2 (CDK2) and anti-Bad (Bad) antibodies. Densitometry was performed inorder to accurately compare the amount of Cdk2 pulled down within the Bad’simmunoprecipated complex. Densitometry measurements of bands correspondingto co-immunoprecipitated Cdk2 at 0 hour and 6 hour after IL-3 stimulation;32974 and 28772 respectively.(B) MC/9 cells were starved of cytokine for 0 (Oh), 2 (2h), 4 (4h), 6 (6h), and 8 hours(8h). Cells were stained with Annexin V and the percentage of cells wasdetermined using FACS.985.2.3 Apoptosis mediated by CaMKII inhibitor is dependent upon Bad S170The possible involvement of CaMKII activity as a regulator of cytokinedependent cell survival, and the correcsponding phosphorylation of Bad at Ser170, wastested by use of the inhibitor KN93. The pharmacological compound with selectiveinhibitory activity against CaMKII, the methoxybenzene sulfonyl derivative KN-93 (336-341), which prevents the activation of CaMK-II by antagonizing CaM binding, ismembrane permeant and has been used in numerous functional studies on living cells(342, 343). As shown in Figure 5.3, when FDC-P1 cells, in the presence of completegrowth medium with IL-3, were incubated with KN93 for 24 hours, at 10 and 50..tMconcentrations, there was a dramatic increase in the level of apoptosis as measured by theextent of annexin V and P1 staining. However, when FDC-Pl cells expressing Bcl-xLwere treated in the same way, cell death was reduced considerably. This finding confirmsthat the apoptosis caused by KN93 was due to its effects on the mitochondrial deathpathway. As previously shown (Fig. 3.2) Bad expression in the cells expressing Bcl-xLcaused them to revert to being dependent upon cytokine for their survival. When thesecells expressing Bad were incubated with KN93 (and still in the presence of cytokine),they also regained sensitivity to the effects of the CaMKII inhibitor. This provides strongevidence in support of the suggestion that the effect of KN93 in causing apoptosis is dueto its inhibition of CaMKII-mediated phosphorylation of Bad at Seri7O. We next testedthe effect of the inhibitor on cells in which Bad Si 70D was expressed, and on these cellsthere was much less effect of KN93, further supporting the hypothesis. Statistical analysisshows that the level of apoptosis caused by KN-93 is significantly different between BadSi 70D expressing cells, and Bad wt cells or parental cells (p<0.05), but not compared tothe cells expressing Bcl-xL alone. It should be noted that the inactive analog, KN92, didnot induce apoptosis at the same concentrations. Together, these results suggest that atleast part of the pro-survival effect of cytokines in blocking Bad’s pr.o-apoptotic activityis due to phosphorylation of Bad by CaMKII at the Serl7O site.99KN93 KN92100I**B0..LiüControlaio40 •SOuM20__n_r::1Hk(FDC-P1 FDCP14dxL FDCP1BcIxL FDCPiBcIxL FDCP1Bad wt Bad 51700*t4est p<0.05Figure 5.3 Induction of apoptosis by the CaMKII inhibitor, KN93KN93 and KN92 was added to cells at OjiM (Control), 10iM (grey bars) or50 1iM (black bars) overnight, after which cells were assayed for P.1 and AnnexinV stain. Results were compiled from 3 experiments and indicate average values+1- standard deviation. Statistical analysis shows that the level of apoptosis causedby KN-93 is significantly different between Bad-Si 70D expressing cells and Badwt cells (p<O.05), but not compared to the cells expressing Bcl-xL alone.Experiment repeated three times (n=3).1005.2.4 Kinase activity against Serl7O peptide increases in S phase of cell cycleA few studies have suggested that CaMKII may play a cell cycle regulatory roleat both the G2-M and Gl-S transitions. For instance, CaMKII inhibitory drugs have beenshown to arrest cells in Gi, and S (336, 344). Other studies, for example, using peptidesthat inhibit CaMKII activity, block sea urchin eggs at the G2-M transition (345) andexpression of a constitutively active CaMKII arrests certain mold and mammalian cells inG2 (346, 347). CaMKII activity and its inhibition is likely to have different cellular andphysiological effects due to the differential expression profiles of CaMKII isoforms ineach of the cell types. Nevertheless, studies have amounted sufficient data to support aconnection between CaMKII activity and cell cycle progression. Together with our dataindicating CaMKII phosphorylates Bad at Ser-170, a site shown to effect cell cycleprogression, we were interested in examining CaMKII activity at different stages of