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B-Raf is an essential component of the mitotic machinery critical for activation of MAPK signaling during mitosis in Xenopus egg extracts
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Borysov, Sergiy I
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MEK kinase
extracellular signal regulated kinase
ERK
Cdk1/cyclin B
Cdc2/cyclin B
M-phase
Signal transduction
Cell cycle
Phosphorylation
Dissertations, Academic -- Molecular Medicine -- Doctoral -- USF   ( lcsh )
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non-fiction   ( marcgt )

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ABSTRACT: Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase responsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activation of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf.^ ^Cdk1/cyclin B associates with and phosphorylates B-Raf in M-phase arrested extracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 residue. Phosphorylation at Ser-144 is critical for M-phase dependent activation of B-Raf and for B-Raf's ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic B-Raf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Further, inhibition or over-activation of MAPK during mitosis enhances or diminishes B-Raf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active mitotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase.^ ^Future studies are necessary to elucidate the involvement of these proteins in regulating B-Raf mitotic functions. In summary, my dissertation studies demonstrate that B-Raf activates MAPK signaling at mitosis and undergoes an M-phase dependent regulation. I propose that B-Raf has important functions at mitosis that contributes to its overall role in promoting cell proliferation.
Thesis:
Dissertation (Ph.D.)--University of South Florida, 2006.
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B-Raf is an Essential Component of the M itotic Machinery Critical for Activation of MAPK Signaling During Mitosis in Xenopus Egg Extracts by Sergiy I. Borysov A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy Department of Molecular Medicine College of Medicine University of South Florida Major Professor: Thomas M. Guadagno, Ph.D. Huntington Potter, Ph.D. Larry P. Solomonson, Ph.D. Hong-Gang Wang, Ph.D Jerry Wu, Ph.D. Date of Approval: August 31, 2006 Keywords: MEK kinase, extracellular signal regulated kinase (ERK), Cdk1/cyclin B, Cdc2/cyclin B, M-phase, signal trans duction, cell cycle, phosphorylation Copyright 2006 Sergiy I. Borysov

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Acknowledgements I thank Dr Guadagno for mentorship and members of the Guadagno lab for camaraderie. I thank my Committee members for scientific guidance. I thank Anthony Cheng for cloning Xenopus B-Raf, developing conditions for Xenopus myc-B-Raf expression and contributing in studying phos phorylation of recombinant myc-B-Raf by ERK. I thank Omar Hammad for his contribution in cloning Xenopus B-Raf. I thank the Chellappan lab (Moffitt Cancer Center) for in troduction to B-Raf antibodies, Dr Ferrel (Stanford University ) for providing antiXenopus MAPK antibody, Dr Maller (University of Colorado) for providing antiXenopus cyclin B antibody, Dr Rey-Huei Chen (University of Cornell) for providing the pGEM transcript ion vector, the Bhalla lab (Moffitt Cancer Center) for providing Fl avopiridol and Chris Marohnic (USF) for valuable assistance in using the FPLC. I thank Clark Craddock and Kathryn Zhan for academic support. I thank Maria Balasis a nd Brittany Palermo for proofreading this manuscript. I thank the American Heart AssociationFlorida for the predoctoral fellowship. I thank my parents and my wife Meghan for everlasting support and inspiration.

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i Table of Contents List of Tables................................................................................................................. ..viii List of Figures....................................................................................................................ix List of Abbreviations.........................................................................................................xv Abstract....................................................................................................................... .....xix Chapter One General Introduction....................................................................................1 General understanding of mitosis............................................................................1 Protein kinases in regulation of mitosis...................................................................5 Three-kinase module of the MAPK pathway..........................................................9 ERK1/2 cascade and mitosis..................................................................................13 Regulation of ERK1/2 si gnaling by MEK kinases................................................21 Functions and structural organi zation of Raf family members..............................23 Regulation of B-Raf activity by pho sphorylation and protein-protein interactions.......................................................................................................27 B-Raf as an oncogene............................................................................................34 Studying MAPK signaling in the cell-free system of Xenopus egg extracts.............................................................................................................36 Dissertation statement............................................................................................41

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ii Chapter Two Identification of B-Raf as an M-phase MEK Kinase................................43 Introduction............................................................................................................43 Results....................................................................................................................45 Preparation of extracts from parthenogenetically activated Xenopus eggs, which are devoid of a germ-cell specific MEK kinase, c-Mos.............................................................................45 Development of a protocol for purification of mitotic MEK kinase activity......................................................................................47 Purification of MEK kinase ac tivity from M-phase arrested extracts.................................................................................................56 B-Raf is enriched at the final stage of the mitotic MEK kinase purification................................................................................60 Depletion of B-Raf, but not Raf-1, blocks activation of MAPK at mitosis..................................................................................63 Conclusions............................................................................................................66 Chapter Three Characteriza tion of B-Raf at Mitosis......................................................68 Introduction............................................................................................................68 Results....................................................................................................................68 B-Raf activity is elevated during mitosis...................................................68 B-Raf associates with MEK in Xenopus egg extracts................................75

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iii B-Raf does not associate with Raf-1 in mitotic Xenopus egg extracts.................................................................................................76 B-Raf undergoes hyperphosphorylation during mitosis............................78 Mitotic activation of B-Raf stems from phosphorylation..........................78 Xenopus B-Raf is not phosphorylated at the conserved Threonine-633 and Serine-636 residues during mitosis......................81 Conclusions............................................................................................................83 Chapter Four Regulation of Mitotic B-Raf by Cdk1/cyclin B........................................84 Introduction............................................................................................................84 Results....................................................................................................................84 Cdk1/cyclin B triggers activation of B-Raf in Xenopus egg extracts.................................................................................................84 B-Raf associates with active Cdk1/cyclin B complexes during mitosis.......................................................................................85 Cdk1/cyclin B (and MAPK) di rectly phosphorylates B-Raf in M-phase arrested Xenopus egg extracts...........................................89 Cdk1/cyclin B directly phosphorylates Xenopus B-Raf in vitro at a conserved Serine-144............................................................91 Ser-144-Ala B-Raf mutant is not activated in an M-phase dependent manner................................................................................93

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iv Ser-144-Ala B-Raf mutant does not activate MAPK cascade during mitosis.........................................................................95 Ser-144-Ala B-Raf mutant ex erts a dominant-negative effect on MAPK activation during mitosis..........................................98 Conclusions..........................................................................................................100 Chapter Five Negative Feedback Regulation of Mitotic B-Raf by MAPK..................102 Introduction..........................................................................................................102 Results..................................................................................................................103 Mitotic hyperphosphorylation of B-Raf depends on active MAPK................................................................................................103 Mitotic B-Raf associates with MAPK.....................................................105 MAPK directly phosphorylates Xenopus B-Raf in vitro and causes B-Rafs electrophoretic mobility shift....................................107 MAPK directly phosphorylates Xenopus B-Raf in vitro at the conserved C-terminal SPKTP motif............................................107 C-terminal APKAP B-Raf mu tant displays a reduced MAPK-dependent shift at mitosis......................................................109 C-terminal APKAP B-Raf mutant possesses an elevated kinase activity....................................................................................111 Inhibition of MAPK activity with a MEK inhibitor U0126 activates B-Raf in M-phase arrested extracts...........................................113

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v Over-activation of MAPK w ith constitutively active recombinant MEK inhibits B-Raf activity in M-phase arrested extracts.................................................................................113 Conclusions..........................................................................................................116 Chapter Six Characterization of Large Multi-protein Complexes Containing Active Mitotic B-Raf.....................................................................................118 Introduction..........................................................................................................118 Results..................................................................................................................119 Purification of mitotic MEK kinase activity as B-Raf containing large multi-protein complexes.........................................119 Identification of potential components of large multiprotein complexes containi ng active mitotic B-Raf...........................121 Purification of mitotic B-Raf within large multi-protein complexes................................................................................................126 Conclusions..........................................................................................................128 Chapter Seven General Discussion...............................................................................131 A novel role for B-Raf in cell cycle regulation....................................................131 B-Raf directly links Cdk1/cyc lin B and MAPK signaling during mitosis............................................................................................................134 B-Raf, but not Raf-1, regulates MAPK activation at mitosis..............................138

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vi Mitotic B-Raf undergoes a negative feedback regulation by MAPK..................142 Mitotic B-Raf functions in multi-protein complexes...........................................145 Future research directions....................................................................................149 Chapter Eight Materials and Methods..........................................................................155 Preparation of Xenopus egg extracts....................................................................155 Studying c-Mos degradation in fertilized and Ca 2+ ionophore activated Xenopus oocytes.............................................................................156 Immunoblot analysis............................................................................................157 Purification of mitotic MEK kinase activity from M-phase arrested Xenopus egg extracts......................................................................................157 MEK kinase assays..............................................................................................158 In vitro histone H1 kinase assay..........................................................................159 Immunodepletion.................................................................................................160 Immunoprecipitation............................................................................................160 Co-immunoprecipitation analysis........................................................................161 Generation of wild-type and mutant Xenopus B-Raf constructs.........................161 Expression of recombinant my c-B-Raf proteins in CSF Xenopus egg axtracts....................................................................................................162 Purification of recombinant myc-B-Raf proteins from Xenopus egg extracts...........................................................................................................163 Phosphatase treatment..........................................................................................163

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vii In vitro Cdk1/cyclin B kinase assay.....................................................................164 In vitro ERK2 kinase assay..................................................................................164 Native gel electrophore sis and transfer................................................................164 Gel filtration.........................................................................................................165 References........................................................................................................................166 About the Author...................................................................................................End Page

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viii List of Tables Table 1. Purification of MEK kinase activity from M-phase arrested Xenopus egg extracts...........................................................................................58 Table 2. Proteins identified by Mass spectrometry in the final mitotic MEK kinase active fraction..............................................................................124

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ix List of Figures Figure 1. Regulation of MAPK signaling by MEK kinases......................................10 Figure 2. MAPK functions and localization during mitosis......................................14 Figure 3. Domain organizati on of Raf family members...........................................26 Figure 4. Phosphorylation in re gulation of B-Raf activity........................................29 Figure 5. Known partners of B-Raf protein complexes............................................31 Figure 6. Cell-free system of Xenopus egg extracts..................................................39 Figure 7. c-Mos is degraded at similar times for in vitro fertilized ( upper panel ) or Ca 2+ ionophore activated ( lower panel ) Xenopus eggs and undetectable during mitosis.........................................46 Figure 8. M-phase arrested Xenopus egg extracts contain active MAPK and MEK kinase activity...............................................................48 Figure 9. 20% ammonium sulfate sa turation precipitates MEK kinase activity from M-phase arrested Xenopus egg extracts...............................51

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x Figure 10. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate preci pitated fraction by using HiTrap Q Sepharose HP anion-exchange chromatography (0 1.0 M NaCl elution gradient)..............................................................52 Figure 11. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate preci pitated fraction by using HiTrap Q Sepharose HP anion-exchange chromatography (0 0.3 1.0 M NaCl elution gradient).....................................................54 Figure 12. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate preci pitated fraction by using Mono Q anion-exchange chromatography.................................................55 Figure 13. Purification of MEK kinase activity from M-phase arrested Xenopus egg extracts..................................................................................57 Figure 14. Final Mono Q fractions contain M-phase MEK kinase activity........................................................................................................59 Figure 15. B-Raf is enriched at the final stage of the mitotic MEK kinase purification......................................................................................61 Figure 16. Alignment of th e amino acid sequences of Xenopus (AAZ06667) and Human (P15056) B-Raf proteins using MegAlign software....................................................................................62 Figure 17. B-Raf is required for ac tivation of MAPK at mitosis................................64

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xi Figure 18. Raf-1 is not required for mitotic activation of MAPK pathway......................................................................................................65 Figure 19. B-Raf activates the MEK/MAPK cascade at mitosis................................67 Figure 20. B-Raf activity is elevated during mitosis...................................................70 Figure 21. Kinase-dead B-Raf i mmuno-complexes from Sand Mphase arrested extracts do not possess MEK kinase activity.................... 72 Figure 22. c-Mos immunoprecipitates do not possess an M-phase MEK kinase activity..................................................................................74 Figure 23. B-Raf associates with MEK in Xenopus egg extracts................................75 Figure 24. Raf-1 is not co-immunop recipitated with B-Raf and MEK from Xenopus egg extracts.........................................................................77 Figure 25. B-Raf undergoes hyperp hosphorylation during mitosis............................79 Figure 26. Mitotic activation of B-Raf stems from phosphorylation..........................80 Figure 27. Xenopus 95 kDa B-Raf is not phosphorylated at the conserved Threonine-633 and Serine-636 residues during mitosis........................................................................................................82 Figure 28. B-Raf activity in M-phase arrested Xenopus egg extracts depends on Cdk1/cyclin B activity............................................................86

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xii Figure 29. B-Raf associates with active Cdk1/cyclin B complexes during mitosis.............................................................................................88 Figure 30. Cdk1/cyclin B (and MAPK) directly phosphorylates B-Raf in M-phase arrested Xenopus egg extracts.................................................90 Figure 31. Conserved putative Cdk1/cyclin B phosphorylation sites in B-Raf protein.............................................................................................92 Figure 32. Cdk1/cyclin B directly phosphorylates Xenopus B-Raf in vitro at a conserved Serine-144..................................................................94 Figure 33. Ser-144-Ala B-Ra f mutant is not activ ated in an M-phase dependent manner......................................................................................96 Figure 34. Ser-144-Ala B-Raf mutant does not activate MAPK cascade during mitosis...............................................................................97 Figure 35. Ser-144-Ala B-Raf mutant blocks MAPK activation in Mphase extracts.............................................................................................99 Figure 36. Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144 to trigger the B-Raf/MEK/MAPK cascade..............................................101 Figure 37. Mitotic hyperphosphorylat ion of B-Raf depends on active MAPK......................................................................................................104 Figure 38. Mitotic B-Raf associates with MAPK.....................................................106

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xiii Figure 39. MAPK directly phosphorylates Xenopus B-Raf in vitro and causes B-Rafs electrophoretic mobility shift..........................................108 Figure 40. MAPK directly phosphorylates Xenopus B-Raf at the conserved C-terminal SPKTP motif........................................................110 Figure 41. Phosphorylation-defec tive C-terminal APKAP B-Raf mutant possesses an elevated MEK kinase activity.................................112 Figure 42. Inhibition of MAPK ac tivity with a MEK inhibitor U0126 activates B-Raf in M-phase arrested extracts...........................................114 Figure 43. Over-activation of MA PK with constitutively active recombinant MEK inhibits B-Raf activity in M-phase arrested extracts.......................................................................................115 Figure 44. MAPK directly phosphorylates B-Raf at Serine-784 and Threonine-787 to inhibit B-Raf activity..................................................117 Figure 45. Protein profiles of m itotic MEK kinase active fractions throughout progression of th e M-phase MEK kinase purification ..............................................................................................120 Figure 46. Mitotic MEK kinase activity purified from M-phase arrested Xenopus egg extracts resembles a large B-Rafcontaining protein complex......................................................................122 Figure 47. Active mitotic B-Raf is pu rified by gel filt ration as a large 700-400 kDa protein complex.................................................................127

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xiv Figure 48. Protein profile co-immunopurified with B-Raf is changing from Sto M-phases.................................................................................129 Figure 49. The N-terminal Cdk1/cy clin B phosphorylation site is conserved among B-Raf member s of Raf kinase family.........................140 Figure 50. Proposed mechanism for re gulation of the B-Raf/MEK/MAPK cascade at mitosis...................................................146

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xv List of Abbreviations Ala Alanine AP-2 Adaptor protein 2 Apacd ATP binding protein associated with cell differentiation APC Anaphase-promoting complex APKAP Alanine-Proline-Lysine-Alanine-Proline AS Ammonium sulfate ASK Apoptosis signal-regulated kinase ATR Ataxia telangiectasia related BAY 43-9006 N-(3-trifluoromethyl-4-chl orophenyl)-N-(4-(2 -methylcarbamoyl pyridin-4-yl)oxyphenyl)urea BH3 Bcl-2 homology 3 Bim Bcl2-interacting mediator of cell death BSA Bovine serum albumin Bub Budding uninhibited by benzimidazole CAS Cellular apoptosis susceptibility CCt8 Chaperonin containing theta Cdc Cell division cycle Cdk Cyclin-dependent kinase CENP-E Centromere-associated protein E Cn2 Cytosolic non-specific peptidase CNK Connector enhancer of KSR CR Conserved region CRD Cysteine rich domain

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xvi CSE1 Chromosome segregation gene 1 CSF Cytostatic factor DEAE Diethylaminoethyl DMSO Dimethylsulfoxide DTT Dithiothreitol EB Extract buffer ELB Egg lysis buffer EDTA Ethylene diamine tetraacetic acid EGTA Ethylene glycol bis(aminoet hylether)-N,N,N',N'-tetraacetic acid ERK Extracellular signal-regulated kinase FF Fast flow FPLC Fast protein liquid chromatography Glu Glutamic acid Grb2 Growth factor receptor bound protein 2 GST Glutathione S-transferase HABP1 Hyaluronan binding protein 1 HEPES N-(2-hydroxy-ethyl)piper azine-N'-2-ethanesulfonic acid HFF Human foreskin fibroblasts His Histidine HP High performance HPLC High Performance Liquid Chromatography HR High resolution HSP90 Heat shock protein 90 HSP70 Heat shock protein 70 IAP Inhibitor of apoptosis INCENP Inner centromere protein IP Immunoprecipitate JNK Jun N-terminal kinase KSR Kinase suppressor of Ras Mad Mitotic arrest-deficient

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xvii MAPK Mitogen-activa ted protein kinase MBP Myelin basic protein MEK MAPK/ERK kinase MEKK MAPK/ERK kinase kinase MKP1 MAPK phosphatase 1 MLK Mixed lineage kinase MMR Modified Ringers solution Mos Moloney sarcoma MPF Maturation-promoting factor MPM2 Mitotic protein monoclonal 2 Mps1 Mono-polar spindle 1 MS Mass spectrometry MT Mutant Ndrg20 N-Myc downstream regulated Nek2 NIMA-related kinase NIMA Never at mitosis A Nup Nucleoporin PA Proteasome activator PAGE Polyacrylamide gel electrophoresis PAK p21-Activated kinase PD98059 2-Amino-3-methoxyflavone PKI Protein kinase inhibitor PP Protein phosphatase PVDF Polyvinylidene difluoride PXS*P Proline-any amino acid-phospho-Serine-Proline Raf Rapidly accelerated fibrosarcoma Ras Rat sarcoma Rap1 Ribosome associated protein 1 RBD Ras binding domain Rsk Ribosomal S6 kinase

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xviii SNAP SNAP adaptor protein SDS Sodium dodecyl sulfate Ser Serine SGK Serum and glucocorticoid inducible kinase Soc-2 Suppressor of clear homolog Sos-1 Son of sevenless 1 SPKTP Serine-Proline-Lysine-Threonine-Proline S*PXK/L Phospho-Serine-Proline-any amino acid-Lysine or Leucine TAK TGF-activated protein kinase TEY Threonine-Glutamic acid-Tyrosine Tom1 Target of myb1 Tpl-2 Tumor progression locus 2 Thr Threonine U0126 1,4-Diamino-2,3-dicyano-1,4bis( 2-aminophenylthio)butadiene V600E Valine-600-Glutamic acid Val Valine WT Wild-type XB Xenopus buffer

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xix B-Raf is an Essential Component of the M itotic Machinery Critical for Activation of MAPK Signaling During Mitosis in Xenopus Egg Extracts Sergiy I. Borysov ABSTRACT Activation of the MAPK cascade during mito sis is critical for spindle assembly and normal mitotic progression. The underlyi ng regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase re sponsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activa tion of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf. Cdk1/cyclin

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xx B associates with and phosphorylates B-Raf in M-phase arrested ex tracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 resi due. Phosphorylation at Ser144 is critical for M-phase dependent activat ion of B-Raf and for B-Rafs ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic BRaf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Furt her, inhibition or ove r-activation of MAPK during mitosis enhances or diminishes BRaf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active m itotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase. Future studies are necessary to elucidate the involvement of these proteins in regulati ng B-Raf mitotic functi ons. In summary, my dissertation studies demonstr ate that B-Raf activates MA PK signaling at mitosis and undergoes an M-phase dependent regulati on. I propose that B-Raf has important functions at mitosis that cont ributes to its overall role in promoting cell proliferation.

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1 Chapter One General Introduction General understanding of mitosis The purpose of the cell cycle is to produ ce two daughter cells identical to the mother cell. This is achieved through a temporal coordination and execution of the cell cycle events. During G1-S-G2 phases of the cel l cycle a cell commits to and prepares for cell division, whereas at M-pha se the cell division is fi nally executed (Murray, 1992). M-phase is traditionally divided into two stages: mitosis (segregation of the chromosomes) and cytokinesis (division of the cytoplasm) (Satterwhite and Pollard, 1992). Therefore, mitosis is the culminating stage of the cell cycle, when the duplicated genome of the mother cell becomes redi stributed into two daughter cells. Mitosis is traditionally described as a series of intracellular morphological changes involving dynamic rearrangements of chromatin and cytoskeleton structures (Karsenti, 1991; McIntosh and Koonce, 1989; Wadsworth, 1993). The first signs of chromatin condensation within an intact nuc leus mark the initial progression toward mitosis, recognized as prophase (Khodja kov and Rieder, 1999; Swedlow and Hirano, 2003). During prophase, a pair of centrosome s (Fukasawa, 2002), duplicated in S-phase,

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2 undergoes separation defining the poles of the forming mitotic spindle (Raff and Glover, 1989). Recruitment of additional gamma-tubulin ring complexes into centrosomal matrix (Khodjakov and Rieder, 1999) provokes a drama tic increase in micr otubule nucleation activity necessary for dynami c spindle assembly (Kuriyama and Borisy, 1981). The following nuclear envelope breakdown represents the first irreversible point of mitosis, prometaphase (Fields and Thompson, 1995; Rieder and Khodjakov, 1997). The dismantling of the nuclear envelope allows physical interaction be tween growing tubulin fibers of the mitotic spindl e and the kinetochores of th e chromosomes. Pushing and pulling forces of the kinetochore microtubules emitting from opposite poles and bound to the same chromosomal kinetochores position chromosomes halfway between the spindle poles, along the equator of a cell, called a metaphase plate (S onoda et al., 2001; Tanaka et al., 2000). Until this stage of mitosis, sist er chromatids are held together by cohesin (Nasmyth, 2001) selectively enriched in the neighborhood of the centromeres (Waizenegger et al., 2000). The metaphase alignment and proper attachment of chromosomes to the mitotic spindle triggers an abrupt and synchronous cleavage of the centromeric cohesin complexes (Hauf et al., 2001; Uhlmann et al., 2000). This provokes the sudden separation of sister chromatids and represents the second ir reversible stage of mitosis, anaphase. Following the cleavage, chromosomes are separated first due to shortening of the kinetochore microtubules (anaphase A) (Mitchison and Salmon, 1992) and later due to growing of the polar micr otubules and elongation of the whole spindle (anaphase B) (Aist et al., 1991). Mitosis is finished at the telophase when the kinetochore microtubules disappear (Wilson et al., 1994), a new nuclear envelope reforms around

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3 each set of adjacent decondensing chromoso mes (Gerace and Blobel, 1980; Newport, 1987) and the nucleoli are reformed (Dimar io, 2004). The following cytokinesis is temporally and spatially coordinated to th e completion of mitosis (Satterwhite and Pollard, 1992). The midbody, a microtubule-based structure assembled at telophase around the original spindle equator, directs proper cleavage of the cytoplasm during cytokinesis (Otegui et al., 2005). An intricate biochemical network gove rns orderly execution and precision of mitotic events. Protein phosphorylation (Nigg, 2001) and dephosphorylation (Kumagai and Dunphy, 1996), protein-protein associati on (Hardwick, 2005; Kramer et al., 2000; Morgan, 1995; Musacchio and Hardwick, 2002; Visintin et al ., 1997) and protein degradation (Bashir and Pagano, 2004; Hollowa y et al., 1993; Sudo et al., 2004) are the main mechanisms of the mito tic regulatory machinery. Proper order and completion of the mitotic stages are accomplished by incorporating positive and negative feedback loops into the regulation of mitosis and creating two points of no return in mitotic progression (Murray, 1992; Murray and Kirschner, 1989b). The first point of no return overlaps with the prophaseprometaphase transition and is associated w ith a robust activation of Cdk1/cyclin B, the master regulator of mitotic entry (Dunphy et al., 1988). The irreversibility of Cdk1/cyclin B activation is ensured by the positive feedback loop existing between Cdk1/cyclin B and its upstream activator, Cd c25C phosphatase (Strau sfeld et al., 1994), which removes the inhibitory phosphorylations from Thr-14 and Tyr-15 residues (Atherton-Fessler et al., 1993; Kumagai and Dunphy, 1996). This positive regulatory

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4 circuit ensures an abrupt and robust accumu lation of active Cdk1/cyclin B and enables Cdk1/cyclin B activation and progression towa rd mitosis to be independent from any further upstream inputs. Cdk1/cyclin B activation sets up conditions for the next irreversible point in mitosis, activation of the anaphase-pro moting complex (APC), a mitosis-specific ubiquitin-ligase, which targets mitotic proteins for degradation promoting exit from mitosis (Castro et al., 2005). Indeed, activati on of Cdk1/cyclin B via direct and indirect mechanisms triggers formation of the m itotic spindle and its association with chromosomal kinetochores. Completion of this spindle-chromosome assembly during metaphase leads to activation of APC, which in turn ubiquitinates and targets securin and cyclin B for degradation. Securin proteo lysis promotes the disjoining of sister chromatids and represents the main condition for anaphase initiation (Holloway et al., 1993). Degradation of cyclin B leads to inactivation of Cdk1/cyclin B complexes (Wheatley et al., 1997). Thus, activation of Cdk1/cyclin B tr iggers a series of events, which eventually leads to eliminatio n of Cdk1/cyclin B signaling. The spindle assembly checkpoint ensures that progression toward anaphase does not happen prematurely. It is thought that throug hout spindle formation and establishment of kinetochoremicrotubule interactions, the status of the kinetochoremicrotubule attachments can be sensed and re flected in the levels of soluble waitanaphase signaling complexes (Rieder and Salmon, 1998; Wassmann and Benezra, 2001), the presence of which delays activation of APC. Indeed, unattached kinetochores or low-tension kinetochores undergo phosphor ylation and act as sites where spindle

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5 checkpoint Mad2-Cdc20 protein complexes are assembled and released to abrogate APC activation (Chan and Yen, 2003; Hardwick, 2005; Yu, 2002). In conclusion, mitosis is the final stage of the cell cycle, when segregation of replicated chromosomes occurs. To accomm odate separation of the duplicated genomic DNA, the chromatin undergoes condensation an d the cytoskeleton transforms into the mitotic spindle. These processes of mitosi s are tightly regulated by mitotic biochemical machinery, which ensures proper timing, comp leteness and fidelity of mitotic stages. Protein kinases in regulation of mitosis Protein phosphorylation comprises one of the major modes of mitotic regulation (Nigg, 2001). Many kinases have been implicated in mitosis. These include Cdk1/cyclin B, members of Polo-like and Aurora kinase families, NIMA, mitotic checkpoint kinases (Bub1, BubR1 and Mps1), MAPK and others. Cdk1/cyclin B is the main kinase involv ed in the regulati on of mitotic onset (Arion et al., 1988; Gautier et al., 1988; Labbe et al., 1989; Lohka et al., 1988; Masui and Markert, 1971; Nurse, 1975). The contem porary dogma of the cell cycle regulation represents cyclin-dependent kinase activities, which fluc tuate throughout the cell cycle, as a regulatory core of the cell cycle (Morgan, 1995). The identification of Cdk1/cyclin B as an initiator of M-phase and a prototype of Cdk/cyclin complexes was a discovery of Nobel Prize caliber (Paul Nurse, Leland Harw ell and Tim Hunt, Nobel Priz in Medicine, 2001).

