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Cloning and characterization of a mammalian mitotic spindle pole associated protein kinase SpaPK Cheng, Xiaoli 2002

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C L O N I N G A N D C H A R A C T E R I Z A T I O N OF A M A M M A L I A N MITOTIC SPINDLE POLE ASSOCIATED PROTEIN K I N A S E SpaPK by Xiaoli Cheng B.Sc., University of Alberta, 1995  A THESIS SUBMITTED IN P A R T I A L F U L F I L M E N T OF THE REQUIREMENTS FOR THE D E G R E E OF DOCTOR OF PHILOSOPHY in THE F A C U L T Y OF G R A D U A T E STUDIES Department of Biochemistry and Molecular Biology  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH C O L U M B I A December 2002 © Xiaoli Cheng, 2002  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or  by  his or  her representatives.  It  is  understood  that  copying  or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department of The University of British Columbia Vancouver, Canada Date  DE-6 (2/88)  Xh,  I™?-  ABSTRACT  Passage through the cell cycle requires accurate replication of the genome and precise segregation of sister chromatids to daughter cells. Many of the proteins involved in cell cycle control are well conserved throughout evolution, from yeast to humans. The search for a human homolog of Saccharomyces cerevisiae Rad53, an important D N A damage and D N A replication checkpoint protein, led to the isolation of a gene encoding a novel protein kinase, designated SpaPK for spindle pole associated protein kinase. SpaPK, a protein of 454 amino acids with a predicted molecular mass of 52.5 kDa, is a serine/threonine protein kinase. The affinity purified recombinant SpaPK exhibits autophosphorylation activity, predominantly towards serine residues^. Northern and Western blotting analyses indicated that SpaPK is expressed in various tissues and cell lines. It is also expressed at all stages of the cell cycle in proliferating cells, and is possibly phosphorylated at mitosis. During the course of this study, the SpaPK gene has been cloned independently by two other research groups and its gene product is designated ZIP kinase. The findings from their functional analyses related to apoptosis, which are different than those from this study, are discussed. In this study, characterization of the functions of SpaPK revealed that it is not a functional homolog of Rad53; instead, it plays a role in mitosis.  Immunofluorescence  studies showed that the subcellular localization of endogenous SpaPK is cell cycle regulated. SpaPK is first detected on duplicated centrosomes after their separation at prophase, remains on the centrosomes and centrosome-proximal part of the spindle until late anaphase, and redistributes to the spindle midzone and midbody as the cells go through cytokinesis. In addition, SpaPK is associated with microtubule organization centers in cells released from a nocodazole block and in taxol-treated mitotic cells. The subcellular localization of SpaPK is sensitive to a number of protein kinase inhibitors, indicating possible upstream regulators of SpaPK. The distinct spatial and temporal localization pattern of SpaPK suggest that this protein kinase may play a role in regulating spindle function during chromosome segregation in mammalian cells. SpaPK may also play a role at the G2/M transition. Flow cytometry analyses showed that overexpression of wild-type or catalytically inactive recombinant SpaPK led to alteration  11  of cell cycle progression with an accumulation of cells in G2.  Assays using Xenopus  interphase extract showed that addition of purified wild-type or catalytically inactive recombinant SpaPK delays the onset of mitosis.  SpaPK inhibits mitosis most likely by  targeting Xenopus Cdc25C at Ser-287 whose phosphorylation is known to inhibit the activity of Cdc25C, thereby preventing the activation of Cdkl/cyclin B complex required for mitotic entry. Taken together, these data suggest that SpaPK functions as a regulator of mitotic spindle function during chromosome segregation and as a coordinator of mitotic entry at the G / M transition. 2  iii  TABLE OF CONTENTS Page ABSTRACT  ii  TABLE OF CONTENTS  i  LIST OF FIGURES  v  xi  LIST OF TABLES  xii  LIST OF ABBREVIATIONS  xiv  ACKNOWLEDGEMENTS  xvii  CHAPTER 1 INTRODUCTION 1.1  The cell cycle  1  1.2  Cyclin dependent kinases and their regulation  5  1.2.1 1.2.2 1.2.3 1.2.4 1.2.5 1.2.6 1.3  Cyclin dependent kinases The role of cyclins The role of phosphorylation TheroleofCDKIs The role of proteolysis The role of subcellular localization  Major cell cycle transitions 1.3.1 G]/S transition 1.3.1.1 Gi/S promoting pathways 1.3.1.2 Cdk2-cyclin E: convergence of Gi/S promoting pathways 1.3.2 G / M transition  12 12 12 13 15  Cell cycle checkpoints 1.4.1 General organization of the checkpoint response pathway. 1.4.2 D N A damage checkpoint 1.4.2.1 D N A damage sensors 1.4.2.2 Transduction of D N A damage signal 1.4.2.2.A Cell cycle arrest at the Gi/S transition 1.4.2.2.B Cell cycle arrest at the metaphase to anaphase transition 1.4.3 D N A replication checkpoint 1.4.3.1 D N A damage sensors  16 18 19 19 19 22  2  1.4  5 5 8 9 10 11  iv  22 23 23  1.4.3.2  1.5  Transduction of unreplicated D N A repair 1.4.3.2.A Delay of D N A replication 1.4.3.2.B Coupling of S phase and mitosis  Experimental rationale  24 24 24 26  CHAPTER 2 CLONING OF THE SpaPK GENE 2.1  INTRODUCTION  27  2.2  MATERIALS AND METHODS 2.2.1 Chemical, enzymes and plasmids 2.2.2 Bacterial strains 2.2.3 Molecular and biological methods 2.2.3.1 Purification of plasmid D N A from E. coli 2.2.3.2 Polymerase chain reaction (PCR) 2.2.3.3 Ligations 2.2.3.4 Transformation of E. coli 2.2.3.5 Preparation of radioactive D N A probes 2.2.3.5.A Random hexamer extension labeling 2.2.3.5. B 5'end labeling 2.2.3.6 Screening of cDNA library 2.2.3.6. A cDNA cloning of SpaPK 2.2.3.6.B Primer extension 2.2.3.6.C Isolation of 5' end of SpaPK from Marathonready™ c D N A library 2.2.3.7 • Isolation of SpaPK from genomic library 2.2.3.8 Large-scale isolation of A. phage D N A 2.2.3.9 Isolation of total R N A from tissue culture cells 2.2.3.10 Northern blot analysis 2.2.3.11 Southern blot analysis 2.2.3.12 Double-stranded D N A sequencing  28 28 28 28 28 29 29 29 30 30 30 30 30 31  2.3  32 32 33 33 33 34 34  RESULTS 35 2.3.1 Identification of human homolog of S. cerevisiae RAD53 -SpaPK 35 2.3.2 Assembly of the composite SpaPK cDNA sequence 35 2.3.3 General overview of the SpaPK cDNA sequence 35 2.3.4 Tissue distribution of the SpaPK gene 39 2.3.5 Analysis of the 5' region of the SpaPK gene 39 2.3.5.1 Primer extension analysis of the SpaPK transcript 41 2.3.5.2 5'-RACE PCR of the SpaPK c D N A 41 2.3.5.3 Genomic structure of the SpaPK gene 44 2.3.5.4 Identification of the promoter region and 5' end the SpaPK gene.50  2.4  DISCUSSION 2.4.1 Evidence for an incomplete SpaPK c D N A 2.4.1.1 The size of SpaPK transcript is longer than the isolated clone.... 2.4.1.2 Absence of an upstream in-frame stop codon within the 5' U T R of the SpaPK cDNA 2.4.1.3 The sequence surrounding initiation A T G is not an optimal Kozak 2.4.1.4 5' U T R of the SpaPK cDNA is interrupted by genomic sequences 2.4.1.5 Primer extension analysis 2.4.1.6 5 ' R A C E  55 55 55 55 55 56 57 57  2.4.2  Promoter region of the SpaPK gene 2.4.2.1 T A T A box-containing promoters 2.4.2.2 TATA-less promoters  57 57 58  2.4.3  Expression of the SpaPK gene 2.4.3.1 SpaPK is expressed in a wide variety of tissues and cell lines 2.4.3.2 SpaPK gene is not alternatively spliced in brain, breast, and endothelial cells 2.4.3.3 Is SpaPK gene spliced differently in other tissues?  59 59 59 59  Other genes on chromosome 19pl3.3 and relations to disease  60  2.4.4  CHAPTER 3 ANALYSES OF SpaPK 3.1  INTRODUCTION  62  3.2  MATERIALS AND METHODS 64 3.2.1 Chemicals, enzymes, plasmids and bacterial strains 64 3.2.2 Molecular biological methods 64 3.2.2.1 Purification of plasmid D N A , PCR, ligation and transformation ofE. coli 64 3.2.2.2 Site-directed mutagenesis 64 3.2.2.3 Construction of recombinant wild-type and catalytically inactive His-SpaPK proteins for overexpression in E.coli 64 3.2.3 Purification of recombinant His-SpaPK proteins from E.coli 65 3.2.3.1 Overexpression of wild-type His-SpaPK and catalytically inactive His-K42A-SpaPK from E.coli 65 3.2.3.2 Large-scale purification of recombinant His-SpaPK proteins from E. coli 65 3.2.3.3 Circular dichroism spectroscopy 66  vi  3.2.4  3.2.5  3.2.6 3.2.7  Analysis of purified His-SpaPK 3.2.4.1 His-SpaPK kinase assay 3.2.4.2 Phosphoaminoacid analysis 3.2.4.3 Screening of peptide substrate for His-SpaPK phosphorylation Production of affinity purified anti-SpaPK polyclonal antibodies 3.2.5.1 Production of anti-SpaPK antiserum 3.2.5.2 Affinity purification of anti-SpaPK polyclonal antibodies Extraction of protein from mammalian cells. Western blot analysis  RESULTS  3.3.1  3.3.2 3.3.3  3.3.4 3.3.5 3.3.6 3.3.7 3.3.8  3.4.2 3.4.3 3.4.4  67 68 68 68 68 69 70  Sequence 3.3.1.1 3.3.1.2 3.3.1.3  analysis of SpaPK 70 Analysis of primary structure of SpaPK 70 Putative domain structures and motifs of SpaPK 70 Putative phosphorylation and other post-translational modification sites of SpaPK 73 3.3.1.4 Comparison of amino acid sequence of SpaPK with other proteins 75 Construction of catalytically inactive SpaPK by site-directed mutagenesis... 82 Expression and purification of recombinant His-SpaPK proteins 82 3.3.3.1 Overexpression of His-SpaPK and His-K42A-SpaPK in E.coli. ..82 3.3.3.2 Renaturation of His-SpaPK and His-K42 A-SpaPK on the nickel affinity column 83 3.3.3.3 Purification of His-SpaPK and His-K42A-SpaPK under native conditions 83 Circular dichroism analysis of the purified recombinant His-SpaPK proteins 86 Autophosphorylation activities of wild-type His-SpaPK and catalytically inactive His-SpaPK 88 Phosphoaminoacid analysis of autophosphorylated His-SpaPK 88 Identification of potential phosphorylation sequence of His-SpaPK 91 Detection of endogenous SpaPK with anti-SpaPK polyclonal antibodies 91 3.3.8.1 Generation of anti- SpaPK polyclonal antibodies 91 3.3.8.2 Affinity purification of SpaPK antiserum 94 3.3.8.3 Detection of the endogenous SpaPK protein from mammalian cell extracts 94  DISCUSSION  3.4.1  67 67 67  97  Evidence for an incomplete SpaPK 97 3.4.1.1 The size of endogenous SpaPK 97 3.4.1.2 Presence of a nuclear export signal in the 5' U T R of the SpaPK cDNA 97 SpaPK is a serine/threonine-specific kinase 98 Substrate specificity of SpaPK 100 Putative phosphorylation sites on SpaPK 102  vii  3.4.5  3.4.6  Other post-translational modifications and motifs of SpaPK protein 3.4.5.1 The putative prenylation site of SpaPK 3.4.5.2 The putative leucine zipper of SpaPK 3.4.5.3 Nuclear localization signal Homology comparisons with other protein kinases  103 103 104 105 105  CHAPTER 4 CHARACTERIZATION OF THE FUNCTIONS OF SpaPK 4.1  INTRODUCTION 4.1.1 Main events of mitosis and cytokinesis 4.1.2 Microtubules: a brief introduction 4.1.3 Microtubule-associated motor proteins 4.1.3.1 Dynein superfamily 4.1.3.2 Kinesin superfamily 4.1.4 Centrosome 4.1.5 Mitotic kinases 4.1.5.1 Cdkl/cyclinB 4.1.5.2 Polo-like kinases 4.1.5.3 Aurora family kinases 4.1.5.4 N I M A family kinases 4.1.6 Spindle assembly and chromosome movements 4.1.6.1 Spindle assembly 4.1.6.2 Chromosome movements 4.1.7 Anaphase onset and mitotic exit 4.1.8 Spindle assembly checkpoint 4.1.8.1 Spindle assembly checkpoint proteins 4.1.8.2 Sensor of spindle assembly checkpoint 4.1.8.3 Interaction between spindle checkpoint proteins. 4.1.8.4 Current model of spindle assembly checkpoint  4.2  MATERIALS AND METHODS 128 4.2.1 Chemicals, enzymes, plasmids and bacterial strains 128 4.2.2 Yeast strains 128 4.2.3 Molecular biological methods 128 4.2.3.1 Construction of HA-SpaPK for overexpression in yeast 128 4.2.3.2 Construction of recombinant wild-type and catalytically inactive hHis-SpaPK proteins for overexpression in mammalian cells 129 4.2.3.3 Transformation of yeast 129 4.2.4 Culturing of yeast cells 129 4.2.5 Extraction of proteins from yeast 130 4.2.6 Yeast complementation test 130 4.2.7 In vitro phosphorylation of Xenopus Cdc25C fragments by His-SpaPK 130  viii  107 107 108 109 110 110 112 113 113 113 115 117 118 119 120 122 123 123 124 125 126  4.2.8 4.2.9 4.2.10 4.2.11 4.2.12 4.2.13 4.2.14 4.2.15  Mammalian cell culture Drug treatments of mammalian cells Mitotic spread of mammalian cells Cell cycle synchronization of mammalian cells Immunofluorescence microscopy Transient transfection of mammalian cells Flow cytometry Activation of Cdc2 in interphase Xenopus extract by recombinant cyclin B 4.2.16 Farwestern blot  131 131 131 132 132 133 133 134 135  RESULTS 136 4.3.1 Complementation of mec2-l yeast mutant 136 4.3.1.1 Overexpression of HA-SpaPK protein in S. cerevisiae 136 4.3.1.2 Complementation assay 136 4.3.2 Cell cycle profile of SpaPK expression 137 4.3.2.1 Synchronization of HeLa cells and MCF-7 cells 137 4.3.2.2 Gene expression of SpaPK during the cell cycle 141 4.3.2.3 Protein expression of SpaPK during the cell cycle 141 4.3.3 Subcellular localization of SpaPK 144 4.3.3.1 Subcellular localization of SpaPK during mitosis 144 4.3.3.2 Effects of mitotic blockers on the localization of the SpaPK .... 145 4.3.3.2.A Localization of SpaPK in MCF-7 cells treated with nocodazole or taxol 149 4.3.3.2.B Localization of SpaPK in MCF-7 cells released from nocodazole arrest 149 4.3.3.3 Effects of protein kinase inhibitors on the localization of the SpaPK protein 154 4.3.4 Subcellular localization of ectopically expressed recombinant SpaPK proteins in mammalian cells 158 4.3.4.1 Overexpression of hHis-SpaPK and hHis-K42A-SpaPK in mammalian cells 158 4.3.4.2 Localization of ectopically expressed hHis-SpaPK and hHisK42A-SpaPK 158 4.3.4.3 Localization of ectopically expressed recombinant hHis-SpaPK proteins in MCF-7 cells treated with nocodazole or taxol 160 4.3.5 Effects of the recombinant SpaPK proteins on cell cycle progression 160 4.3.5.1 Cell cycle distribution of MCF-7 cells overexpressing hHis-SpaPK or hHis-K42A-SpaPK 160 4.3.5.2 Effects of purified His-SpaPK and His-K42A-SpaPK on the onset of mitosis in Xenopus egg extract 165 4.3.5.3 His-SpaPK phosphorylation of Xenopus Cdc25C fragment and variants 167 4.3.6 Farwestern blotting of MCF-7 cell extracts 169  ix  4.4  DISCUSSION 4.4.1 SpaPK is not a functional homolog of Rad5 3 4.4.2 Possible biological functions of SpaPK 4.4.2.1 A role in apoptosis 4.4.2.2 A role in transcription 4.4.2.3 A role in mitosis 4.4.2.3.A Formation of spindle poles 4.4.2.3.B G / M transition 4.4.2.4 Factors contributing to discrepancies in the results 4.4.3 Potential regulators of SpaPK 4.4.3.1 Protein kinases associated with the centrosome and the mitotic spindle 4.4.3.2 Candidate regulators of SpaPK identified from drug inhibition studies 4.4.4 Potential binding partners of SpaPK 2  171 171 172 172 176 176 176 178 180 180 180 181 184  CHAPTER 5 CONCLUSIONS AND PERSPECTIVES  186  REFERENCES  190  x  LIST O F FIGURES  Figure  Page  Figure Figure Figure Figure Figure Figure  1.1 1.2 1.3 1.4 1.5 1.6  A n overview of the eukaryotic cell cycle Cdk-cyclin complexes during cell cycle progression in mammalian cells A model for GJS transition in mammalian cells A model for G / M transition in mammalian cells A model for D N A damage checkpoint pathway in S. cerevisiae A model for D N A replication checkpoint pathway in S. cerevisiae  Figure Figure Figure Figure Figure Figure  2.1 2.2 2.3 2.4 2.5 2.6  Construction of the SpaPK cDNA Nucleotide and deduced amino acid sequences of the SpaPK c D N A Tissue distribution of the SpaPK mRNA Primer extension analysis of the SpaPK gene Genomic structure of the SpaPK gene Identification of 5' end of the SpaPK c D N A  2  Figure 3.1 Figure 3.2  Domain structures of SpaPK Amino acid sequence comparison of SpaPK and its homologs in mouse and rat Figure 3.3 Comparison of the amino acid sequence of SpaPK and other protein kinases Figure 3.4 Comparison of the amino acid sequences of SpaPK, Rad53 andCdsl Figure 3.5 Overexpression, renaturation and purification of His-SpaPK Figure 3.6 Secondary structure content of His-SpaPK Figure 3.7 Kinase activities of His-SpaPK and His-K42A-SpaPK Figure 3.8 Phosphoaminoacid analysis of autophosphorylated His-SpaPK Figure 3.9 Detection of His-SpaPK using anti-SpaPK antiserum Figure 3.10 Detection of SpaPK from MCF-7 cell extract using affinity purified anti-SpaPK antibodies Figure 3.11 Alignment of the putative NES sequence of SpaPK with that of other proteins  Figure 4.1 Figure 4.2 Figure Figure Figure Figure  4.3 4.4 4.5 4.6  Overexpression of HA-SpaPK in yeast strains Suppression of H U sensitivity of mec2-l mutants by overexpression of HA-SpaPK Cell cycle analysis of gene expression and protein expression of SpaPK Subcellular localization of SpaPK during the cell cycle Specificity of polyclonal ApSpaPK antibodies in immunostaining Subcellular localization of SpaPK in nocodazole and taxol treated MCF-7 cells  xi  4 7 14 17 20 25  36 37 40 ^3 .49 54  71 76 78 80 84 87 89 90 95 96 99  138 139 142 146 148 150  Figure 4.7 Figure 4.8 Figure 4.9 Figure 4.10  Figure 4.11 Figure Figure Figure Figure  4.12 4.13 4.14 4.15  Figure 5.1  Subcellular localization of SpaPK in MCF-7 cells released from a nocodazole block 151 Subcellular localization of SpaPK in staurosporine-treated MCF-7 cells 157 Subcellular localization of hHis-SpaPK 159 Comparison of cell cycle distribution of MCF-7 cells expressing wild-type or catalytically inactive recombinant SpaPK with that of transfected but non-expressing cells 24, 48 and 72 hours post-transfection 162 Cell cycle distribution of SpaPK-expressing MCF-7 cells 24, 48 and 72 hours post-transfection 164 Time course of Cdc2/cyclin B activation in Xenopus extract 166 His-SpaPK phosphorylation of Xenopus Cdc25C fragment and its variants... 