cellcycle progression.Using a Gi chemical cell cycle inhibitor, hydroxyurea, we were able tosynchronize cells (same as Fig. 3 .6A) and over a specified time course determine if thekinase activity against CaMKII substrate (Fig. 5.4A) and Bad Serl7O peptide (Fig. 5.4B)changed throughout the cell cycle. These experiments revealed a consistent, statisticallysignificant, increase in CaMKII kinase activity when cells where synchronized in S phase(3-5 hours after hydroxyurea release). In all cases, when cell extracts were treated withCaMKII inhibitor, KN93, we observed a decrease in kinase activity.101LAOhr 2hr 4hr 6hr 8hrtime course after Flydroea rceaseEUC>U0)Inm• No nhibitorKN93(A) Cell extracts, from corresponding time points after hydroxyurea release,were used to perform kinase assays against CaMKII substrate.(B) Cell extracts, from corresponding time points after hydroxyurea release,were used to perform kinase assays against Bad Serl7O peptide.A• GiS G210 hrINo InhibitorU KN936040200C,00FABFigure 5.40 hr 45 hr 8hrEUnCr>*t4est, p<OO5Ohr 4Shr ShrCells synchronized in S phase show relative increase in CaMKIIkinase activity102In addition to hydroxyurea synchronization, similar kinase assay experiments wereperformed using cells sorted based on cell cycle stage (Fig. 5.6). Again, we observed thatcells in S phase show greater kinase activity against Bad Sen 70 peptide than cells in Giand G2 phases of the cell cycle. Interestingly, the increase in activity at S phasecorresponds to the same stage we originally observed an effect of overexpressing BadSi 70A mutant. Together, these results indicate that CaMKII activity is increased in Sphase, as compared to Gi and G2 phases. Furthermore, together with the cell cycleexperiments shown above with the Bad Serl7O mutants, we can conclude that CaMKIIactivity in phosphorylating Bad at Senl7O may serve a regulatory role in cells progressingthrough S phase of the cell cycle.*t-test,p<0.05Figure 5.5 CaMKII kinase activity increase in S phaseCells sorted on the basis of cell cycle stage: Gi, S or G2 were used to performkinase assays against Bad Ser-170 peptide. Results are representative of threeseparate experiments.ceN sortingEC)x>C)U)(U140120100806040200* *00(U0.E0C.)C)U)(UC)C)C6040200-20-40-60-80I flflIGi S G2 Gi S G21035.3 DISCUSSION:The most convincing evidence we have obtainedto show that CaMKII activitycan mediate phosphorylation of Badat Si 70 and provide a survival effect comes fromincubation of cells with the inhibitorof CaMKII, KN93. In parental cells, or thoseexpressing Bad, KN93 induces apoptosis. Expressionof Bcl-xL largely overcomes thepro-apoptotic effect of KN-93. However, whencells were expressing Bad-Si 70D, theKN93 caused much less apoptosis. Thisresult provides clear evidence to support thesuggestion that CaMKII is responsible forphosphorylation of Bad at the Serl7O site andphosphorylation at that site blocks the pro-apoptoticactivity of Bad. Thus we can firmlysuggest that the effect of KN93 in inducing apoptosisis largely due to its effect onblocking phosphorylation of Bad at Sen 70. Nevertheless,one should note that Badmutant S17OA expression in FDC-P1-BclxL wasonly shown to sensitize cells toapoptosis and requires IL-3 withdrawal for completeinduction of apoptosis. However,these results are difficult to assess as the contribututionof endogenous Bad is notcontrolled for. Moreover, we know thatthe Serl7O site is constitutively phosphorylated,even in starved cells there is some level of Serl7Ophosphorylation (137), which is alsosupported by the CaMKII activity assayspresented.Our results showing KN93 treatment induces apoptosisare supported by a recentstudy using cells transfected with Badand CaMKII. The spiral ganglion neuron (SGN)cells, overexpressing Bad, when grownin reduced serum level (1%) media wouldundergo apoptosis unless co-transfected with CaMKII(206). The authors suggested thatdepolarization of SGN cells increasedCaMKII activity which resulted in the functionalinactivation of Bad, implying that Bad inactivationis a means by which depolarizationand CaMKII promote SGN survival (206,348). These studies also relate to anotherrecent finding that demonstrated KN-93 wasa much more potent inducer of cell deaththan wortmannin, a P13K inhibitor, in prostate cancercells (349). Similarly, it has alsobeen shown that overexpression ofCaMKII-o gene results in resistance to apoptosiswhen treated with doxorubicin, thapsigargin and TRAIL.It was suggested that, based