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6 Cdk1/cyclin B initiates and establishes mitosis via phosphorylation of multiple substrates. For instance, ac tive Cdk1/cyclin B directly phosphorylates lamins to induce dismantling of the nuclear envelope (Peter et al., 1991), condensins to contribute to further chromosome condensation (Kimura et al., 1998), and microtubule-associated proteins and kinesin-related motor proteins to affect centrosome separation and mitotic spindle assembly, respectively (B langy et al., 1995). Besides this, Cdk1/cyclin B directly regulates APC and other components of th e mitotic regulatory network (Patra and Dunphy, 1998; Zachariae et al., 1998). Inactivat ion of Cdk1/cyclin B occurs during the anaphase transition and is required for prope r mitotic exit (Noton and Diffley, 2000). It is achieved through an APC-directed degrada tion of cyclin B, a regulatory partner of Cdk1 (Glotzer et al., 1991; Wheatley et al., 1997). Polo-like kinases are a new emerging class of mitotic regulators. Originally, Polo-like kinases were identified in Drosophila (Sunkel and Glover, 1988) and yeast (Kitada et al., 1993) genomes and linked to mito tic regulation. Ther e are at least three Polo-like kinase members in vertebrates (L owery et al., 2005), one of which, Polo-like kinase 1 (Plk1), has been implicated in mitosi s. It has been shown that Plk1 localizes to the mitotic apparatus (Arnaud et al., 1998; Go lsteyn et al., 1995; Wianny et al., 1998), undergoes an M-phase dependent regulation (Charles et al., 1998; Golsteyn et al., 1995; Hamanaka et al., 1995) and has multifaceted roles during M-phase. It has been proposed that Plk1 triggers the Cdc25C-Cdk1/cyclin B positive feedback loop during the mitotic entry (Nakajima et al., 2003; Roshak et al., 2000; Toyoshima-Morimoto et al., 2002), controls mitotic spindle form ation and functions (Ahonen et al., 2005; Casenghi et al.,

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7 2005; Feng et al., 1999; Lane and Nigg, 1996; va n Vugt et al., 2004; Yarm, 2002) and is involved in the regulation of mitotic exit and cytoki nesis (Zhou et al., 2003). The family of Aurora kinases is anothe r important group of mitotic regulators. Aurora kinases were originally identified in Drosophila as being involved in the regulation of centrosome functions during m itosis (Glover et al., 1995) and in budding yeasts as being involved in coordination of mitosis (Chan and Botstein, 1993). The metazoan genome encodes at least three Auro ra kinases, Aurora A, B and C (Andrews et al., 2003). Thus far only Auroras A and B are directly linked to mitosis. Auroras A and B differ from each other in timing of activation, intracellular localizations and functions during mitosis. Aurora A protein levels and activity peak at G2/M, whereas Aurora B is up-regulated a nd activated during late mitosis after Cdk1/cyclin B inactivation (Bischoff et al ., 1998). Both kinases undergo proteasomemediated degradation at the end of mitosis/ G1 (Shu et al., 2003; Taguchi et al., 2002). Aurora A localizes to the cen trosomes throughout mitosis (S tenoien et al., 2003) and is implicated mainly in centrosomal functions (Andrews et al., 2003; Giet et al., 1999). Additionally, Aurora A may be an important component of the M-pha se onset signaling (Katayama et al., 2004; Krystyniak et al., 2006). Aurora B localizes to the kinetochore regions from prophase until the metaphaseanaphase transition and to the midzone a nd midbody during telophase and cytokinesis, respectively (Andrews et al., 2003). It is thought that Aurora Bs association with INCENP (inner centromere protein) and surviv in (Adams et al., 2000; Yasui et al., 2004)

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8 is important for its roles in spindle fo rmation (Moore and Wordeman, 2004) and the mitotic spindle checkpoint (Ditchfiel d et al., 2003; Hauf et al., 2003). Genetic studies in the filamentous fungus Aspergillus nidulans identified a new mitotic kinase, NIMA ( n ever i n m itosis A ) (Osmani et al., 1988) Functional studies characterized NIMA as one of the key regulat ors of mitotic onset (Osmani et al., 1991; Osmani et al., 1988; Osmani and Ye, 1996). However, the closest mammalian homologue of fungal NIMA, Nek2 (Fry, 2002), does not display a global effect on Mphase progression. It was shown that Nek2 is a centrosomal resident protein involved in centrosome separation (Fry et al., 1998a; Fry et al., 1998b). Another important group of kinases involve d in the regulation of mitosis is a set of kinases directly implicated in the generation of spindle checkpoint signaling. This includes kinases of Mad/Bub families (n amely, Bub1 and BubR1/Mad3) and Mps1 kinase. It is proposed that Bub1 (Roberts et al., 1994) and BubR1 (Taylor et al., 1998) facilitate kinetochore recruitment of other members of the Mad and Bub families (Chen, 2002; Sharp-Baker and Chen, 2001) and participate in their rearrangements leading to the formation of spindle checkpoint protein co mplexes, which inhibit APC and postpone anaphase onset (Brady and Hardwick, 2000; S udakin et al., 2001). Checkpoint kinase Mps1 (Weiss and Winey, 1996), similar to Bub1 and BubR1, facilitates kinetocore localization of the Mad proteins (Abrieu et al., 2001). However, unlike Bub1 and BubR1, Mps1 has not been shown to be a co mponent of the wait anaphase complexes (Hoyt, 2001).

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9 In conclusion, many kinases are involv ed in the regulation of mitosis. Cdk1/cyclin B is the major among them. Its activation and inactivation mark two of the most important transitional points in m itotic progression, commitment and exit from mitosis, and ensure the irreve rsibility of the process. The involvement of many other kinases in the mitotic regulatory network reflects the necessity of multilayered and branched control over the fidelity and integrity of cell division. Three-kinase module of the MAPK pathway The MAPK cascade is one of the most well characterized kinase pathways implicated in cellular signali ng. It governs cellular respons es to a variety of stimuli including cytokines, growth factors and hormones, and c onditions, such as cellular stresses, cell adhesion and others (Widma nn et al., 1999). The MAPK signaling module consists of three kinases that sequentially phosphorylate and activat e one another. The up-stream kinase, MAPK kinase kinase (MEK kinase), phosphoryl ates and activates MAPK kinase (MEK), which in turn phos phorylates and activates MAPK (Fig. 1A) (English et al., 1999; Widmann et al., 1999) The MAPK cascade is unique for eukaryotes and conserved from yeast to humans (English et al., 1999; Widmann et al., 1999). Mammalian cells express at least twenty members of the MAPK family (Pearson et al., 2001b). The most studied among them ar e classified into four distinct subfamilies (Widmann et al., 1999). These include extr acellular signal-regulated kinases 1 and 2

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Cell growth Cell proliferation Cell differentiation Inflammation Stress responses Apoptosis Cell proliferation JNK1/2/3 p38 Ra f Mos Tpl-2 MEKK11 MEKK1 MEKK4/5 MEKK2/3 TAK1 MLK ASK1 PAK MEK3/6 MEK4/7 MEK5 MEK1/2 Cell survival ERK1/2 Cell survival ERK5 MEKK A B MEK MAPK Figure 1. Regulation of MAPK signaling by MEK kinases A. General representation of the MEKK/MEK/MAPK signaling module. B. MEK kinase network in the regulation of MAPK p athway functions 10

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11 (ERK1/2), Jun amino-terminal kinases 1, 2 and 3 (JNK 1/2/3), p38 proteins alpha, beta, gamma and delta (p38 / / / ) and extracellular signal-re gulated kinase 5 (ERK5) (Widmann et al., 1999). Each MAPK subfamily is distinctly regulated and carries out distinct biological functions (Fig. 1B). ER K1/2 are predominantly implicated in the regulation of cell proliferat ion, growth, differentiation a nd survival. JNK and p38 are stress-response MAPKs, involved in apoptos is, stress responses to different cellular conditions or agents as well as cellular differentiation and some immune functions (Widmann et al., 1999). ERK5 is proposed to regulate cell survival and proliferation (Dong et al., 2001; Kato et al ., 1998; Pearson et al., 2001a). To ensure the integrity of intracellular processes, activation of a given MAPK cascade within a certain biological context must be precisely regulated (Pearson et al., 2001b; Widmann et al., 1999). Several bioche mical mechanisms within a MAPK module ensure the specificity of MAPK signaling. Firs t of all, the preferen tial phosphorylation of a certain set of intracellular targets by a given MAPK is conditioned by the intrinsic structural properties of a part icular MAPK subfamily and its substrates. Despite the fact that all MAPKs phosphorylate their substrat es at a similar consensus Ser/Thr-Pro sequence (Davis, 1993), substrate recogniti on by particular MAPKs varies depending upon the availability of appropr iate docking sites (Fantz et al., 2001; Gonzalez et al., 1991; Gupta et al., 1996; Tanoue and Nishida, 2003). The specificity of MAPK signaling is cont rolled as well on the level of MEK. Very specific combinations of MEK and MAPK are formed within the MAPK modules. MEK 1/2 activates ERK1/2, MEK 4/7 activ ates JNKs, MEK 3/6 activates p38s and

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12 MEK5 activates ERK5 (Fig. 1B) (Pearson et al., 2001b; Widmann et al., 1999). This remarkable feature of the MAPK cascade or ganization prohibits cr oss-signaling on the level of MAPK kinases, ther eby contributing to selective activation of a particular MAPK. It is well established that MAPK needs to be phosphorylated at both tyrosine and threonine residues within the TEY activation loop for its full activation (Payne et al., 1991). This phosphorylation is exerted by MEKs, dual specific kinases, which phosphorylate hydroxyl side chains of serine/threonine and tyro sine residues (Nakielny et al., 1992; Zheng and Guan, 1993). Interestingl y, this dual phosphorylation of MAPK is exerted in two phases (Ferrell and Bhatt, 1997) and, thus, serves as a threshold mechanism in activation of MA PK signaling (Ferrell, 1999). The specificity of MAPK signaling regulation on the level of MEK kinases appears to be much more complex. There is a vast variety of ME K kinases, which are defined as enzymes that catalyze transfer of phosphate from ATP to hydroxyl side-chains of serine and threonine re sidues within MEKs activati on segment, inducing their activation (Zheng and Guan, 1994). Cumulative evidence demonstrates that some MEK kinases can activate more than one MEK as well as a certain MEK/MAPK cascade can respond to more that one MEK kinase (Fange r et al., 1997; Widmann et al., 1999; Xia et al., 2000; Yujiri et al., 1998) (Fig. 1B). Theref ore, it is thought th at MEK kinases create a signaling network that allows diversity of signaling inputs to trigger specific MAPK pathways (Widmann et al., 1999). In summary, the MAPK pathway represen ts an evolutionary conserved signaling module implicated in the regulation of a vari ety of cellular processes. The core module

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13 of the MAPK pathway consists of three ki nases (MEK kinase, MEK and MAPK) that are sequentially activated via phos phorylation. The MAPK family is divided into four subfamilies, which are distinctly regulated and serve distinctive functions. Several intramodular layers of biochemical regulati on ensure signaling sp ecificity of MAPK pathways. These include selectivity of the MAPK-substrate reaction, activation of MAPK by a specific MEK and the capacity of the MAPK pathways to be linked to diversity of up-stream signals via a variety of MEK kinases. ERK1/2 cascade and mitosis ERK1/2 signaling has traditionally been im plicated in the regulation of cell cycle initiation and G1/S transition following mit ogenic stimulation (Kol ch, 2000; Pearson et al., 2001b). However, evidence from different experimental systems demonstrates that the ERK1/2 pathway is an important regulator of the later stage of the cell cycle, mitosis. It is proposed that the ERK1/2 cascade re gulates G2/M transition, formation of the mitotic spindle, the mitotic spindle checkpoint, exit from mi tosis, Golgi fragmentation and the duration of mitosis (Fig. 2). The first evidence implicating ERK1/2 in M-phase came from studies in Xenopus egg extracts. It has been shown that activation of the Xenopus homologue of ERK2, p42 MAPK, occurs during M-phase of the cell cy cle (Gotoh et al., 1991a; Gotoh et al., 1991b; Minshull et al., 1994; Takenaka et al., 1997). Later works ha ve demonstrated that the

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Spindle Assembly Metaphase Checkpoint G2 Golgi Fragmentation ERK1/2 at Midbody Centrosomes regulates localizes to MitoticOnset Anaphase/ Telophase Metaphase G1 Prometaphase Cytokinesis Spindle poles, kinetochores Spindle poles, midzone Figure 2. ERK1/2 functions and localization during mitosis ERK activity is necessary for proper spindle formation, metaphase/anaphase transition and overall mitotic timing. Localization of active (phosphorylated) MAPK to mitotic structures is shown schematically in red. Note that localization of monophospho-Y-ERK to Golgi is not shown. 14

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15 ERK1/2 cascade is activated as well during mitosis in somatic cells (Harding et al., 2003; Roberts et al., 2002; Shapiro et al., 1998; Willard and Crouch, 2001; Zecevic et al., 1998). Functional studies in somatic cell systems provided evidence that ERK1/2 signaling is important for prope r transition to mitosis. Three different experimental settings have been used to study mitotic entry under the MEK/ER K1/2 loss-of-function conditions. Specifically, it was shown that expression of dominant-negative MEK (Wright et al., 1999), treatment of sync hronized cell populations with PD98059 or U0126, MEK specific inhibitors, (Roberts et al., 2002; Wright et al ., 1999) and depletion of MEK1/2 or ERK1/2 from synchronized ce lls by RNA interference (RNAi) technique (Liu et al., 2004) induced a G2/M arrest (Liu et al., 2004; Roberts et al., 2002; Wright et al., 1999), delayed and reduced activation of C dk1/cyclin B (Liu et al., 2004; Wright et al., 1999), decreased nucl ear translocation of cyclin B (Roberts et al., 2002), decreased reactivity with MPM-2 antibodies that re cognize non-specific mitotic phosphoproteins (Wright et al., 1999) and reduced phosphoryl ation of histone H3 (Liu et al., 2004; Roberts et al., 2002). Thus, it was proposed that MEK/ERK signali ng is important for Cdk1 activation and m itotic initiation. Contrary to this, studies in Xenopus egg extracts showed that activation of Cdk1/cyclin B is not affected in p42 MAPK depleted ex tracts (Guadagno and Ferrell, 1998; Takenaka et al., 1997). Furthermore, constitutive activa tion of p42 MAPK in Xenopus egg extracts prior to mitosis inhibits Cdk1/cyclin B and delays M-phase onset (Bitangcol et al., 1998; Walter et al., 1997). Additionally, it was demonstrated that p42

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16 MAPK can directly phosphorylate and activ ate Wee1, a kinase that inactivates Cdk1/cyclin B (Walter et al., 2000). Thus, involvement of ERK1/2 in regulation of Cdk1/cyclin B activity and mitotic onset remains disputable. Early works have characterized ERK1/2 as a potential regula tor of cytoskeleton functions. It was shown that active ERK1/2 phosphorylates components of the cytoskeleton (Hoshi et al., 1992; Ray and Sturgill, 1987; Verlhac et al., 1993) and largely associates with microtubule cytoskeleton in vivo (Reszka et al., 1995). Functional studies demonstrated that an increase in microtubul e dynamics, typical for M-phase, depends on the presence of active ERK1/2. Specificall y, it was shown that introduction of active p42 MAPK from M-phase to S-phase Xenopus egg extracts dramatically increases tubulin depolymerization (Gotoh et al., 1991b). Cons istently, the abrogation of p42 MAPK signaling in M-phase extracts via inhibition of MEK, immu nodepletion of MEK or p42 MAPK dramatically decreases tubulin de-polymerization (Guadagno and Ferrell, 1998; Horne and Guadagno, 2003). Fluorescent studies further supported im plication of ERK signaling in the regulation of the mitotic apparatus. In 3T 3 and Ptk1 cells, it was shown that active forms of ERK1/2 and MEK localize to the kinetoch ores from early prometaphase through midanaphase, to the spindle and spindle poles throughout mitosis, and the midbody during anaphase (Shapiro et al., 1998; Willard and Crouch, 2001; Zecevic et al., 1998) (Fig. 2). A similar pattern of intracellular p42 MAPK distribu tion was observed on mitotic spindles formed in Xenopus egg extracts (Horne and Gu adagno, 2003) as well as in fertilized sea urchin eggs (Zhang et al., 2005).

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17 The involvement of ERK1/2 signaling in formation of the mitotic spindle was directly shown by loss-of-function studies. Specifically, it was demonstrated that immunodepleting of p42 MAPK or blocking its activation during mitosis with a MEK inhibitor, U0126, induces abnormal spindle formations in mitotic Xenopus egg extracts and compromises stability of already form ed spindles (Horne and Guadagno, 2003). Further, abrogation of mitoti c spindle formation was observe d as well in somatic cells pre-treated with U0126 at the late G2 (Horne and Guadagno, 2003). Recently, an altered formation of mitotic spindles and chromoso me attachment was reported as well in sea urchin fertilized eggs treated with domina nt-negative MEK 1/2 or U0126 (Zhang et al., 2005). It was suggested that one of the substrates through which p42 MAPK may exert its spindle formation function is a kinetoch ore motor protein CENP-E (Zecevic et al., 1998). CENP-E is essential for bi-polar att achment of chromosome to microtubules (Yao et al., 1997). During mitosis p42 MAPK dir ectly associates with and phosphorylates CENP-E at sites important for its association with microtubules (Zecev ic et al., 1998). More than a decade ago it was shown th at an injection of thiophosphorylated ERK1/2 (Haccard et al., 1995) or c-Mos (Sagata et al., 1989) into Xenopus embryos induces metaphase arrest. Further, the addition of constitutively active MEK (Takenaka et al., 1997) or c-Mos (Chau and Shibuya 1998; Guadagno and Ferrell, 1998) into mitotic Xenopus egg extracts maintains an M-phase-like state even following cyclin B degradation and inactivation of Cdk1/cyclin B. Consis tently, blocking mitotic p42 MAPK activation by MEK immunodepletion or MEK inhibition shorte ns the duration of

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18 M-phase in cycling Xenopus egg extracts (Guadagno and Ferr ell, 1998). Thus, ERK1/2 signaling can induce a state similar to mitotic arrest and control the timing of mitosis. Xenopus egg extracts have been utilized to address the involvem ent of p42 MAPK in the mitotic checkpoint. Two different e xperimental settings e used to mimic the mitotic spindle arrest. First, Xenopus egg extracts were supplemented with high concentrations of sperm nucle i and treated with nocodazo le (Minshull et al., 1994). Second, UV-irradiated nuclei were added to extr acts, which then were driven into mitosis by the addition of constitutively active Cd c25C phosphatase (Chau and Shibuya, 1999). These manipulations arrested extracts in an M-phase like state with high levels of Cdk1/cyclin B and ERK1/2 activities. Im portantly, in both experimental systems p42 MAPK activity was critical for the maintenance of the mitotic arrest, which was confirmed by MAPK immunoprecipitation, treat ment with a MAPK specific phosphatase (MKP1) (Minshull et al., 1994) and treatme nt with a MEK inhibitor, PD98059 (Chau and Shibuya, 1999). Another study, which linked ERK1/2 to m itotic arrest functions, was a genetics analysis of Drosophila s rolled/MAPK gene (Inoue and Glover, 1998), which showed that loss-of-function mutations of this gene abrogate the mitotic arrest upon treatment with colchicine (an inhibitor of microtubule polymerization). It was proposed that ERK1/2 may exer t its spindle chec kpoint function via regulation of Cdc20, an activat or of APC (Visintin et al., 1997) and a component of the anaphase-inhibitory protein complexes (Chan and Yen, 2003; Hardwick, 2005; Yu, 2002). It was shown that during mitotic arrest in Xenopus egg extracts ERK1/2 directly

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19 phosphorylates Cdc20 at Thr-64 and Thr-68 residues (Chung and Chen, 2003). A Cdc20 mutant non-phosphorylatable at these (and tw o other) phosphorylation sites malfunctions during induction of the mitotic arrest: it does not form complexes with BubR1 and Mad2 and causes a decline in Cdk1 activity under th e mitotic spindle checkpoint conditions. The same phenotype was observed when ERK1/2 activity was blocked with MEK inhibitors, U0126 or PD98059, which further li nks these Cdc20 functions to ERK1/2 signaling (Chung and Chen, 2003). Regulation of Golgi fragmentation is another function of ERK signaling during mitosis. It was shown that phosphorylated ME K associates with membrane fractions in mitotic cells (Harding et al., 2003), localizes to Golgi during prophase (Colanzi et al., 2003) and is necessary for Golgi disassembly (Acharya et al., 1998). Further, a Golgi reassembly stacking protein of 55 kDa (GRA SP55) was reported to be one possible in vivo target of the MEK/ERK pathway during mito sis (Jesch et al., 2001 ). Interestingly, further studies revealed that a non-traditiona l ERK cascade regulates Golgi fragmentation during mitosis. First, it was shown that MEK1, associated with membranes during mitosis, undergoes a Cdk1/cyclin B dependent cleavage (Harding et al., 2003), which blocks its interaction with full-length ERK (Harding et al ., 2003). Subsequent studies showed that a novel alternatively spliced va riant of ERK1, ERK1c, is involved in the regulation of Golgi fragmentation during m itosis (Aebersold et al., 2004; Shaul and Seger, 2006). ERK1c is regulated in an M-phase dependent manner, undergoes a preferential mono-tyrosine phos phorylation within the TEY ac tivation loop and localizes to Golgi during early stages of mitosis (Shaul and Seger, 200 6). Two speculations can be

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20 made based on these studies. First, the mechanisms of ERK1/2 pathway activation during mitosis may be different from those duri ng cell cycle entry. Second, activation of different functional pools of mitotic ER K may be regulated differentially. In summary, ERK1/2 signaling displays a va riety of roles in mitotic regulation. It is involved in the control of the G2/M transition, spindle formation, the mitotic checkpoint as well as Golgi fragmentation. Therefore, disruption of ERK1/2 functions during mitosis can impair the mitotic regulati on at different levels and compromise the fidelity and/or order of mitotic events. I ndeed, the constitutive activation of ERK1/2 signaling in v-mos or v-ras infected 3T3 fibroblasts provokes genomic instability via disregulation of mitosis (F ukasawa and Vande Woude, 1997; Saavedra et al., 1999). Further, it is proposed that the high inciden ce of cancer in patien ts with hereditary tyrosinemia type I (Weinberg et al., 1976) and hepatitis B (Margolis et al., 1991) might be a result of genomic instability caused by non-controlled ERK1/2 activation at mitosis (Jorquera and Tanguay, 2001; Yun et al., 2004). Therefore, deciphering functions and re gulation of the ERK1/2 pathway during mitosis is important for generation of a co mprehensive vision of its role throughout the cell cycle. It will also c ontribute to understanding biochemical mechanisms underlying pathologies, which is necessary for future development of efficient medical diagnostics and treatment approaches.

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21 Regulation of ERK1/2 signaling by MEK kinases MEK kinases represent a key regulatory level in the MAPK module, where many different stimuli can converge to allow sp ecific activation of a certain MEK/MAPK pathway (Fanger et al., 1997; Widmann et al., 1999; Xia et al., 2000; Yujiri et al., 1998), 2000). Consistent with this activation of the MEK/ERK1 /2 pathway under different cellular circumstances is regulated by different MEK kinases. Indeed, the ERK1/2 signaling during cell cycle entry and meio sis is activated by Raf-1 and c-Mos, respectively. The cell cycle initiation is triggered by engagement of extracellular growth receptors and their oligomerization (Yarden and Schlessinger, 1987). This, in turn, stimulates conversion of a small GTP-ase pr otein Ras from an inactive GDP-bound to an active GTP-bound form (Kolch, 2000; Pears on et al., 2001b). Ras-GTP mediates membrane translocation and activation of Raf1, a classical MEK kinase that triggers the MEK/ERK/1/2 cascade during the G0-G1-S transition. Activate d MAPK directly phosphorylates cytoplasmic and cytoskeletal substr ates or translocates into the nucleus to modulate transcription factors, which togeth er promote progression into S-phase (Kolch, 2000; Pearson et al., 2001b). Mechanism of ERK activation and its f unctions are strikingly different during meiosis. It is well established that hormonal stimulation triggers oocyte maturation, at least partially, via induction of synthesis of c-Mos, a MEK kinase that activates the MEK/ERK pathway during meiosis (Castro et al., 2001). With slight variations among

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22 species, activated ERK during meiosis is invol ved in activation of MPF (Cdk1/cyclin B), maintenance of MPF activity between MI and MII of meiosis and, finally, in induction of the CSF (cytostatic factor) arrest of mature oocytes (Castro et al., 2001). Thus, activation of the ERK pathway by different MEK kinase s may condition specificity of its cellular functions. The underlying biochemical mechanisms th at control activatio n of the MEK/ERK cascade during mitosis are poorly understood. It is unclear whether Raf-1 is involved in the regulation of the ERK pathway during m itosis. It was shown that Raf-1 undergoes hyperphosphorylation and activa tion in cells arrested at mitosis with nocodazole treatment (Laird et al., 1995; Pathan et al., 1996; Ziogas et al., 1998). Interestingly, this phosphorylation and activation of Raf-1 is t hought to be directly regulated by Src and occur in a Ras-independent (Ziogas et al ., 1998), but Rac/Cdc42/Pak-dependent manner (Zang et al., 2001). However, activation of Raf-1 at mitosis does not correlate with activation of the MEK/ERK cascade (Laird et al., 1999; Ziogas et al., 1998). Furthermore, none of the published works provide conclusive evidence for mitotic activation of Raf-1 in naturally cycling somatic cells. As well, it has not been clearly demonstrated that Raf-1 in mitotic cells is linked to some of MAPK s mitotic functions. A study by Yue and Ferrell detected small am ounts of germ-cell specific MEK kinase, cMos, in Xenopus egg extracts and provided eviden ce that c-Mos was important for activating the p42 MAPK pathway at mitosis (Yue and Ferrell, 2004). However, earlier studies show that c-Mos is efficiently degrad ed following egg fertilization (Nishizawa et al., 1993; Watanabe et al., 1991; Watanabe et al., 1989) and its expression is repressed in

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23 somatic tissues (Xu and Cooper, 1995; Zinkel et al., 1992). Therefor e, the involvement of Raf-1 and c-Mos in mitotic MAPK signali ng is speculative and perhaps another MEK kinase is required for activation of the MAPK pathway at mitosis. In conclusion, a variety of MEK kinases allows activation of the MAPK pathways by different stimuli. It is established that Raf-1 triggers activation of ERK1/2 signaling following mitogenic stimulation, whereas cMos activates the p42 MAPK cascade during meiosis. Circumstantial evidence indicates th at neither Raf-1 nor cMos are involved in the regulation of the ERK cascade during mitosi s. Thus, the identity of the mitotic MEK kinase remains elusive. Functions and structural organiz ation of Raf family members Rafs are serine/threonine kinases, which are broadly implicated in regulation of many basic cellular processes, such as cell gr owth, proliferation, survival, differentiation and migration (Chong et al., 2001; Chong et al., 2003; Pearson et al., 2001b). In mammals, the family of Raf kinases is compri sed of three members, Raf-1 (Kan et al., 1984; Rapp et al., 1983), A-Raf (Huleihel et al., 1986) and B-Raf (Calogeraki et al., 1993; Eychene et al., 1992), which are charac terized by a substantial degree of homology and some overlapping functions. Biochemical analyses demonstrate that the vast majority of intracellular Raf functions are exerted via MAPK signaling. Ra f regulates cell grow th and proliferation through MEK/ERK dependent promotion of the cell cycle progression (Roovers and

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24 Assoian, 2000). It is demons trated that at the G0/G1 border of the cell cycle the Raf/MEK/ERK pathway is involved in down-regulation of Cdk inhibitor p27, transcriptional up-regulation of cyclin D and other events that favor activation of the cyclin-dependent cell cycl e machinery (Kerkhoff and Rapp, 1998). The cell survival functions of Rafs are exerted via the MEK/ERK-mediated activation of anti-apoptotic kinase Akt (von Gise et al., 2001), transcriptional repression of pro-apoptotic BH3-only members of the Bcl2 family (for instance, Bim) (Weston et al., 2003) and up-regulation of IAPs (Wiese et al., 2001). The lo ngevity of the intracellular ERK signaling conditioned by kinetics of Raf activation determines the proliferation/differentiation decision of a cell (Brummer et al., 2002; York et al., 1998). It is pr oposed that prolonged ERK activation, directed by B-Raf, induces cell differentiation, whereas transient ERK activation via Raf-1 causes their proliferation (York et al., 1998). Raf family members as well are implicated in the regulation of cyto skeleton rearrangements necessary for cell migration, which is critical for embryogene sis, wound healing, angiogenesis, immune functions and tumor metastasis (Ehrenreiter et al., 2005; Pritchard et al., 2004). Analysis of Raf functions on an organism al level demonstrated that members of the Raf family are critical for life. Ind eed, A-Raf knockout mice di splay intestinal and neurological abnormalities, which can lead to postnatal death or survival with severe neurological defects (Wojnow ski et al., 1998). Complete Raf-1 knockout mice display serious embryonic aberrations: growth re tardation, placenta anomalies and underdevelopment of the liver due to massive apoptosis of hepatocytes. These mice die at midgestation (Mikula et al., 2001). B-Raf-de ficient mouse embryos are characterized by

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25 massive apoptosis, causing aberrant vasculature development and vascular hemorrhage. These mice as well die at midgestation (Wojnowsk i et al., 2000; Wojnow ski et al., 1997). All three members of the Raf kinase family, Raf-1, B-Raf, and A-Raf, share a common architecture of domain organization. They all c ontain three large conserved regions: CR1, CR2, and CR3, and are functionally divided into the Nterminal regulatory domain and the C-terminal catalytic domain (Fig. 3) (Daum et al., 1994; Morrison and Cutler, 1997). CR1 and CR2 conserved regions are located in the N-terminal regulatory portion of Raf protein, whereas CR3 houses the C-terminal kinase domain. The Nterminal CR1 and CR2 conserved regions with in the regulatory domain harbor protein binding and regulatory phosphoryl ation sites. Specifically, CR1 contains a Ras binding domain (RBD) and a cysteine rich domain (CRD). These sites are critical for Raf interaction with up-stream re gulators: small GTP-ases, such as Ras and Rap1 (Chuang et al., 1994) (Fig. 3). The CR2 region contains a phosphorylation site, which is involved in negative regulation of Raf activity (Guan et al., 2000; Morrison et al., 1993; Zimmermann and Moelling, 1999) and repres ents a 14-3-3 binding phospho-epitope (Michaud et al., 1995) (Fig. 3). The kinase domain within th e C-terminal conserved CR3 portion of Raf protein harbors two importa nt phosphorylation regul atory sites: the Nregion (Fabian et al., 1993; King et al., 1998; Mason et al ., 1999) and activation loop (Chong et al., 2001; Zhang and Guan, 2000) (Fig. 3). Analogous to ot her kinases, it is thought that the phosphorylation within the ac tivation loop of Raf induces conformational changes, which favor interaction of the ATP binding site with ATP (Huse and Kuriyan, 2002; Johnson and Lewis, 2001). Interestingly, unlike other Raf family members,

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Regulatory Domain Kinase Domain A ctivation loo p N-re g ion Ras C Ras P P P P CR1 CR3 P P P P P CR2 CR1 CR3 CR1 CR3 NC P P P P P CR2 CR2 B-Ra f N N C Ra f -1 A-Ra f Figure 3. Domain organization of Raf family members All members of the Raf family possess three conserved regions CR1 and CR2, which comprise an N-terminal regulatory domain, and CR3, which comprises the catalytic domain. Note that B-Raf, unlike other members of the family, contains as well a unique N-terminal domain (shown in shaded box). 26

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27 B-Raf contains a unique N-te rminal domain, which may be involved in B-Raf specific regulation or functions (Fig. 3) (Kalmes et al., 1998). In conclusion, Raf kinases are broadly imp licated in a variety of cellular processes and are critical for proper organismal deve lopment and function. Members of the Raf kinase family share a significant sequence homology and a similar functional domain organization, with some exceptions for B-Raf. Regulation of B-Raf activity by phosphory lation and protein-protein interactions It is traditionally assumed that all member s of Raf family are regulated by similar mechanisms. Indeed, all three Rafs have the same basic functional organization: Nterminal regulatory domain a nd the N-region and ac tivation loop in the C-terminal kinase domain. It is believed that in an inactive state the N-terminal re gulatory domain of Raf directly binds to the C-terminal kinase dom ain, blocking its accessibi lity (Heidecker et al., 1990). Thus, activation of Raf can be in terpreted as opening of Rafs tertiary structure by disrupting association between the N-terminal and C-terminal domains (Kolch, 2000). This is achieved by a seri es of coordinated re gulatory phosphorylation and protein-protein association events. It is proposed th at active GTP-bound Ras directly binds to the RBD and CRD within CR1 regi on (Fig. 3) (Chuang et al., 1994; Morrison and Cutler, 1997; Nassar et al., 1995; Okada et al., 1999; Vojtek et al., 1993) and provokes Rafs translocation to the plasma membrane and removal of the inhibitory dephosphorylation from the CR2 (Jaumot a nd Hancock, 2001; Kubicek et al., 2002;

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28 Mitsuhashi et al., 2003). Th ese predispose Raf for positiv e regulatory phosphorylations at the catalytic domain, which finally induce the open active conformation. Despite the over-all similariti es in regulation of Raf1 and B-Raf activation, the accumulated evidence indicates that regulator y mechanisms involved in activation of Raf-1 and B-Raf differ in many important aspe cts. First, Raf-1 and B-Raf require a different dose of the regulatory phosphoryl ation to achieve full activation. Indeed, following Ras-mediated stimulation, activ ation of Raf-1 occurs as a two-set phosphorylation (Marais et al., 1997). Th is includes Ras and Src dependent phosphorylation of Ser-338 and Tyr-341 residue s in the N-region (Diaz et al., 1997; Fabian et al., 1993; Marais et al., 19 95; Mason et al., 1999) and Ras dependent phosphorylation of Thr-491 and Ser-494 residues in the activation loop (Fig. 3) (Chong et al., 2003). In contrast to this, B-Raf does not undergo regulation in the N-region: its Ser445 residue is constitutively phosphorylat ed (Mason et al., 1999) and the residue corresponding to Raf-1s Tyr-341 is a phos pho-mimicking Asp-448 residue (Fig. 4). Hence, Ras-mediated activation of B-Raf requires only phosphorylation at The-599 and Ser-602 residues within the activation loop (Fig. 4) (Zha ng and Guan, 2000). It is proposed that the constitutive presence of th e negative charge in the N-region of B-Raf inhibits intramolecular associa tion between the regulatory a nd catalytic domains(Tran et al., 2005), which explains the higher basal le vel activity of B-Raf compared to Raf-1 (Marais et al., 1997).