168 Detection of binding partners of His-SpaPK 170 Schematic representation of structural organization of D A P kinase family members 173 A model for SpaPK signaling at the G2/M transition  xii  188  LIST OF TABLES Table  Page  Table 1.1 Main discoveries in the field of cell cycle division Table 1.2 Mammalian cyclin dependent kinases  Table Table Table Table Table Table Table Table  2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8  List of bacterial strains Oligonucleotide primers for 5' R A C E PCR of the SpaPK c D N A Summary of 5' R A C E P C R of the SpaPK cDNA Oligonucleotide primers for sequencing of GIII5ASst genomic D N A Oligonucleotide primers for sequencing of GIII5Pst/EcoO 109 genomic D N A . . . . Exon/intron structure of the SpaPK gene Oligonucleotide primers for Northern blotting Predictions of T A T A promoters and 5' end of the SpaPK gene  Table 3.1 Predicted phosphorylation sites on SpaPK Table 3.2 Peptide sequences used for screening of potential phosphorylation sequences of His-SpaPK Table Table Table Table Table  4.1 4.2 4.3 4.4 4.5  2 6  List of mitotic microtubule-associated motor proteins and their functions List of mitotic kinases and their functions List of yeast strains Inhibitors of protein kinases Effects of inhibitors of protein kinases and phosphatases on the subcellular localization of SpaPK in mitotic cells  xiii  28 45 45 47 47 48 52 53  74 92 Ill 116 128 155 156  LIST OF ABBREVIATIONS A Amp APC ATP BSA bp °C CKI CKII CAK CaM kinase II CDK cDNA Ci CKI cm C-NAP1 C-terminal DAPK dATP dCTP DEPC dGTP Dlk DMSO DNA DNA-PK DNase dNTP dTT dTTP EDTA EST EtBr EtOH FHA FITC g GSK3 h His HU IPTG Kbp  absorbance ampicillin anaphase promoting complex adenosine triphosphate bovine serum albumin base pair degree Celsius casein kinase I casein kinase II CDK-activating enzyme calmodulin-dependent protein kinase II cyclin dependent kinase complementary deoxyribonucleic acid Curie cyclin dependent kinase inhibitor centimeter centrosomal Nek2-associated protein 1 carboxy-terminal death associated protein kinase 2' -deoxyadenosine 5' -triphosphate 2'-deoxycytidine 5'-triphosphate diethylenepyrocarbonate 2' -deoxyguanosine 5' -triphosphate death-associated protein like kinase dimethyl sulfoxide deoxyribonucleic acid D N A dependent protein kinase deoxyribonuclease deoxyribonucleoside triphosphate 1,4-dithiothreitol 2' -deoxythymidine 5' -triphosphate ethylenediaminetetraacetic acid expressed sequence tag ethidium bromide ethanol forkhead-associated fluorescein isothiocyante gravity glycogen synthase kinase 3 hour histidine hydroxyurea isopropyl-P-D-thiogalactopyranoside kilobase pair  xiv  kDa LB M mg MLK MTOC NES NLS uCi Hg  ul min ml mM MOPS mRNA MW Nek ng NP-40 nt N-terminal OD PAGE PBS PCM PCR PIP 3-kinase PKA PKC PKG Plk pmol PMSF PCNA PVDF RACE RNA RNase rRNA rpm SDS  ssc  SpaPK TB TBE  kiloDalton Luria broth molar milligram myosin light chain kinase microtubule organization center nuclear export signal nuclear localization signal microCurie microgram microliter minute milliliter milliMolar 3-(N-morpholino)-propanesulfonic acid messenger ribonucleic acid molecular weight NIMA-related kinase nanogram Nonidet P-40 nucleotide amino-terminal optical density polyacrylamide gel electrophoresis phosphate buffered saline pericentriolar material polymerase chain reaction phosphoinositide 3-kinase protein kinase A protein kinase C protein kinase G polo-like kinase picomole phenylmethylsulfonyl fluoride proliferating cell nuclear antigen polyvinylidene fluoride rapid amplification of cDNA ends ribonucleic acid ribonuclease ribosomal ribonucleic acid revolutions per minute sodium dodecyl sulphate standard saline citrate spindle pole-associated protein kinase Terrific Broth Tris-borate-Na E D T A  XV  TBP TBS  TGF-P Tris tRNA U UTR UV V v/v  T A T A binding protein Tris buffered saline transforming growth factor- (3 tris(hydroxymethyl)aminomethane transfer R N A units untranslated region ultraviolet volt volume/volume  xvi  ACKNOWLEGEMENTS  I would like to thank my supervisor, Dr. Michel Roberge, for giving me this project and for giving me the freedom during the course of my research, and my committee members, Dr. George Mackie and Dr. Peter Candido, for their input and ideas. M y thanks also go to past and present members of the Roberge lab, in particular, Duncan Ho, Geoff Stone and Tamsin Tarling. A very special thanks to my parents, whose tireless work ethics and drive for achievements have inspired me numerous ways. To my brother and Songyang who are always understanding and witty, and to my niece Miaobo who has always given me a reason to smile, thank you! I am eternally grateful to my grandparents, who raised me and taught me to become the person I am. And finally to one of the most important person in my life X man, who has shown me the true meaning of love, thank you for standing by me and supporting me in every possible way.  xvn  CHAPTER 1 INTRODUCTION  1.1  T h e cell cycle  Eukaryotic cells, with chromosomes encased in a nucleus, appeared on earth approximately two billion years ago. Organisms consisting of such cells are either unicellular such as yeasts, or multi-cellular such as plants and animals. As one of the most complex examples, the human body consists of about a hundred trillion cells, on the average about one billion cells per gram tissue. The formation and maintenance of such a complex multicellular organism require enormous rounds of cell division. Cell division refers to the process by which new cells originate from other living cells, and is the fundamental means by which all living cells are propagated. A n adult human is estimated to undergo more than 25 million cell divisions each second. Such an enormous output of cells is necessary to replace cells that have aged or died. A better understanding of such a fundamental biological process has led to numerous breakthroughs in the field of cell cycle research, with far-reaching implications. In 2001, the 100 Nobel Prize in Physiology or Medicine was awarded to th  Leland Hartwell, Sir Paul Nurse and Timothy Hunt, for their ground-breaking work in identifying key regulatory molecules of the cell cycle. These and other important discoveries in cell cycle research are listed in Table 1.1. The cell cycle refers to the stages through which a cell passes from one cell division to the next. A standard cell cycle is divided into two major phases: the mitotic (M) phase and the interphase (Figure 1.1).  The M phase includes mitosis during which duplicated  chromosomes segregate, and cytokinesis during which a cell divides into two daughter cells. Interphase is further divided into two gap phases (Gi and G ) separated by a D N A synthesis 2  (S) phase. The Gi and G phases provide additional time for cellular growth and production 2  of new proteins. Cells in the Gi phase do not always continue through the cycle. Instead, they can exit from the cell cycle and enter a resting stage (Go). The duration of the cell cycle varies between different cell types. Whereas M phase typically lasts 30 to 60 minutes, interphase may exist for various time periods depending on the cell type and the conditions. In most mammalian cells, the cell cycle lasts between 10 and 30 hours.  1  Table 1.1 Main discoveries in the field of cell cycle division. Year  Discovery  Investigators  1902  Mitotic spindle  Boveri, T  (Boveri, 1902)  1951  Chiasmata  Ostergren G  (Ostergren, 1951)  1953  D N A replication  Watson, J D & Crick, F H Howard, A & Pelc, S Meselson, M & Stahl, F  (Watson and Crick, 1953) (Howard and Pelc, 1953) (Meselson and Stahl, 1958)  1968  Dynamic instability  Borisy, G.G & Taylor, E W Mitchison, T & Kirschner, M  (Borisy and Taylor, 1967) (Mitchison and Kirschner, 1984)  1970  Cdc mutants  Hartwell, L. H  Cell cycle control  Rao, P N & Johnson, R T  (Hartwell etal, 1970), (Hartwell, 1971) (Hartwell etal, 1974) (Rao and Johnson, 1970)  1971  Maturation promoting factor  Masui, Y Gerhart, J & Kirschner, M Lohka, M J & Mailer, J M  (Masui and Markert, 1971) (Gerhart et al, 1984) (Lohka et al, 1988)  1974  The restriction point  Pardee, A B  (Pardee, 1974)  1975  Regulation of Cdc2  Nurse, P Nurse, P & Thuriaux, P Russell, P & Nurse, P Gould, K L & Nurse, P  (Nurse, 1975) (Nurse and Thuriaux, 1980) (Russell and Nurse, 1986) (Russell and Nurse, 1987) (Gould and Nurse, 1989) (Clarke and Carbon, 1980) (Lechner and Carbon, 1991)  8  References  1980  Yeast centromeres  Clarke, L & Carbon, J Lechner, J & Carbon, J  1982  Cell cycle conservation  Beach, D & Nurse, P Lee, M G & Nurse, P  1983  Cyclin characterization  Evans, T & Hunt, T Murray, A W & Kirschner, M W  (Evans etal, 1983) (Murray et al., 1989) (Murray and Kirschner, 1989)  1987  Replication origins  Stinchcomb, D & Davis, R W Brewer, B J & Fangman, W L Huberman, J. A & Davis, L R Bell, S P & Stillman, B  (Stinchcomb et al, 1979) (Brewer and Fangman, 1987) (Huberman et al, 1987) (Diffley and Cocker, 1992)  1988  Checkpoint control Retinoblastoma/E2F  Weinert, T A & Hartwell, L H DeCaprio, J A & Livingston, D M Whyte, P & Harlow, E Goodrich, D. W & Lee, W H Hieber, S W & Nevins, J R  (Weinert and Hartwell, 1988) (DeCaprio etal, 1988) (Whyte et al, 1988) (Goodrich et al, 1991) (Hiebert et al, 1992)  1989  p53  Baker, Nigro, Baker, Edgar,  (Baker (Nigro (Baker (Edgar  Body plan regulation  S J et al. JM S J & Vogelstein, B B A & O'Farrell, P H  2  1  (Beach etal., 1982) (Lee and Nurse, 1987)  etal, 1989) etal, 1989) et al, 1990) and O'Farrell, 1989)  1991  The mitotic checkpoint A new class of cyclins  The A P C and proteolysis  Hoyt, M A & Roberts, B T L i , R & Murray, A W Matsuhime, H & Sherr, C J Xiong, Y & Beach, D Lew, D & Reed, S I Koff, A & Roberts, J M Motokura, T & Arnold, A Glotzer, M & Kirschner, M W Hershko, A & Cohen, L H King, R, W & Kirschner , M W Sudakin, V & Hershko, A Cohen-Fix, O & Koshland, D Funabiki, H & Yanagida, M Ciosk, R & Nasmyth, K  (Hoyt etal., 1991) (Li and Murray, 1991) (Matsushime et al, 1991) (Xiong et al., 1991) (Lew etal., 1991) (Koff etal., 1991) (Motokura et al., 1991) (Glotzer et al., 1991) (Hershko etal, 1991) (King etal, 1995) (Sudakin etal, 1995) (Cohen-Fix etal, 1996) (Funabiki etal, 1996) (Ciosk et al, 1998)  1993  C D K inhibitors  Xiong, Y & Bead, D el-Deiry, W S & Vogelstein, B Harper, J W & Elledge, S J Serrano, M & Beach, D Gu, Y & Morgan, D O Polyak, K & Massague, J  (Xiong et al, 1993) (el-Deiry et al, 1993) (Harper et al, 1993) (Serrano etal, 1993) (Gu etal, 1993) (Polyak et al, 1994)  1994  SCF and F-box proteins  Schwob, E & Nasmyth, K Bai, C & Elledge, S J  (Schwob etal, 1994) (Bai etal, 1996)  1997  Sister chromatid cohesion  Michaelis, C & Nasmyth, K Guacci, V & Strunnikov, A Losada, A & Hirano, T Uhlmann, F & Nasmyth, K  (Michaelis et al, 1997) (Guacci etal, 1997) (Losada et al, 1998) (Uhlmann etal, 1999)  a  Investigators whose names are in bold-type received Nobel Prize in Physiology or Medicine in 2001.  3  /  Cell  grows arc  DNA replication  F i g u r e 1.1 A n overview of the eukaryotic cell cycle. The cell cycle can be divided into M phase and interphase. M phase consists of successive stages of mitosis and cytokinesis. The interphase consists of Gap 1 (G,), D N A synthesis (S) and Gap 2 (G ) phases. Diagram is modified from Cell and Molecular Biology: Concepts and 2  Experiments.  4  1.2 Cyclin dependent kinases and their regulation 1.2.1 Cyclin dependent kinases The orderly progression of different phases of the cell cycle must be precisely coordinated. A family of protein kinases, known as the cyclin dependent kinases (Cdks), controls the ordered transition through the various stages of the cell cycle in all eukaryotes. The founding members of the Cdk family are products of CDC28 of the budding yeast, Saccharomyces cerevisiae, and CDC2 of the fission yeast, Schizosaccharomyces pombe, identified from genetic screens in the 1970s by Leland Hartwell and Paul Nurse, respectively (Hartwell, 1971; Hartwell et ah, 1970; Nurse, 1975). appears to be controlled by a single Cdk.  In yeasts, cell cycle progression  In metazoans, however, different cell cycle  transitions require distinct Cdks. The first human Cdk, termed Cdc2, was discovered by Paul Nurse in 1987 (Lee and Nurse, 1987), and was later given the name C d k l .  Subsequently,  Cdc28, Cdc2, and Cdkl were shown to be homologous to the catalytic subunit of a universal inducer of mitosis, named M phase promoting factor (MPF) initially identified from Xenopus oocytes (Arion et ah, 1988; Gautier et ah, 1988; Lohka et ah, 1988). Since then, additional mammalian Cdks have been isolated (Table 1.2).  The activities of Cdks, whose protein  levels are relatively stable during the cell cycle, are cooperatively regulated by a number of factors. These include cyclins, Cdk phosphorylation state, Cdk inhibitors (CKIs), controlled proteolysis, and subcellular localization. 1.2.2 The role of cyclins The activities of all Cdks require binding of positive regulatory subunits known as the cyclins. Cyclins constitute a large family of evolutionarily conserved proteins. The first cyclin was discovered from sea urchin in 1983 by Tim Hunt (Evans et ah, 1983). These proteins were named cyclins due to their periodic pattern of synthesis and destruction during the cell cycle, although some of the cyclins identified more recently are expressed at constant levels (Table 1.2) (Figure 1.2). The cell cycle dependent protein expression of cyclins represents the primary mechanism by which the activities of Cdks are regulated (Figure 1.2) (Amon et ah, 1994; Amon et ah, 1993; Hayles et ah, 1994; Knoblich et ah, 1994; Nurse, 1994). Some cyclins and Cdks undergo combinatorial interactions which function at different stages during the cell cycle. Cyclins are largely divided into Gi cyclins, including D-type cyclin and cyclin E,  5  Table 1.2 Mammalian cyclin dependent kinases (Cdks) . a  Name  Regulatory subunit(s)  Proposed function  Cdkl = Cdc2  Cyclins A , B l , B2  G /M transition  Cdk2  Cyclins A , E, D l , (D2, D3?)  Gi/S transition and S phase  Cdk3  ik3-l,ik3-2  G]/S transition  Cdk4  Cyclins D1,D2, D3  G0/G1 and G\/S transitions  Cdk5  p35 , ik3-l,ik3-2  Neurofilament phosphorylation  Cdk6  Cyclins D 1 , D 2 , D 3  Gn/Gi and Gi/S transitions  Cdk7  Cyclin H , p36  Required to activate Cdkl-6 Regulation of transcription  Cdk8  Cyclin C  Regulation of transcription  Cdk9  Cyclins T l , T 2 a , T2b Cyclin K  Regulation of transcription via D N A Regulation of transcription via R N A Cdk activating activity  CdklO  ?  Inhibits Ets2 transactivation  a b c d  2  b  d  0  data taken from (Nigg, 1995), (Sato et al, 2002), (Matsuoka et al, 2000), (Rickert et al, 1999), (Edwards et al, 1998), (Lin et al, 2002), (Kasten and Giordano, 2001). p35 does not show sequence similarity to cyclins, and is a brain-specific activator of Cdk5. A role of Cdk5 in cell cycle control is unclear. Instead, this kinase clearly phosphorylates neurofilaments in postmitotic neurons, p36 does not show sequence similarity to cyclins.  6  Figure 1.2 Cdk-cyclin complexes during cell cycle progression in mammalian cells. Cdk4 and Cdk6 in complex with D-type cyclins drives cell cycle from mid G, phase. Cdk2-cyclin E complex controls G , to S transition. Cdk2-cyclin A complexes control S phase progression. The G to M transition is primarily driven by C d k l 2  cyclin B complex. Degradation of cyclin A and cyclin B, causing inactivation of C d k l , is required for exit from mitosis, allowing the cell to re-enter to cell cycle. Diagram is modified from Cell and Molecular Biology: Concepts and Experiments.  7  and mitotic cyclins, including cyclin A and B-type cyclins. Upon stimulation of mammalian quiescent cells with growth factors, the first cyclins to be expressed are the D-type cyclins (Matsushime et al, 1991; Sherr, 1995; Won et al., 1992). D-type cyclins form complexes with Cdk4 or Cdk6, and are considered to be key regulators of Gi progression. Cyclin E is expressed from mid G i to late G i phase (Botz et al, 1996; Geng et al, 1996). Cyclin E, in association with Cdk2, is required for Gi/S transition (Geng et al., 1996; Koff et al., 1992). Cyclin A is expressed at the Gi/S boundary (Tsai et al, 1991), (Elledge et al, 1992). Cyclin A also forms a complex with Cdk2, and the resulting kinase activity is essential for control of D N A replication and progression through S phase (Cardoso et al., 1993; Ishimi et ah, 2000; van den Heuvel and Harlow, 1993).  Cyclin B l is expressed in late S and G phases. 2  However, Cdkl/cyclin B l complexes are inactive until late G when their activation is 2  required for entry into mitosis (King et al., 1994; Mailer, 1991; Nurse, 1990). 1.2.3 The role of phosphorylation The full activation of Cdks requires phosphorylation on a conserved threonine residue located in the T-loop of Cdks. The T-loop blocks access of ATP to the catalytic domain of the kinase (Kaldis, 1999). Phosphorylation of Thr-172 in Cdk4/6, Thrl60 in Cdk2, and Thr161 in C d k l appears to occur after Cdk-cyclin binding (Kato et al, 1994; Solomon et al, 1990). Association of Cdk-cyclin is thought to cause a conformational change in the Cdk, making the T-loop more accessible for the activating phosphorylation.  