onthese finding, CaMKII expressionplays an important role in prostate cancer cellresistance to apoptosis (208, 349).104In order to elucidate the mechanisms controlling Bad’s association with Cdk2, weperformed a series of experiments examining the effects of DNA damage through the useof UV radiation. Observing the effects of increasing radiation, we were unable to detect achange in the levels of Bad/Cdk2/Cyclin F association. These results however weredifficult to assess as there was a very narrow window of radiation where DNA damagecould be caused without triggering high levels of apoptosis. These findings are similar tothose observed when testing Whether cytokine starvation affected the Bad/Cdk2/Cyclin Ecomplex. We did not observe a significant change in the levels of association during thestarvation time course; however, these experiments are difficult to assess as cells began toundergo apoptosis within 8 hours of starvation (data not shown). The early onset ofapoptosis limited the time course to six hours and, as mentioned above, depending on thestage of the cell cycle cells may respond differently to cytokine withdrawal.We did however show that the kinase activity phosphorylating the Serl7O site isgreater in the S phase of the cell cycle (Fig. 5.4). This was initially done using cellsblocked at GuS by hydroxyurea incubation, then released from the block by washingcells. The kinase activity is consistently higher in the time that most cells are in S phase,and decreases as cells progress into G2. The same type of analysis was also done withnormal cells (not blocked by use of drug) that were sorted into Gi, S and G2 populations.Again, consistently higher Sen 70 kinase activity can be detected when cells are in the Sphase of the cell cycle. Thus, we have this additional evidence that the phosphorylation ofBad at the Sen 70 site is cell cycle regulated, as is the kinase that phosphorylates that site.The activity was also shown to be decreased following cytokine starvation ofcells, and activated by treatment of starved cells with cytokine. Unlike some otherdownstream kinases regulated by cytokine, such as PKB or erkl/2, we did not see acomplete reduction in CaMKII activity during the 4h of cytokine starvation. This result isconsistent with the observation we made previously showing that the S170 site in Bad isalways phosphorylated to some extent in cytokine-starved cells, and its phosphonylationincreases, along with other sites in Bad, upon cytokine stimulation.105In summary, we discovered that in FDC-P 1 cells CaMKII activity is increased inresponse to cytokine treatment, and inhibition of CaMKII correlates with enhancedapoptosis, which is dependent upon the ability of CaMKII to phosphorylate Bad atSeri7O. It is important to specify the cell line used, since it has been shown thatCaMKII’s activity, the expression profile of CaMKII isoforms, and its cellular effectsdiffer between cell types. Nevertheless, these findings provide insight into a novelmechanism regulating Bad and its cell survival and cell cycle functions.Future studies will need to address the question of what protein interactions maybe mediated by the domain of Bad encoding the Serl 70 site, and how phosphorylation ofthat site affects those interactions. For example, we know that Bad-Si 70D, althoughmuch less pro-apoptotic, can still bind to Bcl-xL (137). However, we have notdetermined whether these interactions occur in the cytosol, or at the mitochondrialmembrane. Another interesting question is whether Bad phosphorylated at Serl 70 effectsthe binding to specific 14-3-3 isoforms. The interactions that Bad makes with these, orperhaps other, proteins will likely provide clues as to how the Sen 70 site controls Bad’sapoptotic and cell cycle effects. Furthermore, it would be of interest to determine ifexpression of constitutively active CaMKII, specifically CaMKII-y, de-sensitizes FDCP1 cells from IL-3-withdrawal-related apoptosis or whether the cell cycle is perturbed.106Chapter 6CONCLUSIONApoptosis is controlled by two major pathways,initiated either via cell surfacereceptors (extrinsic) or via disruption of mitochondriaand release of contents into thecytosol (intrinsic) (162, 350). The disruption versusthe maintained integrity of themitochondria is a highly regulated cellular mechanismcontrolled in part by the Bcl-2family of proteins. At the mitochondria, ‘pro-survival’Bcl-2 family members maintainmitochondrial integrity, while pro-apoptotic