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P P P P P P P S750 T753 S428 T439 S445 D448 T599 S602 S364 P C N CR 123 CR CR Figure 4. Phosphorylation in regulation of B-Raf activity Activity of Human B-Raf is regulated both positively and negatively via phosphorylation. Positive regulation by p hosphorylation includes: 1) constitutive phosphorylation at the N-region (constitutively phosphorylated Serine-445 and phosphorylation-mimicking Aspartic acid-448); and 2) Ras-dependent phosphorylation at the activation loop (Threonine-599 and Serine-602 residues). Both regulatory units are located within the C-terminal CR3 (catalytic) domain of B-Raf (shown in yellow). Phosphorylation at Serine-364, Serine-428 and Threonine-439 residues negatively regulates B-Raf and is executed by Akt. B-Raf is also phosphorylated by ERK at the C-terminal Serine-750 and Threonine-753 residues, but the effect of this phosphorylation on B-Raf activity is unknown. 29

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30 Both B-Raf and Raf-1 undergo an inhibito ry phosphorylation by Akt. However, Akt targets B-Raf at three residues (Se r-364, Ser-428 and Thr-439) (Chong et al., 2001; Guan et al., 2000) (Fig. 4), whereas Raf-1 at only one site (Zimmermann and Moelling, 1999). Interestingly, one of Akts phosphoryla tion sites in B-Raf, the Ser-364 residue, can as well be phosphorylated by SGK, a se rum and glucocorticoid inducible kinase, which demonstrates homology to Akt (Zhang et al., 2001). Recent work demonstrated that human B-Raf undergoes a feedback phosphorylation by ERK at Ser-750 and Thr-753 re sidues unique to B-Rafs C-terminal (Brummer et al., 2003) (Fig. 4). The effect of this phosphorylation on B-Raf activity was not determined. However, it is suggested that phosphorylation of B-Rafs C-terminus may negatively affect B-Raf functions, such as cell differentiation (Brummer et al., 2003; Rushworth et al., 2006). B-Raf activity is regulated as well on the level of protein-protein interactions. Several B-Rafs interactors have been identified (Fig. 5), which can be classified in two groups. The first group comprises associati ons with the signaling and structural components of the MAPK module. These includ e B-Rafs direct associations with its upstream activators, small GTP-ases, and downstream target, MEK, association of B-Raf with other components of MAPK cascade via scaffolding proteins and formation of BRaf/Raf-1 heterodimers. The second group of B-Rafs protein partners includes nonenzymatic proteins outside of the MAPK cascade module, such as 14-3-3 protein and chaperone protein HSP90.

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30 kD29 kD44 kD71 kD90 kD21 kD21 kD B-Ra f HSP90 14-3-3 Rap1 Ras PA28 alpha MEK1/2 Raf-1 Figure 5. Known partners of B-Raf protein complexes Proteins known to interact with B-Raf are shown and their molecular weights are indicated. As mentioned above, B-Raf and Raf-1 directly associate with GTP-ases Ras and Rap1 (Berruti, 2000; Houslay and Kolch, 2000; Papin et al., 1996; Vossler et al., 1997; Yamamori et al., 1995; York et al., 1998). Interestingly, B-Raf and Raf-1 differ in their response to up-stream regulation by Rap1. Notably, Rap1 activates B-Raf and B-Raf-dependent activation of MAPK, but inhibits the Raf-1/MEK/ERK cascade (Bos et al., 2001; Houslay and Kolch, 2000; York et al., 1998). B-Raf directly associates with MEK (Papin et al., 1996; Papin et al., 1995) and is characterized by a higher affinity to it than Raf-1 (Papin et al., 1998), which can explain the fact that B-Raf is a much stronger activator of MEK, compared to Raf-1 or A-Raf 31

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32 (Mason et al., 1999; Zhang and Guan, 2000). Interestingly, B-Raf undergoes a unique mode of additional regulati on, alternative splicing (Bar nier et al., 1995), which can modulate affinity of different B-Raf isof orms toward MEK (Papin et al., 1998). The concept of integration of all com ponents the MAPK signaling pathway within one functional unit was developed through st udying mating and osmosensor pathways in budding yeast (Faux and Scott, 1996). Metazoan scaffolding proteins were identified by genetic screens in Drosophila and C.elegans They include KSR (kinase suppressor of Ras), Soc-2 (suppressor of clear homolog) and CNK (connector enhancer of KSR). All of these proteins serve a structural framework for efficient interac tion of Raf, MEK and ERK. To accomplish this they have to fulfill three criteria: 1) accommodate simultaneous interaction with all component s of the MAPK module (Cacace et al., 1999; Stewart et al., 1999); 2) facilitate activation wi thin the module (Li et al., 2000; Roy et al., 2002) and 3) may not possess any kinase activity (Cacace et al., 1999; Morrison, 2001; Stewart et al., 1999; Yu et al., 1998). It is assumed that B-Raf signa ling, like Raf-1s, is facilitated by scaffolding proteins. Early work showed that a forced Raf o ligomerization induces Ras dependent Raf activation (Luo et al., 1996). Furthermore, B-Raf was purified as a component of a Rasdependent activator of Raf-1 (Mizutani et al., 1998). Thus, mutual association and regulation among Raf members was suggested. A more recent study directly showed that endogenous Raf-1 and B-Raf form heterodimers in response to Ras stimulation (Weber et al., 2001). Biological significance of this inte raction is not fully understood. However, it

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33 was shown that B-Raf/Raf-1 heterodimers possess a higher activity than corresponding monodimers (Rushworth et al., 2006). 14-3-3 protein is important for regula tion of B-Raf activity (Kolch, 2000). Phosphorylation of B-Raf at Ser-364 resi due creates a 14-3-3 binding motif, which analogously to Raf-1, may be necessary for locking B-Raf in an inactive conformation (Morrison and Cutler, 1997). Additionally, it is proposed that 14-33 protein contributes to activation of B-Raf signaling at least by facilitating B-Raf heterodimerization with Raf-1 (Garnett et al., 2005; Rushworth et al., 2006). B-Raf has been shown to associate with heat-shock protein 90 (HSP90) (Grammatikakis et al., 1999; Jaiswal et al., 1996). It is accepted that association of HSP90 with B-Raf maintains B-Rafs prope r folding and functionality (Cissel and Beaven, 2000; Schulte et al., 1995). In summary, regulation of B-Raf activati on and function is exerted via controlled coordination of B-Raf phosphoryl ation and its association with regulatory, structural and effector proteins. Similar to other members of the Raf kinase fam ily, activation of B-Raf involves unfolding of its inactive conformati on. However, B-Raf stands apart due to several unique regulatory features. Unlike Raf-1, B-Raf is constitutively phosphorylated within the regulatory N-region, can respond di fferentially to an up-stream signaling by GTP-ases and can undergo alte rnative splicing, which may regulate B-Rafs affinity toward MEK. B-Rafs association with ot her proteins significantly contributes to regulation of its activity and functions. Th e most well studied interactions include: association with Ras and 14-3-3, forma tion of Raf/MEK/ERK signalosomes via

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34 scaffolding proteins, B-Raf/Raf-1 hetero dimerization, and stab ilization of B-Raf molecules by association with chaperone HSP90. B-Raf as an oncogene Members of Raf kinase family were iden tified as proto-oncogen es (Jansen et al., 1984; Sutrave et al., 1984). However, exte nsive studies of Raf-1 mutations did not provide strong evidence for Raf-1 being involved in tumorogene sis. This situation was drastically changed in 2002 when B-Raf wa s claimed to be a potential cause of a significant proportion of human cancers (Davie s et al., 2002). This key study, which performed a wide-range genomic screening of human tumors, demonstrated that B-Raf is mutated in 8% of all human tumors, including nearly 70% of melano mas, 14% of ovarian cancers, 14% of liver cancers a nd 12% of colorectal tumors (Davies et al., 2002). All of the detected B-Raf mutations were within its kinase domain. Interestingly, the overwhelming proportion of these mutations was represented by a single amino acid substitution: Val-600-Glu (B-Raf V600E ). It accounted for 80% of all mutations and for more than 90% mutations in melanomas (Davies et al., 2002). As described in the previous section, phosphorylation of th e residues Thr-599 and Ser-602 flanking the mutated site is critical for full activation of B-Raf (Zhang and Guan, 2000). Therefore, it is not surprising that the phospho-mimicking Val-600-Glu mutation in the activation loop dramatically increases B-Rafs act ivity (Davies et al., 2002). B-Raf V600E induces transformation of NIH3T3 cells and cultured melanocytes via over-activation of the

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35 MEK/ERK pathway (Davies et al., 2002; Wellbro ck et al., 2004) and tumorigenicity in nude mice (Wellbrock et al., 2004). Further, treatment of melanoma cell lines harboring B-Raf V600E with Raf specific inhibitor BAY 43-9006 i nduces cell cycle arrest or apoptosis (Sharma et al., 2005). Since the B-Raf V600E mutant displays all of major features of an oncogene and is expressed in a substantial percentage of melanomas, extensive studies focused on elucidating how it may be involved in the oncogenicity of melanoma. The accumulated evidence suggests that mutation of B-Raf o ccurs during the early benign stages of melanocyte dysplasia (nevi formation) and th e B-Raf mutation must be combined with other genetic abnormalities to become malignant. Indeed, B-Raf V600E mutation was detected in more than 80% of nevi, which can remain quiescent for decades without progressing into malignancy (Michalogl ou et al., 2005). Expression of B-Raf V600E mutant in human melanocytes resulted in their sene scence-like arrest (Michaloglou et al., 2005) or in formation of non-malignant nevi in transgenic animals (Patton et al., 2005). Importantly, transgenic zebrafish carr ying the oncogenic B-Raf on a p53 deficient background progressively developed invasive melanomas (Patton et al., 2005). In conclusion, B-Raf has recently been recognized as a cellular oncogene. Its most frequently mutated version, B-Raf V600E is expressed in a la rge proportion of human cancers, ranking the highest in melanomas. Th is B-Raf mutant displa ys all the hallmarks of a classical oncogene: it po ssesses a greatly elevated kinase activity, promotes constitutive non-controllable ERK signaling, induces transfor mation of cultured cell lines and is required for survival of these tumors. It is proposed that B-Raf mutation occurs at

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36 the early stages of cellular dysplasia and can provoke tumorigenesis in combination with other mutations. Identification of B-Raf as a critical oncogene highlights the importance of research focused on the elucidation of B-Raf regulatory mechanisms and B-Raf functions. Studying MAPK signaling in the cell-free system of Xenopus egg extracts Since the introduction of amphibian egg ex tracts as a research tool (Lohka and Masui, 1983), they have been shown to faith fully mimic many aspects of the cellular cell cycle and have been used to study a variety of cell cycle regulated processes. These include DNA replication (Arias and Walter, 2004; Blow and Laskey, 1986; Tutter and Walter, 2006), nucleus formation (Chan and Forbes, 2006; Hutchison et al., 1988; Lohka, 1998), vesicle fusion (Tuomikoski et al., 1989), cytoskeleton rearrangements (Belmont et al., 1990; Mandato et al., 2001), mitotic spi ndle formation and function (Desai et al., 1999; Horne and Guadagno, 2003; Maresc a and Heald, 2006; Tremethick, 1999), apoptosis (Deming and Kornbl uth, 2006) and oocyte matura tion (Crane and Ruderman, 2006; Ohsumi et al., 2006). The main advantage of the cell-free system of Xenopus egg extracts is that it permits many technical manipulations not feasib le in tissue culture cells. This greatly increases the range of biochemical approaches which can be used for cell cycle analysis. Indeed, egg extracts are homogenous and synchronized for cell cycle progression, which is not the case for proliferati ng tissue culture cells. Importantly, extracts are much more

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37 suitable for biochemical fractionation, imm unoprecipitation and im munodepletion, and morphological analysis of sub-cellular structures. The cell-free system of Xenopus egg extracts is a great experimental model for studying MAPK signaling. It has been repe atedly shown that the MAPK cascade is activated throughout meiosis in Xenopus oocytes (Abrieu et al., 2001; Gotoh et al., 1995; Gotoh et al., 1991a; Gotoh et al., 1991b; Kosako et al., 1994) and during M-phase of the subsequent embryonic cycles (Guadagno and Ferrell, 1998; Ha rtley et al., 1994; Takenaka et al., 1997). This implie s that extracts prepared from Xenopus oocytes at different maturation stages or after ferti lization can recapitulate MAPK signaling under different biological contexts, meio sis or mitosis respectively. Several standard protocols have be en developed in order to prepare Xenopus egg extracts that represent MAPK signaling during different stages of meiosis. Specifically, extracts prepared from immature oocytes arre sted in prophase I of meiosis are used for studying MAPK regulation and functions during the G2/M transition and meiosis (Crane and Ruderman, 2006). Extract pr eparations derived from fully developed CSF (cytostatic factor) arrested Xenopus oocytes (Posada et al., 1993; Sa gata et al., 1989) are a valuable research tool for studying MA PK signaling involved in meiotic metaphase arrest (Fig. 6) (Lohka and Maller, 1985; Nebreda and Hunt, 1993). Extracts prepared from activated Xenopus eggs are routinely used for recapitulating the early embryonic cell cycles of Sand M-phases. The transition of a Xenopus oocyte to the first mitotic cell cycle is triggered during fertilization by a transient increase in intracellular Ca 2+ concentration (Cuthbertson and Cobbold, 1985;

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38 Lorca et al., 1993). To reproduce th is biochemical trigger, mature Xenopus oocytes are parthenogenetically activated in vitro by a brief treatment with Ca 2+ ionophore (Fig. 6) (Chen et al., 1998) or by subjecting them to a quick electro-shock (Murray, 1991). Since extracts prepared from these eggs recap itulate embryonic cell cycling, which does not include G1 and G2 phases, act ivation of MAPK in cycling extracts is restricted to Mphase. Thus, mitotic MAPK activation does not overlap with MAPK activation at the G1/S, as it occurs in somatic cells. Therefore, the cycling Xenopus egg extracts is an ideal biochemical model to study regulati on of the MAPK cascade at M-phase. The synchronous embryonic cell cyc ling during the early stages of Xenopus embryogenesis as well as in Xenopus egg cycling extracts is conditioned primarily by oscillation of mitotic cyc lin B levels (Murray and Kirschner, 1989a). Since Xenopus egg extracts are very amenable to biochemical mani pulations, it is relatively easy to arrest them in Sor M-phase. Specifically, prepar ation of extracts from parthenogenetically activated oocytes in the presence of cyclohe ximide blocks synthesis of cyclin B and arrests them in S-phase (Fig. 6). Supplementing of these extracts with recombinant nondegradable cyclin B (which lacks a ubi quitination D-box) (G lotzer et al., 1991) permanently arrests them in M-phase (Fig. 6). This approach is routinely used to prepare unlimited quantities of experimental material synchronously arrested at the Sor Mphases of the cell cycle.

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A ctivated eggs S-phase arrested extract M-phase arrested extract CSF-arrested oocytes Cycloheximide GST-cyclin B Ca 2+ ionophore CSF extract Cycling extract Figure 6. Cell-free system of Xenopus egg extracts Mature CSF-arrested oocytes (arrested in the second metaphase of meiosis) are collected and directly processed into CSF extracts or parthenogenetically activated with Ca 2+ ionophore A23087 prior to processing into S-, M-phase arrested or cycling extracts. 39

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40 In summary, the cell-free system of Xenopus egg extracts is a powerful experimental model, which surpasses tissu e culture cells by the scope of feasible biochemical manipulations. The system is based on the natural ability of Xenopus oocytes to autonomously sustain the cell cycl ing processes. Many different aspects of cell cycling and signal transduction m echanisms can be addressed by using Xenopus egg extracts. Particularly, the cell-free system of Xenopus egg extracts is an optimal biochemical model to dissect functions a nd regulation of the MAPK signaling during mitosis.

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41 Dissertation statement The MAPK cascade is an important regulat or of the cell cycle (Widmann et al., 1999). It is evident that MAPK signaling is implicated in contro l of at least two stages of the somatic cell cycle, namely, G1/S transi tion (Roovers and Assoian, 2000; Widmann et al., 1999) and mitosis (Gotoh et al., 1991b; Guadagno and Ferrell, 1998; Harding et al., 2003; Minshull et al., 1994; Takenaka et al ., 1997; Willard and Crouch, 2001; Zecevic et al., 1998). Regulatory mechanisms and func tions of the MAPK pathway during cell cycle initiation are well understood and represent a classical dogma of molecular biology (Roovers and Assoian, 2000; Widmann et al., 19 99). In contrast, roles for MAPK and its activation during mitosis are much less defi ned. Nevertheless, data from different experimental systems have revealed involveme nt of MAPK signaling in the regulation of a variety of mitotic functions: mitotic onset (Liu et al., 2004; Wright et al., 1999), formation of the mitotic spindle (Horne a nd Guadagno, 2003; Zhang et al., 2005), mitotic metaphase arrest (Chau and Shibuya, 1999; Mi nshull et al., 1994), Golgi fragmentation (Aebersold et al., 2004; Shaul and Seger, 2006) and the duration of mitosis (Guadagno and Ferrell, 1998; Roberts et al., 2002). Therefore, to obtain a comprehensive understanding of how MAPK signaling is integrated into the cell cycle regulatory network, it is critical to decipher the mech anism of MAPK activa tion during mitosis. Furthermore, understanding how MAPK is controlled during mitosis may shed light on the biochemical mechanisms behind some pat hological states, such as cancer. Indeed, the MAPK pathway is constitutively activated in over 30% of human cancers (Hoshino et

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42 al., 1999). Based on the fact that MAPK plays a variety of roles throughout mitosis, it is plausible to suggest that di sregulation of mitotic functi ons of the MAPK pathway may cause genomic instability and provoke tumorogenesis. The activation of the MAPK cascade unde r certain biological contexts is conditioned by specific activation of partic ular MEK kinases (F anger et al., 1997; Widmann et al., 1999). Ther efore, I hypothesize that a specific MEK kinase is activated in an M-phase dependent manner and activates the MEK/MAPK pathway at mitosis To address this hypothesis I am going to use M-phase arrested Xenopus egg extracts to purify and identify mitotic MEK kinase activity. Subsequently, I will utilize biochemical approaches to study how this MEK kinase is regulated during mitosis. The results of my dissertation studies will determine the mechanism of regulating the activation of the MAPK cascade during mitosis and expa nd our understanding of how MAPK signaling is implicated in overall cell cycle regulation.

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43 Chapter Two Identification of B-Raf as an M-phase MEK Kinase Introduction In order to decipher the regulatory m echanisms involved in activating the MEK/MAPK pathway at mitosis, the M-phase MEK kinase must be identified. To achieve this, I utilized biochemical approaches in order to purify a nd directly identify the mitotic MEK kinase. This research strategy requires an appropriate experimental model that fulfills several requirem ents. First, it should fairly represent the biochemistry of mitotic activation of the MAPK cascade. Secondly, this experimental system should be amenable for biochemical manipulations. And thirdly, the experimental material should be available in quantities su itable for extensive biochemical purification. The cell-free system of Xenopus egg extracts surpasses the tissue culture cells in all of the abovementioned requirements and represents an ideal biochemical experimental model for studying mitotic MAPK signaling. Therefore, my biochemical studies were performed by using the cell-free system of Xenopus egg extracts. For this part of my studies, Xenopus egg extracts that are arrested in M-phase were utilized. To prepare these extracts, mature (CSF-arrested) Xenopus oocytes were

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44 collected and parthenogenetically activated in vitro by a brief incubation with calcium ionophore (Chen et al., 1998). The activated eg gs were processed into extracts and permanently arrested in M-phase by the addi tion of non-degradable recombinant cyclin B (Glotzer et al., 1991). First, I ascertained that my Xenopus extracts preparations represent an M-phase specific activation of the MAPK cascade. Sp ecifically, it was checked whether they are void of meiotic MEK kinase, c-Mos (Castro et al., 2001; Watanabe et al., 1989), and contain high levels of active MEK kinase, MEK and MAPK (Takenaka et al., 1997). Second, M-phase arrested Xenopus egg extracts were subjected to different fractionation approaches in order to isolat e mitotic MEK kinase activity. MEK kinases activate the down-stream MEKs by phosphoryl ating them at Serine and Threonine residues within the activation loop (Z heng and Guan, 1994). Thus, monitoring phosphorylation status of these residues in recombinant MEK following its incubation in vitro with aliquots of samples was used as a sc reening tool to detect the presence of endogenous MEK kinase activity and visual ize its redistribution among different fractions. The outcome of this part of the study was the developm ent of a pur ification scheme leading to an enrichment of mitotic MEK kinase activity. Up-scaled quantities of the M-phase arrested extracts were subjected to the developed purification protoc ol. The final fractions containing active mitotic MEK kinase were analyzed for their compositi on by Western blotting a nd Silver staining. Loss-of-function approach was undertaken to validate involvement of the identified candidate MEK kinase into mitotic activation of the MAPK cascade.

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45 Results Preparation of extracts from parthenoge netically activated Xenopus eggs, which are devoid of a germ-cell specific MEK kinase, c-Mos It is well established that activation of the MAPK pathway during Xenopus oocyte maturation is directed by a germ-cell specific MEK kinase, c-Mos (Castro et al., 2001). A temporal increase in intracellular calciu m concentration induced by egg fertilization triggers degradation of c-Mos (Nishizawa et al., 1993; Watanabe et al., 1989) and inactivation of MAPK si gnaling (Ferrell et al., 1991), which contribute to the transition to the embryonic cell cycling (Cas tro et al., 2001). As shown in Figure 7, fertilization of mature Xenopus oocytes in vitro led to efficient degradati on of c-Mos protein. By 50 min, c-Mos was undetectable by immunoblot analysis with anti-c-Mos antibodies. To recapitulate proper transition from CSF arrest to embryonic cycling in my experimental system, the time require d for c-Mos degradation following Ca 2+ ionophore treatment was determined. To do this, mature Xenopus oocytes were parthenogenetically activated with Ca 2+ ionophore A23187, collected at 10 min intervals and analyzed for the presence of c-Mos protein by Western blotting. As shown in Figure 7, Ca 2+ ionophore activation of Xenopus oocytes led to a complete disa ppearance of c-Mos within 40-50 min after activation. Importantly, the first mitotic cleav age, observed by 80 min postactivation, occurred in the absence of c-Mos. Therefore, I concluded that the activated eggs must be incubated at room temper ature for 50-60 min before processing into extracts. Using this protocol, I was able to consistently prepare Xenopus egg extracts that

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are devoid of detectable amounts of c-Mos protein and recapitulate in vitro early embryonic cell cycles. are devoid of detectable amounts of c-Mos protein and recapitulate in vitro early embryonic cell cycles. Fertilization First embr y onic cleava g e c-Mos c-Mos 46 Figure 7. c-Mos is degraded at similar times for in vitro fertilized (upper p anel) or Ca 2+ ionophore activated (lower panel) Xenopus eggs and undetectable during mitosis Mature oocytes were fertilized or activated with Ca 2+ ionophore, collected at the indicated time points and lysed. Lysates were separated by SDS-PAGE and immunoblotted with c-Mos antibodies. Note that the first embryonic cleavage occurred by 80 min in fertilized eggs. Equal loading was verified by Coomassie staining. 10 20 30 40 50 60 70 80 min 10 20 30 40 50 60 70 80 min Unfertilized e gg s c-Mos Ca 2+ iono p hore

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47 Development of a protocol for purifica tion of mitotic MEK kinase activity S-phase extracts were prepared from activated Xenopus eggs using the protocol described above. Then, S-phase extracts were cycled into a stable M-phase state by the addition of non-degradable cyclin B. Due to the absence of growth factor control, MAPK activation is restricted to M-phase in Xenopus egg extracts (Fig. 8). Note that Xenopus oocytes and early embryos contain only one isoform of ERKs, ERK2 (Zaitsevskaya and Cooper, 1992). Since it is the first identified Xenopus s MAPK (Gotoh et al., 1991a), it is traditionally referred as to p42 MAPK. A biochemical assay for detecting MEK kinase activity in Xenopus egg extracts was developed in Dr Guadagno laboratory. Aliquots of extracts were diluted in the kinase buffer and incubated in vitro in the presence of recombinant unactive GST-MEK. The products of the reaction were separa ted by SDS-PAGE and phosphorylation of GSTMEK at the activation segmen t (Ser-217/Ser-221) was studied by Western blotting with phospho-MEK specific antibodies. Despite the absence of c-Mos, MEK kinase activity was strongly detected in M-phase egg extracts compared to S-phase egg extracts (Fig. 8) implying that some other MEK kinase respons ible for activation of the MAPK pathway at M-phase. Several MEK kinases have been shown to directly activate MEK1/2. This includes Raf family members (Hagemann and Rapp, 1999), MLK3 (Hartkamp et al., 1999), MEKKs 1-3, c-Mos, and Tpl-2 (Fange r et al., 1997; Lewis et al., 1998). Unfortunately, at the time when this project was initiated, research tools (antibodies, siRNA technique, etc) which would enable me to sp ecifically target a certain MEK kinase in order to elicit its effect on m itotic activation of the MAPK pathway, were

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Figure 8. M-phase arrested Xenopus egg extracts contain active MAPK and MEK kinase activity Equal amounts of Sand M-phase Xenopus egg extracts were subjected to an in vitro MEK kinase assay with recombinant GST-MEK as a substrate. Phospho-MEK antibodies were used to analyze phosphorylation of GST-MEK. Activation of endogenous MAPK was analyzed by phospho-MAPK Western blotting. Equal sample loading was confirmed by immunoblotting for MAPK protein (data not shown). not readily available. Therefore, to facilitate identification of the mitotic MEK kinase, I decided to purify MEK kinase activity from M-phase arrested Xenopus egg extracts. In many regards, Xenopus egg extracts represent an ideal model system for purifying and identifying the MEK kinase responsible for mitotic activation of the MAPK cascade. First, Xenopus eggs provide a rich source of components of the MAPK cascade. Secondly, activation of the MAPK cascade is restricted to mitosis in Xenopus egg extracts that autonomously undergo synchronous cell cycles of Sand M-phases (Guadagno and Ferrell, 1998; Takenaka et al., 1997). Finally, the Xenopus egg extracts are amenable to biochemical manipulations not feasible with tissue culture cells. 48

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49 As a first purification step, the crude M-phase arrested Xenopus egg extracts were separated by ultracentrifugation (100, 000 g 1.5 hr, twice) into cytosolic and membrane fractions. By applying equivalent amounts of crude and cytosolic fractions to an in vitro MEK kinase assay, I showed that the majority of the total M-phase MEK kinase activity is present in the cytosolic fr action (Table 1). This suggest s that the mitotic MEK kinase activity is cytosolic and not membrane-bound. As a second purification step, I fracti onated the M-phase cytosol by ammonium sulfate (AS) precipitation. The M-phase cytosolic fraction was supplemented with an appropriate volume of EB buffe r containing 50% ammonium su lfate to reach a final 25% salt saturation. After this, samp les were rotated for 1.5 hr at 4 0 C and precipitated proteins were pelleted by a high-speed cen trifugation (10, 000 g 15 min at 4 0 C). The protein pellet was resuspended and proteins remaining in the supernatant were transferred to a new tube and subjected to sequential 50% and 75% AS precipitations as described above. Finally, to monitor the redistribution of the MEK kinase activity among different fractions, equal amounts of protein from all three AS cuts (0-2 5%, 25-50% and 50-75% saturation) as well as the final supernatant were subjected to an in vitro MEK kinase assay. It was determined that the majority (>90%) of the mitotic MEK kinase activity from the cytosolic fraction was precipitated in the 0-25% AS cut (data not shown). Thus, mitotic MEK kinase activity from cytosolic fraction of M-phase arrested Xenopus egg extracts can be precipitated by low AS saturation, which provides a convenient purification step.