The activating  phosphorylation causes a further conformational change in the T-loop, making the catalytic domain fully accessible to ATP (Jeffrey et al., 1995). The CDK-activating kinase ( C A K ) is responsible for catalyzing the activating phosphorylation on the conserved threonine (Kaldis, 1999). The activity of C A K appears to be constant throughout the cell cycle (Tassan et al, 1994). The catalytic subunit of C A K , Cdk7, is itself a member of the Cdk family. The active human C A K contains two additional subunits: cyclin H and M A T 1 (Fisher and Morgan, 1994; Tassan et al, 1994). The activity of C A K is opposed by the action of a specific phosphatase known as K A P whose function and regulation remain largely unknown (Hannon et al., 1994). Whereas phosphorylation on the conserved threonine activates Cdks, phosphorylation at sites near the N-termini of all Cdks inhibits their activities even in the presence of C A K catalyzed activating phosphorylation (Morgan, 1997). These inhibitory phosphorylation sites  8  are on Tyr-15 of Cdkl and Cdk2 and on Tyr-17 of Cdk4 and Cdk6, and an additional site on Thr-14 of Cdkl and Cdk2 (Morgan, 1997).  Weel and M y t l have been shown to  phosphorylate Cdkl and Cdk2 on Thr-14 and Tyr-15 (McGowan and Russell, 1993; Mueller et al, 1995; Parker and Piwnica-Worms, 1992). The kinase responsible for phosphorylating Tyr-17 of Cdk4 and Cdk6 has not yet been identified. The inhibitory phosphorylations on Cdks are removed by the actions of dualspecificity Cdc25 family of protein phosphatases (Nilsson and Hoffmann, 2000).  The  activity of Cdc25A is required for the activation of Cdk2/cyclin E for Gi/S transition and Cdk2-cyclin A for progression through S phase (Hoffmann et al., 1993; Hoffmann et ah, 1994) (Section 1.3.1.2). The activities of Cdc25B and Cdc25C are necessary for activation of Cdkl-cyclin B l required for mitotic entry (Section 1.3.2). 1.2.4 TheroleofCDKIs Cdk/cyclin activity can be blocked by interaction with inhibitory proteins.  Seven  mammalian CDKIs have been identified, and can be divided into two families: the inhibitors of Cdk4 (Ink4) family consisting of pi5, p i 8 and p i 9, and the Cip/Kip family consisting of p21, p27 and p57 (Xiong, 1996). The Ink4 family of CDKIs specifically targets Cdk4 and Cdk6. These inhibitors were originally shown to prevent or diminish binding of Cdk4 or Cdk6 to cyclin D (Coleman et al, 1997; Guan et al, 1994; Hannon et al, 1994; Hirai et al, 1995), but more recently, ternary Cdk/cyclin D/Ink 4 complexes have been found under high expression levels of p i 5 or p l 9 (Jeffrey et al, 2000). Transcription of p i 5 is induced by treatment with transforming growth factor-p (TGF-P) (Hannon and Beach, 1994).  Other members of Ink4 family of  inhibitors are also thought to mediate cellular response to anti-proliferative signals (Serrano etal,  1993). The Cip/Kip CDKIs have been described as universal Cdk inhibitors. Cip/Kip family  members bind to multiple pre-formed Cdk/cyclin complexes, and appear to function by preventing CAK-catalyzed activating phosphorylation (Hitomi et al, 1998; Rank et al, 2000; Xiong et al, 1993). The level of p21 is low in quiescent cells and rises in response to mitogenic stimuli (Li et al, 1994b). p21 expression is also upregulated by p53 in response to D N A damage (el-Deiry et al, 1994; L i et al, 1994b). It inhibits cyclin D and cyclin Econtaining complexes, promoting G i arrest with hypophosphorylated pRb (Dulic et al,  9  1994). p21 also has been found to interact with proliferating cell nuclear antigen (PCNA), suggesting a role in D N A replication and/or D N A repair (Li et al, 1994a; Waga et al., 1994). p27 is expressed in quiescent cells and its level decreases in response to mitogenic treatment (Koff et al., 1993).  p27 has been implicated in mediating TGF-(3 and contact-induced  inhibition of proliferation (Polyak et al., 1994). p27 can prevent the activation of Cdk4cyclin D and Cdk2-cyclin E, leading to cell cycle arrest at the Gi/S transition (Polyak et ah, 1994). The availability of p27 for inhibition of Cdk2-cyclin E appears to be dependent on the sequestration of p27 by Cdk4-cyclin D complex (Ladha et al., 1998; Perez-Roger et al., 1999). Interestingly, recent reports have described the presence of p21 and p27 in active Cdk/cyclin complexes, suggesting that these CDKIs may function as either assembly factors or inhibitors (LaBaer et al., 1997; Zhang et al., 1994). p57 appears to have a similar role to p27, although its expression is restricted to certain tissues (Matsuoka et al., 1995). 1.2.5 The role of proteolysis Elimination of Cdk regulators is important for orderly cell cycle progression. The ubiquitin-proteasome pathway is responsible for the degradation of cell cycle components. This pathway comprises two steps: the covalent attachment of ubiquitin molecules to the protein substrate and the degradation of the polyubiquitinated protein substrate by the 26S proteasome. The attachment of multiple ubiquitin molecules to a target protein substrate is mediated by a ubiquitin-activating enzyme (El), a ubiquitin-conjugating enzyme (E2), and in some cases a ubiquitin ligase (E3). Two major types of E3 enzymes, SCF complex and the anaphase-promoting complex or cyclosome (APC/C), target specific cell cycle components for ubiquitination (Peters, 1998). The SCF complex is active at the end of G i , through S and into early G phase, and 2  mediates ubiquitination of short-lived Gi cyclins and CDKIs (Peters, 1998). The degradation of cyclin D l and cyclin E in complex with their Cdks is initiated with phosphorylation of Thr-286 by GSK-3p and Thr-380 by Cdk2-cyclin E, respectively (Clurman et al, 1996; Diehl et al, 1998; Diehl et al, 1997; Won and Reed, 1996). Cyclin D l and cyclin E can be also  degraded  independently  of binding to  Cdks and  of the  above  mentioned  phosphorylations (Germain et al, 2000; Singer et al, 1999). Degradation of p27 is initiated by phosphorylation of Thr-187 catalyzed by Cdk2-cyclin E complex (Morisaki et al, 1997; Nguyen et al, 1999). This reaction appears to occur only when p27 is bound to Cdk2-cyclin  10  E (Xu et al, 1999).  Phosphorylated p27 is transported to the cytoplasm, where p27 is  ubiquitinated for degradation (Tomoda et al., 1999). A P C is active from late G2 to mid Gi, and plays an important role in mitosis (Peters, 1999). Mitotic cyclins are targeted for ubiquitin-proteasome mediated proteolysis by an N terminal nine amino acid motif known as the destruction box (Klotzbucher et al., 1996; Sudakin et al, 1995). Cyclin A is required throughout G2, and is degraded after nuclear envelope breakdown (den Elzen and Pines, 2001; Furuno et al., 1999).  Cyclin B l  degradation occurs during the metaphase to anaphase transition until the completion of anaphase, and is required for exit from mitosis (Clute and Pines, 1999). 1.2.6 The role of subcellular localization Proper cell cycle progression requires appropriate subcellular localization of Cdkcyclins and their regulators.  During Gi phase, cyclin D, E and A are synthesized and  associate with their respective Cdks. Cdk complexes containing cyclin D l , E and A are predominantly or exclusively localized in the nucleus (Baldin et al., 1993; Ohtsubo et al., 1995; Pines and Hunter, 1991). Cdk6-cyclin D3 has been found in both the nuclear and the cytoplasmic fractions, although only the nuclear complex displayed kinase activity (Mahony et al., 1998).  At the onset of S phase, Cdk-cyclin D l complexes are exported to the  cytoplasm for proteolysis (Baldin et al, 1993).  Cyclin A and E-containing complexes  remain in the nucleus during S phase (Ohtsubo et al., 1995; Pines and Hunter, 1991). By contrast, cyclin B l and cyclin B2, which bind to Cdkl upon synthesis during S phase, accumulate almost exclusively in the cytoplasm (Pines and Hunter, 1994). Preceding the onset of mitosis, cyclin B l translocates to the nucleus and this accumulation in the nucleus is required for initiation of mitosis (Jackman et ah, 1995). Cyclin A and cyclin B2 remain in the nucleus and the cytoplasm, respectively, until nuclear envelope breakdown (Jackman et al, 1995; Pines and Hunter, 1991). Functional Cdk activity predominantly resides in the nucleus where the majority of C A K activity has been detected. However, no obvious nuclear localization signal (NLS) can be found on the primary sequences of vertebrate cyclins or their Cdk partners. Cdk-cyclin complexes appear to utilize different mechanisms for nuclear import. The association with NLS-containing proteins such as p21 or p27, or a recently discovered SEI-1 protein may provide a mechanism for nuclear import of Cdk-cyclin D l (LaBaer et ah, 1997; Sugimoto et  11  al., 1999).  Cyclin E appears to contain a cryptic N L S , and binds to the N L S receptor  importin-a, and the nuclear transport factor importin-P, for localization to the nucleus. Nuclear import of cyclin A requires the activity of its associated Cdk (Moore et al., 1999). Finally, Cdkl-cyclin B l complex is thought to interact with cyclin F, and the resulting interaction provides the necessary N L S signal for entering the nucleus, perhaps through direct binding to importin-P in the absence of importin-a (Kong et al., 2000; Moore et al., 1999). The predominantly cytoplasmic localization of Cdkl-cyclin B l prior to mitosis depends on the balance between ongoing nuclear import and rapid nuclear export. The N terminus of cyclin B l contains a cytoplasmic retention signal (CRS) which includes a leucine-rich nuclear export signal (NES) (Hagting et al, 1998; Toyoshima et al., 1998; Yang et al., 1998). Cyclin B l export is mediated by interaction of the N E S with a nuclear export factor CRM1 (Yang et al., 1998). A model for the control of cyclin B l localization at the G / M transition proposes that phosphorylation of cyclin B l N E S by a yet undetermined 2  kinase alters the balance of import/export activities in favor of cyclin B1 accumulation in the nucleus and consequential entry into mitosis (Yang et al., 1998). The CRS of cyclin B l and cyclin B2 are conserved, but the NES region within the human cyclin B2 CRS differs from that of human cyclin B l (Yang and Kornbluth, 1999).  The mechanism for cytoplasmic  localization of cyclin B2 is unclear.  1.3 Major cell cycle transitions The progression of a eukaryotic cell through its cell cycle is regulated primarily at two transitions, one occurring near the end of Gi when the cell becomes committed to initiation of D N A replication, and the other occurring near the end of G2 when the cell determines to enter mitosis. 1.3.1 Gj/S transition 1.3.1.1 Gi/Spromoting pathways The growth of cancer cells is independent of mitogenic stimuli. However, most nontransformed cells require growth factor induction to enter the cell cycle from a quiescent Go state (Hanahan and Weinberg, 2000). Mitogenic signaling is required only until a point in late Gi, known as the restriction point, after which the cell is committed to undergo D N A  12  synthesis (Pardee, 1974). The Gi/S transition is regulated by two parallel and cooperating pathways (Figure 1.3). While silencing of either pathway does not completely prevent D N A synthesis, inhibition of both pathways block entry into S despite the presence of growth factors (Santoni-Rugiu et al, 2000). The traverse through the restriction point requires inactivation of retinoblastoma (pRb) family of proteins (Weinberg, 1995). In its hypophosphorylated state, pRb binds to and inhibits the E2F family of transcription factors. Upon phosphorylation of pRb, E2F is released, leading to transcription of genes whose products are required for the completion of Gj/S transition and the progression of S phase (Harbour and Dean, 2000; Nevins, 1998). Phosphorylation of pRb is initiated by the Cdk4/6-cyclin D complexes (Hinds et al., 1992). In quiescent cells, the basal level of cyclin D is low, and the activity of Cdk4/6-cyclin D is inhibited by p l 6 (Serrano et al., 1993). The presence of growth factor induces accumulation of cyclin D, resulting in the formation of active Cdk4/6-cyclin D and phosphorylation of pRb (Sherr, 2000). Another Gi/S promoting pathway involves the c-myc transcription factor.  The  expression of c-myc is induced by mitogens and its ectopic expression enables entry into S phase in quiescent cells (Bouchard et al., 1998). Among target genes of Myc, relevant to d / S control, are those encoding cyclin D l , D2, E and Cdc25A protein phosphatase (SantoniRugiu et al, 2000). 1.3.1.2 Cdkl'-cyclin E: convergence of Gi/S promoting pathways One of the primary goals of the two parallel Gi/S promoting pathways involving pRbE2F and Myc is regulation of the activity of Cdk2-cyclin E, an essential factor for Gi/S transition (Figure 1.3). Both pathways contribute to the timely activation of this key S phase kinase promoting kinase by transcriptional induction of genes encoding cyclin E and Cdc25A phosphatase (Bouchard et al, 1998; Santoni-Rugiu et al, 2000). The combined action of cyclin E, which is a positive regulator of Cdk2, and Cdc25A, which removes inhibitory phosphorylations on Thr-14 and Tyr-15 of Cdk2, result in activation of Cdk2 kinase. One of the substrates of Cdk2-cyclin E is pRb and its related proteins.  Further  phosphorylation and thereby inactivation of pRb provides a positive feedback loop which  13  Mitogens  E2F  Active  1  NPAT  \^Cdc45^  Origin firing  Histone biosynthesis  F i g u r e 1.3 A model for Gj/S transition in mammalian cells. Entry into S phase depends on the activation of Cdk2-cyclin E complex.  Mitogen-induced  accumulation of cyclin D results in the formation of active Cdk4/6-cyclin D complexes which phosphorylate pRb, leading to the release of E2F. E2F activates transcription of genes required for S phase initiation and progression, including cyclin E. Mitogen-induced Myc can also activate E2F and Cdk2-cyclin E. Cdk2cyclin E amplifies E2F activity by phosphorylating and further inactivating pRb, and amplifies its own kinase activity by phosphorylating and targeting p27 CDKI for ubiquitin-proteasome mediated degradation.  Activated Cdk2-cyclin E facilitates  DNA synthesis possibly by promoting Cdc45-mediated origin firing and by promoting histone biosynthesis through direct phosphorylation of NPAT.  14  amplifies both E2F and Cdk2-cyclin E activities (Harbour and Dean, 2000). Cdk2-cyclin E also targets phosphorylation of the p27 inhibitor to facilitate subsequent ubiquitin-dependent proteolysis and allow timely increase of Cdk2 activity essential for G\/S transition (Montagnoli et al., 1999; Sheaff et al., 1997). In addition, Cdk2-cyclin E plays a role in promoting biosynthesis of histones which must be provided for D N A replication. Cdk-cyclin E directly phosphorylates p220NPAT, whose modification is essential for activation of histone gene promoters at the onset of S phase (Ma et al., 2000; Zhao et al., 2000). Cdk2cyclin E additionally stimulates D N A synthesis, presumably by facilitating firing of D N A replication origins through Cdc45 dependent recruitment of D N A polymerase a onto the preinitiation complex (Arata et al., 2000; Zou and Stillman, 1998). In summary, Cdk2-cyclin E plays a crucial role at the G]/S transition. 1.3.2 G?/M transition The transition from G2 into mitosis requires the activity of the Cdkl kinase (Figure 1.4). As introduced earlier, the activity of Cdkl is regulated at several levels. Cdkl exists as an inactive hypophosphorylated monomer during the early phases of the cell cycle, and becomes associated with B-type cyclins during S phase.  Cyclin binding facilitates  phosphorylation of Cdkl on three regulatory sites: Thr-161 whose phosphorylation is required for full enzymatic activity, and Thr-14 and Tyr-15 whose phosphorylations maintain Cdkl in an inactive state. Therefore, Cdkl-cyclin B remains inactive from S to G2 phases due to inhibitory phosphorylations by Weel and M y t l .  Throughout interphase, Cdkl-cyclin B  complexes shuttle between the nucleus and the cytoplasm with net cytoplasmic localization (Section 1.2).  Weel is a nuclear protein, and is thought to protect the nucleus from  prematurely activated Cdkl (Heald et al, 1993). M y t l is localized to Golgi and endoplasmic reticulum and is believed to maintain the inactivating Cdkl phosphorylations in the cytoplasm during interphase (Liu et al., 1997). Removal of the inhibitory phosphorylations of Cdkl is a rate-limiting step for entry into mitosis (Krek and Nigg, 1991).  Dephosphorylation is accomplished by the dual  specificity protein phosphatases, Cdc25B and Cdc25C (Draetta and Eckstein, 1997). Several studies suggest that Cdc25B is responsible for triggering mitosis.  Overexpression of  Cdc25B, but not of Cdc25C, caused premature mitosis, and inhibition of Cdc25B activity by microinjection of specific antibodies blocked mitotic entry (Karlsson et al., 1999; Lammer et  15  al, 1998). On the one hand, the level and the activity of Cdc25B increase in G (Lammer et 2  al, 1998). The accumulation and activation of Cdc25B in the cytoplasm are thought to mediate the initial activation of cytoplasmic Cdkl-cyclin B l whose dephosphorylation provides a signal for nuclear import. On the other hand, the level of Cdc25C is constant throughout the cell cycle.  During G , the activity of Cdc25C increases, partially due to 2  Cdc25B-dependent activation of Cdkl-cyclin B (Draetta and Eckstein, 1997). Activated Cdc25C translocates to the nucleus where it dephosphorylates and activates translocated Cdkl-cyclin B , which further activates Cdc25C (Gabrielli et al, 1997; Izumi and Mailer, 1995; Izumi et al, 1992).  