proteinssuch as Bax and Bak, play a role inperturbing the mitochondrial wall. Disruption ofthe mitochondrial wall results in therelease of mitochondrial contents including cytochromec and numerous other proteinsthat play a role in initiating caspase activation. Theactivation of caspases results in thedegradation of intracellular molecules which eventuallyleads to the death of the cell(162).Bcl-2 was first characterized as an oncogene that wasover-expressed in B-celllymphomas (15). However, unlike the growth-promotingoncogenes that had beendescribed at that time, the oncogenic function of Bcl-2was shown to be due to its abilityto prevent apoptosis (124). Once researchers demonstratedthat Bcl-2’ s form and functionwere evolutionarily conserved, this gave way to a plethoraof studies investigating boththe mechanisms controlling and the importance of apoptosisin the development ofdiseases such as cancer and neurological degenerative disorders.With respect to cancer,many of the chemotherapeutic agents or radiation treatmentsthat were found to beefficacious stem from their ability to cause apoptosisof cancer cells (28, 35 1-355).Further interest was fuelled by findings indicatingp53 kills cells mainly by a Bcl-2-dependent mechanism, since Bcl-2 overexpressioncan block most forms of cell deathsinduced by p53 (257, 356, 357). Both clinical observationsand mouse model experimentssuggest that inhibition of apoptosis (e.g. p53 mutation, Bcl-2overexpression) (283, 358)greatly promote oncogenic transformation causedby mutations that promote cellularproliferation alone (e.g. c-Myc overexpression,Ras mutations). Thus, reversing theprocess of tumorigenesis by promoting cell death, suchas by activating p53 function orby inhibiting Bcl-2 function, may allow novel waysto complement our current treatments107for malignancies. Thus, agents that directly mimic the BH3-only proteins would bepredicted to induce cell death and since they are known to antagonize the pro-survivalBcl-2 family members, therefore, be of value therapeutically. In particular, since many ofthe oncogenic mutations, such as those to p13 results in defects in sensing cellulardamage that would normally result in cell death by a Bcl-2-dependent mechanism,directly targeting Bcl-2 and its homologs may circumvent such mutations. This may alsopermit an alternative route to overcome tumor resistance to current treatments.To date, the solution structures of Bcl-xL (37) and Bcl-2 (38) have been solved,and NMR structural analysis of Bcl-xL complexed with the BH3 domains of Bak (39) andBad (359) shows that the BH1, BH2, and BH3 domains of Bcl-xL form a hydrophobicgroove which envelops the o-helica1 BH3 domain of Bad or Bak. Given this level ofunderstanding it has been possible to design BH3 peptidomimetics (86, 89, 90) such asABT-737 (91) which is currently in clinical trials (360). ABT-737 (BH3 mimmetic)development represents an overall shift towards rational, targeted approaches to inducingapoptosis.As such, there has been a large effort directed towards understanding the preciserole of Bcl-2 family members in controlling apoptosis. Stemming from such studies,Bad’s function in the regulation of apoptosis has become well established (287, 361).Knock-in mice expressing Bad with Ser mutated to Ala at 112, 136 and 155 sites showedeffects in growth factor responsive or apoptosis-inducing conditions, affecting thethreshold for mitochondrial disruption (287). The complete knockout of Bad is viable, butthose mice confirmed the role of Bad as a ‘sensitizer’ BH3 molecule, since there weresigns of altered growth factor survival signaling (288). Studies have also shown thatanimals with non-functional forms of Bad or those with reduced Bad expression are moresusceptible to developing cancer with increased age, and in response to sublethalradiation (287, 288). Thus, the lack of functional Bad can a have a major effect inhomeostasis and therefore, understanding how Bad is regulated may have majortherapeutic implications.108It has been shown that phosphorylation ofBad at multiple serine residues controlswhether it is able to bind withpro-survival proteins such as Bcl-xL(65, 96, 130), andconsequently neutralize their pro-survivalfunction. The interaction andconsequences ofBcl-2 member interactions and functionalcontrol of Bad through phosphorylationlead toseveral studies investigatinghow the presence of survival cytokinesand growth factorsare able to promote the phosphorylationof