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50 To narrow down further AS concentration necessary for precipitation of the MEK kinase activity, I performed the following AS cuts with M-ph ase cytosol: 0-10%, 1015%, 15-20%, 20-25% and 25-30%. The precipitated protein fr actions were resuspended and subjected to an in vitro MEK kinase assay. As shown in Figure 9, the majority (>90%) of MEK kinase activity was precipitated in the 15-20% AS cut. Note, that the 20% ammonium sulfate saturation precipitated only 10% of the total proteins from the original M-phase cytosol fraction (Fig. 9). Thus, 20% ammonium sulfate saturation can be used as an efficient step in purific ation of the mitotic MEK kinase activity. Next, a chromatography approach was util ized to further purify the MEK kinase activity contained within the 20% AS cut. Several chromatography exchangers were screened for the ability to efficiently se parate MEK kinase activity. The following chromatography columns proved not to be useful in purifying the MEK kinase activity: a Mono S HR 5/5 column (a strong cation exch anger), a HiTrap DEAE Sepharose column (a weak anion exchanger), a Butyl Sepha rose 4FF column (a hydrophobic interaction exchanger, aliphatic interactions), a Phenyl Sepharose 6FF column (a hydrophobic interaction exchanger, aromatic interactions). Fortunately, I was able to show that ani onic exchange columns were effective for purifying the MEK kinase activity. As shown in Figure 10, I was able to separate mitotic MEK kinase activity by using a HiTrap Q Sepharose HP column (a strong anion exchanger). Specifically, by us ing a straight gradient of 0 1 M NaCl, the MEK kinase activity eluted at a range of 19-36% salt saturation. To furt her focus the elution point for the MEK kinase activity and eliminate contamination of the MEK kina se active fractions

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0102030405060708090100 MEKK activity (GS T-ME K -P ) ctrl total 10% 15% 20% 25% 30% supernatant Ammonium sulfate saturation % of totalprotein in cytosolic fraction Figure 9. 20% ammonium sulfate saturation precipitates MEK kinase activity from M-phase arrested Xenopus egg extracts Cytosolic fraction of M-phase arrested Xenopus egg extracts was sequentially subjected to protein precipitation with increasing concentration of ammonium sulfate. Precipitated proteins were recovered and subjected to an in vitro MEK kinase assay. Protein concentration of the obtained fractions was determined by using Bio-Rad Protein Assay kit. Note that previously it was shown that concentrations of ammonium sulfate higher that 25% do not precipitate MEK kinase activity from M-phase cytosolic fraction. 51

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MEK kinase active fractions 2 5 6 7 8 9 10 11 12 13 14 15 52 16 17 18 19 20 21 22 23 24 25 26 27 28 GST-MEK-P GST-MEK-P Figure 10. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate precipitated fraction by using HiTrap Q Sepharose HP anion-exchange chromatography (0 1.0 M NaCl elution gradient) 1.5 mg of 20% ammonium sulfate precipitated fraction resuspended in buffer A (50 mM HEPES, pH 7.5, 10 mM MgCl2) was applied to a 5 ml HiTrap Q Sepharose HP column (Amersham Biosciences). Proteins were eluted with a straight gradient of 1 M NaCl (buffer B: 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 M NaCl). Aliquots of eluted fractions were subjected to an in vitro MEK kinase assay. Note that MEK kinase activity was eluted at a 0.19 0.36 M NaCl range.

PAGE 75

53 with irrelevant proteins with similar desalting properties, I decided to increase the elution resolution in the range of 0 30% of salt saturation. To do this, proteins bound to a HiTrap Q Sepharose HP column were eluted with a step-wise gradient of 1 M NaCl: 0 30% saturation (4 column volumes) and 30 100% saturation (1 column volume). This significantly increased the efficiency of purif ication (Fig. 11). The MEK kinase activity, eluted at 0.22 M NaCl, was concentrated in a fewer number of fr actions and contained less total protein. Another anion-exchange column (Mono Q HR ) also turned out to be a promising tool for purification of mitotic MEK kina se activity. By applying the 20% AS precipitated fraction to a M ono Q HR 5/5 column and eluting bound proteins with a straight gradient of 0 1 M NaCl, I was able to collect mitotic MEK kinase activity in six discrete 0.5 ml fractions eluted at a 0.36 0.51 M NaCl (Fig. 12). Mono Q resin is composed of finer beaded particles than Hi Trap Q Sepharose and t hus is suitable for a more refine protein separati on. Therefore, I decided to use a Mono Q column as a purification step following separation on HiTrap Q Sepharose column. Indeed, application of MEK kinase activ e fractions eluted from a HiTrap Q Sepharose column to a Mono Q column further purified the MEK kinase activity (not shown). To increase the efficiency of separation of eluted proteins on a Mono Q column, I modified the elution conditions to a step-wise gradient of 1 M NaCl: 0 30% (3 column volumes), 30 50% (15 column volumes) and 50 100% (2 column volumes).

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MEK kinase active 11 12 13 14 15 16 17 18 19 20 21 22 23 24 fractions GST-MEK-P Figure 11. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate precipitated fraction by using HiTrap Q Sepharose HP anion-exchange chromatography (0 0.3 1.0 M NaCl elution gradient) 3.0 mg of 20% ammonium sulfate precipitated fraction resuspended in buffer A (50 mM HEPES, pH 7.5, 10 mM MgCl2) was applied to a 5 ml HiTrap Q Sepharose HP column (Amersham Biosciences). Proteins were eluted with a step-wise gradient of 1 M NaCl (buffer B: 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 M N aCl). Aliquots of eluted fractions were subjected to an in vitro MEK kinase assay. N ote that MEK kinase activity was eluted at a 0.22 M NaCl range. 54

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MEK kinase active fractions 3 4 5 6 7 8 11 15 16 23 27 32 35 36 fractions 55 37 38 39 40 41 42 43 44 45 50 55 60 fractions GST-MEK-P GST-MEK-P Figure 12. Partial purification of mitotic MEK kinase activity from 20% ammonium sulfate precipitated fraction by using MonoQ anion-exchange chromatography 1.9 mg of 20% ammonium sulfate precipitated fraction resuspended in buffer A (50 mM HEPES, pH 7.5, 10 mM MgCl2) was applied to a 1 ml Mono Q HR column (Amersham Biosciences). Proteins were eluted with a straight gradient of 1 M N aCl (buffer B: 50 mM HEPES, pH 7.5, 10 mM MgCl2, 1 M NaCl). Aliquots of eluted fractions were subjected to an in vitro MEK kinase assay. Note that MEK kinase activity was eluted at a 0.36 0.51 M NaCl range.

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56 Thus, by subjecting M-phase arrested Xenopus egg extracts to different purification approaches and using an in vitro MEK kinase assay as a screening tool, I worked out several efficient purification t echniques, which can be combined in one purification protocol to separate mitotic MEK kinase activity. Purification of MEK kinase activi ty from M-phase arrested extracts Based on results described in the prev ious section, I designed a four-step purification scheme to purify MEK kinase activity present in M-phase arrested Xenopus egg extracts (Fig. 13A). Crude S-phase ex tracts prepared from parthenogenetically activated Xenopus eggs were cycled into a stab le M-phase by the addition of nondegradable recombinant cyclin B. The M-pha se state of the extrac ts was confirmed by analyzing an in vitro histone H1 kinase activity and/or observing the nuclear envelope breakdown and chromatin condensation under fluorescent microscopy. Then, the crude M-phase extracts were separated into cytosolic and membrane fractions by ultracentrifugation. The cytosolic fraction, containing nearly all of the MEK kinase activity (Table 1), was separated by ammoni um sulfate precipita tion. Approximately 90% of the MEK kinase activity was precipita ted in the 0-20% ammonium sulfate cut and purified further by anion exchange chromat ography on HiTrap Q Sepharose and Mono Q columns. Collectively, these purification st eps enriched MEK kinase activity by ~ 260fold (Table 1). At the final purification step, a single protein peak eluted from the Mono Q column (Fig. 13B) contained MEK kinase activity as assessed by both phosphorylation

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Figure 13. Purification of MEK kinase activity from M-phase arrested Xenopus egg extracts A. Scheme for purification of an M-phase MEK kinase activity. B. Mono Q elution p rofile over a three-step 0 1.0 M NaCl gradient. C, Mono Q fractions #10-16 contain an M-phase MEK kinase activity. MEK kinase activity of Mono Q fractions # 4-60 was measured in an in vitroMEK kinase assay. MEK kinase activity was not detected in the flow through volume (not shown). 57

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58 Table 1. Purification of MEK kinase activity from M-phase arrested Xenopus egg extracts Total protein, mg Total activity, a.u. Specific activity, a.u./mg Yield, % Fold purification Crude M-phase extract 1156.8 246.2 0.21 1 Cytosol fraction 302.0 260.0 0.86 100 4.1 20% AS cut 28.0 236.8 8.46 91.1 40.3 HiTrap Q Sepharose 3.8 105.75 27.83 40.7 132.5 Mono Q 0.405 22.05 54.44 8.5 259.2 a.u. arbitrary units; fractions from each purification step were subjected to an in vitro MEK kinase assay, the levels of GSTMEK phosphorylation were determined by Western blotting and quantified by using ImageQuant software of recombinant MEK (Fig. 13C) and ac tivation of the MAPK cascade in an in vitro linked kinase assay (Fig. 14). Therefore, my results suggest that the kinase activity purified from M-phase arrested Xenopus egg extracts represents a bona fide MEK kinase. This protocol was applied at least three times and consistently led to an enrichment of the mitotic MEK kinase activity.

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04080120160200 MEK kinase activity, a.u. Fractions 8 9 10 11 12 13 14 15 16 17 18 Figure 14. Final Mono Q fractions contain M-phase MEK kinase activity Equal aliquots of Mono Q fractions # 8 18 were subjected to an in vitro linked kinase assay as described in Materials and Methods chapter. Briefly, aliquots of Mono Q fractions # 8 18 were incubated in vitro with recombinant unactive GST-MEK and GST-ERK. Next, the reaction mix was supplemented with myelin basic protein (MBP) as a substrate for ERK and incubation was continued. The levels of ERK activation in the linked kinase reaction were determined by the levels of MBP phosphorylation. To assess contribution of endogenous MAPK in the purified fractions to MBP phosphorylation, the aliquots of the fractions were incubated with MBP alone, without addition of GST-MEK and GST-ERK. The levels of MEK kinase activity were represented as a difference between MBP p hosphorylation in the cascade kinase reaction and MBP phosphorylation by the fractions itself. The levels of MBP phosphorylation were visualized by autoradiography and quantified by using ImageQuant software. 59

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60 B-Raf is enriched at the final stage of the mitotic MEK kinase purification Silver staining analysis of th e final MEK kinase active fracti ons revealed several protein bands. An example of one of the active fracti ons is shown (Fig. 15, lane 1). A prominent protein band between 90-100 kDa correlated with M-phase MEK kinase activity throughout the progress of purifi cation (see Fig. 45) and migrated at a similar molecular weight range as B-Raf. Therefore, I used a polyclonal antibody ra ised against a highly conserved N-terminal portion of Human B-Raf (Fig. 16) to assess whether the protein band might represent B-Raf. As shown in Fi gure 15 (lanes 5 and 6), this antibody readily recognized Xenopus B-Raf in crude Sand M-phase egg extracts as a 95 kDa doublet. When a purified fraction containing mitotic MEK kinase activity was immunoblotted with the B-Raf antibody, B-Raf was strongly detect ed (Fig. 15, lane 2 versus lane 6). On the other hand, neither c-Mos nor Raf-1 were detected by Western blotting (Fig. 15, lanes 3 and 4). Similar results were observed for other fractions contai ning partially purified MEK kinase activity. Thus, I conclude that B-Raf, not Ra f-1 or c-Mos, is enriched during purification of the m itotic MEK kinase activity.

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Figure 15. B-Raf is enriched at the final stage of the mitotic MEK kinase purification Mono Q fraction #12 (see Fig.11) was separated on 8% SDS-PAGE and analyzed by either silver staining (lane 1) or Western blotting with anti-B-Raf (lane 2), anti-Raf-1 (lane 3), or anti-c-Mos (lane 4) antibodies. Similar results were obtained with fraction #11. Equal amounts of total protein from crude S-phase (lane 5), M-phase (lanes 6, 7, 8), and CSF extracts (lane 9) were immunoblotted for B-Raf (lanes 5, 6), Raf-1 (lane 7), and c-Mos (lanes 8, 9). Note that the asterisk indicates recombinant GST-cyclin B recognized by Santa Cruz B-Raf polyclonal antibodies, raised against a GST-conjugated B-Raf p e p tide 61

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Figure 16. Alignment of the amino acid sequences of Xenopus (AAZ06667) and H uman (P15056) B-Raf proteins using MegAlign software sc9002 antibody epitope comprising an N-terminal region of Human B-Raf is shown in red dashed line. 62

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63 Depletion of B-Raf, but not Raf-1, blo cks activation of MAPK at mitosis Based on the purification resu lts, I hypothesized that BRaf might be required for activation of the MEK/MAPK pathway at mito sis. To test this directly, endogenous BRaf was quantitatively removed (~99%) from S-phase extracts by two rounds of immunodepletion (Fig. 17A) and the depleted extracts were cycled into mitosis by the addition of recombinant non-degradable cyclin B. At indicated time points, aliquots of egg extracts were collected to assess MAPK activation. Entry into mitosis was monitored by nuclear envelope breakdown and chromatin condensation, which in control and BRaf-depleted extracts occurred at 20 min after cyclin B addition. Following mitotic entry, MAPK became activated at 30 min in control extracts (Fig. 17B, fist and second lanes). In contrast, mitotic MAPK activati on was strongly inhibited in B-Raf depleted extracts (Fig. 17B, third lane). The addition of recombinant B-Raf protein to B-Rafdepleted extracts was sufficient to restore MA PK activation at levels similar to control extracts (Fig. 17B, fourth lane). Thus, I conclude that B-Raf is essential for activation of the MAPK pathway during mitosis in Xenopus egg extracts. Raf-1, another member of Ra f MEK kinase family, is a classical activator of the MAPK cascade after mitogenic stimulation. Interestingly, studies in mammalian cells have implicated a possible role for Raf-1 in G2/M progression (Laird et al., 1995; Pathan et al., 1996; Ziogas et al., 1998). Since Raf-1 is expressed in Xenopus egg extracts (Fig. 18A, first lane), I decided to test whet her activation of MAPK at mitosis in Xenopus egg extracts depends on Raf-1. In order to do this, Raf-1 was removed from extracts by immunodepletion (Fig. 18A). After this, the Ra f-1 depleted extracts were driven into

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Figure 17. B-Raf is required for activation of MAPK at mitosis A. B-Raf protein levels in S-phase extracts after mockand B-Raf-depletion, and adding back of recombinant His-tagged B-Raf. B. MAPK activation at mitosis is blocked in B-Raf-depleted extracts and restored after addition of recombinant B-Raf. Mockand B-Raf-depleted extracts were driven into mitosis with non-degradable cyclin B and assessed for MAPK activation by phospho-MAPK Western blotting at indicated times. 64

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Figure 18. Raf-1 is not required for mitotic activation of MAPK pathway A. Raf-1 protein levels in S-phase extracts after mockand Raf-1-immunodepletion. B. Raf-1 is not required for activation of MAPK during mitosis. Mockand Raf-1-depleted extracts were driven into mitosis with non-degradable cyclin B. 1-ml aliquots of the egg extract were collected at the indicated times to monitor MAPK activation by phospho-MAPK immunoblotting. Equal loading of samples was confirmed by Ponceau S staining of membranes. 65

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66 M-phase by the addition of recombinant non-de gradable cyclin B. Aliquots of the extracts were taken at the indicated times, and time-course of MAPK activation was studied by phospho-MAPK Western blot. As it shown on Figure 18B, depletion of endogenous Raf-1 protein had no effect on m itotic activation of the MAPK pathway. Thus, I conclude that Raf-1 is not required for mitotic activation of MAPK signaling in Xenopus egg extracts. Conclusions The primary aim of this study was to id entify the MEK kinase responsible for mitotic activation of the MEK/MAPK pathway in Xenopus egg extracts. To address this, I developed a procedure for prep aration of M-phase arrested Xenopus egg extracts devoid of a meiotic MEK kinase, c-Mos. By usi ng these extracts as a starting material, I developed a purification protoc ol leading to an enrichment of the mitotic MEK kinase activity. Fractionation of M-phase arrested Xenopus egg extracts led to the purification of a single peak of MEK kinase activity that was identified by Western analysis as B-Raf. Neither c-Mos nor Raf-1 were detected in th e purified MEK kinase active fraction. The results from my immunodepletion experiments de finitively show that B-Raf is critical for activating the MAPK pathway during mitosis in Xenopus egg extracts. In contrast, the related Raf family member, Raf-1, is not re quired for M-phase ac tivation of the MAPK pathway. Thus, I conclude that B-Raf is the major MEK kinase responsible for activation of the MAPK signaling at mitosis (Fig. 19)

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M-phase MEK B-Raf MAPK Figure 19. B-Raf activates the MEK/MAPK cascade at mitosis 67

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68 Chapter Three Characterization of B-Raf at Mitosis Introduction In Chapter Two, I demonstrated that B-Raf functions as a critical activator of the MAPK pathway during mitosis. Presently, li ttle is known about the cell cycle regulation of B-Raf at M-phase. In this Chapter I propos e to characterize the mitotic regulation of B-Raf activity using the cell-free system of Xenopus egg extracts. Specifically, data describing possible cell cycle dependent ch anges in B-Raf activity, posttranslational modification by phosphorylation a nd protein interactions with its down-stream target, MEK, will be presented. Results B-Raf activity is elevated during mitosis To examine the regulation of the BRaf/MEK/MAPK cascade at mitosis, I decided to characterize B-Raf activity in Xenopus egg extracts. Based on the requirement for B-Raf in activation of the MAPK cascad e during mitosis, I predicted that B-Raf activity would be highest during M-phase. To measure B-Raf activity, similar amounts

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69 of B-Raf protein were immunopr ecipitated from Sand M-phase arrested egg extracts and subjected to an in vitro linked kinase assay. The data showed that B-Raf activity is markedly elevated (4-6 fold) in M-phase arre sted extracts compared to S-phase extracts (Fig. 20A). A modest amount of B-Raf activ ity detected in S-phase extracts likely represents its high basal activity due to both constitutive phosphorylation and the presence of a phospho-mimicking aspartic ac id residue in the positive regulatory Nregion (Mason et al., 1999). Controls that either lack recombinant MEK and ERK or measure background kinase activity associated with rabbit IgG complexes demonstrated the specificity of the in vitro cascade reaction. Next, I examined B-Raf activity in Xenopus egg cycling extracts that naturally oscillate between Sand M-phases. Fi rst, extracts prepared from activated Xenopus eggs were incubated at room temperature to init iate cycling. At 10-min intervals, equal aliquots of cycling extracts were collected, snap-frozen on dry ice and stored at -80 o C. Extracts cycling between Sand M-phase was monitored by observing nuclear envelope breakdown/chromatin condensation and meas uring Cdk1/cyclin B activity in an in vitro histone H1 kinase assay. To measure B-Raf activity thr oughout the cell cycle, equal amounts of B-Raf protein we re immunoprecipitated from Xenopus egg cycling extracts, washed several times and subjected to an in vitro linked kinase assay. I consistently observed a fluctuation of B-Raf activity dur ing the cell cycle peaking highest during Mphase (Fig. 20B). Thus, my data shows that B-Raf is activated in an M-phase dependent manner in Xenopus egg extracts.

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Figure 20. B-Raf activity is elevated during mitosis A. B-Raf is activated in M-phase arrested extracts. B-Raf immunoprecipitates from S-and M-phase egg extracts were subjected to an in vitrolinked kinase. Equal loading of immunoprecipitated B-Raf per kinase reaction was confirmed by Western analysis. B. B-Raf activity is up-regulated during mitosis in Xenopus cycling extracts. B-Raf was immunoprecipitated from aliquots of cycling egg extract at indicated times and subjected to an in vitro linked kinase assay. In vitro histone H1 kinase activity of the same aliquots was measured in parallel as a marker for Cdk1/cyclin B activation and mitotic entry. Activation of MAPK was studied by phospho-MAPK Western blotting. 70

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71 In a recent report, high amounts of B-Raf activity were detected in Xenopus egg extracts but the authors were unable to meas ure differences in B-Raf activity between Sand M-phases (Yue et al., 2006). I speculate that the negative data could be caused by functional limitations of the two-component in vitro MEK kinase assay used in their study to measure B-Raf activity. I utilized an in vitro B-Raf/MEK/ERK/MBP linked kinase assay, which allows greater signal am plification, and used at least 5-fold more recombinant MEK. Thus, saturation of th e reaction by purified active B-Raf complexes is less likely to occur under my assay conditi ons. Therefore, I conclude that my cascade kinase reaction is more sensitive and reliab le for detecting changes in B-Raf activity. To rule out the possibility that a ME K kinase associated with B-Raf might contribute to the activation of ERK in the linked kinase reaction, I decided to compare in vitro kinase activities of catalytically inactiv e and wild type B-Raf immunoprecipitates from Sand M-phases. First, a site-direc ted technique was used to create a kinase inactive mutant of Xenopus B-Raf by substituting Lysine re sidue in the ATP-binding site to Methionine (Guan et al., 2000). Next, wild type and kinase dead recombinant myc-BRaf proteins were expressed in Xenopus CSF extracts and introduced for incubation in Sand M-phase arrested extracts (see Materials and Methods for details). Finally, the recombinant myc-B-Raf proteins were recovered by myc-tag immunoprecipitation and subjected to an in vitro linked kinase assay as described a bove. My results show that the catalytically inactive B-Raf immunoprecipita tes do not display any MEK kinase activity in an in vitro linked kinase assay compared to wild type B-Raf (Fig. 21). This result

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Figure 21. Kinase-dead B-Raf immuno-complexes from Sand M-phase arrested extracts do not possess MEK kinase activity Kinase-dead (KD) and wild-type (WT) myc-B-Raf were expressed in Xenopusegg extracts as described. Aliquots containing the recombinant myc-B-Raf were introduced in a 1:20 ratio to S-phase arrested extracts, which after one-hour incubation at room temperature were driven into M-phase with non-degradable GST-cyclin B. Myc-B-Raf proteins were purified by means of myc-tag antibodies and subjected to an in vitro linked kinase assay. Loading of immunopurified myc-B-Raf per kinase reaction was verified by myc-tag Western blotting. Cdk1/cyclin B activity of the corresponding extracts was measured in an in vitro histone H1 kinase assay (not shown). 72

PAGE 95

73 clearly demonstrates that the activ ation of the MAPK cascade by B-Raf immunoprecipitates in the in vitro linked kinase assay re flects B-Raf activity. I would like to point out that my results implicating B-Raf at mitosis conflict with a recent study suggesting that c-Mos might regulate M-phase activation of the MAPK cascade in Xenopus egg extracts (Yue and Ferrell, 2004) I speculate that the main reason for these conflicting results stem from differen ces in how the egg extracts were prepared. In contrast to Yue and Ferrells study, I do not detect any c-Mos protein in my egg extract preparations due to allowing the activated eggs incubating at room temperature for 50 min prior to processing them into extracts (s ee Fig. 7). Moreover, I do not detect any MEK kinase activity associated with c-Mo s immuno-complexes isolated from M-phase arrested Xenopus egg extracts (Fig. 22). Therefore, I am unable to assess any biological significance for c-Mos in my egg extract sy stem. The absence of c-Mos in my egg extracts closely mimics its disappearance shortly following fertilization that was demonstrated in earlier studies (Nishizawa et al., 1993; Watanabe et al., 1991; Watanabe et al., 1989) and in this study (Fig. 7). Furthe rmore, the proposal that c-Mos could trigger the transient activation of the MAPK pathway at mitosis does not comply with its wellestablished role as a cytostatic factor during ooctye matu ration (Castro et al., 2001) or from studies showing that micro-injection of even small amounts of c-Mos can mediate a metaphase arrest in cleaving Xenopus embryos (Sagata et al., 1989). Finally, gene knockout studies in mice argue against an essent ial role for c-Mos at mitosis in somatic tissues since mos (-/-) mice are viable wit hout any tissue abnormalities (Colledge et al., 1994; Hashimoto et al., 1994). On the othe r hand, homozygous knockouts for B-Raf are

PAGE 96

embryonically lethal (Wojnowski et al., 2000; Wojnowski et al., 1997). Thus, I propose that B-Raf, rather than c-Mos, is the mitotic MEK kinase that plays an essential role during mitosis in activating the MAPK cascade. Figure 22. c-Mos immunoprecipitates do not possess an M-phase MEK kinase activity Santa Cruz and Abcam c-Mos antibodies were used to immunoprecipitate MEK kinase activity from M-phase arrested extracts (c-Mos immunoprecipitates 1 and 2 respectively). The immunoprecipitates were washed and subjected to an in vitro linked kinase assay with recombinant unactive MEK and ERK. Since M-phase arrested extracts have undetectable levels of c-Mos, Coomassie staining for IgGs was used as a loading control. Note that equivalent amounts of B-Raf antibodies immunoprecipitate significantly higher levels of mitotic MEK kinase activity. 74

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B-Raf associates with MEK in Xenopus egg extracts Next, I examined whether M-phase activation of B-Raf correlates with its ability to associate with MEK, the direct target of B-Raf. To do this, co-immunoprecipitation assays were performed for B-Raf and MEK. B-Raf or MEK complexes were immunopurified from Sand M-phase Xenopus egg extracts and subjected to Western analysis for detection of both MEK and B-Raf. As shown in Figure 23, MEK and B-Raf were found in a complex in both Sand M-phase egg extracts suggesting that their association is independent of B-Raf activation at mitosis. Figure 23. B-Raf associates with MEK in Xenopus egg extracts Aliquots of Sand M-phase arrested extracts were incubated with B-Raf, MEK, or rabbit IgG (mock control) antibodies. Protein complexes purified on protein A beads were subjected to Western analysis for both B-Raf and MEK. 75

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76 B-Raf does not associate with Raf1 in mitotic Xenopus egg extracts Recent work suggested that B-Raf/Raf1 heterodimerization following mitogen stimulation is an important mechanism that ensures proper signaling via the Raf-MEKERK cascade (Rushworth et al., 2006). However, my data indicate that Raf-1, unlike BRaf, is not involved in the regulation of the MAPK pathway during mitosis in Xenopus egg extracts. Indeed, Raf-1 was not pur ified as a mitotic MEK kinase from Xenopus egg extracts (Fig. 15) and its i mmunodepletion did not affect MAPK activation at mitosis (Fig. 18). These results sugge st that B-Raf and Raf-1 are not implicated in the same signaling cascades in Xenopus egg extracts. To obtain a di rect evidence for this, I performed B-Raf/Raf-1 co-immunoprecipitation to analyze B-Raf/Raf-1 interactions. Equal amounts of M-phase arrested extracts were subjected to B-Raf, Raf-1 or mock immunoprecipitation. Antibody complexes reco vered on protein A beads were washed and analyzed for the presence of Raf-1 protei n by Western blotting. As shown in Figure 24A, anti-Raf-1 antibody readily immunoprecipitated Raf-1 from M-phase arrested extracts. Contrary, B-Raf immunoprecipitate s did not show immunoreactivity with Raf-1 antibodies, indicating that mitotic BRaf does not interact with Raf-1 in Xenopus egg extracts. Interestingly, analysis of Raf-1 and MEK interaction in Xenopus egg extracts showed that Raf-1 immunoprecip itates from Sand M-phase extracts failed to co-purify MEK, whereas B-Raf antibodies reproducibly co-immunoprecipitaed MEK (Fig. 24B and Fig. 23). This further suppor ts that Raf-1 is uncoupled from MAPK signaling during mitosis as shown in this (Fig. 18) and previ ous studies (Laird et al., 1999; Ziogas et al., 1998).