This positive feedback loop between C d k l and Cdc25C is  necessary for the full activation of the Cdkl-cyclin B complex, leading to irreversible entry into mitosis. The regulation of Cdc25C activity is complex.  Cytoplasmic localization may  contribute to the negative regulation of Cdc25C activity. Cdc25C is predominantly localized in the cytoplasm during interphase and enters the nucleus prior to mitosis (Dalai et al, 1999). During interphase, Cdc25C is actively exported from the nucleus due to a nuclear export signal, and is retained in the cytoplasm through interaction with 14-3-3 family proteins (Kumagai and Dunphy, 1999; Yang et al, 1999). Phosphorylation of Cdc25C at Ser-216 within its nuclear localization signal (NLS) is thought to generate a binding site for 14-3-3 proteins (Dalai et al, 1999; Peng et al, 1997). Cdc25C activity can also be negatively regulated by the prolyl isomerase Pinl (Crenshaw et al,  1998; Shen et al,  1998).  Stimulation of Cdc25C activity during mitosis requires hyperphosphorylation (Izumi et al, 1992; Kumagai and Dunphy, 1992). This activation of Cdc25C requires Cdkl-cyclin B , and probably other upstream kinase(s). Cdc25C can be phosphorylated and activated by Cdk2cyclin E, Cdkl-cyclin A , and Xenopus polo-like kinase (Plxl) in vitro (Izumi and Mailer, 1995; Kumagai and Dunphy, 1996). Studies also revealed that Plx and Cdc25C form a positive feedback loop at the onset of mitosis, since injection of Cdc25C causes activation of P l x l , and P l x l is required for hyperphosphorylation of Cdc25C and activation of C d k l cyclin B at the G / M transition (Abrieu et al, 1998; Qian et al, 1998a). 2  16  Cytoplasm Figure 1.4 A model for G / M transition in mammalian cells. Entry into mitosis 2  requires activation of the Cdkl-cyclin B complex. During interphase, Cdkl-cyclin B, which shuttles between the cytoplasm and the nucleus with a net cytoplasmic localization, is kept inactive by the kinase activities of cytoplasmic M y t l and nuclear Weel. Cdc25C also is cytoplasmic until prior to mitosis, when the rate of nuclear import exceeds that of nuclear export. In G , the accumulation and activation of 2  Cdc25B results in the removal of the inhibitory phosphates in Cdkl-cyclin B, leading to its initial activation. Activated Cdkl-cyclin B translocates to the nucleus, where translocated nuclear Cdc25C maintains dephosphorylation of Cdkl at the inhibitory sites and further activate Cdkl-cyclin B , which in turn further activates Cdc25C.  This positive feedback loop between Cdkl/cyclin B and Cdc25C is  necessary for entry into mitosis.  17  1.4 Cell cycle checkpoints Cell cycle progression must be tightly monitored as the loss of this control can lead to genomic instability, contributing to the onset and the development of many cancers. Cells have developed surveillance mechanisms, known as checkpoints, to halt cell cycle progression in response to D N A damage (DNA damage checkpoint), unreplicated D N A (DNA replication checkpoint), and chromosome misalignment (spindle assembly checkpoint, see Chapter 4). This delay in cell cycle progression is thought to provide time for the repair of abnormalities.  In addition to imposing a cell cycle arrest, D N A damage and D N A  replication checkpoint pathways also regulate D N A repair, transcription and programmed cell death. 1.4.1 General organization of the checkpoint response pathway The checkpoint pathways are organized into three components: sensors which detect abnormalities and then emit the appropriate checkpoint signal; transducers which transmit and amplify the signal along the proper pathway; and effectors which control the biological consequences of triggering the pathways. Activation of the D N A damage checkpoint pathway and the D N A replication checkpoint pathway induces arrest at three or more stages in the cell cycle, including the Gi/S transition, S phase progression and the G2/M transition. The common and the primary molecular mechanism of cell cycle arrest in most eukaryotes is the inhibition of activities of Cdks required for cell cycle passage. However, a second mode of regulation exits at the G2/M transition in S. cerevisiae, due to the difference in spindle assembly pathway.  In  mammals and fission yeast, the mitotic spindle does not assemble until after the completion of G2 phase. In budding yeast, spindle assembly, regulated by Cdc28, begins during S phase, thereby effectively initiating mitosis. Therefore, in response to D N A damage or incomplete replication, instead of inhibiting Cdc28 kinase activity at the G2/M transition, S. cerevisiae blocks cell division by preventing sister chromatid separation at the metaphase to anaphase transition during mitosis. Checkpoint responses are not simple linear pathways, but rather are made up of complex networks of interacting regulators.  Some of the checkpoint proteins function in  more than one stage of cell cycle arrest, and regulate or interact with multiple proteins. Genes mediating the checkpoint response pathways appear to be conserved from yeast to  18  human. Here, we focus on checkpoint pathways induced by D N A damage and unreplicated D N A in S. cerevisiae (Figure 1.5 and Figure 1.6). 1.4.2 D N A damage checkpoint 1.4.2.1 DNA damage sensors In budding yeast, the proteins involved in sensing D N A damage are divided into two groups which function additively, primarily in G i and G2 (Figure 1.5).  One of the two  groups is defined by Rad24, Rad 17, Mec3 and D d c l , and the other group is defined by Rad9 (Lowndes and Murguia, 2000). Mutation of any of these proteins results in full checkpointdefective phenotype.  Of the first group, Rad24 shares sequence homology with the five  subunits of replication factor C (RFC) (Griffiths et al, 1995).  Radl7, Mec3 and Ddcl  exhibit sequence homology with trimeric proliferating cell nuclear antigen (PCNA) (Aravind et al, 1999). R F C functions as a "sliding clamp loader", and physically loads P C N A , the "sliding clamp", onto the primer-template junction during D N A replication, allowing processive D N A synthesis by polymerase 8 (Mossi and Hubscher, 1998). The sequence similarity of Rad24 and Radl7/Mec/Ddcl to R F C and P C N A , respectively, prompted speculation that these complexes may localize to the sites of D N A damage in a manner analogous to the RFC-mediated loading of P C N A onto sites of D N A replication. In support of this idea, Rad24 appears to substitute for Rfc 1, and form a five subunit complex with Rfc2, 3, 4, and 5 (Green et al, 2000; Shimomura et al, 1998). In addition, both the Rad24 complex and the Radl7/Mec3/Ddcl complex translocate to the sites of D N A damage, and the recruitment of the latter complex is dependent on the Rad24 complex (Kondo et al, 2001; Melo et al, 2001). These data suggest that the Rad24 complex functions as an RFClike loading factor, allowing loading of additional PCNA-like checkpoint proteins onto sites of D N A damage.  The checkpoint function of Rad9 requires a C-terminal B R C T domain  necessary for oligomerization and activation of the protein (Soulier and Lowndes, 1999). In contrast to the Rad24 group, the mechanism of D N A damage detection by Rad9 is unclear. 1.4.2.2 Transduction of DNA damage signal The sensor proteins activate a set of signal transducers.  Two principal protein  kinases, M e c l and Rad53, are known to be involved in the transduction of D N A damage signal (Figure 1.5). M e c l , a member of phosphoinositide 3-kinase-like family of proteins,  19  D N A damage  Dunl  Swi6  X  1  Cdc28  •  Figure 1.5  Chkl  *  Swi4  Induction of transcription  Cdc5  GJS arrest  Mitotic exit  Pdsl  1 Anaphase  A model for D N A damage checkpoint pathway in S. cerevisiae. D N A  lesions are sensed by two groups, the Rad9 branch, and the Rad24, Rad 17, Mec3 and Ddcl branch.  The checkpoint signals converge on Mecl/Led 1 complex and are  transmitted to Rad53 and C h k l .  Rad53 regulates the activity of D u n l , a protein  kinase involved in induction of transcription of D N A repair genes; Swi6, a transcriptional regulator targeted in G,/S checkpoint; and Cdc5, a polo-like kinase which regulates exit from mitosis. Chkl targets Pdsl, a regulator of sister-chromatid cohesion at the metaphase to anaphase transition. Linear pathways are drawn for simplicity. 20  exists in complex with L c d l which is required for all functions of M e c l (Paciotti et al., 2000; Rouse and Jackson, 2000; Wakayama et al., 2001). M e c l is probably the most upstream kinase of the transduction pathway (Gardner et al., 1999; Sanchez et al., 1996; Sun et al., 1996). However, some evidence suggests that M e c l also acts as an sensor for D N A damage. M e c l - L c d l complexes localize to sites of D N A damage (Kondo et al., 2001). In addition, M e c l has been shown to be absolutely required for damage-induced phosphorylation of D d c l , and partially required for damage-induced phosphorylation of Rad9, placing M e c l upstream of the putative D N A damage sensors (Emili, 1998; Longhese et al,  1997).  Therefore, M e c l may function as a mediator between the sensor complex and downstream checkpoint components.  As is the case for mutants of the rad9 and the rad24 group, the  D N A damage checkpoint response is completely abolished in mecl mutants (Gardner et al., 1999; Sanchezes/., 1999). Rad53 plays an essential role in the D N A damage checkpoint pathway.  It is  phosphorylated and activated in response to D N A damage. The initial phosphorylation of Rad53 is dependent on M e c l , but once activated by M e c l , autophosphorylation of Rad53 occurs in the absence of other proteins. (Pellicioli et al., 1999). Rad53 contains two forkheadassociated (FHA) domains, one located at the C-terminus and the other located at the N terminus of the protein (Li et al., 2000). F H A domains are phosphoprotein-binding modules and are found in a wide variety of proteins (Durocher and Jackson, 2002; L i et al., 2000). FHA  domains  of  Rad53  mediate  a  specific  interaction  between  Rad53  and  hyperphosphorylated Rad9 (Durocher et al., 1999; Sun et al., 1998). The binding between the Mecl-dependent hyperphosphorylated form of Rad9 and the C-terminal F H A domain of Rad53 is required for the checkpoint response (Sun et al., 1998). Therefore, in addition to sensing D N A damage, Rad9 may function in amplification of the checkpoint signal by recruiting Rad53 to the site of D N A damage. M e c l and Rad53 also activate Dunl, a protein kinase which induces transcription of genes involved in D N A repair (Allen et al., 1994). This transcriptional response is partially initiated by Crtl for cell cycle arrest at the metaphase to anaphase transition (Huang et al., 1998). Crtl is a D N A binding protein which recruits the general repressors Ssn6 and Tupl to the promoters of D N A damage-inducible genes. Hyperphosphorylation of Crtl, dependent  21  on the Mecl-Rad53-Dunl pathway, prevents binding of Crtl  to D N A , leading to  transcriptional induction. 1.4.2.2.A Cell cycle arrest at the Gi/S transition In response to D N A damage during Gi phase, budding yeast delays cell cycle progression at or before START, the equivalent of the restriction point in mammalian cells, and thereby prevents entry into S phase (Figure 1.5).  This cell cycle arrest is, in part,  achieved by Rad53-mediated phosphorylation of transcriptional regulator, Swi6 (Sidorova and Breeden, 1997). Swi6 is also directly phosphorylated by Rad53 in vitro. Phosphorylated Swi6 is thought to inhibit the transcriptional activator Swi4 required for the transcription of Gi cyclins. However, ectopic expression of Gi cyclins does not override the checkpoint, suggesting that there are other mechanisms controlling Gi/S arrest (Siede et al., 1994). 1.4.2.2.B Cell cycle arrest at the metaphase to anaphase transition The D N A damage-induced metaphase arrest is mediated by two parallel pathways downstream from M e c l : the Rad53-Cdc5 pathway and the Chkl-Pdsl pathway, each contributing approximately 50% of the checkpoint arrest (Gardner et al, 1999) (Figure 1.5). The existence of two parallel pathways was first demonstrated with kinetic studies. They showed that in the presence of irreparable D N A damage, rad53 and pdsl single mutants can not fully override the induced arrest. rad53 and pdsl single mutants delayed anaphase for approximately 90 minutes, compared to no delay in mecl mutants and 8 to 10 hours of delay in wild-type cells. However, rad53 and pdsl double mutants showed a full arrest-defective phenotype, similar to that of mecl mutant (Gardner et al, 1999). Other biochemical data revealed that D N A damage-induced phosphorylation of Pdsl depends on M e c l activity, but not Rad53 whose phosphorylation also depends on M e c l (Cohen-Fix and Koshland, 1997). The data support an order-of-function between M e c l , Rad53 and Pdsl. The activation of the Chkl-Pdsl signal transduction pathway leads to stabilization of Pdsl.  Pdsl is an anaphase inhibitor which is degraded at the metaphase to anaphase  transition to allow separation of sister chromatids (Cohen-Fix and Koshland, 1997; CohenFix et al., 1996). Expression of non-degradable pdsl prevents anaphase, while deletion of pdsl compromises D N A damage-induced arrest (Yamamoto et al, 1996). D N A damageinduced phosphorylation of Pdsl has been suggested to stabilize the protein, and requires Chkl kinase. The level of phosphorylated Pdsl was high in D N A damage-induced arrested  22  cells, whereas the amount declined before completion of mitosis in chkl mutants after D N A damage. Chkl can also bind and phosphorylate Pdsl in vitro (Sanchez et al., 1999). Similar to Pdsl, Chkl is also phosphorylated in response to D N A damage in a Mecl-dependent but Rad53-independent manner (Sanchez et al, 1999). In addition, mutation in both Rad53 and Chkl is required to completely abolish the checkpoint, further strengthening the notion that Mecl-Chkl-Pdsl function in the same pathway (Sanchez et al., 1999). The purpose of this pathway is to control the stabilization of Pdsl, thereby inhibiting anaphase entry. The activation of the Rad53-Cdc5 pathway leads to maintenance of Cdc28 activity, thereby preventing mitotic exit after D N A damage. Cdc28 kinase activity is required for the checkpoint since mutations which inactivate Cdc28 compromised metaphase arrest (Li and Cai, 1997). Kinetic studies of anaphase entry revealed that the Rad53-dependent anaphase delay might function through inhibition of Cdc5, which in turn, inhibits Cdc28 kinase activity (Sanchez et al., 1999). Cdc5 is a polo-like kinase, whose activity is required for activation of mitotic cyclin degradation required for mitotic exit (Charles et al., 1998). Consistent with this, a Cdc5 loss of function mutant suppressed the Rad53 checkpoint effect, and overexpression of Cdc5 could drive the damaged cells through checkpoint arrest (Sanchez et al, 1999). In addition, Cdc5 itself is phosphorylated in a Rad53-dependent manner (Cheng et al., 1998). Taken together, these data indicate that Chkl-Pdsl and Rad53-Cdc5 function in independent pathways which cooperate to ensure efficient arrest at the metaphase to anaphase transition. 1.4.3 D N A replication checkpoint Cell cycle arrest in S phase ensures that the onset of anaphase is dependent on the completion of D N A replication.  During S phase, D N A replication checkpoint proteins  prevent entry into anaphase when D N A is damaged or D N A synthesis is inhibited. In the presence of genotoxic agents such as hydroxyurea (HU) and methylmethansulfonate (MMS), cells containing loss of function mutations in these checkpoint proteins enter mitosis with aberrant D N A leading to growth defects. 1.4.3.1 DNA damage sensors During S phase, both D N A damage and inhibition of D N A synthesis can be sensed by the replication machinery which resides at the replication forks (Figure 1.6). These putative  23  sensors are D N A polymerase s, Rfc5 which is the small subunit of replication factor C involved in recruiting polymerase to the replication forks, D p b l l and D r c l (Araki et al, 1995; Navas et al, 1995; Sugimoto et al, 1996; Wang and Elledge, 1999). 1.4.3.2 Transduction of unreplicated DNA signal As in the D N A damage checkpoint pathway, M e c l and Rad53 are components of the signal transduction pathway induced by incomplete replication (Figure 1.6). These signaling proteins respond to replication inhibition by inducing transcription of genes involved in D N A repair. 1.4.3.2. A Delay of D N A replication In addition to induction of D N A repair genes, studies have shown that M e c l and Rad53 prevent D N A synthesis from late origins when the replication fork is either stalled or encounters D N A damage during early S phase (Santocanale and Diffley, 1998; Shirahige et al,  1998) (Figure 1.6).  In this regard, Rad53 inhibits the phosphorylation of D N A  polymerase a/primase complex required for the initiation of D N A synthesis (Pellicioli et al, 1999), and negatively regulates the binding of replication protein A (RPA), a three subunit complex which promotes origin firing by stabilizing unwound D N A , to the origins (Tanaka andNasmyth, 1998). 1.4.3.2.B Coupling of S phase and mitosis Completion of S phase and completion of mitosis are coupled by distinct checkpoint mechanisms which appear to occur sequentially (Figure 1.6).  During early S phase, cell  cycle arrest is M e c l and Rad53-dependent since cells lacking mecl and rad53 proceed into anaphase when they are blocked in early replication (Weinert et al, 1994). This arrest in early S phase is independent of Pdsl, since pdsl null mutants can inhibit anaphase when replication is blocked with H U (Weinert et al, 1994).  