Bad (112). Stemming from thesestudies, fivemain sites of phosphorylation have beencharacterized. Phosphorylationof Serli2 andSer136 were shown to promote associationof Bad with cytosolic 14-3-3 proteins,thusinhibiting Bad’s ability to interactwith Bcl-xL at the mitochondria(125).Phosphorylation at Sen 55 appearsto represent another mechanism controllingBadIBclxL interaction. Sen 55 which lies withinthe BH3 domain, has been shownto disrupt theinteraction of Bad with Bcl-xL(135, 136). More recently,phosphorylation of Bad atSer128 was reported to promote apoptosis(77), but the significance of this is unclearsince the sequence of murine and humanBad are not completely conservedat that site.Investigators in Duronio’s lab reporteda fifth site of phosphorylationon Bad at Seri7O(137). Interestingly, this site was shownto alter a novel growth inducing characteristicofBad. Though the mechanism by whichphosphorylation at Bad-Serl7O functionstocontrol the activity of Bad is not yet clear,the thesis does present data indicating thatCaMKII-gamma phosphorylates Bad-Serl7O,therefore providing insightinto themechanism controlling phosphorylationstate of this site. Furthermore,we show throughthe use of Bad mutants mimickingdephosphorylation at Ser 112, 136,and 155, thatdephosphorylation of Sen 70 isable to transform the Bad protein intoa more potentinducer of cell death. Conversely,Bad mutant mimicking phosphorylationat Ser 170 wasshown to be less able to promote apoptosisregardless of whether the otherthree sites(Si 12, S136, and S155) were dephosphorylated.Moreover, we were intriguedby the fact that the Bad-Sen7OAsp mutant canpromote an increase in cell number(as well as blocking normal apoptoticactivity ofBad); thus, we further investigated thepotential connection between phosphonylationatSerl7O and cell cycle regulation. Thework presented in chapter 4shows that theexpression of Bad Si 70A resultsin a slowing of cell cycle progressionthrough S phase109and this effect appears to be unrelated to Bad’s apoptotic functions. We speculate thatthis intersection of Bad’s pro-apoptotic and cell cycle regulatory activity may represent asensory mechanism by which Bad acts as signaling node where apoptotic and cell cyclesignals converge. The work in this thesis has provided some evidence to further explorethe potential role of the Seri7O site, which may regulate protein associations viaphosphorylation of Seri7O. Considering we have shown that CaMKII (specificallyCaMKII-y) kinase activity against Bad Serl7O increases not only in response to pro-survival IL-3 signalling but at specific stages of the cell cycle, this may represent asurvival signal threshold prior to cells commiting to undergoing DNA replication.Experiments examining the effects of CaMKII inhibition on cell cycle stage transition,specifically at the GuS transition, may provide insight into a mechanism involving Badand cell cycle regulation.Bad’s cell cycle effect is supported by work describing Bad affecting cellproliferation (321) and similar studies suggesting a role for Bcl-2 or Bcl-xL in cell cyclere-entry from G0 into GuS (248). The premise for several of the studies presented in thisthesis originated from other work being pursued in our lab. Work by Jamil et al. showedthat Mci-i, which is known to suppress cell growth when overexpressed, interacts withinactive Cdkl in the nucleus (298). This interaction, and other interactions, includingwith the checkpoint kinase, Chki, has been recently shown to be involved in DNAdamage response signaling (320). Based on some of these findings, we were interested inlearning whether Bad was able to interact directly with proteins known to regulate cellcycle progression. Since we had data showing Bad’s effect was specific to the S phase,we examined Bad’s ability to interact with known S phase regulators. From theseexperiments we demonstrate that Bad is able to interact with Cdk2 and Cyclin E. It islikely very significant that Cdk2 together with Cyclin E are known to play a key role inthe entry into S phase of the cell cycle, and we show that the kinase activityphosphorylating the Sen 70 site is greatest in the S phase of the cell cycle. Thus, we havethis additional evidence that the phosphorylation of Bad at the Sen 70 site is cell cycleregulated, as is the kinase that phosphorylates that site.110As detailed in Chapter 4 we have shown that the kinase that phosphorylatestheBad-Serl7O site, and which is responsible for the pro-survival