PAGE 99

A Total extract B-Raf IP Mock IP Raf-1 IP Raf-1 B S-phase M-phase M-phase S-phase Mock Raf-1 B-Raf I Mock S-phase M-phase S-phase M-phase P B-Raf Raf-1 MEK MEK Figure 24. Raf-1 is not co-immunoprecipitated with B-Raf and MEK from X enopus egg extracts A. Aliquots of M-phase arrested extracts were incubated with Raf-1, B-Raf antibodies or rabbit IgG (mock control). Protein complexes purified on protein A beads were subjected to Western analysis for Raf-1. B. Aliquots of Sand M-phase arrested extracts were incubated with B-Raf or Raf-1 antibodies or rabbit IgG (mock control). Protein complexes purified on protein A beads were subjected to Western anal y sis for B-Raf/MEK and Raf-1/MEK co-immuno p reci p itation. 77

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78 B-Raf undergoes hyperphosphorylation during mitosis As revealed by Western blotting, B-Raf undergoes a prominent electrophoretic shift in M-phase arrested egg extracts (F ig. 25A) and during mitosis in cycling egg extracts (Fig. 25B, 60-70 and 140 min). It is well established that phosphorylation comprises one of the major mechanism of post-translational regulation of B-Raf activity (Chong et al., 2003). Therefore, I tested whethe r the electrophoretic shift of B-Raf during mitosis is due to phosphorylati on. To do this, B-Raf immunocomplexes isolated from Sand M-phase egg extracts were subjected to an in vitro treatment with lambda protein phosphatase. After completion of the reacti on, proteins were separated by 8% SDSPAGE and changes in B-Raf electrophoretic mobility were analyzed by Western blotting. The results show that phosphatase treatment progressively eliminated the shift of B-Raf isolated from M-phase extr acts (Fig. 25C) demonstra ting that it stemmed from phosphorylation. B-Raf isolated from S-pha se egg extracts was also sensitive to phosphatase treatment since it is constitutively phosphorylated in the positive regulatory N-region (Mason et al., 1999). Therefore, I conclude that Xenopus B-Raf is constitutively phosphorylated in S-phase extracts and becomes hyperphosphorylated during mitosis. Mitotic activation of B-Raf stems from phosphorylation Is mitotic phosphorylation of B-Raf importa nt for B-Raf activation? To answer this question, I studied how dephosphorylation a ffects activity of mitotic B-Raf. Similar to the above described experiment, equal am ounts of Sand M-phase immunoprecipitated

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Figure 25. B-Raf undergoes hyperphosphorylation during mitosis A. The electrophoretic mobility of B-Raf shifts up at M-phase. Equal aliquots of Sand Mphase arrested extracts were separated by 8% SDS-PAGE and subjected to immunoblot analysis. Cdk1 activity was measured in an in vitro histone H1 kinase assay. Coomassie blue staining was used to confirm equivalent loading of histone H1. B. B-Raf undergoes an electrophoretic mobility shift during mitosis in Xenopus egg cycling extracts. Aliquots of cycling egg extracts were collected at indicated times over two cell cycles and immunoblotted for B-Raf or MEK (loading control). C. B-Raf hypershift stems from phosphorylation. B-Raf immunoprecipitated from Sor M-phase egg extracts was treated with lambda protein phosphatase (lambda-PP) for indicated times, separated by SDS PAGE, and subjected to immunoblot analysis. 79

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B-Raf were subjected to an in vitro dephosphorylation with lambda protein phosphatase. After the completion of the reaction, B-Raf immunoprecipitates were washed with copious amounts of buffer containing phosphatase inhibitors (25 mM NaF and 10 mM Na 3 VO 4 ) and B-Raf activity was measured in an in vitro linked kinase assay. The results showed that dephosphorylation of mitotic B-Raf abolished its kinase activity (Fig. 26). Besides this, the basal activity of B-Raf isolated from S-phase extracts was also sensitive to phosphatase treatment. Thus, this data demonstrates that phosphorylation is critical for regulating M-phase specific activation of B-Raf. Figure 26. Mitotic activation of B-Raf stems from phosphorylation B-Raf immunopurified from Sor M-phase extracts was treated with lambda protein p hosphatase (lambda-PP) and subjected to immunoblot analysis (top panel) or an in vitro linked kinase assay (bottom panel). 80

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81 Xenopus B-Raf is not phosphorylated at the conserved Threonine-633 and Serine636 residues during mitosis Phosphorylation of Threonine-599 and Seri ne-602 located within an activation segment of Human B-Raf is critical for B-Raf activa tion, specifically for Ras-dependent induction of B-Raf activity (Zhang and Gua n, 2000). Since these phosphorylation sites are conserved to Xenopus B-Raf (Threonine-633 and Se rine-636, respectively, see Fig. 16), I asked whether phosphorylation at the i ndicated sites occurs during mitosis. To answer this question, equal amounts of Sand M-phase extracts were probed with anti-BRaf phospho-Threonine-599 and phos pho-Serine-602 antibodies. The results showed that Xenopus 95 kDa B-Raf is not phopsphorylated at th e indicated residues neither in Snor in M-phases (Fig. 27). Interestingly, the small B-Raf isoform (~68 kDa) present in Xenopus egg extracts and previously descri bed in human cells (Moodie et al., 1994; Oshima et al., 1991; Sithanandam et al., 1990) is highly phosphorylat ed in an M-phase dependent manner at the conserved Threonine -633 and Serine-363 within the activation segment. While beyond the scope of this study, it would be extremely interesting to determine functions and regulation of the smaller 68 kDa B-Raf isoform at mitosis by developing B-Raf antibodies capable of i mmunoprecipitation of the smaller B-Raf isoform.

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Mp hase Mp hase Mp hase Sp hase 82 115.5 kDa 82.2 kDa 64.2 kDa 115.5 kDa 82.2 kDa 64.2 kDa 68 kDa B-Raf 95 kDa B-Raf 1 2 3 Figure 27. Xenopus 95 kDa B-Raf is not phosphorylated at the conserved Threonine-633 and Serine-636 residues during mitosis Aliquots of Sand M-phase extracts were separated by 8% SDS-PAGE and immunoblotted for phospho-Thr-599/Ser-602 B-Raf (1), 68 kDa B-Raf (2) and 95 kDa B-Raf (3). Note that Threonine-599 and See activation segment of Human B-Raf correspond to Threonine-633 and Serine-636 in Xenopus B-Raf (see rine-602 residues in th Fig. 16).

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83 Conclusions In this Chapter, I provide evidence th B-Raf is regulat ed in an M-phase dependent manner in Xenopus egg extracts. First of all, I show that consistent with its requirement in mitotic activation of the MAPK pathway, B-Raf activity is remarkably and tran an Mitogen t at siently increased during mitosis. Furthermore, Xenopus B-Raf undergoes phase specific hyperphosphorylation. Importa ntly, mitotic phosphoryl ation of B-Raf is critical for its activation during M-phase. Thus, I speculate that up-stream kinase(s) target B-Raf at mitosis to allow for the activation of the B-Raf/MEK/MAPK pathway. Interestingly, 95 kDa Xenopus B-Raf is not phosphorylated at the conserved Threonine633 and Serine-363 within the activation l oop during mitosis, contrary to its Rasdependent activation following mitogenic s timulation (Zhang and Guan, 2000). This indicates that the mechanism of B-Raf activation at mitosis is princi pally different from that described for B-Raf during the cell cycle entry, which occu rs in response to m stimulation via activated Ras (Roovers and A ssoian, 2000)). Finally, I demonstrate tha B-Raf does not form heterodimers with Raf1 and associates with endogenous MEK, its direct target, validating that B-Ra f functions as a MEK kinase in Xenopus egg extracts.

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84 Chapter Four Regulation of Mitotic B-Raf by Cdk1/cyclin B In this chapte and B-Raf activation in Xenopus egg extracts that cycle from S-phase to mitosis as well as a requirement of Cdk1/cyclin B activity f B-Raf in mito tic extracts. Second, Cdk1/c ill ring Cdk1/cyclin B triggers activation of B-Raf in Xenopus egg extracts From my analysis of temporal ac f Cdk1/cyclin B and B-Raf in Xenopus egg cycling extracts, I show that B-Raf ac tivation during mitosis follows Cdk1/cyclin B activity clin B and BIntroduction r I propose to analyze the timing of Cdk1/cyclin B in activ ation o yclin B association with B-Raf in mitotic Xenopus egg extracts will be characterized based on data obtained in co -immunoprecipitaion studies Lastly, I w present data that demonstrate that B-Raf is phosphorylated by Cdk1/cyclin B and that this phosphorylation is important for activation of B-Raf and the MAPK cascade du mitosis in Xenopus egg extracts. Results tiv ation o (Fig. 20B). To study the biochemical relationship between Cdk1/cy

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85 Raf, I a tion r hapiro, 2004) and then analyzed B-Raf activity as outlined above. Following inhibiti activation of B-Raf could be under control of Cdk1/cyclin B. Interestingly, Cdk1, reco mbinant GST-cyclin B, and B-Raf were all detecte the Chapter sked whether Cdk1/cyclin B could trigger B-Raf activity in Xenopus egg extracts cycled into a stable mitotic state. Specif ically, after the additi on of recombinant nondegradable GST-cyclin B to Sphase arrested extracts, I colle cted aliquots of extract at 10 min intervals. Cdk1/cyclin B activity was measured by an in vitro histone H1 kinase assay. In parallel, B-Raf wa s immunoprecipitated from 5 l of the same time-point aliquots and subjected to an in vitro linked kinase assay to determine its activity. The data shown in Figure 28A demonstrates that activation of B-Raf follows Cdk1/cyclin B activation suggesting that Cdk1/cy clin B directly or indirectly controls mitotic activa of B-Raf. Is Cdk1/cyclin B activity necessary for BRaf activity during mitosis? To answe this question, I treated M-phase arrested extracts with Flavopiridol, a Cdk specific inhibitor (S on of Cdk1/cyclin B, I observed a re duction in activity of mitotic B-Raf (Fig. 28B). This indicates that th e enzymatic activity of Cdk1/cyc lin B complexes is involved in the control of B-Raf activation during mito sis. Therefore, the data presented here strengthens the idea that Cdk1/cyclin B serves as a direct or indirect activator of mitotic B-Raf. B-Raf associates with active Cdk1/cyclin B complexes during mitosis My data suggest that M-phase specific d in the final step of the mitotic MEK kinase purification, described in

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Cdk actiity B-Rf actity 86 A ctivity, a.u. 05101520253035 Cdk1/cyclin B activity B-Raf activity 0 10 20 30 40 50 60 70 80 120 min Cdk1 activity B-Raf activity A B 1vaiv 0102030 Cdk1/cyclin B activity B-Raf activity A ctivity, a.u. S-phaseM-phaseM-phaseFlavopiridFigure 28. B-Raf activity in M-phase arrested Xenopus egg extracts depends on Cdk1/cyclin B activity A. Time-course of Cdk1/cyclin B and B-Raf activation in Xenopus egg extracts after GST-cyclin B addition Cdk1/cyclin B activity is necessary for B-Raf activation during mitosis. Cdk1 activity in M-phase arrested exts inhibited with Flavopiridol. B-Raf was immuno-purified and its activity was measured in an in vitroEqual loading of B-Raf immunoprecipitates per kinase assay was confirmed by B-Raf Western bloactivity was measured in in vitro histone H1 kinase assay. + olB. tracs wa link kinase assay. tting. Cdk1/cyclin B

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87 Two (Fig. 29A). To determine whether Cdk1/ independently or within a large multi-proteimplex, a glutathione bean from Sand M-phase extracts was performe s sh in Fig. 2 1 and BRaf were readily detectable in GSTB complexes isolated from extracts. In contrast, glut athione beads pull-down from S-phase extracts, which do not contain GST-conjugated cyclin B, failed to precipitate either B-Raf or Cdk1. These studies were extended into Xenopus egg cycling extracts to exam Cdk1/cyclin B and B-Raf interactions dur ing the embryonic cell cycle. C immunoprecipitates from t of cycling extract were analyzed by Western blotting for both B-Raf and Cdk1 protein or subjected to an assays to measure Cdk1 activity. Cell cycle nuclear formation, nuclear envelope breakdowatin condensation. The results clearly show that B-Raf associates wdk1/cyclin B complexes during m interaction parallels the activation of Cdk1 (Fig. 29C). Analogous experim performed by using Cdk1 antibody. Cdk1 im munocomplexes were purified from course aliquots of cycling extracts and an alyzed for the presence of B-Raf and Cdk1 by Western blotting as well as for Cdk1/cy H1 kinase assay. Similar to the results observed an increase in B-Ra f co-immunoprecipitation at the beginning of overlapped with activation of Cdk1/cyclin B complexes (Fig. 29D). Thus, these data reveal that B-Raf directly associates active C dk1/cyclin B at m GST-cyclin B and B-Raf were co-purified n co d. A ds pull-dow 9B, both Cdk M-phase arrested yclin B in vitro H1 kinase acts was assessed by itosis and this ents were timeto an in vitro mitosis, which itosis. own cyclin ine endogenous ime-course aliquots progression of the extr n and chrom ith C clin B activity by applying them obtained with Cyclin B immunoprecipitates, I with

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Figure 29. B-Raf associates with active Cdk1/cyclin B complexes during mitosis A. MonoQ column fraction containing partially purified mitotic MEK kinase activity was analyzed by B-Raf, cyclin B, and Cdk1 Western blotting. B. Cdk1/GST-cyclin Bcomplexes were purified from M-phase arrested extracts by using Glutathione beads.C. Cyclin B immuno-complexes were precipitated from aliquots of cyclingand subjected to B-Raf and Cdk1 Western blotting, and an in vitro H1 kina extracts se assay to measure an associated Cdk1 activity. D. Cdk1 immuno-complexes were precipitated from aliquots of cycling extracts and subjected to B-Raf and Cdk1 Western blotting, and an in vitro H1 kinase assay. 88

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89 Cdk1/cyclin B (and MAPK) directly phosphorylates B-Raf in M-phase arrested Xenopus egg extracts B-Raf directly associates with active C dk1/cyclin B complexes and its activation occurs downstream of and depends on active C dk1/cyclin B. All thes e data suggest that B-Raf could be a direct target of Cdk1/cyclin B. To clarify this, I decided to study whether phosphorylation of B-Raf in mitotic Xenopus egg extracts depends on Cdk1/cyclin B. Specifically, I analyzed the status of B-Raf phos phorylation in M-phase arrested extracts where Cdk1/ cyclin B activity was selectively blocked by Flavopiridol treatment. Under these conditions, I obser ved a minor reduction in B-Rafs mitotic electrophoretic mobility (Fig. 30, third lane). To obtain further evidence that Cdk1/cyclin B phosphorylate B-Raf at mitosis, I deci ded to immunopurify B-Raf from M-phase arrested extracts and subject it to Western blot analysis with phospho-Cdk substrate antibodies. It is well established that ac tive Cdks phosphorylate their substrates at the Serine/Threonine residue within the conserved Se rine/Threonine-Proline-XLysine/Leucine sequence (Nigg, 1991). I deci ded to take advantag e of commercially available phospho-antibodies, which rec ognize both Cdk and MAPK phosphorylated substrates at the consensus sequences S* XK/L and PXS*P respectively. Since these antibodies may recognize both Cdk and MA PK phosphorylation, I prepared M-phase e w in o xtracts inhibited either for Cdk1/cyclin B (t reated with Flavopirid ol) or MAPK (treated ith U0126, a MEK inhibitor) as well as M-phase extract where both kinases were hibited by the treatment with both inhib itors. As shown in Figure 30, phosphorylation f B-Raf at the PXS*P/S*PXK/L sites is dramatically increased from Sto M-phase

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DMSO Flavopiridol U0126 U0126 + Flavopiridol S-phase PXS*P/S*PXK/L B-Raf IP B-Raf Cdk1 activity MAPK-P M-phase B-Raf Figure 30. Cdk1/cyclin B (and MAPK) directly phosphorylates B-Raf in M-phase arrested Xenopus egg extracts MEK correspondently. B-Raf phosphorylation was analyzed by monitoring B-Raf shift and reactivity of B-Raf immunoprecipitates with phospho-Cdk/MAPK substrate Extracts were treated with Flavopiridol and/or U0126 to inhibitCdk1/cyclin B and antibodies (PXS*P/S*PXK/L). 90

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91 (fourth lane). Importantly, inhibition of Cdk1/cyclin B led to a reduction in B-Rafs phosphrylation (Fig. 30, forth lane). This indicates that Cdk1/cyclin B directly phosphorylates B-Raf at Cdks c onsensus phospho-site(s) during mitosis. Interestingly, data presented in Figure 30, fourth lane, dem onstrates also that MA PK as well directly targets B-Raf at mitosis. Studies on MAPKs involv the regulation of mitotic BRaf are presented inpter. dk1/cyclin B directly phosphorylates Xenopus B-Raf in vitro at a conserved residue Serine-144 enopus B-Raf contains two conserved SPXK sequences, which represent potential Cdk consensus sites (Fig. 31). In terestingly, the first N e most closely resembles a sequence of a Cdk1/cyclin B preferential phosphor ylation site, which was characterized as of K/R-S/T-P-Polar amino acid-K/L (Moreno and Nurse, 1990). Based on the results presented in the previous section, Serine residues within these two sites (namely, Serine-144 and Serine-328) are possible target s for Cdk1/cyclin B phosphorylation. To address this directly, I prepared recombinant non-phosphorylatable X c-tagged B-Raf mutants. Briefly, Xenopus B-raf coding sequence, cloned in the Guadagno lab, was sub-cloned into modifi ed pGEM vector and subjected to sitedirected mutagenesis to produce Ser144Ala Ser-328-Ala and Ser-144-Ala/Ser-328-Ala mutants. Corresponding mRNAs were synthesized by an an extracts as describe d under the Materials and M eme nt in Cha Five C X -proximal sit enopus my in vitro transcription reaction d translated in CSF Xenopus egg ethods. The mutant as well as non-mutated myc-B-Raf proteins were purified by the

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92B-Raf protein Figure 31. Conserved putative Cdk1/cyclin B phosphorylation sites in Xenopus

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93 means of myc-tag antibodies and used as substrates for an in vitro phosphorylation by active recombinant Cdk1/cyclin B in the pres ence of radioactive ATP. As shown in Figure 32, non-mutated myc-B-Raf was readily radiolabeled by Cdk1/cyclin B, demonstrating that B-Raf can be directly phos phorylated by Cdk1/cyc lin B. Importantly, the only Ser-328-Ala mutant displayed a phosp horylation by Cdk1/cyclin B (Fig. 32). This data indicates that BRafs Serine-144 is the main target of Cdk1/cyclin B phosphorylation in vitro This is the first evidence that B-Raf can be directly phosphorylated by Cdk1/cyclin B in vitro and that this phosphorylation occurs preferentially at the conser ved Serine-144 residue within the Cdk consensus sequence. Note that to reduce the possibi lity of irrelevant myc-B-Raf radiolabeling because of BRaf autophosphorylation activity, all recombinant myc-B-Raf proteins us ed in this assay were kinase dead (KD) due to mutation in the ATP binding domain. Ser-144-Ala B-Raf mutant is not activ ated in an M-phase dependent manner Data described in the previous sectio n implies that Cdk1/cyclin B mediated phosphorylation at Serine-144 may regulat e mitotic activation of B-Raf in Xenopus egg extracts. If this prediction is true, than the M-phase specific activation of nonphosphoralatable Ser-144-Ala myc-B-Raf mutant should be blocked. To test this, I directly measured activities of wild type (WT) and Ser-144-Al a myc-B-Raf proteins in Sand M-phase arrested extrac ts. Briefly, the recombinant WT and mutant myc-B-Raf proteins were expressed in Xenopus CSF extracts. Aliquots of th e extracts containing the recom binant myc-tag B-Raf were introduced into S-phase arrested extracts. After 1 hr

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94 S144 A S144 A S328 A S328 A S144 A T328 A S144 A T328 A KD KD Myc-B-RafP32 M y c-B-Ra f Myc-B-Raf IP No Cdk1 + Cdk1 Figure 32. Cdk1/cyclin B directly phosphorylates Xenopus B-Raf in vitro at a WT and mutant myc-B-Raf proteins were expressed in Xenopus egg extracts, purified by recombinant Cdk1/cyclin B. Phosphorylation of myc-B-Raf proteins was visualized by conserved Serine-144 means of myc-tag antibodies and subjected to an in vitro phosphorylation by active autoradiography. Myt-tag Western blotting was performed as a loading control.

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95 incubation the extracts were driven into M-phase by the addition of recombinant nonegradable cyclin B. myc-B-Raf proteins we re purified from Sand M-phase extracts with myc-tag antibodies and subjected to an in vitro linked kinase assay to assess their ctivities. As expected, wild type myc-BRaf underwent a 3-4 fold activation at M-phase ig. 33). Importantly, the mutant non-phosphorylatable Ser-144-Ala myc-B-Raf was not ctivated and its activity remained essentially the same from Sto M-phase (Fig. 33). his shows that Ser-144-Ala B-Raf mutant is not respons ive to an M-phase specific ctivation. Ser-144-Ala B-Raf mutant does not ac tivate MAPK cascade during mitosis Is phosphorylation at Serine-144 important for B-Rafs funct EK kinase? To answer this, I studied wh ether the mutant Ser144-Ala myc-B-Raf can scue mitotic activation of the MEK/MAPK cascade in B-Raf depleted extracts. ndogenous B-Raf was immunodepleted form Sphase arrested extracts as described arlier (see Chapter Two). Then recombinant wild type or Ser-144-Ala myc-B-Raf roteins were introduced into B-Raf depleted extracts (Fig. 34A) and the extracts were driven into M-phase by the addition of reco mbinant non-degradable cyclin B. 1.0 l very 20 min a nd analyzed for MAPK activation by phosphodition f recombinant wild type myc-B-Raf reconsti tuted mitotic activation of MAPK at levels milar to those observed in control (non-de pleted) extracts (Fig. 34B, first and third d a (F a T a ioning as a mitotic M re E e p aliquots were collected e MAPK Western blotting. As expected, depletion of endogenous B-Raf abrogated activation of MAPK in M-phase arrested extr acts (Fig. 34B, second lane). The ad o si

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96 010 2030B-Raf activity, u.a. 40 S M S M Cdk1 activity myc-B-Raf activmyc-B-Raf WT S144A myc-B-Raf ity Figure 33. Ser-144-Ala B-Raf mutant is not activated in an M-phase dependent manner in Sand M-phase arrested B-Raf depleted extracts, immunopurified by means of myc-tag antibodies and subjected to an in vitro linked kinase assay. Wild type (WT) and Ser-144-Ala (S144A) myc-B-Raf proteins were incubated

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973er-144-Ala m-Raf mutant does not activate MAPK cascade during mitosis elB-Raf protock and B-Raf depleted Xenegg extracts, and in B-Raf depleted extracts e with recombt wild-tyr Ser-1a mu myc-B-Raf. B. Ser-144-Ala myc B-Raf mutant, wipe myc B-M-phase B-Raf-depleted extracts. Mock and B-Raf d acts, and B-Rpleted ets suppbinant wild-type or Ser-144-Ala myc-B-Raf were itosis with reinant non-the indicated times aliquots were collected and MAPK as assessed by phospho-MAPK West opus tatedn in nted with recom Figure A. Levsupplemunlike depletedriven into mactivation w 4. Ss of ntedld-tyextr yc-Bein in minanaf decomb pe oRaf, does not rescue MAPK activatioxtrac 44-Allemedegradable cyclin B. At ern blotting.

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98 lanes). Importantly, the addi tion of equivalent amount of non-phosphorylatable Ser-144Ala myc-B-Raf mutant failed to rescue MAPK activation at mitosis d extracts (Fig. 34B, fourth la ne). Thus, phosphorylation of Serine-144 residue within the Cdk phosphorylation sequence contributes to ac tivation of B-Raf, and enables B-Raf to function as an M-phase MEK kinase. Ser-144-Ala B-Raf mutant exerts a dominant -negative effect o during mitosis Since Ser-144-Ala myc-B-Raf does not act ivate MAPK cascade at m Raf depleted extracts, I decided to study whether this mutant can com endogenous B-Raf and interfere with mitotic activation of MAPK in non-depleted extracts. Similar amounts of wild type and Ser-144-Ala myc-B-Raf proteins (Fig. 35, first lane), equivalent to the levels of e ndogenous B-Raf, were phase arrested Xenopus egg extracts. Following 1.0 hr in cubation, extracts were analyzed for the levels of MAPK ac tivation by phospho-MAPK Wester Figure 35 (second lane), MAPK phosphorylation in M-phase ex Ser-144-Ala myc-B-Raf mutant was significantly reduced comp supplemented with wild type myc-B-Raf. Note that activation of Cdk1/cyclin B resembled a normal pattern in both extracts (Fig. 35, third lane s the importance of phosphorylation at the Ser-144 for B-Rafs ability to function as an Mphase MEK kinase. Furthermore, it suggests th at the Ser-144-Ala m in B-Raf deplete n MAPK activation itosis in Bpete with ented with yc-B-Raf mutant can introduced into Sand Mn blotting. As shown in tracts supplem ared to M-phase extracts ). This data reconfirm

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99 99 eM-phase S-phase M-phase has WT S144A myc-B-Raf S-p Myc-B-Raf MAPK-P Cdk1 activity e Figure 35. Ser-144-Ala B-Raf mutant blocks MAPK activation in M-phasextracts Equal amounts of wild-type and mutant Ser-144-Ala myc-B-Raf proteins were incubated in Sor M-phase arrested Xenopus egg extracts. MAPK activation was assessed by phospho-MAPK Western blotting. Note that addition of myc-B-Raf p roteins did not affect activation of Cdk1/cyclin B.

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100 e utilized as a potential research tool to selectively inhibit MAPK signaling at mitosis in enopus egg extracts and, possi bly, tissue cell cultures. Conclusions The dataented in this Chapter rev a novel mechanism of regulation for e B-Raf/MEK/MAPK cascade. For the first tim e, I demonstrate that B-Raf serves as a irect link between the mitotic cell cycle machinery and the MAPK pathway. Indeed, dk1/cyclin B associates and phosphorylates B-Raf at mitosis in Xenopus egg extracts. ata from an in vitro kinase reaction show that ac tive Cdk1/cyclin B directly hosphorylates B-Raf at its c onserved Serine-144 residue. Biochemical analysis emonstrated that phosphorylation of B-Raf at this residue is critical for the M-phase ependent activation of B-Raf as well as a functionality c MEK inase. In conclusion, I identi fy a new B-Raf regulatory site and provide insights into the echanism that regulates the activation of the MAPK pathway during mitosis (Fig. 36). b X eals pres th d C D p d d of B-Raf as a mitoti k m

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101 Figure 36. Cdk1/cyclin B directly phosphorylates B-Raf at Serine-144 to trigger the B-Raf/MEK/MAPK cascade S144 M-phase B-Raf MEK MAPK S144 Cdk PCyclin B

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102 Chapter Five Negative Feedback Regulation of Mitotic B-Raf by MAPK n The data presented in the previous chapte rs indicate that dur ing mitosis B-Raf can e regulated by MAPK. F ll, I repeatedly observe d that the pronounced B-Rafs hypershift during mitosis coincides wi activation (see Fig. 20B and Fig. 30). A similar correlation was recently reported in activated Human B lymphocytes (Brummer et al., 2003). More importantly, the authors showed that Human B-Raf is directly phosphorylated by MAPK at the conserved C-te inal sequence and that this causes BRaf hypershift (Brummer et al., 2003). Sin ce the C-terminal MAPK phosphorylation site is conserved in Xenopus B-Raf, it is important to eluc idate whether the same mechanism operates during mitosis. Indeed, blocking of MAPK activation in M-phase arrested Xeno K inhibitor U0126 redu ces the levels of B-Rafs mitotic hyperphosphorylation (see Fig. 30). In addition, the treatment with a MEK inhibitor U0126 reduces B-Rafs reacti ity with Cdk/MAPK phospho-substrate antibodies (see Fig. 30). Thus, th e goal of the studies presented in this Chapter is to further explore the MAPK-dependent phosphorylation of B-Raf in Xenopus egg extracts Introductio b irst of a th MA PK rm pus egg extracts by pretreatment with a ME v

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103 during mitosis and characterize its role in regulating activity of mitotic B-Raf. Specifically, data that describe interac tions between endogenous B-Raf and MAPK during M-phase and phosphorylation of Xenopus B-Raf by MAPK will be presented. Finally, by observing changes in B-R conditions when the MAPKmediated feed ylation on -Raf activity will be characterized. Results xtracts is af activity under back is blocked or enhanced, the effect of this MAPK phosphor B Mitotic hyperphosphorylation of B-Raf depends on active MAPK B-Raf was previously reported to under go an electrophoretic mobility shift during B lymphocyte activation through feedback phosphorylation by ERK (Brummer et al., 2003). Therefore, I asked whether B-Rafs hyper-shift in mitotic Xenopus egg e dependent on MAPK. To answer this, Xenopus egg extracts were treated with a MEKspecific inhibitor U0126 prior to cycling them into mitosis. The immunoblot analysis confirmed inhibition of MAPK, which led to blocking M-phase specific hyperphosphorylation of B-Raf (Fig. 37A, see as well Fig. 30). Similar results were observed in Xenopus cycling extracts trea ted with the MEK inhibitor U0126 (Fig. 37B). Note that the MEK inhibitor U0126 does not affect Cdk1 activation in Xenopus egg extracts, as assessed both by microsc opic analysis of nuclear envelope breakdown/chromatin condensation and by measuring Cdk1 activity in an in vitro H1

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104e mitotic kinase assay (see Fig. 30). Thus, MAPK feedback substantially contributes to thhypershift of B-Raf. Figure 37. Mitotic hyperphosphorylation of B-Raf depends on activS-phase extracts were cycled into M-phase with recombinant GST-cyclin B in the e MAPK A. Mitotic hyper-shift of B-Raf is blocked in the presence of the MEK inhibitor U0126. p resence of 50 mkM U0126 or DMSO. Equal aliquots of the egg extracts were blotting. Raf during mitosis. Aliquots of cycling extracts were separated by 8% SDS-PAGE and separated by 8% SDS-PAGE and analyzed by B-Raf and phospho-MAPK Western B. Treatment of Xenopus cycling egg extracts with U0126 blocks hyper-shift of B-analyzed by Western blotting.