Partway through S phase, the  centromere cohesion between the sister chromatids is established, and Pdsl  becomes  essential for maintaining cohesion thereby preventing sister chromatid separation before the completion of D N A replication. Kinetic studies, using H U at a concentration which does not block D N A replication but slows down D N A synthesis, showed that pdsl mutants continued with cell division when roughly 50% of the genome has been replicated.  In the same  experimental setting, mecl mutant cells began anaphase when little or no D N A had been replicated, suggesting the presence of a Pdsl-independent checkpoint system in early S phase  24  D N A replication fork lesions  I  D N A polymerase £ Rcf5 Dpbll Drcl  N  •  M e c l LLcd •  Dunl  Induction of transcription Figure 1.6  D N A pol al primase  RPA  D N A synthesis from late origin  Anaphase  A model for D N A replication checkpoint pathway in S. cerevisiae.  During S phase, D N A damage and inhibition of D N A synthesis are sensed by replication machinery, and transmitted  to M e c l / L c d l  complex.  Mecl/Lcdl  activates Rad53, which then regulates Dunl-mediated induction of transcription of D N A repair genes, and inhibits D N A polymerase a/primase complex and replication protein A (RPA) to prevent D N A synthesis from late origins. To arrest cells at the metaphase to anaphase transition, the Mecl-Rad53 pathway is required during early S phase, and Mecl-Pdsl pathway is required partway through S phase. pathways are drawn for simplicity.  25  Linear  (Clarke et al., 1999). Therefore, during S phase, two pathways exist to coordinate ongoing D N A replication and mitosis. 1.5 Experimental rationale The fact that cells multiply through cell division has been known since the late 1800's. However, the molecular mechanisms that regulate the cell cycle have only begun to be elucidated during the past two decades.  These findings show that the fundamental  components of the cell cycle are highly conserved through evolution and operate in a similar manner in all eukaryotic organisms. Many components of the cell cycle machinery from mammalian cells can functionally substitute for their yeast counterparts. In this regard, we were interested in Rad53, a dual specificity protein kinase from Saccharomyces cerevisiae. Rad53 is an essential regulator of the D N A damage checkpoint and D N A replication checkpoint, whose equivalent in humans was not known at the beginning of this project. The aims of the project were to identify and clone the human homolog of the yeast cell cycle checkpoint protein Rad53 and to study its properties and potential role(s) in important cellular processes during the cell cycle.  26  CHAPTER 2 CLONING OF THE SpaPK GENE  2.1 INTRODUCTION In modern molecular biology practice, genes are isolated from an organism using a variety of strategies. We used a homology-based approach that employed an Expressed Sequence Tag (EST) database (GenBank) in an attempt to identify the human homolog of yeast RAD53. ESTs are small portions of D N A sequences generated by sequencing the 5' and/or the 3' end of an expressed gene. These sequences serve as tags to monitor gene expression in certain cells, tissues or organs, and also to facilitate the identification of unknown genes. In this chapter, we describe the identification and analysis of a gene encoding a novel protein kinase, designated SpaPK. A n EST, which showed high similarity to the nucleotide sequence encoding the protein kinase domain of yeast Rad53, was used to screen a human brain c D N A library. Positive clones obtained from the c D N A library were then used as nucleic acid hybridization probes to screen a human genomic library. A SpaPK c D N A clone was constructed based on the screening results.  Tissue-specific and cell line-specific  expression of SpaPK was also studied. A n alignment of the c D N A and the genomic D N A generated information on the exon-intron organization of the SpaPK gene. In addition, an analysis of the 5' end region was performed to identify the promoter region of the SpaPK gene.  27  2.2 MATERIALS AND METHODS 2.2.1 Chemicals, enzymes and plasmids A l l chemicals were of reagent grade and were purchased from common commercial sources. Various application kits for molecular biology were also obtained commercially from the denoted manufacturers. Restriction enzymes were obtained commercially from many companies: AmershamPharmacia; New England Biolabs (NEB); Gibco-BRL; Stratagene etc. They were used as specified by the manufacturers. Recombinant plasmid vectors were purchased from commercial sources or were kindly provided by other laboratories as indicated below.  2.2.2 Bacterial strains E. coli strains and their genotypes are listed in Table 2.1. DH5awas used for propagation of plasmid D N A .  K802 was used for propagation of genomic D N A . BL21  (DE3) was used for overexpression of recombinant proteins. XL-Blue was used in sitedirected mutagenesis.  Table 2.1 List of bacterial strains. Strain DH5a  Genotype F \ O80 lacZ Ml 5, (lacZYA-argF), U169, deoR, rec Al, endM, hsdRll (r ~, m ), phoA, supE44, X', thi-\, gyrA96, relAX  K802  hsdR2~(vK~, mK ), hsdM , gal", metBl, supE44, rncrA', mcrB'  BL21 (DE3)  F~, ompT, hsdS(rs , m ~), dcm, galX(DE3) recAl, endAl, gyrA96, thi-l, AsdR17(r ~, m ) , supE44, X-, lac, [F'proAB, lacl ZAM15, Tnl0(Tet )l  +  K  K  +  +  B  +  K  XL-Blue  q  K  r  2.2.3 Molecular and biological methods 2.2.3.1 Purification of plasmid D N A from E. coli Plasmid D N A was purified from overnight cultures using either a Qiagen Miniprep Kit or a Qiagen Maxiprep Kit according to the manufacturer's instructions.  28  2.2.3.2 Polymerase chain reaction (PCR) Unless otherwise specified, amplifications were performed in a 50 ul or 100 ul reaction mix, containing 1 ul template D N A (-50 ng), 50 pmol of each oligonucleotide primer, 50 u M of each deoxynucleotide triphosphate, and 4 U of ID-Proof D N A polymerase (ID-Proof Incorporated) in a P C R buffer as described by the manufacturer.  Depending on  the length and the G C content of the oligonucleotide, the amplifications consisted of 30 cycles of 94 °C for 30 seconds, 50-70 °C for 60 seconds, and 72 °C for 90 seconds. A l l amplified D N A products were purified using the QIAquick Gel Extraction Kit (Qiagen) according to the manufacturer's instructions, digested with appropriate restriction enzymes and then further purified using the QIAquick PCR Purification Kit (Qiagen) according to the manufacturer's instructions. 2.2.3.3 Ligations A l l plasmid vectors were linearized with appropriate restriction enzymes and then gel-purified using the QIAquick Gel Extraction Kit (Qiagen). A l l inserts of interest were purified using either QIAquick Gel Extraction Kit (Qiagen) or QIAquick P C R Purification Kit (Qiagen).  Ligations were performed at room temperature for 1 hour or at 16 °C  overnight, with 1 U of T4 D N A ligase (Life Technologies) in 20 ul of 1 X T4 ligation buffer (Life Technologies). 2.2.3.4 Transformation of E. coli Transformations were performed either by electroporation or by the heat shock method (Sambrook et al., 1989). For transformation by electroporation, 1 ul of D N A was mixed with 50 ul of electrocompetent cells on ice for 5 minutes.  The mixture was  electroporated at 1.8 k V , 200 ohms and 25 uF using a Pulse Controller II (BioRad). For transformation by the heat shock method, 1 u.1 to 3 u.1 of D N A were mixed with 100 pi of competent cells on ice for 30 minutes, heat shocked at 42 °C for 45 seconds, then chilled on ice for 5 minutes. In both methods, 1 ml of L B mediun (10 g/1 Bacto™ Tryptone [DIFCO], 5 g/1 Bacto™ Yeast Extract [DIFCO], 10 g/1 NaCl) was added to each transformed sample and the suspension was incubated at 37 °C for 1 hour. Cells were then grown at 37 °C overnight on L B agar plates containing the appropriate antibiotic. Electrocompetent cells and heat shock-competent cells were prepared using standard methods (Sambrook et al., 1989).  29  2.2.3.5 Preparation of radioactive D N A probes 2.2.3.5.A Random Hexamer Extension Labeling The random priming labeling method was based on the kit provided by N E B . Ten u.1 of double-stranded D N A fragment (50-100 ng) were boiled for 10 minutes and chilled quickly on ice. The denatured D N A was combined with 3 u.1 of Reaction Buffer (20 m M HEPES [pH 6.6], 20 m M Tris [pH 7.1], 0.1 m M E D T A , 4 mg/ml B S A , 3.5 OD/100 ul pdN  6  [Promega]), 3 ul of Labeling Mix C Buffer (333 m M Tris [pH 8.0], 33.3 m M M g C l , 10 m M 2  (3-mecaptoethanol, 0.5 m M dATP, 0.5 m M dGTP, 0.5 m M dTTP), 30 uCi of [a- P] dCTP, 32  and 1 U of Klenow D N A polymerase (Life Technologies) to a final volume of 20 ul. The mixture was incubated at 37 °C for 30 minutes, and the reaction was terminated by the addition of 0.2 M EDTA. The unincorporated nucleotides and short D N A fragments were separated from the D N A probes on an 1 ml Sephadex G50 (Pharmacia) spin column. The purified D N A probes were denatured with 0.12 N NaOFf at 37 °C for 5 minutes before probing. 2.2.3.5. B 5'end Labeling Each primer (5 pmol) was labeled with 10 U of T4 polynucleotide kinase (Life Technologies) and 50 uCi of [Y- P] A T P in 20 pi of I X T4 polynucleotide kinase buffer 32  (Life Technologies). The labeling reaction was conducted at 37 °C for 45 minutes and terminated at 65 °C for 5 minutes. The 5'-end labeled oligonucleotide was precipitated with 2 M N H O A C and 2.5 volumes of 95% EtOH at -20 °C for 45 minutes, and recovered by 4  centrifugation at 13,000X g at 4 °C for 10 minutes. The pellet was washed with 80% EtOH, centrifuged as before, dried in a Speedvac evaporator and resuspended in 10 jul of water. 2.2.3.6 Screening of c D N A library 2.2.3.6. A. cDNA cloning of SpaPK A brain c D N A library (kindly provided by Dr. Mario Filon, University of Montreal) was screened with a probe corresponding to 814 base pairs of the SpaPK c D N A from position 192 to 1005. The library was transformed into E.coli strain DH5a, and plated onto ampicillin-containing L B plates (90cm) to yield approximately 50,000 colonies per plate. The plates were incubated at 37 °C overnight, and colony lift was performed for each plate using nylon membranes (Schleicher & Schuell). The membranes were sequentially washed in a denaturation buffer (0.5 M NaOH), a neutralization buffer (1 M Tris-HCl, 1.25 M NaCl), 30  and a washing buffer (2X SSC). After the membranes were air dried on Whatman® paper, the D N A was immobilized on the membrane by baking at 80 °C in vacuo. Membranes were then incubated at 42 °C for 4 hours in a prehybridization buffer (6X SSPE, 10X Denhardt's solution, 1% SDS, 50% deionized formamide, 25 pg/ml salmon sperm D N A , 62.5 pg/ml yeast RNA), and hybridized at 42 °C overnight with the same buffer containing the randomly primed radiolabeled probes. The blots were then washed twice at room temperature for 15 minutes each in a buffer containing 6X SSPE and 1% SDS, then washed twice at 37 °C for 15 minutes each in a buffer containing I X SSPE and 1% SDS. Positive signals were detected by exposing the membranes to Kodak X-Omat films with an intensifying screen at -70 °C overnight.  Agar plugs containing potential positive clones were excised from the plates.  Plasmid DNAs were purified and screened by restriction digestion and Southern blotting. Clones that were positive from the southern blot were further subjected to a second and third round of screening as described above. The resulting clone, designated III7CA, contained the largest insert but lacked the 5' end of the gene. Further overlapping EST clones were identified in subsequent searches of the dbEST section of GenBank. One of the EST clones containing the most 5'-end region of the gene is AA557328. Because of the presence of an internal Seal site in the SpaPK coding region, a wildtype construct using the EcoR l-Sca I fragment of EST AA557328 containing the 5' end of the SpaPK cDNA, and the Sea l-Xho I fragment of clone III7CA containing the 3'end of the SpaPK cDNA were cloned into EcoR l-Xho I-digested pBluescript II KS(-). This construct, designated pBluescript-SpaPK, comprised the SpaPK coding region, 143 nucleotides of 5' UTR and 665 nucleotides of 3' UTR.  A l l subsequent constructs were made using  pBluescript-SpaPK as a template. 2.2.3.6..B Primer extension The primer 5' - G G A C C T C A G G A G T C C C C T A A T G G C - 3 ' which is complementary to base pairs 54 to 77 of the SpaPK 5' UTR was used in the primer extension analysis. The primer was labeled by the 5'-end labeling method and purified by precipitation with NH40Ac/EtOH as described in Section 2.2.3.5 B. A l l primer extension reactions were assembled in a final volume of 10 pi. Two pmol of the labeled primer was mixed with 5 pg of brain total R N A (Clontech), denatured at 70 °C for 10 minutes and chilled quickly on ice. The mixture was then incubated at 42 °C for 2 minutes in I X First Strand Buffer (Life  31  Technologies) containing 50 p.g/ml actinomycin D, 10 m M DTT, and 0.5 m M each of dNTPs. One hundred units of Superscriptll™ reverse transcriptase (Life Technologies) were added to the above mixture, and the primer extension reaction was carried out at 42 °C, 44 °C, 47 °C or 50 °C for 45 minutes. Six ul of stop buffer (90% formamide plus tracking dyes) were added after the reaction was terminated at 70 °C for 15 minutes. Five ul of the reaction mix were loaded onto an 8% D N A sequencing gel and subjected to electrophoresis.  The  sizes of the primer extension products were determined by comparing with a D N A sequence ladder generated using the same primer and the clone pBluescript-SpaPK as a template. 2.2.3.6.C Isolation of 5' end of SpaPK from Marathon-ready ™cDNA library 5' Rapid Amplification of c D N A Ends (RACE) was performed using the human brain M A R A T H O N RACE-ready ™ c D N A library (Clontech) and the Advantage™ CloneTaq Polymerase M i x (Clontech). Amplifications were carried out in two rounds. The first round was performed with 10 u M of Adapter Primer 1 (Clontech) and 10 u M of a gene-specific primer (GSP5"#3) in the following PCR conditions: denaturation at 94 °C for 3 minutes, 10 cycles of 94 °C for 30 seconds, 63 °C (-0.5 °C per cycle) for 30 seconds and 68 °C for 2 minutes, 25 cycles of 94 °C for 30 seconds, 56 °C or 58 °C for 30 seconds and 68 °C for 2 minutes, and a final extension period at 68 °C for 10 minutes.  The second round of  amplification was performed using 5 ul of 1:250 dilution of the first round PCR products as the template, 10 u M of Adapter Primer 2 (Clontech) and 10 u M of nested gene-specific primers. The following P C R conditions were used: denaturation at 94 °C for 3 minutes, 30 cycles of 94 °C for 30 seconds, 68 °C for 30 seconds and 72 °C for 2 minutes, and a final extension period at 72 °C for 10 minutes. A l l resulting PCR products were gel purified and cloned into pSTBlue-TA vector (Novagen).  Potential positive clones were screened by  restriction digestion and confirmed by D N A sequencing. 2.2.3.7 Isolation of SpaPK from genomic library A human genomic library in E M B L 3 SP6/T7 phage (Clontech) was screened using a probe corresponding to the first 865 base pairs of the SpaPK transcript. For each 150 mm plate, a bacteriophage library that would yield approximately 50,000 plaque forming units (pfus) was diluted in 100 pi of Lambda Dilution Buffer (35mM Tris-HCl [pH 7.5], 100 m M NaCl, 20 m M MgS04, 0.01% gelatin) and combined with 600 ul of overnight culture of E.  32  coli K802 that was grown in L B containing 10 m M MgSC»4 and 0.2% maltose. The mixture was incubated at 37 °C for 15 minutes. Seven ml of melted L B soft top agarose containing 10 m M MgSC>4 were added to the cell suspension and poured onto pre-warmed L B agar containing 10 m M MgSCu. The 150 mm plates were incubated at 37 °C for 5 to 6 hours until the plaques were just about to make contact with one another, and chilled at 4 °C overnight. The next day, plaque lifts were performed using nylon membranes. (Schleicher & Schuell). The filters were soaked in a denaturing solution (1.5 M NaCI, 0.5 N NaOH) for 5 minutes, a neutralizing solution (1.5 M NaCI, 0.5 M Tris-HCl [pH 8.0]) for 5 minutes, and a 3X SSC washing solution for 10 minutes. The filters were dried, prehybridized, hybridized, washed, and subjected to autoradiography as described in Section 2.2.3.6 A . Agar plugs containing potential positive plaques were incubated with 1 ml of Lambda Dilution Buffer at room temperature for 2 hours. Those potential positives, plated at approximately 200 pfu per 90 mm plate, were further subjected to a second and third round of screening as described above. 2.2.3.8 Large-scale isolation of A, phage D N A Bacteriophage containing D N A of interest was plated at approximately 100,000 pfu per 150 mm plate. Lambda phage D N A was purified from 10 confluent 150 mm plates using Qiagen Lambda Purification Kit, according to manufacturer's instructions. 2.2.3.9 Isolation of total R N A from tissue culture cells A l l tissue culture cells, grown in monolayer, were scraped and homogenized in 0.75 ml of TRIzol solution (Life Technologies) at room temperature for 5 minutes. R N A was extracted with 0.2 ml of chloroform at room temperature for 2 minutes, and centrifuged at 13,000X g at 4 °C for 10 minutes. R N A was then precipitated from the upper aqueous phase with 0.5 ml of isopropanol at room temperature for 10 minutes, and recovered by centrifugation as above. The resulting R N A pellet was washed with 1 ml of 75% EtOH, recovered by centrifugation, air-dried and finally dissolved in RNase-free water.  