effectof thatphosphorylation, is calmodulin-dependent kinaseII gamma (CaMKII--y). Furthennore,we show that CaMKII acts as a cytokine-dependent survival kinase, as wedemonstrateCaMKII activity levels increase when cells are stimulated with IL-3. In addition,morerecent work done by lab colleagues have gone on to show that IL-3stimulationpecifically increases the activity level CaMKII-y. Recent experiments donein ourlaboratory have has also shown that CaMKII-’y immunoprecipitated fromcell lysates,shows activity against Bad Serl7O peptide. Together, all of our data supportsa role forCaMKII (more precisely CaMKII-y) in mediating cytokine-dependent survivalby virtueof its phosphorylation of Bad at Serl7O.CaMKII’s effects on both apoptosis and cell cycleappear to be cell type specific.It is likely that the variability observed in different cell types isa consequence ofdifferential expression patterns of CaMKII isoforms. It is knownthat CaMKIIholoenzymes have altered functions depending on their size and isoformcomposition(196). Thus a careful characterization of the CaMKII isoforms expressed willlikely benecessary before predictive cellular consequences of increased or decreasedCaMKIIactivity can be made. From this, it will be interestingto determine the effects ofoverexpressing CaMKII-’y in response to IL-3 survival signalingand cell cycleprogression.Another aspect of our studies that will be importantto pursue in future is themechanism by which a cytokine such as IL-3 can activate CaMKII activity.As shown,we found that IL-3 stimulated phosphorylationof CaMKII activation site (Thr286) andBad Serl 70. Understanding which signaling pathways and kinases are responsibleforinitiating the phosphorylation and activation of CaMKIIin haemopoietic cells will be ofmuch interest. Furthermore, understanding how CamKII-y is regulated in haemopoieticcells and what are the cellular consequences of alteringCamKII-y expression andactivity, through knockdown siRNA or overexpression experiments andJorexpressingconstitutively active mutant forms of CaMKII-y.111The potential role of Bad in cell cycle regulation combined with findingsassociated with CaMKII as the Sen 70 kinase represents a new direction in the study ofthis protein. Though the cell cycle effect of Bad may not represent its main function theredoes appear to be a growing level of interest into the intricate connections betweenproteins that were formerly thought to be solely responsible for either cell cycle orapoptosis regulation.Future work exploring the cellular consequences of Bad/CyclinE/Cdk2, as itrelates to cell cycle progression and cell death would be of great interest. In severalattempts, experiments were carried out to examine the effect of Bad phosphorylation atSen 70 on the association with Cdk2 and CyclinE; however we were unsuccessful inachieving a clear result. This was mostly due to the difficulty of working with CyclinEgiven its unstable characteristics along with inconsistent results associated with non-detection due to poorly functioning antibodies. However, given sufficient time totroubleshoot with additional reagents, these results may shed light on a novel mechanismwhich may involve the signaling convergence between what is thought of as two separatemechanisms: apoptosis and cell cycle. Moreover, understanding the cellular signals thataffect these interactions may also provide insight into the mechanism by which Badaffects cell cycle. Stemming from studies in progress in our lab, examining the potentialinvolvement of Bad in DNA surveillance and GuS or S phase checkpoint may also resultin a greater understanding of Bad’s cell cycle related role.Understanding how Bad Serl7O is able to play a key regulatory role in bothapoptosis and cell cycle, the two most widely studied aspects of cancer progression, mayhold therapeutic promise. Peptidomimetics based on the specific sequence surroundingSerl7O may be able to elicit a similar effect as what has been observed with full lengthBad Si 70A. That is cells under stressful conditions, similar to IL-3 starvation, undergoapoptosis while normally growing cells are relatively unaffected. The other therapeuticavenue relates to CaMKH inhibition, which we have shown to cause apoptosis. Since wehave shown evidence that specifically CaMKII-’y phosphorylates Bad, gamma isoformspecific inhibitors may represent another therapeutic strategy.112REFERENCES1. Tata, J. R. 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