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105 ctly phosphorylates B-Raf at mitosis, then it might be possible to detect the association between these two proteins. To test this possibility, equivalent amounts of MAPK were immunoprecipitated fr om Sand M-phase egg extracts and subjected to Western analysis with B-Ra f antibodies. The levels of B-Raf coimmunoprecipiated with MAPK were much higher in the MAPK complexes isolated from M-phase arrested extracts compared to S-phase extracts (Fig. 38A). To analyze dynamics of B-Raf/MAPK inte raction during mitosis, I studied how B-Raf/MAPK association is changing in Xenopus egg cycling extracts. MAPK was immunoprecipitated from frozen time-course aliquots and obtained immunoprecipitates were subjected to B-Raf Western blotting. Interestingly, I dete cted a two-phase BRaf/MAPK association throughout mitotic progression (Fig. 38B). First, B-Raf was associated with MAPK at the on-set of m itosis, which overlapped with Cdk1/cyclin B activation. Secondly, B-Raf was bound to MA PK during middle-late mitosis, which coincided with robust MAPK activity. Since I repeatedly observe the appearance of BR is te PK m b Mitotic B-Raf associates with MAPK If MAPK dire afs mitotic hyper-shift during middle-late stages of mitosi s (Fig. 20B, Fig. 25B), it mpting to speculate that during the sec ond phase of B-Raf/MAPK interaction, MA ay exert a direct B-Raf phosphorylation. Taken together, my data show that B-Raf ecomes associated with MAPK in egg extracts specifically during M-phase.

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Mp hase Sp hase Mp hase Sp hase MAPK B-Ra f 106Figure 38. Mitotic B-Raf associates with MAPK ntrol immunoprecipitates from Sand M-phase extracts were separated on 10% SDS-PAGE and analyzed by B-Raf and MAPK Western blotting. B. B-Raf associates with MAPK twice throughout mitosis in cycling extracts. MAPK immunoprecipitates from aliquo of cycling extracts collected at the indicated time-points were separated on 10% SDS-PAGE and analyzed by B-Raf and MAPK Western blotting. Cdk1 activity of the corresponding aliquots was assessed in an in vitro histone H1 kinase assay. A. B-Raf associates with MAPK in M-phase arrested extracts. MAPK and co Mock IPMAPK IP A ts B-Ra f MAPK 20 40 60 80 min Cdk1/cyclin B activity MAPK-P mitosis MAPK IP B

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107 directly phosphorylates Xenopus B-Raf in vitro and causes B-Rafs electrophoretic mobility shift he associationAPKh B-ugge at it might contribute directly to B-Raf hyperphosphorylation. To study this, I perforan in vitro kinase assay using endogenous Xenopus B-Raf isolated from S-phase extr acts as a potential substrate for active recombinant ERK2. The results show that active ERK2 readily phosphorylated BRaf in vitro (Fig. 39A). Moreover, phosphoryl ation of B-Raf by ERK2 caused a reduction in the electrophoretic mobility of B-Raf (Fig. 39B, forth and fifth lanes). Therefore, I conclude that MA PK can directly phosphorylate Xenopus B-Raf and contribe-Rafs mitotic shift. directly phosphorylates Xenopus B-Raf in vitro at the conserved Cterminal SPKTP motif uman B-Raf is phosphorylated directly by ERK2 at both MAPK consensus S / T P sites within the C-terminal S PK T P motif (Brummer et al., 2003). However, the effect of this phosphorylation on B-Raf activ ity has not been described. Since the SPKTP motif is conserved between Human and Xenopus B-Raf (Fig. 40A), I asked whether the same phosphorylation sites in Xenopus B-Raf protein are targeted by MAPK. T r inal S ild ty sted M APK T of M wit Raf s st s th med ut to B M APK H o do this, I utilized a site-directed mu tagenesis technique a nd introduced alanine esidues at the two phospho-acceptor sites (serin e and threonine) within the C-term PKTP motif to created a B-Raf non-phosphorylatable A PK A P mutant. mRNAs of w pe (WT) and mutant (MT) myc-tagged Xenopus B-Raf were translated in CSF-arre

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108 immunoprecipitated from S-phase arrested egg extracts and subjected B-Raf IP S-phase B-Raf-P ERK2 + 32-P-ATP + + A Figure 39. MAPK directly phosphorylates Xenopus B-Raf in vitro and causes B-Rafs electrophoretic mobility shift A. MAPK directly phosphorylate immunopurified B-Raf in vitro. B-Raf was Xenopusto an in vitro phosphorylation by active recombinant ERK2 in the presence of radioactive ATP. Radiolabeling of B-Raf protein was visualized by autoradiography. B. Phosphorylation of B-Raf by MAPK causes B-Raf hypershift. B-Raf was immunopurified from S-phase arrested extracts and incubated in vitro with active recombinant ERK2 in the presence or absence of ATP. Reaction mixer was separated by 8% SDS-PAGE and B-Raf was analyzed by B-Raf immunoblotting. B

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109 xtracts (see Materials and Methods for details). Then, the recombinant myc-tagged Baf proteins were re-isolated by ipitati bje to an in vitro phosphoron ctive r K2 in the presence of dioactive ATP. As shown in Figure 40B, radio-labeling of the Xenopus myc-tagged Baf mutant in the presence of active ERK2 was strongly suppressed compared to wild pe myc-B-Raf demonstrating that thes e conserved sites are targeted by ERK2. -terminal APKAP B-Raf mutant displays a reduced MAPK-dependent shift at itosis To study whether MAPK-mediated phosphor ylation at B-Rafs C-terminal PKTP motif occurs during mitosis, I anal yzed the phosphorylati on status of the Crminal B-Raf mutant in M-phase arrested Xenopus egg extracts. The nonhosphorylatable myc-B-Raf APKAP mutant intr oduced to M-phase arrested extracts. fter 1.0 hr incubation, the electrophoretic mobility of mutant B-Raf was analyzed by % SDS-PAGE. The data showed that the mitotic hypershift of B-Raf (APKAP) mutant as m ourth nes g xtra as igh (Fig. 40C, ird hos e R immunopr ec on with myc-tag antibodies and su cted ylati by a ecombin ant ER ra R ty C m S te p A 8 w arkedly reduced compared to that of wild type B-Raf (Fig. 40C, third and f la ) suggesting that these MA PK sites are phosphorylated in Xenopus M-phase eg cts. Interestingly, the electrophoretic mobility of the B-Raf (APKAP) mutant w tly increased following inhibition of MAPK with a MEK inhibitor U0126 and sixth lanes). This indicates th at perhaps some other B-Raf site(s) are phorylated in an MAPK-depende nt manner during mitosis. e sl th p

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110Figure 40. MAPK directly phosphorylates Xenopus B-Raf at the conserved A. Amino acid alignment of the C-terminal Human (P15056) and Xenopus and phosphorylation sites are indicated. B. The conserved C-terminal SPKTP wild type (WT) and the C-terminal APKAP mutant (MT) myc-B-Raf proteins epitope antibodies and subjected to an in vitro kinase reaction with recombinant by autoradiography (top panel). Levels of myc-B-Raf per reaction were estimated Mutant (MT) B-Raf displays a reduced M-phase electrophoretic mobility. ere incubated in S-, M-phase extracts, or M-phase extracts pre-treated with U0126. analyzed by Western analysis. C-terminal SPKTP motif (AAZ06667) B-Raf regions using MegAlign software. Putative MAPK binding motif in Xenopus B-Raf is phosphorylated by ERK2. Recombinant full-length were expressed in Xenopus egg extracts as described, immunopurified by myc-active Erk2 and gamma-32P-ATP. Phosphorylation of myc-B-Raf was detected by Western analysis using myc epitope antibodies (bottom panel). C. APKAP Recombinant wild type (WT) or APKAP mutant (MT) myc-B-Raf proteins wPhosphorylation status of the recombinant B-Raf proteins was

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111 C-terminal APKAP B-Raf mutant posse sses an elevated MEK kinase activity Currently, it is unknown whether the MAPK-mediated feedback phosphorylation of B-Raf at the conserved C-terminal SPKTP motif plays a role in regulating its kinase activity. Therefore, I analyzed the activity of the myc-tagged B-Raf (APKAP) mutant in Xenopus egg extracts. Myc-tagged B-Raf wild type and (APKAP) mutant proteins were incubated in either Sor M-phase egg ex tracts, re-isolated by means of myc-tag antibodies, and subjected to an in vitro linked kinase assay. As expected, wild type BRaf kinase activity was 2-4-fold higher in Mphase egg extracts than in S-phase extracts (Fig. 41, bars 1 and 3). Interestingly, the kinase activity of the non-phosphorylatable BRaf APKAP mutant isolat ed from M-phase egg extracts wa s 2-3 fold higher than activity of wild type B-Raf (Fig. 41, bars 3 and 4). This suggests that phosphorylation of the Cterminal SPKTP motif renders a negative e ffect on B-Raf activity. Importantly, the Cterminal APKAP B-Raf mutant was still activ ated in an M-phase specific manner (Fig. 41, bars 2 and 4). This implies that the C-te rminal mutation does not interfere with the abi ed as in Spha ing the k reg y an ele pho ack phosphorylation at these sites negative ly regulates B-Raf during mitosis. lity of the mutant B-Raf to respond to activation mitotic signali ng inputs. I observ well that the C-terminal APKAP B-Raf muta nt possesses a higher kinase activity se extracts compared to wild type B-Raf (Fig. 41, bars 1 and 2). Since it is miss MAPK phosphorylation sites, which potentia lly are involved in negative feed bac ulation, this B-Raf mutant may be more re sistant to inactivation and, thus, displa vated kinase activity at S-phase. Collectively, these results show that MAPK sphorylates B-Raf at the Cterminal SPKTP sequence a nd suggest that feedb

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Figure 41. Phosphorylation-defective C-terminal APKAP B-Raf mutant possesses an elevated MEK kinase activity X enopus wild-type (WT) or APKAP mutant (MT) full-length myc-B-Raf proteins epitope antibodies and subjected to an in vitro linked kinase assay. Levels of were incubated either in Sor M-phase extracts, immunopurified by means of myc-myc-B-Raf per reaction were estimated by Western blotting. 112

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113 Inhibition of MAPK activity with a ME K inhibitor U0126 activates B-Raf in Mphase arrested extracts If MAPK feedback negatively regulates B-Raf activity in Xenopus mitotic egg extracts, then I predict that blocking MAPK activity would l ead to an increase in B-Raf activity at mitosis. To test this prediction, I pretreated Xenopus egg extracts with a MEK specific inhibitor U0126 before driving them into M-phase. Inhibition of MEK blocks activation of MAPK and, thus, eliminates the MAPK-mediated feedback loop. Then endogenous B-Raf was immunopurif ied from control (treat ed with DMSO) and U0126 treated extracts and subjected to an in vitro linked kinase assay. The results show that BRaf activity is enhanced in the absence of MAPK feedback compared to non-treated extracts (Fig. 42). Over-activation of MAPK with constituti vely active recombinant MEK inhibits BRaf activity in M-phase arrested extracts Likewise, I tested whether B-Raf activity would decrease un der conditions of constant MAPK feedback. Indeed, the addi tion of a constitutively active recombinant MEK to M-phase arrested egg extracts mainta ined high levels of MAPK activity and led to a ate MA Raf substantial decrease in B-Raf activity (Fig. 43). Together, these results implic PK in a feedback loop, which phosphorylates the C-terminal SPKTP sequence of B to negatively regulate its activity.

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Figure 42. Inhibition of MAPK activit y with a MEK inhibitor U0126 activates To inhibit MAPK activation during mitosis, S-phase arrested extracts were M-phase with recombinant non-degradable cyclin B. After 1.0 hr incubation at B-Raf in M-phase arrested extracts supplemented with a MEK inhibitor, U0126, concomitantly with driving them into room temperature extracts were quick frozen. MAPK activation was assessed by p hospho-MAPK Western blotting and activity of B-Raf was measured in an in vitro linked kinase assay. Loading of immunopurified B-Raf per kinase reaction was verified by B-Raf Western blotting. Note that the addition of the MEK inhibitor did not affect Cdk1/cyclin B activity (not shown). 114

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y Figure 43. Over-activation of MAPK with constitutively active recombinant MEK inhibits B-Raf activity in M-phase arrested extracts To over-activate MAPK during mitosis, M-phase arrested extracts were supplemented with recombinant constitutively active MEK. After 1.0 hr incubationat room temperature extracts were quick frozen. MAPK activation was assessed b p hospho-MAPK Western blotting and activity of B-Raf was measured in an in vitrlinked kinase asssay. Loading of immunopurified B-Raf per kinase reaction was verified by B-Raf Western blotting. Note that the addition of constitutively active MEK did not affect Cdk1/cyclin B activity. o 115

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116 Conclusions The experiments presented in this Chapter demonstrate that mitotic B-Raf in Xenopus egg extracts undergoes a direct regulat ion by MAPK. First, B-Raf associates with MAPK specifically dur ing M-phase and its mitotic hyperphosphorylation depends on active MAPK. Secondly, active MA PK can directly phosphorylate Xenopus B-Raf in vitro and induce its electrophoretic mobility shift at levels si milar to that of mitotic BRaf. Interestingly, the in vitro phosphorylation of B-Raf by MAPK as well as MAPKdependent phosphorylation of B-Raf in mitotic extracts occurs predominantly at the Cterminal conserved SPKTP motif. Eliminati on of this phosphorylation site enhances activity of mitotic B-Raf. Fu rther, inhibition or over-ac tivation of MAPK in M-phase arrested Xenopus egg extracts oppositely affects B-Raf activity: enhances or diminishes it, respectively. These results together demonstrate that the MAPK-mediated feedback phosphorylation at the C-terminus negatively regulates B-Raf activity during mitosis. Thus, I propose the negative MAPK-media ted feedback loop contributes to downregulation of the B-Raf/MEK/MAPK at the late mitosis (Fig. 44).

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117 M-phase Figure 44. MAPK directly phosphorylates mitotic B-Raf at Serine-784 and Threonine-787 to inhibit B-Raf activity B-Raf S784 T 787 P P MEK MAPK

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118 Chapter Six Characterization of large multi-protein c mplexes containing active mitotic B-Raf Introduction B-Raf is known to be regulated l of protein-protein interaction. Identification of proteins in teracting with B-Raf under diffe rent biological contexts is important for deciphering how B-Raf erse cellular processes. The majority of known B-Raf interacting proteins were identified by co-immunoprecipitation technique or the yeast two-hybrid system (Berruti, 2000; Papin et al., 1996). On the other hand, some studies report that B-Raf can be purified within large multi-protein comple implies that regula bly may be much more complex than currently understood. Proteomics is a powerful approach for analyzing multi-protein complexes. The goal of research presented on this Chapter is to ch aracterize the partially purified large molecular weight multi-protei n complexes that contain active mitotic BRaf. In addition, proteomics data obtaine d by using mass spectrometry analysis will be presented to describe the candidate components of the mitotic B-Raf complexes. o at the leve regulates div xes (Jaiswal et al., 1996; Mizutani et al., 2001; Mizutani et al., 1998). This tion of BRaf by protein-protein assem

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119 Results Purification of mitotic MEK kinase aity as B-Raf containing large multiprotein complexes ig.13A), which leads to an enrichment of MEK kinase activity present in M-phase arrested Xenopus egg extracts. Briefly s M-phase egg extracts were subjected to two rounds of ultracentrifugation (100,000 g for 1.5 hr) to obtain the cytosol vations eins are of ivity ay be represented by multi-protein complex(s) containing B-Raf. To obtain a direct ctiv As described in Chapter Two, I have de veloped a purification protocol (see F crude Xenopu ic fraction. The M-phase cytosol was fractionated by ammonium sulfate precipitation. A 0-20% ammonium sulfate cut, containing 90% of the MEK kinase activity, was sequentially subjected to sepa ration on two anion-exchange columns, HiTrap Q Sepharose HP and Mono Q HR 5/5. During this purification two obser relevant to this Chapter were made. First, th e MEK kinase activity elut es as a single peak from the final Mono Q column (Fig. 13B), which may imply that the eluted prot purified within one large complex. Secondly, Silver stain analysis of equal amounts protein (5 g) at each of the purification st ep detected several pr otein bands, some of which are increased in intensity at each su ccessive purification step (Fig. 45) indicating that they may be associated with the purified MEK kinase activity. Western analysis shows that B-Raf (95 kDa) is enriched in the final purified frac tion (see Fig. 15, second lane) and most likely represents one on these protein bands. Thus, there are at least two indirect observations indicating that the pa rtially purified mitotic MEK kinase act m

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120 crude c 110 kDa 130 kDa y tosol 20% AS HiTrapMono Q 75 kDa 220 801609070120605030100190 kDa 95 kDa 55 kDa 35 kDa 120 kDaprogression of the M-phase MEK kinase purification GE and their protein compositions were visualized by Silver staining. Figure 45. Protein profiles of mitotic MEK kinase active fraction throughout Equal amounts (5 g) of MEK kinase active fractions were separated by 8% SDS-PA

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121 evidence whether thiv p u separately or together in a comple at the final ( Qma rag of proteins of the purified MEK kinase active fraction on a 5-20% gradient native polyacrylamide gel under nondenaturonditions to allow protein complexes to remain together. After completion of the run, I visualized a patte rn of protein separation by Silv er staining. A single strong broad band was detected (with a few weak mi nor bands below) suggesting that most of the proins in mitotic MEK kinase activ e fraction are togethecomplex (Fig. 46, first lane). Is B-Raf a part of th is large protein complex?er this, Western analysis was performed on the same sa mple. The results show that the majority of B-Raf resided in the major higher molecu lar weight band (Fig. 46, second lane). Collectively, these results suggest that the purified mitotic MEK kinase activity represents large molecular weight complex( s), which include B-Raf and several other unknown proteins. entifion of potential components of large multi-protein complexes containing active mitotic B-Raf I postulate that components of the purifi ed mitotic MEK kinase active complex(s) mig it is i that both Cdk1 and cyclin B are present in the purified MEK kinase active fraction (see Fig. 29A) and show that they asso ciate with B-Raf at mitosis in Xenopus egg extracts (see Fig. 29B, C and D). To further characterize th is protein complex, I used a proteomics ese ind Mono idual colu rotei n) st ns co -p ge, I rified n 5 x ing c te the r in a large To answ Id cati ht serve a role in regulating B-Raf activatio n or function during mitosis. Therefore mportant to identify these proteins. Fo rtuitously, I determined by Western analysis

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Non-denaturing PAGE Silver Staining B-Raf Western 122 122 F igure 46. Mitotic MEK kinase activity purified from M-phase arrested X enopus egg extracts resembles a large B-Raf-containing protein complex Final MEK kinase active fraction was run on a gradient non-denaturing PAGE and analyzed by Silver staining and B-Raf Western blotting.

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123 pproach to identify the other proteins purified with the mito tic MEK kinase activity. To o this, I performed a large-scale purification of the MEK kinase activity from Xenopus itotic egg extracts using an established fou r-step protocol (Fig. 13A). 50 g of purified EK kinase activity wa tained with colloidal blue nvitrogen). Fourteen protei n bands detected by colloidal blue were carefully excised om the gel and pooled into three groups for Mass spectrometric (MS) analysis. equence analysis of trypsin-digested peptides was performed at the Harvard icrochemistry Facility by microcapillar y reverse-phase HPLC nano-electrospray ndem mass spectrometry on a Finnigan quadrupole ion trap mass spectrometer. The dividual MS/MS pep tide spectra were then correlate d with known protein sequences. addition to B-Raf, Cdk1, and cyclin B, over 25 new proteins were id entified (Table 2). Many of the proteins identified in the plex have stablished roles in actin cytoskeleton regulation (actin, filamin, talin 2, gelsolin), ceptor-mediated endocytosis (Adaptin, Epsi n1, beta-adaptin 1 subunit, clathrin heavy hain, HABP1, and AP-2), nuclear transport and nuclear pore complex formation (CAS/Cse1, Nup93, importin beta 3, Nup 155) mitotic regulation (CAS/Cse1, APACD) and c t PA28 was identified by MS as a potential compone nt of the B-Raf containing mitotic MEK inase active protein complex(s). The identification of PA28 proteasome subunit pports the validity of my approach for disc overing novel proteins that comprise a large multiprtein complex containing B-Raf. a d m M s separated by SDS PAGE and s (I fr S M ta in In purified MEK kinase active com e re c hromatin-related functions (RPA, ATR). The proteoso me activator alpha subuni previously shown to be a B-Raf-specifi c interacting protein (Kalmes et al., 1998), k su o

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124 Name MW MS/MS Comments Table 2. Proteins identified by Mass spectrome try in the final mitotic MEK kinase active fraction spectra Cytoskeletal proteins Beta-Actin 42 kD 53 Structural component of cytoskeleton Nup93 93 kD 35 Nucleoporin, a nuclear pore complex protein Filamin 280 kD 28 Non-muscle actin-binding scaffold protein Gelsolin 86 kD 17 Actin-binding de-polymerizing factor Nup155 155 kD 14 Nuclear pore complex protein Talin 2 220 kD 2 Adaptor between integrin and actin cytoskeleton Cellular Trafficking proteins Adaptin 90 kD 42 Mediator of in tracellular Golgi trafficking Importin 120 kD 37 Mediator of nuclear transport beta 3 CAS/CSE1 110 kD 27 Regulator of nuc lear/cytosolic reshuffling of importin alpha AP-2 48 kD 9 Adaptor protein required for uptake of cargo proteins BetaAdaptin 1 105 kD 7 A subunit of AP-1 complex involved in clathrin assembly in Golgi Clathrin, 184 kD 4 Structural compone nt of Golgi trafficking heavy chain vesicles HABP1 57 kD 4 Nucleus/mitochondria and extracellular trafficking agent Tom1 54 kD 2 Sorts ubiquitinated proteins into multivesic ular bodies SNAP 32 kD 2 Golgi protein Epsin1 90 kD 2 Regu lator of endocytosis

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125 ontinued) Name MW spe Comments Table 2. Proteins identified by Mass spectro metry in purified mitotic MEK kinase active fraction (c MS/MS ctra M itotic proteins CAS/CS E1 110 kD 27 Mitotic spindle checkpoint protein Apacd 29 kD 10 to Microtubule dynamics regulator; localizes centrosomes and midbody Epsin1 90 kD 2 Endocytosis; Cdk1/cyclin B su bstrate during mitosis DNA Metabolism proteins Replicatio n n Factor A 70 kD 22 Assists T-antigen in initiating the replicatio system Wellco me 4 Nucleotide metabolism CRC pSK ATR 292 kD 3 heckpoint regulator DNA damage c Protein Sta bility Regulatory factors CCt8 60 kD 66 of the Cct chaperonin complex Subunit HSP7 0 70 kD 24 Chaperone protein PA-28-beta 28 kD 3 tor subunit Proteosome activa Tom1 54 kD 2 cular Sorts ubiquitinated proteins into multivesi bodies Intrllul ace ar Metabolism Proteins Ndrg20 40 kD 16 lase Alpha/beta hydro Aldolas e 40 kD 10 Glucose metabolism Cn2 52 kD 9 lic dipeptidase Cytoso

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126 Described above approaches for characte rization of mitotic MEK kinase active co) do not prn n ular weight. To get some insights into the size of the B-Raf contitotic MEK kinase active complex(s), I used gel filt atoghichws on their sizes under native conditions. First, thten 00 HR 10/30) was pre-calibrated with a mix cular weights. After this, 2.6 mg of 20% ammonium sulfate p ed fraction containing B-Raf activity was applied to the column and s e conditions. Aliquots of the ctionsubj to Western blotting to analyze si ze-wise distribution B-Raf protein respectively. As shown in Figure 47, both mitotic MEK kinase activity anf were locahigh00actions (# 28-38). This data is consistent with the id ea that active mitotic B-Raf exists in large proplexes. nglyRaf middle-range molecular we (90-), butal M actions was signiftly lower. Sincitotic MEK kinase a represented by B-Raf (Fig. tic B-Raf functions in higlar weigin coexe Purification of mitotic B-Raf with in large multi-protein complexes mplex(s ovide a estimatio aining m of its molec ration chrom raphy, w allo for the separation of proteins based e re tion capacity of the column in use (Superdex 2 of pr oteins of known mole reci pitat eparated under the same nativ collected fra s were ected an in vitro MEK kinase assay and B-Raf of the mitotic MEK kinase activity and d B-Ra ted in a er 4 700 kD molecular weight ra nge of fr tein com Interesti Bwa s also detected in ight fractions 300 kD t to E K kinase activity of these fr ican e m ctivity in Xenopus egg extracts is 17), this may indi cate that active mito h molecu ht prote mpl s.

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12 7 or Figure 47. Active mitotic B-Raf isa large 700-400 kD2.6 mg of 20% amiuSd0 0fiocolumfor levels of MEK kinase activity by an in vitroMke y fthe presence of B-Rystcsiftn capacity was calibr purified by gel filtration as a protein complex monm sulfate precipitated fraction was separated on a uperex 20 HR10/3 gel ltratin n. Collected fractions were analyzed EK inasassaand af b Weern blotting. To determine moleular zes o the eluted proteins the column reentioated with a mix of proteins with known molecular weights.

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128 If protein-protein interactions are impor tant for B-Raf signaling at mitosis in Xenopus egg extracts, than I would speculate that formation of these higlecular BRaf containing protein complex(s) should be dynamic throughout the. To obtain some preliminary evidence for this, I decided to compare protein p immunopurified with B-Raf antibodies from S-phase (inactive B-Raf) and M-phase (active B-Raf) Xenopus egg extracts. Equal amou nts of B-Raf protein were immunoprecipitated from S and M-phase arrested Xenopus egg extracts, separated by a 10% SDS PAGE, and co-immunoprecipitated protei ns were visualized taining. As shown in Figure 48, there is a significant di fference in a set of proteins, which interact with B-Raf at Sand M-phases. Specificall y, 120 kD and 45 kD prot B-Raf preferentially during S-phase, whereas 65 kD and 77 kD proteins interacted exclusively with mitotic B-Raf. This prelim inary data indicates tha complexes changes from Sto M-phases. Conclusions In this Chapter, I show that mitotic MEK kinase activity p egg extracts is purified as B-Raf containing multi-protein complex(s). Gel ftimates the size of these active mitotic B-Raf protei n complex(s) to be in the range of 400-700 kDa. Protein composition of B-Ra f immunoprecipitated complexes extracts changes from Sto M-phase, i ndicating that formati on of B-Raf protein associations is controlled in the cell cycl e dependent manner and may be important for her mo cell cycle rofiles coby Silver s eins associated with mbly of B-Raf Xenopus iltration es Xenopus egg t asse resent in in

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129 160 12080705060120 kD 95 kD 77 kD 904065 kD 45 kD B-Raf IPSM Figure 48. Protein profile co-immunoprecipitated with B-Raf is changing B-Raf immuno-complexes precipitated from equal amounts of Sand M-phase ofrom Sto M-phases arrested extracts were washed and separated on 10% SDS-PAGE. Proteins c p urified with B-Raf antibodies were visualized by Silver staining.