The  concentration of R N A was determined by spectrophotometry at 260 nm. 2.2.3.10 Northern blot analysis Fifteen pg of total R N A or 3 pg of poly(A)+ R N A were mixed with an equal volume of R N A loading buffer (56% deionized formamide, 2.2 M formaldehyde, 7.5% glycerol. 0.3% bromophenol blue in I X MOPS buffer), heat denatured at 65 °C for 15  33  minutes, and separated by electrophoresis on a 1% formaldehyde (2.2 mM) agarose gel. The R N A was vacuum-transferred at 55 psi for 3 hours in 10X SSC onto Hybond-N nylon +  membrane (Amersham Pharmacia Biotech) and U V cross-linked to the membrane at 60 mJ using a GS Gene Linker U V Chamber (BioRad). For blotting with a c D N A probe, the membrane was prehybridized, hybridized and washed as described in Section 2.2.3.6 A . For blotting with oligonucleotide probes (Table 2.2), the membrane was incubated at 37 °C for 4 hours with an oligonucleotide prehybridization buffer ( I X SSC, 5X Denhardt's solution, 4 m M Pipes [pH 6.4], 25 u.g/ml salmon sperm D N A , 62.5 pg/ml yeast RNA), and hybridized at 37 °C overnight with the same buffer containing the 5'-end labeled oligonucleotide. The blot was then washed twice at 37 °C for 15 minutes each in a buffer containing 2X SSC and 1% SDS. A l l signals were visualized by autoradiography. 2.2.3.11 Southern blotting analysis D N A samples were separated by agarose gel electrophoresis and vacuumtransferred onto a nylon membrane (Amersham) at 50 psi. During the transfer process, the gel was incubated sequentially in 50 ml of a depurination buffer (0.2 N H O ) for 30 min, 50 ml of a denaturation buffer (0.5 M NaOH, 0.5 M NaCl) for 30 min, 50 ml of a neutralization buffer (1 M Tris [pH 7.5], 1.5 M NaCl) for 30 min, and 50 ml of a transfer buffer (20X SSC) for 1 hour. D N A was U V cross-linked onto the membrane at 150 mJ. The blot was then prehybridized, hybridized and washed as in Section 2.2.3.6 A . The signals were detected by autoradiography. 2.2.3.12 Double -stranded D N A sequencing Double-stranded D N A sequencing was performed using standard methods (Sambrook et. al), or by the D N A Sequencing Laboratory at the University Core D N A and Protein Services at the University of Calgary, or by the Nucleic Acid Protein Service Unit at the University of British Columbia.  34  2.3 RESULTS 2.3.1 Identification of human homolog of S. cerevisiae Rad53 -SpaPK To identify the human homolog of the S. cerevisiae cell cycle checkpoint protein Rad53, also known as Mec2, Sadl and Spkl, the amino acid sequence of the yeast protein kinase was used to search the human EST database at the National Center for Biotechnology Information. A human EST cDNA clone that is highly similar (61%) to the protein kinase domain of S.cerevisiae Rad53 was identified from the database using the T B L A S T N homology search program (default setting). This clone, EST n23936, was obtained from the Integrated Molecular Analysis of Genomes and their Expression (I.M.A.G.E.) Consortium, and nucleotide sequence analysis of the 814 nucleotide insert revealed a partial open reading frame. Using this EST cDNA as a probe, several clones were found from a human brain cDNA library. The D N A sequence analysis of the clone with the largest size of insert, designated III7CA, revealed a partial open reading frame that lacked an initiation codon.  2.3.2 Assembly of the composite SpaPK cDNA sequence To obtain the cDNA sequence of the human homolog of Rad53, the EST database was searched again using clone III7CA as the query. This B L A S T N (default setting) search resulted in the identification of an EST clone, AA557328, that encoded more of the 5' end of the gene including a potential initiation codon which is in-frame with the sequence homologous to Rad53. The combined D N A sequence information from all cDNA clones allowed inference and the construction of the SpaPK cDNA.  The SpaPK cDNA was  assembled from EST AA557328 which contained the 5' end portion of the cDNA, and the clone III7CA isolated from the human brain cDNA library which contained the 3'end portion of the c D N A (Figure 2.1). The gene was given the name SpaPK, for spindle pole associated protein kinase (see Chapter 4), and cloned into pBluescriptll K S (-) vector. In the following sections, this version of the gene is referred to as SpaPK cDNA.  2.3.3 General overview of the SpaPK cDNA sequence The resulting composite sequence of SpaPK c D N A consists of 2172 nucleotides which contains an open reading frame that potentially codes for a 454 amino acid protein of 52.5 kDa (Figure 2.2). The putative start codon is located from nucleotide 144 to 146. The  35  SpaPK cDNA  5'  3'  EcoRl  Not I EST n23936  EcoRl  Not I  EST AA557328  EcoRl  Xhol Clone III7CA  EcoRl  Seal  Xhol  pBluescript IIKS <-> 2958 bp  Figure 2.1 Construction of the SpaPK cDNA. The EcoR l-Sca I fragment of EST AA557328 containing the 5' end portion of the SpaPK cDNA, and the Sea l-Xho I fragment of clone III7CA containing the 3'end portion of the SpaPK cDNA were cloned into EcoR I-Xho I-digested pBluescript II KS . The top line represents the ( )  SpaPK cDNA. 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CJ 4J CJ 4J 4-1 CJ CJ CJ  Ol  CD  g- £  4-1  CJ CJ  4J 4-1 CJ 4J  CJ CJ CJ  4J CJ  in  01 01 (0 01 Dl  [H  (N  CJ  CJ CJ  4J CJ  n)  4J  01  CJ 4-1  01 Ol nl 0)  CO  ns  Dl Dl  Dl  CJ CJ  CJ CJ  CJ CJ  4J  4J  Dl Dl  CJ CJ  01  01 01 01  01  to Dl Dl Dl  IB  4J  4J  CJ  CJ CJ CJ CJ 4J  4J 4J  CJ  ns  01 01 01 nj 01 Dl 01 Dl  Ol  '3  CD  H—»  _cd CO  & tn cd  143 nucleotide 5'-untranslated region (UTR) of SpaPK c D N A does not contain an in-frame stop codon.  The sequences flanking the putative translation start site of SpaPK  ( C C G G C C A C C A U G U ) show similarity to the optimal sequence for the initiation of translation known as the Kozak consensus sequence, G C C G C C ( A / G ) C C A U G G , in which the A of the A U G codon is designated +1, with positive and negative integers proceeding 3' and 5' of A , respectively. The most conserved positions in the Kozak motif are a purine in position -3 and G in position +4. This configuration has been experimentally determined to support the highest efficiency of translation (Kozak, 1991). The SpaPK sequence, however, contains a purine at -3 but lacks a G at +4. The putative protein kinase domain which is homologous to the Rad53 protein kinase is encoded from nucleotides 144 to 968. The 3'untranslated region of SpaPK cDNA has 664 nucleotides. It contains a poly(A) tail that starts at position 2153 and is preceded by one polyadenylation signal, A A T A A A .  2.3.4 Tissue distribution of the SpaPK gene The level of expression of SpaPK mRNA in different tissues and cell lines was examined by Northern blot analysis. As shown in Figure 2.3, P-labeled random-primed 32  cDNA corresponding to nucleotide 192 to 1005 of the SpaPK c D N A hybridized to a single fragment of 2.4 K b from total R N A (Panel A and Panel B, lanes 1-3). To distinguish positive signals from non-specific hybridization to the 18S ribosomal R N A which migrates at 1.87 Kb, Northern blot analysis was also performed using poly(A) m R N A isolated from human endothelial cells. A band of 2.4 Kb was detected from the poly(A) mRNA sample (Panel A and Panel B, lane 4), indicating that the signal is generated from an mRNA transcript. The size of the detected SpaPK transcript is approximately 230 nucleotides longer than the size of the cloned SpaPK transcript. The SpaPK gene was expressed in all tissue and cell lines examined. The level of SpaPK expression was relatively higher in MCF-7 breast cancer cells and endothelial cells than in the brain (Fig 2.3).  2.3.5 Analysis of the 5' region of the SpaPK gene Several lines of evidence raised the possibility that the 5' end of SpaPK cDNA might be incomplete. First, the context of the putative Kozak sequence for initiation of translation  39  Figure 2.3 Tissue distribution of the SpaPK mRNA.  (A) A n ethidium bromide  stained formaldehyde-agarose gel containing total R N A (15 pg/lane) isolated from human brain (lane 1), MCF-7 (lane 2) and Jurkat tissue culture cell lines (lane 3), or poly(A)+ R N A (3 pg/lane) isolated from human endothelial cells (lane 4). (B) A Northern blot of (A) probed using a [ P] labeled random-primed c D N A fragment, 32  corresponding to nucleotides 192 to 1005 of the SpaPK cDNA. The sizes of R N A markers (2 pg/lane) are indicated (in Kb) on the left of each panel. The R N A bands recognized by the probes derived from SpaPK are indicated by an arrow on the right.  40  is not optimal. Second, there is no in-frame stop codon located in the 5' U T R region of the SpaPK cDNA. Third, the size of SpaPK transcript as detected by Northern blotting is larger than the composite nucleotide sequence derived from clones.  To further analyze the 5'  region of the SpaPK gene, several approaches, including primer extension, 5'-rapid amplification of c D N A ends (RACE), and cloning from a genomic library, were taken. 2.3.5.1 Primer extension analysis of the SpaPK transcript Primer extension experiments were carried out to determine the precise length of the SpaPK transcript.  A 5'-end  32  P-labeled primer ( 5 ' - G G A C C T C A G G A G T C C C C T A  ATGGC-3') complementary to position 54 to 77 of SpaPK (5' U T R region) cDNA was hybridized to brain total R N A and extended by the Superscriptll™ reverse transcriptase. The products of the extension were resolved on a sequencing gel (Fig. 2.4 A). To determine the size of the primer extension products, a D N A sequencing ladder using the same primer and the pBluescript-SpaPK clone as a template was electrophoresed adjacent to the extension products (Fig. 2.4 A , lanes A , C, G and T). Six shorter products of 46 bp, 47 bp, 48 bp, 49 bp, 50 bp, 53 bp, and 55 bp were observed from extensions performed at 42 °C, 44 °C, 47 °C, and 50 °C (data not shown). Several additional bands of 84 bp, 89 bp, 101 bp, 113 bp and 164 bp (Fig 2.4 A , lanes 1-4, arrows), as well as some possible signals at 131 bp, 135 bp, 147 bp and 154 bp, were detected from products extended at 50 °C (Fig 2.4 A , lane 4, question marks). The length of the largest product is 164 nucleotides. This suggests that the SpaPK transcript is potentially 87 nucleotides longer at the 5'end than the cloned SpaPK cDNA (Figure 2.4 B). Thus, the size of the SpaPK transcript is potentially at least 2259 nucleotides, where the transcription initiation start site is 230 bases upstream of the potential translation initiation codon. 2.3.5.2 5'-RACE PCR of SpaPK cDNA To identify and isolate the missing sequence of the 5' end of the SpaPK cDNA, 5'R A C E was performed using a human brain M A R A T H O N RACE-ready™ c D N A library. Several primers specific for the SpaPK cDNA sequence were used (Table 2.2). Initially, 5'R A C E touch down P C R was performed at 56 °C or 58 °C with primer 5'RaceGsp5'3 which is complementary to nucleotide 1043 to 1070 of the SpaPK cDNA.  To increase the  specificity of the reaction, 5'-RACE was performed with nested primers which anneal to the 3' of primer 5'RaceGsp5'3. These primers are Gsp5'2, Gsp5'4 and Primerext which are  41  M A T G C  1 2  42  3 4  B  1508  144  2173 "AAAAAAA  54  ?  77  144  — primer extension oligonucleotide *- primer extension product (164 nucleotides) Figure 2.4 Primer extension analysis of the SpaPK gene. (A) Primer extension of SpaPK.  A primer complementary to nucleotides 54 to 77 of the 5'-untranslated  region of the SpaPK mRNA was hybridized to 10 ug of total brain R N A . The same oligonucleotide was used to prime D N A sequencing of pBluescript-SpaPK and the products of the reaction (lanes A , T, G and C) were run on the same sequencing gel. The primer extension  reactions  were  catalyzed by  Superscriptll™  reverse  transcriptase at 42 °C (lane 1), 44 °C (lane 2), 47 °C (lane 3) and 50 °C (lane 4). The positions of molecular size D N A markers (lane M) are indicated on the left. The positions of definitive and possible primer extension products are indicated by arrows and questions marks, respectively. (B) Schematic representation of the 5' end of the SpaPK transcript. The region in the open rectangular box is enlarged to illustrate the results of the primer extension reactions. Comparing to the size of the primer extension product which is 164 nucleotides, there appears to be 86 nucleotides missing from the 5' end of the cloned SpaPK cDNA. The position of the missing nucleotides is represented by a question mark over a dotted line. The rectangular box (diagonal stripe) represents the potential open reading frame encoded by the SpaPK cDNA starting from position 144 and ending at position 1508. The uni-directional arrow indicates the position of the oligonucleotide for primer extension.  The bi-directional arrow indicates the position of the primer  extension product.  43  complementary to nucleotide 252 to 269, 146 to 167 and 54 to 77 of the SpaPK cDNA, respectively (Table 2.2). PCR products of or close to predicted sizes were gel purified and sub-cloned into pSTBlue-TA vector followed by sequencing. A summary of the resulting 5'RACE PCR products is presented in Table 2.3. The sequence of the largest PCR product generated by primer Gsp5'2 matches its corresponding SpaPK c D N A sequence from nucleotide 56 to 269. Gsp5'4  and  The sequences of the largest PCR products generated by primers  Primerext  both  revealed  16  additional  nucleotides  (5'-  G G C T T C T G G A G G C G G C - 3 ' ) at the 5'end. However, they are 70 nucleotides shorter than the predicted sizes (see Discussion). The new sequence combined with the existing SpaPK cDNA sequence still shows a lack of an in-frame stop codon upstream of the potential initiation codon A T G . Subsequent searching of the nucleic acid database (GenBank™) and the EST database using the newly identified 16 nucleotides as the query did not result in any matches. 2.3.5.3 Genomic structure of the SpaPK gene To further determine the 5' region and also to map the promoter region of the SpaPK gene, a probe corresponding to the first 865 nucleotides of the SpaPK c D N A clone was used to screen a human genomic library. The isolated phage D N A from potential positive clones was subjected to restriction enzyme digestions followed by Southern blotting probed with the same cDNA fragment used for cloning from the genomic library. D N A fragments that displayed positive signal were purified, sub-cloned into pBluscriptll KS(-) vector, and sequenced.  Sequencing of plasmids containing inserts was carried out in both directions  using M l 3 Forward and M13 Reverse primers, and subsequently by primer walking using other internal primers derived from the sequenced region (Table 2.4 and 2.5). Clones that revealed the most sequence information were GIII5ASst, GIII5Pst and GIII5Eco0109. Since sequences of the latter two clones overlap by 710 bases, they were combined and the resulting sequence was designated GIII5Ecol09/Pst. Clone GIII5ASst consists of 4225 nucleotides, and its sequence from nucleotide 1933 to 3330 matches nucleotides 1 to 205 of the SpaPK cDNA (Figure 2.5).  The sequence of GII15Eco0109/Pst consists of 4819  nucleotides, and its sequence from nucleotide 3748 to 4495 matches nucleotides 206 to 696 of the SpaPK cDNA (Figure 2.5). During the course of this study, the sequence of the human genome was determined (listed, 2001). Sequence comparison of the genomic clones with  44  Table 2.2 Oligonucleotide primers for 5' R A C E PCR of the SpaPK cDNA.  Name  Nucleotide sequence  Coordinates  5'RaceGsp5'3  5 '-CGACTTGATGGTGTACTCCTTCAGACGC-3'  1043 - 1070  Gsp5'2  5 '-CTTGGCTGCGTACTCCTT-3'  252 - 269  Gsp5'4  5' - C T C C T C C T G C C T G A A C G T G G A C - 3 '  146- 167  Primerext  5 '-GGACCTCAGGAGTCCCCTAATGGC-3'  54-77  Table 2.3 Summary of 5' R A C E PCR of the SpaPK cDNA. Name of oligonucleotide primer  Predicted size of PCR product  Actual size of PCR product  Presence of new sequence  Gsp5'2  339 bp  300 bp  no  Gsp5'4  233 bp  182 bp  yes  Length of new sequence  16 bp  5' end position  3  Length of missing sequence  56  143 bp  -16  70 bp  141 bp Primerext 92 bp yes 16 bp -16 70 bp a The 5' extremity of the PCR products relative to the SpaPK cDNA. The first nucleotide of the 5'-untranslated region of the SpaPK cDNA is defined as nucleotide 1.  45  High-Throughput Genomic Sequences (HTGS) using the B L A S T N program (default setting) resulted in the identification of clone CTB-171N13 from chromosome 19. The 132817 nucleotides of clone CTB-171N13 is a working draft sequence, and is made of four contigs separated by four gaps.  The SpaPK gene is located within the last contig (26906 bp),  between nucleotide 105912 and 132817. The sequences of GIII5ASst and GIII5Eco0109/Pst match the sequences on the "minus" strand of clone CTB-171N13, from nucleotide 117937 to 122162 and 113078 to 117891, respectively (Figure 2.5). The exon-intron boundaries of the SpaPK gene were determined by aligning the SpaPK cDNA sequence with clone CTB-171N13, GIII5Asst and GIII5Ecol09/Pst (Table 2.6). The SpaPK gene extends over 11.3 Kb of the human genome. It is organized into 9 exons separated by 8 introns (Figure 2.5).  