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130 gulation of B-Raf functions. To generate a broader view of regulation of B-Raf during M-phase, proteins potentially comprising the mitotic B-Raf containing MEK kinase active protein complex(s) were id ass spectrometry. These include 25 proteinwith known functions invod i tin cytoskeleton regulation, receptormediated endocytosis, nuclear/cytosol tra fficking and nuclear pore complex formation, mitotic regulation and proteins with chroma tin-related functions. Future studies are necessa to validate interac these proteins within Bing multi-protein complex(s) and disclose their involveme nt in regulation of B-Raf functions. re entified by m s lve n ac ry tion of -Raf contain

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131 The central discovery of this dissertation is the demonstration that B-Raf is the ajor MEK kinase required for activation of th e MAPK pathway at mitosis. This work rovides the first evidence that B-Raf functi ons at mitosis and expands our understanding f B-Rafs involvement in the regulation of th e cell cycle. In part icular, it is proposed at B-Raf signaling controls progression thr ough the cell cycle at two different stages: uring the G1/S transition and at mitosis. MAPK signaling has several roles at mitosis, including regulation of the G2/M ansition (Liu et al., 2004; Wr ight et al., 1999), control of the spindle formation (Horne nd Guadagno, 2003; Zhang et al., 2005), mainte nance of the spindle assembly arrest hau and Shibuya, 1999; Minshull et al., 1994), regulation of Golgi fragmentation ebersold et al., 2004; Shaul and Seger, 2006) and the duration of mitosis (Guadagno nd Ferrell, 1998; Roberts et al ., 2002). I speculate that at least some of these MAPK nctions at mitosis are regulated by B-Raf. Indeed, a num ber of recent studies by the uadagno laboratory have extended my work of B-Raf to human somatic cells. First, BChapter Seven General Discussion A novel role for B-Raf in cell cycle regulation m p o th d tr a (C (A a fu G

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132 Raf was shown to localize to the mitotic apptus in HFF (human foreskin fibroblasts) cells in a pattern similar to one observed for active MEK and ERK (Shapiro et al., 1998; Willard and Crouch, 2001; Zecevic et al., 1998). Specifically, it was shown that B-Raf localizes to the centrosomes through indle microtubule and kinetochores during metaphase. Further, phosp Ser-602) B-Raf was detected in the areas of condensed chromatin during propha se, at the centrosomes, kinetochores and chromosomes during aphase and at the midbody during cytokinesis (Borysov a MK, Guadagno TM, 2006, manuscript submitt he mplicat ed in the regulation of mitotic spindle checkp ara out mitosi s and sp horylated (T hr-599 and metaphase, at the spindle midzone during late an ed). Loss-of-function studies showed that reduction of endogenous B-Raf by RNA interference treatment led to the accumulation of abnormal mitotic spindles (Borysova MK, Guadagno TM, 2006, manuscript submitted). Thus, these data support the idea that B-Raf directs MAPK signaling duri ng mitosis in somatic cells to regulate t mitotic spindle. In addition, B-Raf may as well be i oint signaling. Specifica lly, it was shown that expre ssion of the human mitotic checkpoint kinase Mps1 (Weiss and Wine y, 1996) is dependent on B-Raf/MEK/ERK signaling (Cui Y, Guadagno TM, manuscript in preparation). Thus, Mps1 may be an important target of B-Raf signaling during mitosis to regulate th e spindle checkpoint Further studies are required to explore the possible implication of B-Raf in the mechanism of spindle checkpoint. Based on the data presented in my dissert ation, I speculate that mitotic B-Raf is not involved in the regulation of Golgi fragment ation during mitosis. In the experimental

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133 h tation is shown to be dependent on MAPK signaling in somatic cells, is discuss d that Bwas attr ibuted to its pro-survival functions (Wojno system used in my studies the vast majority of mitotic MEK kinase (B-Raf) activity was detected in the cytosol fraction void of memb ranes. Thus, it is unlikely that mitotic BRaf localizes to Golgi apparatus. However, this was not specifically addressed in my experiments. A recent study proposed that Raf-1 activates Golgi-associated pools of MEK during mitosis thereby regulating Golgi fr agmentation at the mitotic onset (Colanzi et al., 2003). However, this conclusion is based on the data obtaine d by inhibition of Raf1 activity with the autoinhib itory domain of Raf-1 (Brude r et al., 1992) or Raf kinase inhibitory protein (RKIP) (Yeung et al., 2000), both of which may interfere wit activation of the other members of the Ra f family, A-Raf and B-Raf. Thus, the involvement of a particular Raf family memb er in the regulation of Golgi fragmen during mitosis is not conclusively defined yet. The potential implication of mitotic B-Raf in the regulation of the G2/M transition, which ed in the next section (B-Raf dire ctly links Cdk1/cyclin B and MAPK signaling during mitosis). Identifying a role for B-Raf at mitosis further expands our u nderstanding of the prominent role that B-Raf has in mediati ng cell proliferation during embryogenesis. Gene knockout of B-Raf in mice demonstrated that B-Raf is critical for proper fetal development (Wojnowski et al., 2000; Wojnowsk i et al., 1997). It was suggeste Rafs involvement in embryogenesis wski et al., 2000; Wojnowsk i et al., 1997). However, my new data indicates that

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134 which g of B-Rafs involvement in the regulat af will r of Raf in Xenopus egg extracts occurs subsequent to the activation of Cdk1/cyclin B (Fig. regulation of cell division during early embryogenesis could be another important role for B-Raf. B-Raf is an important oncogene which is mutated and activated in 8% of all human tumors and in nearly 70% of me lanomas (Davies et al., 2002). Activating mutations in the B-Raf gene lead to c onstitutive B-Raf signaling and elevated ERK activity. It is tempting to speculate that constitutive B-Raf signaling could promote mitotic errors that contribute to B-Raf-driven tumorigenesis. In fact, deregulation of B Rafs mitotic functions may provoke the forma tion of aberrant mitotic structures, can lead to genomic instability. In summary, identification of B-Raf as the mitotic MEK kinase is a novel finding, which expands our comprehensive understa ndin ion of the cell cycle. Further explor ation of potential mitotic roles for B-R shed insights into the mechanisms of B-Rafs biological functions, such as its involvement in embryogenesis, cell proliferation and oncogenesis. B-Raf directly links Cdk1/cyclin B and MAPK signaling during mitosis The data in my dissertation demonstrate th at Cdk1/cyclin B, a major regulato mitotic entry, directly phosphorylates B-Raf a nd that this phosphorylation contributes to the activation of B-Raf at mitosis. Seve ral lines of eviden ce support this novel mechanism as a regula tor of mitotic B-Raf activity. First, activation of endogenous B-

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135 dependent manner (Fig. 29) and unde rgoes a Cdk-dependent phosphorylation at some o lin B e-144 ot he dk1/cyclin B directly phosphorylates and activates mitotic r ing cell n extracellular signals trigger activation of B-Raf. Bindi ng of mitogen proteins to the appropriate growth e plasma membrane and induces its activati on. At mitosis an intrinsic mechanism involvi hat B-Raf t 28). Second, endogenous B-Raf associates w ith active Cdk1/cyclin B complexes in an M-phase f its Cdk-phosphorylation consensus site (s) (Fig. 30). Thir d, active Cdk1/cyc directly phosphorylates in vitro recombinant Xenopus B-Raf at its conserved Serin residue (Fig. 32). Finally, a non-phosphoryl atable Ser-144-Ala B-Raf mutant does n undergo activation in an M-phase dependent manner (Fig. 33) and does not activate t MAPK pathway at mitosis (Fig. 34); furtherm ore, the mutant exerts a dominant-negative effect by blocking mitotic activation of MAPK in Xenopus egg extracts (Fig. 35). Together, these data suggest that C B-Raf. A role for Cdk1/cyclin as an activator of B-Raf opens new perspectives fo understanding how B-Raf is regulated during the cell cycle. The mechanism of B-Raf activation at M-phase appears to be distin ct from its activation mechanism dur cycle entry (Roovers and Assoian, 2000). Du ring cell cycle initi atio -factor receptors induces activation of Ras, which in turn recruits B-Raf to th ng the mitotic machinery leads to BRaf activation. B-Raf accommodates mitotic signals from Cdk1/cyclin B that promote activation of the MAPK pathway. Based on what is known about B-Raf regulation by phosphor ylation, it can be proposed t is receptive to two distin ctive regulatory phosphorylati ons. Specifically, following extracellular mitogenic stimulation B-Raf undergoes a Ras dependent phosphorylation a

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136 e dies are necessary to el ucidate the molecular basis for how phosph ding at erine-Raf t if ortion of yclin its Threonine-599 and Serine-602 residues w ithin the activation loop (Zhang and Guan, 2000). Interestingly, my data show that mitotic 95 kDa B-Raf is not phosphorylated at these sites in mitotic Xenopus egg extracts (Fig. 27). Howe ver, the activation of mitotic B-Raf in the Xenopus egg extract system requires phos phorylation at its Cdk/cyclin B phosphorylation site. Further analysis in soma tic cells will need to determine whether th same Serine-144 site is phosphorylated at mito sis. This could be analyzed by developing phospho-site specific antibodies and perfor ming immunofluorescent studies. Thus, during cell cycle entry and mitosis B-Raf can be subjected to two different sets of phosphorylation, both of which cont ribute to B-Rafs activation. Future stu orylation at the Serine-144 residue induce s B-Raf into an active state. Accor to the existing dogma, activation of Raf kinase s results from conformational changes th open the C-terminal kinase domain from th e inhibitory N-terminal regulatory domain (Kolch, 2000). Therefore, I sp eculate that the presence of a negative charge at the S 144 residue can promote the aforementioned in tramolecular changes necessary for B activation, similar to other know n regulatory phosphorylations. It should be noted tha this prediction is correct, this would be the first identified site in the N-terminal p B-Raf protein that undergoes a positive re gulatory phosphorylation. Alternatively, my data do not exclude that the phosphorylation of B-Raf at the Ser-144 residue creates a binding site for a B-Raf interactor, whic h in turn mediates B-Raf activation. The proposed biochemical mechanism of B-Raf activation by a direct Cdk1/c B phosphorylation helps explain how MAPK signaling and the cell cycle regulatory

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137 was cell MAPK activities during ay be under tic gs tic machin machinery are coordinated during M-phase. From the present study, it appears that activation of the MAPK pathway during M-pha se occurs down-stream of Cdk1/cyclin B and that B-Raf serves a direct link that in tegrates activation of MAPK signaling to the initiation of M-phase. Therefore, it can be proposed that activati on of mitotic B-Raf is coordinated to the completion of DNA replic ation and occurs after commitment to mitosis. The role for B-Raf as a link between C dk1/cyclin B and the MAPK pathway described by using the Xenopus egg extracts, which represent the early embryonic cycle. It is likely that a similar m echanism coordinates C dk1 and the somatic cell cycle. Circumstantia l evidence indicates th at B-Raf m the regulation of Cdk1/cyclin B in tissue culture cells. Indee d, both B-Raf and active Cdk1/cyclin B localize to the same mitotic stru ctures. Similar to B-Raf, at the early Mphase Cdk1/cyclin B is found at the centrosom es (Jackman et al., 2003) and at the mito spindle and condensed chromatin at metaphase (Huo et al., 2005; Lee et al., 2003; Stiffler et al., 1999). In conclusion, I demonstrate that Cd k1/cyclin B phosphorylation of B-Raf is required for its activation at mitosis. The M-phase activation of BRaf via Cdk1/cyclin B phosphorylation provides a novel mechanism of B-Raf regulation. It expands our understanding of how B-Raf signaling is contro lled throughout the cell cycle and begin to elucidate how activation of the MAPK cascade is coordinated with the mito ery.

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138 ed in ion of MAPK signa ling during mitosis. In fact, immunodepletion of Raf-1, unlike B t xtracts, whereas the B-Raf/MEK interactions were reproducibly detected. Next, B se f Bher, monstrate that B-Raf, but not Raf-1, functions as th e mitotic MEK kinase. B-Raf, but not Raf-1, regulates MAPK activation at mitosis The studies herein provide evidence that the role of mitotic MEK kinase is specific to B-Raf within the Raf kinase fa mily. My data demonstrate that another member of the Raf family, Raf-1, does not disp lay features of a MEK kinase involv the regulat -Raf, from Xenopus egg extracts does not affect MAPK activation at M-phase, indicating that that the presence of Raf-1 is irrelevant for triggering the MAPK pathway during mitosis (this study and Yue and Ferrell, 2004). This is consistent with the previously published data show ing that activation of Raf-1 in nocodazole-arrested mitotic cells does not lead to activation of the dow n-stream components of the MAPK cascade (Laird et al., 1999; Ziogas et al., 1998). The present study pr ovides further evidence tha Raf-1 is uncoupled from mitotic MAPK signaling. Co-immunoprecipitation analyses showed that Raf-1 does not associate with its classical down-stream target, MEK, in Xenopus egg e -Raf, but not Raf-1, was enriched dur ing purification of th e mitotic MEK kina activity, demonstrating that B-Raf is the majo r MEK kinase present in M-phase arrested Xenopus egg extracts. Finally, a recent study de monstrated that down-regulation o Raf, but not Raf-1, via RNA interference (RNAi ) abrogates normal spindle formation in somatic cells (Borysova MK, Guadagno TM, 2006, manuscript submitted). Toget these data de

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139 Recen ers is important for activation of the MAPK pathway following mitogenic stimulation (Weber et al., 2 itotic ediated f-1 fic he 11S regulator with the 20S proteasome core complex affects the specificity of the tly it has been proposed that formation of B-Raf/Raf-1 heterodim 001). However, I did not detect interactio ns between B-Raf and Raf-1 in m Xenopus egg extracts. Therefore, I conclude that Raf-1 is no t involved in B-Raf m activation of the MAPK cascade at M-phase. Several biochemical mechanisms may ensure the specificity of B-Raf versus Ra 1 as the mitotic MEK kinase. As mentioned above, specificity can be controlled by a mechanism that favors the preferential associ ation of mitotic MEK with B-Raf versus Raf-1. Secondly, Raf-1, unlike B-Raf, ma y be unreceptive to mitotic up-stream regulatory inputs necessary to trigger its activ ation as a MEK kinase. My work revealed that phosphorylation of Xenopus B-Raf at its Cdk1/cyclin B phosphorylation site (Serine 144 residue) is critical for act ivation of B-Raf and for B-Ra fs capacity to trigger the MAPK cascade at M-phase. Interestingly, this Cdk1/cyclin B phosphorylation site is conserved only among B-Raf members of the Ra f kinase family (Fig. 49). Thus, Raf may not be implicated into the Cdk1/cyclin B MAPK pathway due to the absence of this regulatory site. The Serine -144 residue, targeted by Cdk1/cyclin B, is located in the N-proximal portion of B-Raf, which does not resemble homology to other members of the Raf family. It is intriguing that regulat ion of this domain may enable B-Rafs speci functions. This idea is supported as we ll by another study which showed that the Nterminal domain of B-Raf is critical for B-Ra f, but not Raf-1, specific interaction with t alpha-subunit of the 11S proteasome regulator (Kalmes et al., 1998). Association of the

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140Figure 49. The N-terminal Cdk1/cyclin Bhoshoryatiosite conerved amng BRaf family The-terinalorti of minocid queces o B-Raf (AAZ06667), -Raf ppln isso-membef ke Nm pona asenf XenopusHumanB (P150 rs oRaf inas 56), Mouse B-Ra f Raf-1 man A-Raf (CAB81555), CickeB-ZebafishB-Ra (BA1678), umaRaf(P0049),(NP084056), CckenRaf-(CA3009), Xnop Raf1 (P0560 Raf-1 (BAD34647), (TVUA) andMou A-af (P4627 wer aliged businMegAlign hn Raf (Q04982), r fD2Hn 14 Mousehi 1 A6eus-9),ZebrafishHuHF seR0)eny g software. The Cdk1/cyclin B phosphorylation site iswrath s h o n i n e d f r m e; e N H T P S D P S RINPRSPQKPIVRVFLPNKQ R T VVPARSG132Xenopus B-RafQP VPKVK R T VG3HumaB-RafQP APKVKVG7MousB-RafP RIVLK VG3cP RIVLK VG4rhRA DDSVKVG4aRaf-1RA DDSVKVG4sRaf-1DDSVK VG4cDDSVK VG4oSD-SVQ VG3rhAG P-VVK VGaA-RafVG P-VVK VGsA-Raf N T D A RSNKSPQPIVRFLPNQVPARC19 T T D S RNNKSPQPIVRFLPNQ R T VPARC3Q N T D M S NNPKSPQKPVRFPNQ RT VPARC19ChiQ T T D A T GNPRSPQKPVRFPNQ RT VPARC19Zeb R SD G KLTPSKTNTIRFLPNQ R T VNVRN0Hum R SD G KLTSSKTNTIRFLPNQ R T VNVRN0MouR R A SD G KISTSKTNTIRFLPNQ RTVNVRN0ChiR R A SD G KLSTSKTSTMRYLPNQ RTVNVRS0Xen R S S D ----SKTRTIRFLPNQ RTVNVRP9Zeb N AE----SRAGTVKYLPNQ RTVTVRD9Hum S AE----SRAGTVKYLPNQ RTVTVRE9Mou n e ken B-Rafafis B-Rafn e ken Raf-1pus Raf-1afis Raf-1n e tnof1iue e r m i a l portion C R is shaded n b l

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141 ubiquitin directed proteolysi s (Dick et al., 1996; Groettr up et al., 1996). However, whether B-Raf regulates the 11S proteasome subunit is not clear subunit is not phosphorylated by B-Raf (Kalmes et at B-Raf and Raf-1 can respond differently P-ase, Rap1, activates B-Raf, but does not y and Kolch, 2000; York et al., 1998). The /cyclin B proposed herein further highlights the nase family. im tracellula r stimulation by growth factors, it is cascade from components of the m h C dk1 in nocodazole-arrested mitotic cells and tion. Impor ted nocodazole stitu tively active H-Ras or Raf-1 (Dangi and e evidence specific to B-Raf within the Raf family. Un like B-Raf, Raf-1 is not required for MAPK activation in mitotic Xenopus egg extracts. Further, Rafur ified as the Msince the 11S alphapacity to activate the MAPK receptors to MAPK itogen-dependent dk1/cyclin B activity inhibits -arrested cells as well as in mitotic MEK kinase is 1 was neither p al., 1998). ulation by growth factors. Since cells growth-factor tantly, C that the role of Notably, it has already been reported th te R to up-stream signaling inputs. A sm 1 ( all GT sla ac tiva afBos et al., 200 1; Hou me cha nis m o f B -Ra f a ctiv atio n b y C dk1 reg ula tor y di ffe renc es wit hin the Ra f ki It app ear s th at t hr oughout the cell cycle, Raf-1s ca pa thw ay is tr igg ere d on ly foll owi ng mitogenic st un der goin g m ito sis do not res pon d to ex pro po sed tha t du rin g G 2/M th e s igna ling via Ra f-1 is b loc ked by Cd k1 /cy clin B activity (Dangi and Shapiro, 2005). Specifically, it w as sho wn tha t R af-1 an d i ts u p-st ream pa thw ay, So s-1 and Gr b2, ass oci ate wit un der go a Cd k1 -de pen den t ph osp ho ryla activation of MAPK in growth -facto r trea no cod azo le-a rre sted ce lls exp res sing con Shapiro, 2005). In co ncl usio n, m y resu lts pro vid

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142 fic Nand a Cdk1/cyclin B mediate ts of my dissertation demons trated that B-Raf undergoes negative feedbac phase MEK kinase or shown to associate with MEK. I propose that the B-Raf speci terminal domain may enable B-Raf to be receptive for mitotic regulatory inputs via Cdk1/cyclin B. Uncoupling Raf-1 from association with MEK d inhibition of the growth-factor r eceptor pathway may as well ensure B-Rafs specificity in functioning as the mitotic MEK kinase. Mitotic B-Raf undergoes a negative feedback regulation by MAPK Feedback loop phosphorylation is broadly im plicated in the regulation of MAPK signaling. The guanine nucleotide exchange factor, Sos-1, is phosphorylated by MAPK following EGF receptor stimulation (Corbalan-Ga rcia et al., 1996; Waters et al., 1996) This phosphorylation is thought to represen t a negative regulatory feedback loop that desensitizes the receptor-mediat ed signaling by inhibiting Sos1 interactions with adaptor proteins, such as Grb2 (Corbalan-Garcia et al., 1996; Waters et al., 1996). The phosphorylation feedback loop operates with in the MAPK module itself. MEK was shown to exert a positive fee dback regulation on Raf-1 (Z immermann et al., 1997). Raf1 was demonstrated to undergo both ne gative (Dougherty et al., 2005) and positive (Balan et al., 2006) feedback regulations by MAPK. The resul k regulation by MAPK during mitosis. My data showed that the hyperphosphorylation of mitotic B-Raf is primarily caused by a MAPK-mediated phosphorylation at Serine784 and Threonine 787 residues within the conserved C-

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143 mics e B-Raf mutant enhances PC12 differentiation (Rushworth et al., 2006). This suggests that phosphorylation of the Cterminal My study is the first to address the eff ct of C-terminal phosphorylation on B-Raf kinase y ates of B-Raf/Raf-1 heterodimers (Rushw rrested Xenopus the terminal SPKTP motif of B-Raf. A similar si tuation was reported for B-Raf in activated human B lymphocytes (Brummer et al., 2003). Furthermore, a B-Raf mutant that mi constitutive phosphorylation at the C-te rminal MAPK phosphorylation sites is characterized by a decreased efficiency in promoting differentiation of PC12 cells (Brummer et al., 2003), whereas a non-phos phorylatabl SPKTP motif negatively a ffects B-Raf biological functions. e activity. I showed that kinase ac tivity of a non-phosphoryl atable B-Raf mutant following incubation in M-phase extracts is significantly higher compared to wild-type B-Raf. Further, inhibition and over-activation of endogenous MAPK in M-phase arrested Xenopus egg extracts enhances or diminishes BRaf activity, respectively. Together, m data support the proposal that B-Raf activity during mitosis is negatively regulated by a feedback loop mediated by MAPK phosphorylation. The mechanism by which phosphorylation of the C-terminal sites down-regul activity of B-Raf requires further studies. It was proposed that MAPK-mediated phosphorylation at the Threonine residue w ithin the C-terminal phosphorylation site abrogates B-Raf signaling by promoting disa ssociation orth et al., 2006). However, it is unlikely that the same mechanism operates during mitosis, since B-Raf/Raf-1 heterodi mers do not form in M-phase a egg extracts. The C-terminal MAPK phosphor ylation sites are lo cated within close proximity to a 14-3-3 binding site (Brummer et al., 2003). Thus, it is possible that

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144 ted over I ediated feedback phosph APK oc curs at the middle-to-late mitosis nt nism MAPK phosphorylation modulates B-Raf associ ation with 14-3-3 protein and thereby induces down-regulation of its activity. Why is a B-Raf activity in M-phase arrested Xenopus egg extracts eleva S-phase levels despite its hyperphosphorylation and the pr esence of active MAPK? speculate that mitotic B-Raf in these extracts is represented by two different species: active B-Raf, which is not phosphorylat ed at the C-terminus, and inactive hyperphosphorylated B-Raf. Another explana tion is that the MAPK-m orylation of B-Raf is not sufficient to completely inhibit B-Raf activity and needs to be combined with other regulatory events for instance inactivation of Cdk1/cyclin B Analysis of temporal fluctuations of Cdk1/cyclin B, B-Raf and MAPK activities and B-Raf hyperphosphor ylation in cycling Xenopus egg extracts shows that inactivation and hyperphosphorylation of B-Raf during mitosis overlaps with inactivation of Cdk1/cyclin B and activation of MAPK (Fig. 20). Based on th ese data, I speculate that the negative feedback phosphoryl ation of B-Raf by M and contributes to inactivation of BRaf at times of cyclin B degradation and inactivation of B-Rafs activator Cdk1/cyclin B. It is plau sible to suggest that this mechanism of B-Raf inhibition via a MAPK-m ediated feedback loop ensures a transie activation of the B-Raf/MEK/MAPK path way necessary to accommodate M-phase progression and exit from mitosis. In summary, mitotic B-Raf undergoes f eedback phosphorylation via activated MAPK that appears to negatively regulate B-Ra f activity. I postulate that this mecha ensures a transient activati on of the B-Raf/MEK/MAPK pathway during mitosis.

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145 tivation of Cdk1/cyclin B at the M-phase onset directly B of of a t tein ork sted ingle peak of MEK kinase activity eluted from a Mono Q Together, my work describes a comprehensive mechanism for the regulation of B-Raf activity during mitosis. I pr opose that ac triggers activation of B-Raf, perhaps at the spindle apparatus, which directs activation of the MAPK cascade. At the latter stages of mito sis, probably after cyclin degradation and inactivation of Cdk1, MAPK feeds back to B-Raf to down-regulate its activity (Fig. 50). Together, these regulatory mechanisms c ontrol transient activation the B-Raf/MEK/MAPK cascade during mitosis. Mitotic B-Raf functions in multi-protein complexes Active B-Raf was reportedly purified within large protein complexes (Jaiswal et al., 1996; Mizutani et al., 2001; Mizutani et al., 1998). Spec ifically, purification Ras-dependent Raf-1 activator from a rat brai n homogenate led to isolation of a 400 kDa B-Raf containing protein complex (Jaiswal et al., 1996; Mizutani et al., 2001; Mizutani e al., 1998) and B-Raf from PC12 cell lysates was isolated within large (>300 kDa) pro complexes (Jaiswal et al., 1996; Mizutani et al., 2001; Mizutani et al., 1998). The w from my dissertation suggests that active mitotic B-Raf exists as well in large multiprotein complexes. Purifica tion of M-phase MEK kinase ac tivity from M-phase arre Xenopus egg extracts resulted in a s chromatography column. Separation of the final MEK kinase active fraction under native non-denaturizing conditions show ed that B-Raf was present in a large protein complex. Analysis of the protein composition of this complex by SDS

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146Fiure 0. Poposd mhanm f reglatio of e B-af/During S-phases of embronic ell cclingB-Ra is inctivand acve Ck1/cclin assiatewithB-Raand irecty phphoractivation of -Rafnd tggerthe -RafMEKMAPK casade.fe-bac phophorlatio of BRaf t its -ternal erine784 itoc MPK gnalinbito one inlue. g5reecisorunthRMEK/MK cascade aoycy fae associawith MEK. A etMstidyBocs f dlosylates Serine-144. cib tB aris B//c At latages of M-phcMKrtedksyn-aCmiS-and Thine-787, whicegeRactivity and probably leads to turn-down of mtiAsiing. Ation states anerw hirys b APted it at er streonctiva t mitsis t theon-s of -phae Thisontruteso ase ative AP exes a h down-rulats B-af d evnts ae shon inred, P B-af R B-Raf B-Raf S-hae ps M-ps hae S784 T 7P 87 MEK MP MEK MEK MPK A S144P Cdk 1 AK cyclin B

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147 PAGE revealed that it consists of ma ny proteins, many of which were enriched throughout the purificsing gel filt ration, it was estimae molecular weigth range of the active mitotic B-Raf containing complex is between 400-700 kDa. Preliminary studies demonstrate that a set of proteins that a immunoprecipitates in etractsrs sligh 48). Thus, the formation of B-Raf complexes in Xenopus s to be dynamay be an important mechanism that controls B proper functioning at M-phase. The biochemical analysis of the purified B-Raf complex described several proteins that may associ ate with B-Raf ity and signaling during mitosis. My work demonstr ated that B-Raf associates w Cdk1/cyclin B compled Mn an association between B-R s detected at the beginning of mitosis in Xenopus ion with MAPK was visualized twice throughout mitosis, at the beginning and during middle-late M-phase. B shown that B-Raf forms complexes with MEK both at SAdditionally, over 25 proteins were identif ied as potential com Raf containing protein compTable 2). Several of the identified proteins have already been linked to the re ce, beta-actin was identified as one of the pre dominant com itotic MEK kinase active complexes. It has been shown that B-Raf (P MEK (Pawlak and Helfman, 2002) and MAPK (Barros and in the regulation of actin cytosk eleton functions. Future stud lucidate atio n. B y u ted that th ssociate with B-Raf from Sto M-phases (Fig. -Rafs regulation and/or ctiv ith active anner. Interestingly, esides this, it was M-phases. ponents of the Bnts of the purified m ritchard et al., 2004), Ma rshall, 2005) are involved ies are necessary to e Xenopus gg ex diffe tly egg extracts seem to regulate its a and MAPK cascade. For instan pone ic and m xes an APK i and M-phase dependent m af wa Cdk1/cyclin B egg extracts, w hereas B-Rafs associat lexes ( gulation or functions of the

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148 occurs at mitotic s a 996), a n of active ERKs f ion whether the B-Raf/MEK/MAPK de pendent reorganization of ac tin cytoskeleton M-phase. This can be addressed by studyi ng how reduction in B-Raf, MEK and MAPK levels following specific siRNA treatments affe cts the actin cytoskeleton structures in cells. Some of the identified candidate proteins are known substrates and/or interactor of B-Raf, MEK, MAPK or Cdk1/cyclin B. Specifically, PA-28-beta, a proteasome activator subunit, is shown to be a B-Raf in teractor (Kalmes et al., 1998). CAS/CSE1, protein essential for proper chromosome se gregation in yeast (Brinkmann et al., 1995; Xiao et al., 1993), which localizes to mitotic spindle in somatic cells (Scherf et al., 1 is thought to be a non-traditional substrate for MEK1 (Scherf et al., 1998). HABP1, regulator of intracellular traffic, is a s ubstrate for MAPK (Majumdar et al., 2002). Finally, Epsin1, a protein that regulates endocytosis (Rosse et al., 2003) and replication factor A (Dutta and Stillman, 1992) ar e substrates for Cdk1/cyclin B. Other proteins identified by Mass spectro metry are linked to MAPK signaling. It is proposed that importin 3 beta is involved in the intracellular tr anslocatio ollowing injury of neurons (Perlson et al., 2005). Filamin A, an actin-binding protein, has been implicated in the regulat ion of MAPK activation following stimulat of extracellular receptors (He et al., 2 003; Scott et al., 2006). ATRs nuclear translocation upon DNA damage was shown to be regulated by ERK (Wu et al., 2006). Future studies are necessary to validate associ ation of the identified proteins within the mitotic B-Raf complexes and directly assess th eir involvement in the regulation of B-Raf activity or functions at mitosis.

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149 tic f B-Raf regulation and function. This work provides some insights into the nature o nd es the tions for s. In conclusion, the data presented in th is study demonstrate th at mitotic active BRaf exists in large protein complexes. Identi fication of proteins a ssociated with a large molecular weight complex with B-Raf during Mphase will aid in deciphering the mito mechanisms o f B-Raf protein-protei n interactions that may cont ribute to B-Raf signaling during mitosis. I showed that mitotic B-Raf direc tly associates with C dk1/cyclin B, MEK a MAPK. Additionally, a proteomics approach wa s utilized to identify other candidate BRaf interactors at mitosis. Over 25 prot eins, which are structural or regulatory components of the cytoskeleton and cellular tr afficking or implicated in mitosis, DNA metabolism or regulation of prot ein stability, were identified. Some of these proteins have been already linked to MAPK or Cdk1/cy clin B signaling. This fact encourag use of the candidate protein list as a good st arting point for future investigations that elucidate the regulation and functions of the mitotic B-Raf complexes. Future research directions Identification of B-Raf as a mitotic ME K kinase is a novel finding that opens a broad spectrum of new research interests. I propose three main research direc future studies of B-Raf signaling at mitosis. First is the elucid ation of biochemical mechanisms that regulate mitotic B-Raf. Second is defining B-Rafs roles during mitosi And third is investigating the eff ects of oncogenic B-Raf at mitosis.