Sizes of the exons range from 27 to 361  nucleotides, while introns vary from 201 to 4682 nucleotides. As shown in Table 2.6, the exon/intron boundary sequences conform to the consensus splice donor and acceptor sequences (Alberts et al., 1994). Each splice donor at the 3' intron sequence begins with GT and each acceptor site at the 5' intron sequence ends with an A G preceded by a polypyrimidine tract (Alberts et al., 1994). There are three types of intron splice phase (Rogers, 1985). The intron that occurs between codons is type 0, the intron that interrupts the first and second bases of the codon is type I, and the intron that interrupts the second and third bases of the codon in type II. In the SpaPK gene, the intron splice phase is type 0 for introns 3 and 8, type I for intron 4, and type II for introns 1, 2, 5, 6 and 7. The potential translation initiation codon (ATG) is located within the second exon.  However, the  interruption of the 5'-UTR region by 1189 bp of genomic sequence suggests that the cloned SpaPK cDNA (Section 2.3.3) is incomplete at 5' end and that translation possibly starts upstream of the predicted initiation codon. Cloning of GIII5ASst, GIITSPst and GlII5Eco0109 revealed  1932 additional  nucleotides upstream of the 5' end of the SpaPK cDNA, while the genomic sequence of clone CTB-171N13 contains an additional 10655 nucleotides upstream of the 5' end of the cloned SpaPK cDNA.  Therefore, clone CTB-171N13 was used in subsequent analysis to  study the 5'-end region of the SpaPK gene. The 16 additional nucleotides identified from 5'R A C E PCR (Section 2.3.5.2) matched the genomic sequence immediately upstream of the 5' extremity of the SpaPK cDNA, and does not match genomic sequences located elsewhere on  46  Table 2.4 Oligonucleotide primers for sequencing of GIII5ASst genomic D N A .  Name  Nucleotide sequence  Coordinates"  Geno9  5 '-CTACTTCAAGTGACCTGCCCGCC-3'  284 - 304  Genol3  5 '-GTGGTGGCGTGCACCTGTAGTCCC-3'  528 - 551  Genol2  5 '-AATGCAAATGAGGTCAATCTGGGG-3'  1407- 1429  Genol1  5 '-AGCGCCGCTCTGGCCCCACCC-3'  2017- 2037  Geno7  5' -CTGC A G C C T G G A A C T C C A A G G C - 3 '  2644 - 2665  Gsp5'4  5 '-GTCCTCCTGCCTG AACGTGGAC-3'  3270 - 3291  GenoS  5' -GCC A T T A G G G G A C T C C T G A G G - 3 '  3178 - 3198  Geno6 a  5 '-CTACAGGCATG AGCTACCACACC-3' 3706 - 3728 Nucleotide numbered according to GIII5ASst genomic D N A .  Table 2.5 Oligonucleotide primers for sequencing of GIII5Pst/Eco0109 genomic D N A . a  Name  Nucleotide sequence  Coordinate  Geno2  5 '-CACCCCGCTGGGAACAGCAGCC-3'  513 - 534  Geno5  5'-ACGGAATGTGA A ACGTGCCTC-3'  1806 - 1826  Geno3  5 '-CCCGGCTCCACGCTGTCTCCTG-3'  2450-2471  Geno 1  5'-GCAAGGCAAGTGCCAGATCCGC-3'  3085 - 3106  Gsp5'2  5 '-CTTGGCTGCGTACTCCTT-3'  3791 - 3811  Gsp5' a b  5  5 '-CGATCTTGTGCGCGATGCCG-3' 4428 - 4447 Combined sequence of GIIIEcoO 109 and GIII5Pst. Nucleotide numbered according to GII15Eco 109/Pst genomic D N A .  47  bo  c -a  >-l  O  *  O  bO  00  OO  X  'rvl  00  c o  oo  CN  o  00  OO CN CN o  s  00  ON  co  ON TT TT l  oo in  Tt-  TT  co  m  co  i  io  ON  CN O  o  m  oo C«~l O N ro oo  CN  U Sb  CO  co  ON  ON TT  TT  CO  ON  oo  CN O  ON  oo  CN  O CO  NO  co CN m OO  o  o  00 <0  ON TT  NO NO  CO  co  co  oo Tt-  T f  m CN  NO  w  C <U 3 ^  v  CN  ON  O  CN  CN  m m  CN  oo  T—H  i—i  CN  '—'  co  O CN  ON 1—1  oo  CO  o '—1  CN  TT  m lO  T f  m NO  ON  o  o  '~  o  CN  ON  NO  o  CN  o  NO 1  o  ,—i  Tt-  H  CN  in  NO  ON  ON  oo o  CN  r~-  O 1  1  '—  1  (—1  s  tu  a o c  tu _o O 73  ^  tu  H—'  too  CO  tu O Cfl  CO  CU  o c c o CL) §1 < u C/l  o  bfj 03  60 03 03 03  bO bo bo 03  CJ) bO o  CU  o c tu tU C/3 c o  CN  o bO o  H—»  bo 03 bo bO bO  o  bO bo bO  bfj  H—»  bO  bfj 03  o  H—'  o  bo  o CJ tu  H—»  bO 03  bO +-» bO bo 03 03  £P  ,x  w  bfj bfj  o  bO bo  bfj  tu  O  bO » +-» bfj  bfj 03 bfj  bl) bfj bO  tu  *  bfj  03  bO  o  03  bo 03 03  03  bfj bO  bl) bD  bfj  ,  CJ  bfj 03 bO  o  03  top I  03 bo  03 CJ  bfj  t«  bfj  bo  +->  bO bO bO bO  o o H->  BP O CU  tu 03  +J  O  03  bO  H—»  CJ  bl)  03 03 03  03 03 toO bO  tu o  CJ 03  03  u  t3  03  -4—*  O O CJ  03  bf)  bD  bfj bO  bfj bO  bfj  c3  bO  03 03 CJ  o CJ o  tu O  M l  bO  o, 03  top|  tu CJ  bO bO bO bO  H—»  bO o3 03  H—•  bO  bO  bO 03 03  03  O CJ  H—» H—>  H—' H—»  o  03 bO bo  bO bfj  o  bO 03 rt  bO ,  bO too bO  o, bO  bO  u  o H—»  03 bo  cj  bO bO 03  tu u o c a o cu CU  O  o o  CU  <u  Q  o CJ  ON  tu -+—»  bo  o  in  o 00 g  03  -4—>  C/J  <"  bo  CJ  CT  ia.s  bo bO 03 bO bO  +-» 03  1)  |T3 O3  CJ CJ  CU  03  -t—'  tu o  bO  o  o CN i  o CO  CJ  NO NO CO I  NO  o CN  bo  bo  O  o,  03  o o o  03  bfj  03 CJ  o CJ  NO ON NO  bfj  H—*  in  CJ  o o o  +-> .  bo  in  CN  TT  bo CJ  o  H—» ,  Tl-  bO 03  o o  bo 03  o  H—»  CJ  bo bO  m CN ON  NO ON NO  60 03 o,  bo  o  i r-~ NO  bO 03  ON  H—* H—*  CJ  o m o m  NO  CN  ON  ON  CN  o ON  48  C«  c o  CQ H U  CD 4—>  in  o IT)  o c CD  1) 43 S3 CD  co  ON  ON '  -3 CD  c cd  O  CM O  .S  C N  > o  T3 C  cd CD  co  33 S3 CD  43  H  O  o  43  CD  43 H—<  cd  CO S3 O  S3  CD CD CO  O O  o u  <N  CD  S3  co PH  W  in  o H->  CD  oo  o  CO CD  43  NO ON  -ON  \0"  H S3 CD OO  NO  in  SO  m  -o CN  i4 PH cd  a oo CD  cd CO S3 O  X  CD  O CD  & 00  w  43  S3  cd  CD  > O 43  cd CD  43  s =3 S3  S3  ^ PH cd  o  cd  H  ON O  CD  43  43  _> CD CD  O  CD  CD  S3 O  CD  43  -o c  cd  £  ON  o  c CD  43  -r-  PH  CD  CD  ON  CD  CD  CD  S3  l-H CO CD  43  X  o  42  CD  tH  \Q  in .CN  ft  CD  CD  r-v.  CD CD  CD  CD  PH  CN  cd  CD  PH  cd  53 O X CD  S3 CD CD  CD  43  m CD  S3  CO cd  -3 CD  -4—*  Q CD PH cd  PH  < < cco  00  CH  O  cd CO  _0  CO  00  CD  CD  43  -4—»  CD  43 H—' <+H  43  O  CD  CD  =3  ON  oo  -4—*  r--  s m  in  ON  r--  •••© CN O  m  CN  •S o c  CD  a  £  *~  H  T CJ CD  c o  CM  o  CD CJ  S3  CM CN  3 OX)  CD  =3  cr CD  co  49  CD  O  CO CD H—»  CD  43  -4—*  -a  cd  H—*  ON  JU  ~o =3 S3 CD  43  "o 33 S3 CD  43  S3  T3  o  4 -—> O  S3  CD  rs CD  43  cd  o o  o S3H  C+-H  CD  co  • ON  cd  O  co S3 O  X  CD  -o S3 o  S3H  CO  t CD  O  CD  chromosome 19.  This suggests that these 16 nucleotides belong to the same exon as  nucleotide 1 to 49 of the SpaPK cDNA (Table 2.6). Further analysis of genomic sequences revealed an in-frame stop codon 37 nucleotides upstream of the new sequence or 53 nucleotides upstream of the SpaPK cDNA. The absence of an in-frame A T G initiation codon between the new sequence and the stop codon implies the existence of another upstream exon. Two potential acceptor splice sites for type I and type II introns were identified within these 37 nucleotides (Alberts et al., 1994; Rogers, 1985). The distance between sites of Type I intron and Type II intron and the last base of exon 1 is 73 and 90 nucleotides, respectively. In summary, the results of the primer extension experiments indicate that the SpaPK transcript extends at least 87 nucleotides beyond the 5' extremity of the SpaPK cDNA. Sixteen of the 87 nucleotides were identified by 5'-RACE P C R and may be part of exon 1, according to the analysis of the genomic sequence.  The unknown 71 nucleotides contain  sequence from part of exon 1 and an upstream exon. 2.3.5.4 Identification of the promoter region and 5' end the SpaPK gene In eukaryotic cells, all genes encoding protein products are transcribed by RNA polymerase II. R N A polymerase II, in concert with other important regulatory proteins, binds to the promoter region of a gene for transcription. Nearly all genes transcribed by RNA polymerase II contain a motif called the T A T A box (Smale, 1997). Using web based programs at the Sanger Center, TSSG and TSSW, which predict the human R N A polymerase II binding region and the start of transcription of a gene, five potential T A T A box promoters were found in sequences upstream of exon 1 of the SpaPK gene in clone CTB-171N13. For each predicted T A T A box, several criteria were used to determine the location of the potential translation initiation codon (ATG) and the position of the splice donor site in the upstream exon. First, the T A T A box is located 25 to 35 bases upstream of the transcription start site.  Second, the sequence surrounding the A T G  immediately downstream of the transcription start site shows similarity to the Kozak consensus sequence for the initiation of translation. Third, the distance between the start of transcription and the A T G is ideally between 17 to 80 nucleotides. Fourth, the intron is either Type I or Type II in order for in-frame read-through of the SpaPK cDNA to occur. Fifth, the overall length between the start of transcription and A T G , A T G and the splice donor site, and splice acceptor site and the last nucleotide of exon 1 is at least 136  50  nucleotides. Following these criteria, five oligonucleotides complementary to the predicted A T G translation initiation codon of the new exon and its upstream sequence were designed for Northern blotting (Table 2.7). A summary of above predictions for each T A T A promoter is presented in Table 2.8. If the sequences surrounding the predicted A T G sites are part of the SpaPK cDNA, then Northern blotting using oligonucleotides complementary to these sequences listed in Table 2.7 against total R N A should yield a positive signal of 2.4 Kb. Northern blotting using the [ T ] labeled random-primed cDNA fragment corresponding to nucleotide 862 to 2172 of the SpaPK cDNA or the 5' end-labeled PrimerExt oligonucleotide served as positive controls. As shown in Figure 2.6, both positive controls yielded a band of 2.4 Kb (lanes 1 and 2), although the signal from the PrimerExt oligonucleotide probe is very weak. Probing using Northern4 (lane 5) and Northern7 (lane 7) did not show any signal, suggesting that their complementary sequences are not part of the SpaPK transcript.  Probing using  Northem3 (lane 4) and Nothern6 (lane 6) detected bands smaller than 1.35 Kb. Probing using Norfhern2 (lane3) did not reveal any specific signal but a smear of products.  In  summary, Northern blotting using oligonucleotides listed in Table 2.7 did not result in a signal of the predicted size.  This suggests that the sequences complementary to these  oligonucleotides are not part of the SpaPK transcript.  51  Table 2.7 Oligonucleotide primers for Northern blotting.  Name  Nucleotide sequence  Coordinates  Nothern2  5 '-CATGTTGGCCAGGATGGTCTC-3'  121377 - 121397  Northern3  5 '-CATTCTCCTGCCTCAGCCTCC-3'  120976- 120996  Northern4  5 '-CATGCTGAGCCGTGCCTCACC-3'  125585 - 125605  Northern6  5' - C A T G C T T G G G C A A G A C C T G G T - 3 '  129914 - 129934  3  Northern7 5' -C A T T C T A A A T A C T T A A A A T G C - 3 ' 120417- 120437 a Nucleotide numbered according to clone CTB-171N13 genomic D N A .  52  T3  c  G  .2 <~ a  °  -pi  <+J  TO  J=  PH  O o C ~ t 2 " C S g T3 O  Cd  , H_J  r-J  100 fa 3 O  rO r-o  PH  PH  X o  PH  G  X  CD  ON  C+H  o  o  CD  c o  CD CD r-<  co  CN  O TO  PH  X  X  ;>>  CD  cj  < z Q  >-H  c o  CD  PH  X  o. •  c  PH  X  PH  X  PH  PH  X  oo  ON  c o  PH  PH  cd  cd I 35  15  CD CO T J 3 CD CO CJ Id CD CD N $ cr ° O O cC oD  X  CO  <l CJ  < < u o  o a  a  <  < U  H  < o a  <  x  O CN  NO  a o  to  1  co  X  PH  o < u  H  U  cd  X o  a  < u  o < u u  O: H  w  CO  PH  CO  PH  X  CM" CO  o.  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CD  Xa  CD  cd  O "cd rg  SH  NO  o  >-H  < F—  CO p i  p  Z  cd  CD  C  CD O rO  G  X  X o  PH  CD CD  T3 CD >-  "G ' o o  cd  0/)  CD cd  o  G  1  1  ^  G O  £  O  -a f— f— O CD o H c d  cd  o  o  a <  D  T3  G  S-H  o o E—i  PH  co O  D CD ^ m C CO f- Gcd •GG sCD_ u rg O CD G C^D X CO -a CD T3  G  TO  — f  .2  G  00 cd CO "to  X  cd co co  cd  I-a  G  O o CD  X  T 3  TO  cd  G CD  !>  G  o  CD  >^ cd  CJ  X  O CO  C D co  OJ ^  G G  PH  CO  CT  ^  i_ -a x  cd  CD  PH  3 G  U~  O  H  D  G C <H_,  PH X  l. 5 CD ^  X  CH  M  G  G  B  Z Z  G C  C^  O TO  X  a  M  c  o  CD  H  X  ag , C  fa .5  G  Z  00  Figure 2.6 Identification of 5' end of the SpaPK cDNA. Northern blotting of total RNA (15 pg/lane) isolated from MCF-7 tissue culture cells, probed using [ P] 5' 32  end-labeled oligonucleotides which are complementary to each of the predicted translation initiation codons and their surrounding sequences. The oligonucleotides, as described in Table 2.7 and Table 2.8. are Northern2 (lane 3). Northern3 (lane 4). Northern4 (lane 5). Northern6 (lane 6). Northern7 (lane 7). [ P] labeled random32  primed cDNA fragment corresponding to nucleotides 862 to 2172 of the SpaPK cDNA (lane 1) and the 5* end-labeled PrimerKxt oligonucleotide (lane 2) were used as positive controls. The RNA fragments recognized by labeled SpaPK cDNA and PrimerKxt oligonucleotide are indicated by an arrow. The sizes of RNA markers (2 pg/lane) are indicated (in Kb) on the left of each panel.  54  2.4 DISCUSSION 2.4.1 Evidence for an incomplete SpaPK cDNA Independent of the cloning of the SpaPK cDNA in our laboratory, another research group isolated a gene encoding a novel protein kinase from two-hybrid screens searching for binding partner(s) of murine activating transcription factor 4 (ATF4) (Kawai et al, 1998). The search resulted in the identification and characterization of human ZIP kinase from a placenta c D N A library and mouse ZIP kinase from a brain c D N A library. ZIP kinase was also isolated from a HeLa cell cDNA library by another research group, in search for a kinase capable of phosphorylating the regulatory light chain of myosin II (MRLC) (Murata-Hori et al., 1999). Screening of the HeLa cell cDNA library was performed using a partial cDNA fragment encoding the kinase and the calmodulin regulatory domains of the bovine stomach smooth muscle M L C K . The putative open reading frames of SpaPK and HeLa ZIP kinase are identical. However, comparison of the nucleotide sequences of SpaPK or HeLa ZIP kinase to those of placenta ZIP kinase revealed three base changes in the coding region (not shown), although these differences did not affect the protein sequence.  Thus, all three  cDNAs encode the same protein. The length of the 5' end of placenta ZIP kinase and HeLa ZIP kinase is 50 nucleotides and 98 nucleotides shorter than that of SpaPK, respectively. Interestingly, the 5' extremity of placenta ZIP kinase c D N A corresponds to the second nucleotide of the second exon of the SpaPK cDNA.  Although neither research group  reported the possibility of a partial cDNA sequence, our results indicate that the SpaPK cDNA is incomplete. 2.4.1.1 The size of SpaPK transcript is longer than the isolated clone The size of the SpaPK transcript, as detected by Northern blotting, is approximately 2.4 Kb.  However, cloning resulted in the identification of a SpaPK cDNA of 2172  nucleotides, which is smaller than expected. Assuming that the poly (A) tract is 80 to 200 nucleotides in length, which is the range of the length of poly (A) tract of eukaryotic mRNAs (Sarkar, 1997), up to 170 nucleotides are missing from the cloned SpaPK cDNA. 2.4.1.2 Absence of an upstream in-frame stop codon within the 5' U T R of the SpaPK cDNA Examination of 144 nucleotides of the 5' UTR of SpaPK c D N A did not reveal an inframe stop codon, raising the possibility of a longer SpaPK open reading frame initiated at an upstream methionine. Although there are proteins which lack an in-frame stop codon within  55  their 5' untranslated regions, the presence of at least one upstream in-frame stop codon does definitively imply that the putative initiation methionine is the first translated amino acid of a gene. Thus, additional unidentified sequences may be located upstream of the 5' extremity of cloned SpaPK cDNA. 2.4.1.3 The sequence surrounding initiation A T G is not an optimal Kozak The optimal Kozak sequence for initiation of translation is GCCGCC(A/G)CC A U G G , where the underlined nucleotides A U G encodes the translational initiation codon (Kozak, 1986; Kozak, 1987b). The sequences surrounding the putative initiation methionine of SpaPK cDNA, C C G G C C A C C A U G U , do not perfectly match the Kozak consensus, suggesting that the A U G is not the site of initiation of translation. However, although 97% of the vertebrate mRNAs have the required purine at position - 3 , very few possess the full consensus sequence (Kozak, 1991). The presence of a modest amount of secondary structure near the start of the coding sequence is important in compensating for the sub-optimal context around the A U G initiation codon (Kozak, 1991).  A hairpin of 20 nucleotides,  located 12 to 15 nucleotides downstream from the A U G initiation codon, contributes to the fidelity of initiation possibly by preventing the 40S ribosome from scanning too fast or too far (Kozak, 1990). This is especially important for mRNAs which initiate translation at an A U G codon in a very weak Kozak context lacking both a purine at position -3 and a G in position +4 (Kozak, 1987a), or at a non-AUG initiation codon (Kozak, 1990). An R N A structure prediction program generated two possible hairpins immediately downstream of the putative initiation A U G codon of the SpaPK cDNA. 2.4.1.4 5' UTR of the SpaPK cDNA is interrupted by genomic sequences A search of the human genome database resulted in the identification of genomic clone CTB-171N13 from chromosome 19. The structure of the SpaPK gene was determined from a comparison of the genomic sequence and the c D N A sequence. It is organized into 9 exons separated by 8 introns (Section 2.3.5.3). Interestingly, the supposedly continuous 5' UTR of SpaPK cDNA is interrupted by nucleotides of genomic sequences.  This strongly  suggests that the sequences upstream and downstream of the interruption site are part of different exons. Although unusual, genes that contain non-coding exons have been reported (Denger et al, 2001; Mummidi et al, 1997; Wilhelm et al, 2001). Non-coding exons, found  56  at the 5' end of a gene, are combined via splicing with downstream exons where the initiation of translation begins. The significance of non-coding exons is unknown. 