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150 lished by ons APK phosphorylations oppositely affect activity of mi totic B-Raf, it will be interesting to understand how these two pho nated throughout mitosis. To address this, phospho-specific antibodies need to be prepared and then used to analyze a timeatory phospho-sites will be important to study. This can be done by analyzing B-Raf activity and There are several aspects of B-Raf regulation at mitosis that can be addressed in future studies. In this di ssertation I show that phosphorylation is the major mode of regulation of mitotic B-Raf. Further studi es are required to co mpletely decipher how phosphorylation regulates B-Raf at M-phase. Specifically, it will be interesting to elucidate the mechanism by which phosphoryla tion of the Cdk1/cyclin B phosphorylation site (Serine-151 residue in Human B-Raf) contributes to activa tion of B-Raf, as well as a mechanism of inhibitory phos phorylation at the C-terminal MAPK phosphorylation sites (Serine-750and Threonine 753 residues in Human B-Raf). This can be accomp analyzing activities of corresponding phospho-mimicking and non-phosphorylatable mutants as well as their associations with re gulatory and effector proteins of the MAPK cascade at mitosis. Analysis of the B-Raf tertiary structure by X -ray crystallography and virtual molecular 3D modeling could provide detailed information how phosphorylati of these sites affect B-Rafs conformation. Since Cdk1/cyclin B and M sphorylation events are coordi course of B-Raf phosphor ylation throughout mitosis. Furthermore, it needs to be addressed whether mitotic B-Raf undergoes regu latory phosphorylation at other sites. A broad analysis of B-Raf phospho-modi fications can be performed by phosphomicrosequencing of B-Raf i mmunopurified from mitotic Xenopus egg extracts. Finally, on the flip side, the phosphatases that dephos phorylate B-Rafs re gul

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151 phosph er an be y from t the re this orylation during mitosis following treatmen t with specific phos phatase inhibitors or in in vitro phosphatase assays. Analysis of the protein complexes that fo rm with B-Raf during mitosis is anoth important area of biochemical studies. The pr oteomics data provided in this study c used as a good starting point for this re search. Co-immunoprecipitation studies and biochemical purification approach es could be used to validate interaction of the identified proteins with B-Raf at mitosis. Validation of particular B-Raf m itotic interactors will refine and focus future studies of B-Ra f regulation and functions at mitosis. B-Raf is known to undergo alternative sp licing, which can modulate its activit (Papin et al., 1998). My data indicate that a small 68 kDa isoform of B-Raf is phosphorylated within the activat ion loop in M-phase arrested extracts, indicating that the 68 kDa B-Raf may function during mitosis along with the 95 kDa B-Raf studied in this work. Interestingly, my data showed th at immunodepletion of 95 kDa B-Raf Xenopus egg extracts does not affect the levels of 68 kDa (not shown) suggesting tha smaller B-Raf isoform is not required for ac tivation of the MAPK cascade at mitosis. However, the contribution of 68 kDa B-Raf in MAPK activation in mitotic extracts was not addressed directly. Thus it still needs to be dete rmined whether 68 kDa B-Raf regulates MAPK-independent functions at m itosis or cooperates with 95 kDa B-Raf in the regulation of mitotic MAPK signaling. It would be very interesting to explo possibility further in future studies by rais ing antibodies that can immunoprecipitate the smaller B-Raf isoform.

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152 tern analysis I have reproducibly observed an enrichm n is -box sequen af dissertati on research suggest that B-Raf can be activated twice throughout the cell cycle: at the G1/S fo llowing mitogenic stimulation and during It needs to be addressed whether B-Raf protein levels are regu lated in an M-phase dependent manner. By usi ng Wes ent of B-Raf during mitosis and decrease of its levels at th e subsequent S-phase (Fig. 20B, 25B, 37B). This implies that B-Ra f protein levels may be up-regulated during M-phase and down-regulated at the mitotic exit The possibility that B-Raf expressio regulated in an M-phase depende nt manner needs to be addressed directly in the future studies. Studying stability of B-Raf throughout the cell cycle will be used to elucidate this question. However, B-Raf protei n does not possess classical Dor KEN ces for ubiquitination, which targets m itotic proteins for proteasome-directed degradation (Pfleger and Ki rschner, 2000). Thus, it is unlikely that mitotic B-R undergoes APC-directed proteolysis. Recent work revealed that B-Raf localizes to mitotic structures, such as the mitotic spindle, centrosomes, condensed chromatin, kinetochores and midbody (Borysova MK, Guadagno TM, 2006, manuscrip t submitted). Proper intracellular localization of B-Raf can play an important role in ensuring specificity of B-Rafs regulation and functions during mitosis. Thus, studying biochemical mechanisms that control intracellular distribution of mito tic B-Raf will contribute to understanding regulation of B-Raf signali ng. To study this, the domains necessary for B-Rafs localization to certain sub-cellular structur es need to be defined. This can be accomplished by analyzing sub-cellular localizati on of different B-Raf truncated mutants The results of my

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153 mitosis nisms ein s of -Raf s. sis are K, nism t ormation the mit d to To generate a comprehensive picture of B-Rafs regulation during cell cycle, it would be interesting to elucidate how thes e two different B-Raf regulatory mecha are coordinated. This can be accomplished by studying a time-course of B-Rafs phosphorylation at the Cdk1/cyclin B-depende nt and Ras-dependent residues throughout the cell cycle in a synchronized population of tissue culture cells. Therefore, several aspects of B-Raf re gulation during mitosis require further study. These include further exploration of phosphorylation and protein complex formation as modes of regulation of mitotic B-Raf, elucidation whether B-Raf prot levels are regulated in an Mphase dependent manner and whet her alternative isoform B-Raf are involved at mitosis and, finally, th e characterizatio n of mechanisms of B intracellular localization. The second direction for future research is studying B-Rafs mitotic function First of all, it should be addressed whethe r the known MAPKs func tions at mito mediated by B-Raf. A recent study demonstrat ed that B-Raf localizes to the mitotic apparatus and is required for proper spindle formation in somatic cells (Borysova M Guadagno TM, 2006, manuscript submitted), a mitotic role which already has been defined for MAPK (Horne and Guadagno, 2003). To further understand the mecha by which B-Raf regulates the mitotic spindl e, it will be necessary to identify wha substrates are targeted by the B-Raf/MEK/MAPK cascade to promote proper f of the mitotic spindle. Am ong other MAPK functions that may be mediated by B-Raf are otic spindle checkpoint, regulation of M-phase entry, and Golgi fragmentation. BRaf loss-of-function and gain-of-function studie s will reveal whether B-Raf is linke

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154 ing n of B-Rafs mitotic functio t ati on of B-Raf mitotic functions may result from expression of the B s n some of the abovementioned mitotic events. There is a possibility as well that B-Raf may have mitotic roles, which are not dependent on MAPK. Tissue cell lines and the cell-free system of Xenopus egg extracts could be utilized to explore B-Rafs functions dur mitosis. The third research direction is to study whether disregulatio ns can provoke tumorigenesis. Recen tly B-Raf was recognized as a prominen oncogene, which is mutated and activated in a large proportion of human cancers, particularly, melanoma (Davie s et al., 2002). Since I have shown that B-Raf activates MAPK signaling at mitosis and is implicated in the regulation of the mitotic spindle in somatic cells (Borysova MK, Guadagno TM, 2006, manuscript submitted), it is plausible to hypothesize that disregul -Raf oncogene. This may lead to improper chromosome segregation and genomic instability, thereby provoking tumorogene sis. Analysis of mitotic abnormalitie and the rate of accumulated chromosomal ab errations in somatic cell lines ectopically expressing the oncogenic form of B-Raf will clarify its role in tumorigenesis. In conclusion, my dissertation research describes B-Raf as the mitotic MEK kinase critical for activation of the MAPK cascade. I demonstrate that B-Raf activation at mitosis is coupled to the mitotic regulat ory machinery, namely Cdk1/cyclin B. I addition, recent studies have demonstrated a role for B-Raf in regulating the mitotic spindle in human somatic cells. Therefore, I propose that B-Raf is an essential regulator of mitosis. Future research is necessary to define and characterize B-Rafs regulation and functions at mitosis in normal cells and in cancer.

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155 Chapter Eight S-phase arrested Xenopus egg extracts were prepared essentially as described (Chen et al., 1998). Briefly, mature Xenopus oocytes were dejellie d in 2% L-cysteine solution (pH 7.8), washed in MMR buffer (5 mM HEPES, pH 7.8, 100 mM NaCl, 2 mM KCl, 1 mM MgCl2, 2 mM CaCl2, 0.1 mM EDTA) and parthenogene tically activated with the Ca ionophore A23187 (0.2 g/ml) for 2.5 minutes. After Ca ionophore withdrawal, the activated eggs were incubated in egg lysis buffer (10 mM HEPES, pH 7.8, 250 mM sucrose, 50 mM KCl, 2.5 mM MgCl2, 1 mM DTT, 50 g/ml cyclohexamide) for another 50-60 min at r oom temperature before processing into extracts by a high-speed centrifugation (10, 000 g 15 min at 4C). This method closely mimics the degradation of c-Mos, a meiosi s specific MEK kinase, which is observed following fertilization (Chen et al., 1998; Nishizawa et al., 1993; Watanabe et al., 1991; Watanabe et al., 1989). M-phase arrested ex tracts were prepared by supplementing crude S-phase egg extracts with recomb inant non-degradable sea urchin 90 cyclin B1 (75-100 nM final) and incubating at room temperat ure for 60 min. Expressi on and purification of GST90 cyclin B was performed as described (Horne and Guadagno, 2003). Note that Materials and Methods Preparation of Xenopus egg extracts 2+ 2+

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156 all preparations of M-phase extracts were checked for the activation of MAPK and the absence of c-Mos protein by immunoblot analysis. Cycling extracts were prepared in a pro cedure similar to described above, except that eggs activated with the calcium 87 were incubated in XB buffer (10 mM HEPES, pH 7.7, 50 mM su MgCl2, 0.1 mM CaCl2) for 45 min before processing them into extracts Cytostatic factor (CSF) arrested Xenopus egg extracts were prepared as described (Murray, 1991). Studying c-Mos degradation in fertilized and Ca2+ ionophore activated Xenopus oocytes Mature Xenopus oocytes were fertilized with sper ms freshly isolated from male Xenopus frogs (Heasman et al., 1991) or activated with Ca2+ ionophore A23187 as mentioned above. Fertilized and Ca2+ ionophore activated eggs we re incubated at room temperature in 0.25X MMR solution. (1.25 mM HEPES, pH 7.8, 25 mM NaCl, 0.5 mM KCl, 0.25 mM MgCl, 0.5 mM CaCl 0.025 mM EDTA). 10 eggs were collected at 10minute intervals and frozen on dry ice. Fro zen eggs were thawed on ice and resuspended in 100 l of extraction buffer (20 mM HEPES, pH 7.2, 0.25 M sucrose, 0.1 M NaCl, 50 mM -glycerol phosphate, 2.5 mM MgCl 1 mM Na VO 25 mM NaF) supplemented with 10 g/ml of each leupeptin, pepstatin, and ap rotinin. Lysates were subjected to centrifugation for 12 min at 4oC in an eppendorf centrifuge. The levels of c-Mos protein in the clarified extracts were visualized by Western analysis with c-Mos antibodies. i onophore A231 crose, 100 mM KCl, 1 mM 2 2 2 34

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157 clonal anti-phospho (T202/Y204) ERK (1 was purchased from Calbiochem, rabbit anti-MEK (1:1000) was prepared by Zymed Laboratories (South San Francisco, 1:1000) were provided by Jim Ferrel (S tanford University) and rabbit anti-cyclin B were ated mM ammonium sulfate to reach fina l 20% saturation. Samples we re rotated for 2.0 hrs at 4C Immunoblot analysis Primary antibodies used include: mouse mono :2000), rabbit polyclonal anti-phos pho (S217/S221) MEK (1:1000), and mouse monoclonal myc-tag (1:1000) were purchased from Cell Signaling; rabbit polyclonal anti-B-Raf (sc9002; 0.02 g/ml), anti-Raf-1 (0.4 g/ml), anti-c-Mos (0.4 g/ml), antiERK2 (0.2 g/ml) and anti-phospho (T599/S602) B-Raf (0.2 g/ml) were purchased from Santa Cruz; mouse monoclonal anti-Cdk1 (0.2 g/ml) CA) against an N-terminal 16 amino acid sequence of Xenopus MEK1, rabbit antiMAPK ( provided by James Maller (University of Colorado). Secondary antibodies included species-sp ecific alkaline phosphatase-conjug anti-mouse (Jackson ImmunoResearch Laboratories) and anti-rabbit (Sigma) IgG that were detected with the CDP-Star chemiluminescence substrate (Roche Diagnostic). Purification of mitotic MEK kinase activity from Xenopus egg extracts Crude M-phase arrested extracts prepared as described above were ultracentrifugated twice at 100,000 g for 1.5 hr to isolate cytosolic fraction. Mitotic cytosol fraction was diluted in ice-cold e gg lysis buffer (10 mM HEPES, pH 7.8, 250 sucrose, 50 mM KCl, 2.5 mM MgCl 2 1 mM DTT) supplemented with 10 g/ml of each leupeptin, pepstatin, and aprotinin and mixe d 3:2 with the same buffer containing 50%

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158 and precipitated proteins were pelleted by a high-speed centrifugation (10, 000g 15 min at 4C) ) Ca stepl) were collected and analyze ed f kinase The pellet was resuspended in buffer A (50 mM HEPES, pH 7.5, 10 mM MgCl 2supplemented with 25 mM NaF and 1 mM Na 3 VO 4 and 10 g/ml of each pepstatin, leupeptin and chymostatin, and subjected to chromatography separation by using FPL AKTA (Amersham Biosciences). First, the pellet was applied to a 10-ml HiTrap Q Sepharose HP column (Amersham Biosciences). The proteins were eluted with a stepwise NaCl gradient in buffer A: 0-0.3 M ( 40 ml), 0.3-1 M (10 ml). Fractions that contained MEK kinase activity (0.17-0.25 M NaCl ) were pooled together and applied to Mono Q HR 5/5 column (Amersham Biosciences). The proteins were eluted with a wise NaCl gradient in buffer A: 0-0.35 M (3 ml), 0.35 0.50 M (15 ml), 0.5-1 M (3 ml). Fractions containing MEK kinase activity (0.37-0.42 M NaC d for protein compositi on by Western blotting and Si lver Staining with GelCode SilverSNAP Stain Kit (Pierce). All pu rification steps were performed at 4 o C and assay for MEK kinase activity. MEK kinase assays To measure MEK kinase activity, samples were incubated in 30 l o buffer (50 mM HEPES, pH 7.5, 10 mM MgCl 2 0.1 mM ATP, 25 mM NaF, 1 mM Na 3 VO 4 1 mM DTT) containing 0.5 g of recombinant unactiv e GST-MEK1 (Upstate) for 20 min at 25 o C. The kinase reaction was stop ped with SDS sample buffer and reaction products were separated by SDS-PA GE. Phosphorylation of recombinant GST

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159 .1 mM 0 l DS-PAGE, and transf erred to a PVDF membrane. The levels of MBP phosphorylation was visualized by autoradiography and quantified by using ImageQ protein (Upstate) and 2 Ci 32P ATP for 20 min at 30C. Reactions were stopped by the MEK1 at serine residues 217 and 221 was analyzed by immunoblotting with phosphoS217/S221 MEK antibodies (Cell Signaling). Alternatively, MEK kinase ac tivity was measured in an in vitro linked kinase assay as described (Guan et al ., 2000). Briefly, aliquots of purified fractions or protein A beads immunocomplexes were incubated with 1.0 g of recombinant unactive GSTMEK1 (Upstate) in 25 l of reaction buffer (25 mM HEPES, pH 7.5, 10 mM MgCl 2 25 mM -glycerophosphate, 5 mM EGTA, 1 mM DTT, 5 mM NaF, 1 mM Na 3 VO 4 0 ATP) for 20 min at room temperature. Th e reaction mix was briefly centrifugated to pellet immunocomplexes. Next, 20 l of the supernatant was mixed with 10 l of the reaction buffer containing 9.0 g of recombinant unactive GST-ERK and reaction was continued for another 15 min. Finally, 3 l aliquot of the reac tion was mixed with 3 of reaction buffer containing 50 g of MPB and 5 Ci of 32 P -ATP and incubated for another 10 min at room temperature. The kinase reaction was stopped with SDS sample buffer, separated on 15% S uant software. In vitro histone H1 kinase assay To measure Cdk1/cyclin B activity, 1 l aliquots of Xenopus egg extracts were incubated in 50 l of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 1 mM EGTA, 2 mM DTT, 0.1 mM ATP, 10 M PKI, 0.01% Brij 35) containing 20 g H1

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160 io n levels of histone H1 were determined by auto recipitations were performed by adding 4 g of rabbit polyclonal B-Raf antibodies (Santa Cruz) to 10 l of Xenopus egg extracts diluted in 40 l of buffer A (50 m l2, 25 mM NaF, 1 mM Na3VO4) supplemented with 10 anta addition of SDS sample buffer and heating at 95C for 5 min. Reaction products were separated by 10% SDS PAGE and phosphorylat radiography. Immunodepletion To immunodeplete B-Raf fr om S-phase extracts, 50 l of extracts were incubated under gently rotation for 1-1.5 hr at 4C with 15 g of rabbit polyclonal B-Raf antibodies (Santa Cruz) pre-bound to 5 l of protein A sepharose 4B Fast Flow beads (Sigma). B Raf immuno-complexes were pelleted by a quick centrifugation, and a second round of immunodepletion was performed. As a control, extracts were mock depleted in parallel using an equivalent amount of rabbit IgG (Sigma). In a similar procedure, Raf-1 was immunodepleted from S-phase extracts with 5 g of rabbit polyclonal Raf-1 antibodies (Santa Cruz). Immunoprecipitation B-Raf immunop M HEPES, pH 7.5, 10 mM MgC g/ml of each pepstatin, leupeptin and chymostatin. To immunoprecipitate cMos-associated MEK kinase activity from M-phase arrested extracts, 5 g of either S Cruz c-Mos antibodies or Abcam cMos antibodies were added to 10 l of extracts diluted in 40 l of buffer A. Following a 2 hr incu bation on ice, immunocomplexes were

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161 directly in in vitro linked kinase or pho sphatase assays or resuspended in SDS sample buffer and analyzed by immunoblotting. chymostatin. The immune comp lexes were recovered on protein A agarose beads (Sigma) and washed three times with EB buffer containing 0.1% Triton X-100. 03 nd recovered on protein A agarose beads (Sigma ), washed twice with the same buffer containing 0.1% Triton X-100, and 3 times with buffer A alone. Washed B-Raf immune complexes were used Co-immunoprecipitation analysis For co-immunoprecip itation studies 20 l of Xenopus egg extracts were mixed with 6.5 g of anti-B-Raf (Santa Cruz), 6.5 g of anti-Raf-1 (SantaCruz), 4 g of antiMEK (customer-designed, Zymed Laboratories), 4 g of anti-MAPK, 4 g of anti-Cdk1 (Calbiochem) or 6 g of anti-cyclin B1. Extracts were diluted 1:1 in EB buffer (80 mM -glycerol phosphate, pH 7.3, 15 mM MgCl 2 20 mM EGTA, 25 mM NaF, 1 mM Na 3 VO 4 ) supplemented with 0.1% Triton X-100 and 10 g/ml of each pepstatin, leupeptin and Generation of wild-type and mut ant Xenopus B-Raf constructs A Xenopus B-Raf cDNA clone was obtained from ATCC (Image Clone I.D. 6860469). DNA sequencing analysis, performed at H. Lee Moffitt Cancer Center DNA facility, confirmed a full-length Xenopus cDNA containing an open reading frame of 8 amino acids. The sequence data of Xenopus B-Raf has been submitted to the Genbank database under accession N o DQ097958. The B-raf coding re gion was excised at Hi III sites and sub-cloned into a modified pGEM transcription vector (kindly provided by

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162 ycprepared and verified by DNA sequencing (H. Lee Moffitt Cancer Center DNA fac ility): Ser-144-Ala, Ser-329-Ala, Ser-144-Ala/Ser-329-Ala, Ser784-Ala/Thr-787-Ala, Lys-517-Met, Lys517-M y as f CSF-arrested egg extracts supplemented with 2.5 l of rabbit reticulocyte lysate (Ambio tle mixing every 15 min in Dr. Rey-Huei Chen from the University of Cornell) down-stream of an N-terminal m tag. Mutant constructs were generated by using the QuikChange site-directed mutagenesis kit (Stratagene). The followi ng myc-B-Raf mutants were et/Ser-144-Ala, Lys-517-Met/Ser-329-Ala, Lys-517-Met/Ser-144-Ala/Ser-329Ala. Expression of recombinant myc-B-Raf proteins in CSF Xenopus egg extracts Expression of wild-type and mutant B-Ra f proteins was performed similarl described (Sharp-Baker and Chen, 2001). Br iefly, wild type and mutant myc-B-ra transcripts were generated using the mMESSAGE mMACHI NE T7 transcription kit (Ambion). 4 g of purified mRNA transcripts were introduced into 21 l of Xenopus n). Protein expression was carried out at 23C for 5 hr with gen Expression of recombinant proteins was confirmed by Western blotting with myc epitope antibodies. In order to reproduce the S-phase phosphoryl ation status of the recombinant B-Raf proteins, aliquots of tran slated WT or mutant myc-B-Raf proteins were introduced into S-phase arrested extrac ts in a 1:20 ratio and incubated for 30-60 m at room temperature. To induce M-phase phosphorylation of the recombinant proteins, aliquots of S-phase extracts containing myc-B-Raf protein we re driven into M-phase by

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163 binant myc-B-Raf proteins undergo dephosphorylation during incubation in Sphase e tracts. in r (25 mM HEPES, pH 7.5, 150 mM NaCl, 25 mM -glycerophosphate, 10 mM MgCl2, 10% glycerol, 5 mM EG TA, 1 mM DTT, 1 mM Na2VO4, 5 mM NaF, 0.1% Triton ed on ice w h and ing to in d adding recombinant non-degradable cyclin B. Western blot analys is revealed that recom xtracts and acquire hyperphosphorylati on during incubation in M-phase ex Purification of recombinant myc-B-Ra f proteins from Xenopus egg extracts Myc-B-Raf recombinant proteins were purified by immunoprecipitation using myc-tag antibodies. Aliquots of egg extracts containing myc-B-Raf were diluted 1:20 buffe X-100 and 10 g/ml of each pepstatin, leupeptin and chymostatin) and incubat ith myc-tag monoclonal antibodies (Cell Signaling) for 2 hrs. Protein A Sepharose beads were added and incubation was continued for anot her 12-16 hrs wit gentle inversion at 4 C. Immuno-complexes were washed twice with the same buffer twice with the reaction buffer (25 mM HEPES, pH 7.5, 25 mM -glycerophosphate, 10 mM MgCl 2 5 mM EGTA, 1 mM DTT, 1 mM Na 2 VO 4 5 mM NaF) before apply vitro kinase assays. Phosphatase Treatment B-Raf immunoprecipitates from Sand M-pha se arrested extracts were incubate with 50 units of recombinant lambda prot ein phosphatase (Upsta te Biotech) in 50 l of phosphatase buffer (50 mM HEPES, pH 7.5, 0.1 % BSA, 100 M EDTA, 2 mM MnCl 2 and 5 mM DTT) for 30 min at 37C. To st op reactions, precipitates were washed with

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164 7.5, vitro Cdk1/cyclin B kinase assay clin B (New of ed by the addition of SDS sample buffer and heating at 950C for 5 min. copious amount of the buffer containing phospha tase inhibitors (50 mM HEPES, pH 10 mM MgCl 2 25 mM NaF, 1 mM Na 3 VO 4 ). In Myc-B-Raf IPs were incubated with 100 un its of recombinant active Cdk1/cy England Biolabs) in 30 l of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 1 mM EGTA, 2 mM DTT, 0.1 mM AT P, 0.01% Brij 35) with radioactive 32 P ATP (5 Ci/reaction) for 30 min at 30C. Reactions were stopped by the addition SDS sample buffer and heating at 95C for 5 min. In vitro ERK2 kinase assay Endogenous B-Raf or myc-BRaf immunoprecipitates were incubated with 20 units of recombinant active ERK2 (New England Biolabs) in 30 l of kinase buffer (50 mM Tris-HCl, pH 7.5, 10 mM MgCl 2 1 mM EGTA, 2 mM DTT, 0.1 mM ATP, 0.01% Brij 35) with or without radioactive 32 P ATP (5 Ci/reaction) for 30 min at 30 0 C. Reactions were stopp Native gel electrophoresis and transfer Samples were run in a 4-25% gradient ge l (BioRad) in SDS-free buffer (250 mM Tris, 200 mM glycine) for 12-14 hrs at 4C. Transferring of separated proteins to PVDF

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165 S, pH 00 l. membrane was performed in the transfer buffer supplemented with 0.5% SDS (20 mM Tris, 150 mM glycine, 0.5% SDS). Gel filtration Size exclusion chromatography was perfor med at 4C using Superdex 200 HP 10/30 column (bed volume ~24 ml; Amer sham Biosciences) with a FPLC-AKTA (Amersham Biosciences) purification system in the buffer containing 50 mM HEPE 7.5, 10 mM MgCl 2 and 10 g/ml of each pepstatin, leupeptin and chymostatin. The flow rate was 0.25 ml/min and the sample volume was 1

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166 References Abrieu, A., Doree, M. and Fisher, D. (2001) The interplay between cyclin-B-Cdc2 kinase 267. Acharya, U., Mallabiabarrena, A., Acharya, J.K. and Malhotra, V. (1998) Signaling via fragmentation during mitosis. Cell 92 183-192. Smythe, C., Gerloff, D.L. and Earnshaw W.C. (2000) INCENP binds the Aurorachromosomes, the central spindle and cleavage furrow. Curr Biol 10, 1075-1078. Aebersold, D.M., Shaul, Y.D., Yung, Y., Yarom, N., Yao, Z., Hanoch, T. and Seger, R. (2004) Extracellular signal-regulated kinase 1c (ERK1c), a novel 42-kilodalton RK, demonstrates unique modes of regulation, localization, and function. Mol Cell Biol, 24 10000-10015. honen, L.J., Kallio, M.J., Daum, J.R., Bolton, M., Manke, I.A., Yaffe, M.B., Stukenberg, P.T. and Gorbsky, G.J. (2005) Polo-like kinase 1 creates the tensionsensing 3F3/2 phosphoepitope and modul ates the association of spindlecheckpoint proteins at kinetochores. Curr Biol 15 1078-1089. ist, J.R., Bayles, C.J., Tao, W. and Berns, M.W. (1991) Direct experimental evidence r the existence, structural basis and f unction of astral forces during anaphase B in vivo. J Cell Sci 100 ( Pt 2) 279-288. Andrews, P.D., Knatko, E., Moore, W.J. and Swedlow, J.R. (2003) Mitotic mechanics: the auroras come into view. Curr Opin Cell Biol, 15, 672-683. Arias, E.E. and Walter, J.C. (2004) Initiation of DNA replication in xenopus egg extracts. Front Biosci, 9, 3029-3045. Arion, D., Meijer, L., Brizuela, L. and Beac h, D. (1988) cdc2 is a component of the M phase-specific histone H1 kinase: evidence for identity with MPF. Cell 55, 37178. Arnaud, L., Pines, J. and Nigg, E.A. (1998) GFP tagging reveals human Polo-like kinase at the kinetochore/centromere region of mitotic chromosomes. Chromosoma 107, 424-429. Atherton-Fessler, S., Parker, L.L., Geahle n, R.L. and Piwnica-Worms, H. (1993) Mechanisms of p34cdc2 regulation. Mol Cell Biol 13, 1675-1685. (MPF) and MAP kinase duri ng maturation of oocytes. J Cell Sci 114, 257mitogen-activated protein kinase kina se (MEK1) is required for Golgi Adams, R.R., Wheatley, S.P., Gouldsworthy, A.M., Kandels-Lewis, S.E., Carmena, M., related kinase AIRK2 and is required to target it to E A A fo 3 1

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About the Author Sergiy I. Borysov was born in Ukraine. He received a Masters Degree in Biochemistry (1998) from Kiev National Ta ras Shevchenko University. While being a student of this University, Sergiy was a re cipient of the International So ros Support for Educational Sciences Grant (1995 and 1997) and a winner of All-Ukrainian Students Contests in Biology (1996, 1997). Sergiy Borysov entered an Interdisci plinary Ph.D. program in Cellular and Molecular Biology at the University of Sout h Florida in January 2001. In June 2001 he affiliated with the Department of Biochemistry and Molecular Biology. While in the Ph.D. program, Mr. Borysov received an Outs tanding Graduate Student Award from the College of Medicine (2005). Sergiy Borysov was a recipient of the American Heart Association Pre-Doctoral Fello wship (2003-2005). His finding s have been presented at the regional and international scientific mee tings and were publishe d in the Journal of Biological Chemistry in June 2006.


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B-Raf is an essential component of the mitotic machinery critical for activation of MAPK signaling during mitosis in Xenopus egg extracts
h [electronic resource] / by Sergiy I. Borysov.
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[Tampa, Fla] :
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ABSTRACT: Activation of the MAPK cascade during mitosis is critical for spindle assembly and normal mitotic progression. The underlying regulatory mechanisms that control activation of the MEK/MAPK cascade during mitosis are poorly understood. The goal of my dissertation research is to identify the MEK kinase responsible for activation of the MAPK cascade during mitosis and to elucidate the biochemical mechanisms that regulate its activity. In the described herein work I purified and characterized the MEK kinase activity present in M-phase arrested Xenopus egg extracts. I demonstrate that B-Raf is the critical MEK kinase required for activation of the MAPK pathway at mitosis. Consistent with this, I show that B-Raf is activated in an M-phase dependent manner. Further, I provide data linking Cdk1/cyclin B to mitotic activation of B-Raf.^ ^Cdk1/cyclin B associates with and phosphorylates B-Raf in M-phase arrested extracts and directly targets Xenopus B-Raf in vitro at a conserved Ser-144 residue. Phosphorylation at Ser-144 is critical for M-phase dependent activation of B-Raf and for B-Raf's ability to trigger activation of the MAPK cascade at mitosis. Finally, I demonstrate that mitotic B-Raf undergoes feedback phosphorylation by MAPK at its conserved C-terminal SPKTP motif. Mutation of both phosphorylation sites within the SPKTP sequence to alanines increases activity of mitotic B-Raf. Further, inhibition or over-activation of MAPK during mitosis enhances or diminishes B-Raf activity, respectively. These results indicate that MAPK-mediated feedback phosphorylation negatively regulates B-Raf activity. Additionally, I show that active mitotic B-Raf exists in large multi-protein complex(s). By utilizing a proteomics approach I identify a set of proteins, which potentially associate with B-Raf at M-phase.^ ^Future studies are necessary to elucidate the involvement of these proteins in regulating B-Raf mitotic functions. In summary, my dissertation studies demonstrate that B-Raf activates MAPK signaling at mitosis and undergoes an M-phase dependent regulation. I propose that B-Raf has important functions at mitosis that contributes to its overall role in promoting cell proliferation.
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Dissertation (Ph.D.)--University of South Florida, 2006.
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MEK kinase.
extracellular signal regulated kinase.
ERK.
Cdk1/cyclin B.
Cdc2/cyclin B.
M-phase.
Signal transduction.
Cell cycle.
Phosphorylation.
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