2.4.1.5 Primer extension analysis Primer extension analyses using human brain total R N A indicate that 87 nucleotides are missing from the 5' extremity of the cloned SpaPK c D N A (Section 2.3.5.1). Surprisingly, the primer extension reactions performed at different extension temperatures generated many products. The shorter products are present in all primer extension reactions. Longer products, in lesser quantity, are present only in reactions performed at the highest extension temperature tested. The heterogeneity of the sizes of the resulting products is most likely due to the presence of secondary structures on the m R N A template. Non-specific annealing of the primer is unlikely since DNA sequencing using the same primer generated the expected sequences.  Thus, there are potentially even longer upstream sequences,  however the reverse transcriptase might have been unable to pass through the secondary structures under the experimental conditions utilized. 2.4.1.6 5' R A C E Despite optimization of PCR conditions, 5' R A C E using a human brain Marathonready™ cDNA library resulted in the identification of only 16 additional nucleotides (Section 2.3.5.2). This size is 70 nucleotides shorter than the size predicted from primer extension analyses. The difficulty of obtaining more sequences could be due to high G C content. The overall G C content of the 5' U T R of SpaPK cDNA is 68%.  The G C content of the  nucleotide sequences upstream and including those complementary to the primer (PrimerExt) used in 5' R A C E is higher, at 74%. The difficulty could also due to an absence of the complete SpaPK cDNA in cDNA library, which is synthesized from mRNAs using reverse transcriptase. Even when taken into account these additional 16 nucleotides, there is still no in-frame stop codon in the supposed 5' UTR of SpaPK cDNA.  2.4.2 Promoter region of the SpaPK gene 2.4.2.1 T A T A box-containing promoters in nearly all eukaryotic genes, accurate initiation of transcription is dependent on a T A T A box located 25 to 30 bp upstream of the transcription start site (Breathnach and Chambon, 1981; Grosschedl and Birnstiel, 1980a; Wasylyk et al, 1980). TATA-binding  57  protein (TBP), together with other TBP-associated factors, is part of a multiprotein complex called TFIID which interacts specifically with the T A T A box (Cavallini et al, 1988; Hernandez, 1993; Parker and Topol, 1984). Binding of TFIID to a T A T A box nucleates the formation of a pre-initiation complex containing R N A polymerase II and several other general factors (Buratowski et al, 1989; Conaway and Conaway, 1993; Dynlacht et al, 1991; Van Dyke et al, 1988; Zawel and Reinberg, 1995; Zhou et al, 1992). Five putative TATA-containing promoters were predicted from sequences upstream of the SpaPK gene on genomic clone CTB-171N13.  The gene immediately upstream of the SpaPK gene on  chromosome 19 is EEF2 whose 3' extremity is 4416 nucleotides upstream of the 5' extremity the SpaPK gene. Two of the putative TATA-containing promoters, most upstream from the SpaPK gene, are located within the EEF2 gene. Northern blotting using oligonucleotides that are complementary to sequences downstream of the predicted T A T A boxes failed to detect signals equivalent to the size of the SpaPK transcript (Section 2.3.5.4), suggesting that these sequences do not belong to the SpaPK transcript. 2.4.2.2 TATA-less promoters The consensus T A T A box directs the transcription of the vast majority of genes, but evidence accumulated over the past years indicates that many promoters do not contain T A T A boxes (Smale, 1997). Promoters of TATA-less genes are categorized into GC-rich and AT-rich. GC-rich promoters are found primarily in housekeeping genes, and AT-rich promoters are often constitutively active and regulate the expression of genes involved in differentiation and development (Smale and Baltimore, 1989).  TATA-less initiation of  transcription within eukaryotic protein coding genes is dependent on an element known as the initiator (Inr) (Chen and Struhl, 1985; Grosschedl and Birnstiel, 1980b; Smale and Baltimore, 1989). Inr can accurately direct transcription initiation by R N A polymerase II in the absence of other control elements and achieve high level of transcription when stimulated by an upstream activator (Smale and Baltimore, 1989). A loose consensus Inr sequence of Py-Py-A-N-T/A-Py-Py, where A is the site of initiation and Py represents pyrimidines, was determined from functional assays using over 80 randomly generated and specific Inr mutants (Javahery et al, 1994). The possibility that the SpaPK transcript is derived from a TATA-less promoter was tested.  A search of the genomic clone CTB-171N13, using TSSG and TSSW search  58  programs, found two highly probable TATA-less start sites. These two start sites are located 136 and 54 nucleotides upstream of the 5' extremity of the SpaPK gene. Oligonucleotides complementary to sequences downstream of these sites were used as probes in Northern blotting.  Neither probe detected a band corresponding the size of SpaPK transcript,  suggesting that transcription of the SpaPK gene is not dependent on these two putative TATA-less initiation sites (data not shown). Recently, another genomic clone, CTD-2622U3, containing the SpaPK gene was found from a search of human HTGS database. This genomic clone carries additional 64525 nucleotides from the 5' extremity of SpaPK cDNA, 53870 nucleotides longer than the previously identified genomic clone CTB-171N13. A detailed analysis of the sequences may reveal insight about the promoter region of the SpaPK gene.  2.4.3 Expression of the SpaPK gene 2.4.3.1 SpaPK is expressed in a wide variety of tissues and cell lines One hundred forty six ESTs corresponding to segments of the SpaPK cDNA have been found from many different tissues and cell lines. Quantification of ESTs from various tissues in Unigene clusters shows that the SpaPK cDNA is most frequently expressed in thymus, followed by germ cells, eye, bone, colon and foreskin (GenCard, 2002). Reverse transcriptase-PCR of the ZIP kinase transcript detected signals from fourteen tissues, but not the spleen (Saito et ah, 1998). These results suggest that the SpaPK gene is widely expressed in various tissues. 2.4.3.2 SpaPK gene is not alternatively spliced in brain, breast, and endothelial cells Northern blotting detected a single band of 2.4 Kb from the brain, MCF-7 breast carcinoma cell line, Jurkat and endothelial cells (Section 2.3.4). A lack of signals of larger or smaller sizes suggests that there are no splice variants in those samples. The SpaPK cDNA was constructed using partial cDNAs from two different sources, a c D N A isolated from a brain cDNA library and an EST sequence obtained from the breast tissue. This is reasonable since the SpaPK gene is expressed as a single species of the same size in both tissues. 2.4.3.3 Is SpaPK gene spliced differently in other tissues? The number of entries in the EST and genomic databases is growing exponentially. A recent search of EST database revealed six ESTs which have longer sequences than the 5'  59  end of the SpaPK gene. These ESTs are from the brain, pancreas and placenta. Although the lengths of the ESTs are different, they do share identical sequences. The longest EST is from the placenta and has additional 30 nucleotides extending from the 5' extremity of the SpaPK gene on the genomic clone. As detailed in Section 2.3.5.3, a Type I intron splice site was predicted from 53 nucleotides upstream of the SpaPK cDNA.  On the one hand, the 5'  extremity of the recently identified placenta EST sequence is located 4 nucleotides upstream of the putative intron Type I, suggesting that this Type I intron may not be a true splice site. On the other hand, whether or not the SpaPK gene is spliced in placenta has not been investigated. Also recently, a c D N A clone of accession number AK027590 was sequenced as part of the New Energy and Industrial Technology Development Organization (NEDO) human cDNA sequencing project and submitted to GenBank™. The amino acids encoded by the open reading frame of clone AK027590 and the SpaPK cDNAs are identical, despite three mismatches in their D N A sequences.  The putative 5' U T R of clone AK027590 is 71  nucleotides longer than that of the SpaPK cDNA, and the sequences downstream of the splice site are identical amongst the two cDNAs. Interestingly, the sequences upstream of the splice site do not match each other. The first 120 nucleotides of clone AK027590 match genomic sequences located at 1090 nucleotides upstream of the splice site, whereas the first 49 nucleotides of the SpaPK gene match genomic sequences located at 1189 nucleotides upstream of the splice site. Clone AK027590 is found from teratocarcinoma NT2 neuronal precursor cells after 2 weeks of retinoic acid induction, whereas the cloned SpaPK gene is found from the brain and the breast tissues. NT2 cells, when induced with retinoic acid, differentiate into postmitotic neurons with characteristic morphologies (Leypoldt et al., 2001).  Taken together, these observations suggest that the SpaPK gene is alternatively  spliced in different sources. Translation of the clone AK027590 c D N A did not show an inframe stop codon upstream of the putative initiation codon.  2.4.4 Other genes on chromosome 19pl3.3 and relations to disease Genes of similar function are often grouped in a cluster on the same chromosome (Ahuja et al., 1992; Gerard et al., 1993; Samson et al, 1996). Genes located upstream of the SpaPK gene are the eukaryotic translation elongation factor 2 (EEF2), protein inhibitor of  60  activated STAT protein PIASy (PIASY), HIV-1 inducer of short transcripts binding protein (FBI 1), and mitogen-activated protein kinase kinase 2 (MAP2K2).  Other known genes  located downstream of the SpaPK gene are muscle-specific beta 1 integrin binding protein (MIBP), megakaryocyte-associated  tyrosine kinase ( M A T K ) and hypothetical proteins.  These flanking genes encode proteins of diverse function and hence do not provide any insight of the function of SpaPK. The mouse ZIP kinase gene is located on chromosome 10 at position 43.0 c M . A search of Mouse Knockout and Mutation Database did not generate phenotype information associated with knockout or classical mutations in mice. The human ZIP kinase gene was mapped to chromosome 19p 13.3 by fluorescent in-situ hybridization (Saito et al, 1998). The 19pl3.3 region has been implicated in several diseases. Psoriasis is a chronic scaling skin disease triggered by genetic and environmental risk factors. Psoriasis susceptibility loci have been mapped to a number of regions including 19pl 3.3 (Bowcock et al., 2001). Asthma is a chronic disease of the lung involving inflammation of the airways.  The contribution of  genetic factors in asthma is widely assessed yet poorly understood.  Non-parametric  multipoint linkage analysis was used to study northeastern Italian families. One of the two D N A markers was found on chromosome 19pl3.3, suggesting that this region contains susceptibility loci associated with allergic asthma (Venanzi et al., 2001).  Moreover, a  genome-wide search using linkage disequilibrium mapping was performed in search for novel susceptibility genes of late-onset Alzheimer's disease in Finland. Several Alzheimer associated chromosomal loci were found, one of which is located in chromosome 19p 13.3 (Hiltunen et al, 2001). Although several lines of evidence suggest that the SpaPK c D N A sequence is incomplete, much effort was devoted to finding the missing sequences but did not generate useful information. Subsequent experiments were performed using the cloned SpaPK cDNA. It is conceivable that translation from the A T G located from position 145 to 147 does not cause drastic change in enzymatic function, particularly the putative kinase activity.  61  CHAPTER 3 ANALYSES OF SpaPK  3.1 INTRODUCTION For regulation of biological processes, living organisms depend on a family of enzymes called protein kinases.  Initially identified in the 1950s in studies on glycogen  phosphorylase, the number of known kinases has risen ever since.  Protein kinases are  enzymes that catalyze the transfer of y-phosphate from adenosine triphosphate (ATP) to a recipient amino acid residue in a reversible process called phosphorylation. Phosphorylation can rapidly stimulate or reduce the activity of different enzymes. Multiple protein kinases successively phosphorylate one another in a cascade fashion, thereby providing a conduit for cellular signals.  In addition, target proteins are often phosphorylated at several sites by  different protein kinases.  This permits integration of different signaling pathways and  coordination of cellular processes. There are two major classes of eukaryotic kinases: those transferring phosphate to serine and threonine residues and those directing phosphorylation to tyrosine residues. Comparison of amino acid sequences and known structures of catalytic domains from different protein kinases show that they all share common features (Hanks et al., 1988). Structurally, the kinase domain is organized into a small and a large lobe. The cleft formed between the lobes is the site of catalysis. Located within the kinase domain are eleven conserved subdomains. The consensus Gly-X-Gly-X-X-Gly is found in subdomain I, and a universally conserved lysine residue and a glutamate residue lie in subdomain II and subdomain III, respectively.  These conserved motifs in the small lobe all contribute to  binding and stabilizing of a- and (3-phosphates of ATP. The function of the large lobe, on the other hand, is to bind substrate, to orient the y-phosphate of ATP for phosphotransfer, and to direct catalysis. These functions are contributed by an invariant Asp residue and a highly conserved Asn residue in subdomain VI, and the consensus triplet Asp-Phe-Gly in subdomain VII and Ala-Pro-Glu in subdomain VIII. Furthermore, the last three subdomains contribute to structural stability of the kinase substrate complex. In this chapter, we show that SpaPK is a serine/threonine-specific protein kinase. Using the recombinant SpaPK, the potential optimal phosphorylation sequence of this protein  62  was determined from a panel of peptides known to be phosphorylated by serine/threoninespecific protein kinases.  In addition, specific anti-SpaPK polyclonal antibodies were  produced, affinity-purified and used in detection of endogenous SpaPK from mammalian cell extracts.  63  3.2 MATERIALS AND METHODS 3.2.1 Chemicals, enzymes, plasmids and bacterial strains Same as in Section 2.2.1 and 2.2.2 unless specified otherwise.  3.2.2 Molecular biological methods 3.2.2.1 Purification of plasmid DNA. PCR, ligation and transformation of E. coli Same as in Section 2.2.3.1 to 2.2.3.4 unless specified otherwise. 3.2.2.2 Site-directed mutagenesis To generate an inactive form of SpaPK kinase, site-directed mutagenesis was performed on the pBluescript-SpaPK construct using the Quickchange Site-Directed Mutagenesis Kit (Stratagene) according to the manufacturer's instructions. Lysine 42 was replaced by an alanine using a pair of complementary oligonucleotides: sense primer: 5'G A G T A C G C A G C C G C A T T C A T C A A G A A G - 3 ' . and antisense primer: 5'- C T T C T T G A T G A A T G C G G C T G C G T A C T C - 3 ' . The bold nucleotides encode the mutated amino acid, and the underlined nucleotides introduce a Bsm I restriction site to facilitate the screening of mutants. PCR was performed in 50 pL of 1X Pfu Buffer containing 5 ng of template D N A , 125 pg of each of the sense and antisense primers, 5 units of Pfu D N A polymerase (Stratagene) and 0.2 m M of each of dNTPs. Sixteen cycles of PCR (denaturation at 95 °C for 30 seconds, annealing at 55 °C for 1 minute and extension at 68 °C for 10 minutes) were performed following an initial denaturation step at 95 °C for 5 minutes. The hemimethylated wild-type parent plasmids were then selectively digested with 20 units of Dpnl restriction enzyme at 37 °C for 1 hour, and the resulting nicked D N A containing the mutation of interest were transformed into heat-shock competent E. coli XL-1 Blue cells. The incorporation of the mutagenic oligonucleotides into the construct was verified by restriction enzyme analysis and confirmed by D N A sequencing. This catalytically inactive SpaPK is designated K42ASpaPK. 3.2.2.3 Construction of recombinant wild-type and catalytically inactive His-SpaPK proteins for overexpression in E.coli The open reading frame of the wild-type or the catalytically inactive SpaPK was amplified by PCR with oligonucleotides XC1 (5 ' - C G C G G A T C C A T G T C C A C G T T C A G G C A G G A G G - 3 ' ) and X C 2 ( 5 ' - C C G G A A T T C C T A C T A G C G C A G C C C G C A C T C - 3 ' )  64  using  the SpaPK or K42A-SpaPK cDNA, respectively, as templates.  X C 1 and X C 2 contain  restriction sites, BamH I and EcoR I, respectively, at their 5'-ends to facilitate cloning. The amplified P C R fragments were digested with BamH I and EcoR I, and cloned into the EcoR 1/BamH 1 sites of the expression vector pRSETA (Invitrogen). The nucleotide sequences of PCR fragments were confirmed by D N A sequencing. The resulting recombinant proteins, designated His-SpaPK and His-K42A-SpaPK, both contained an N-terminal 6X histidine tag. All subsequent experiments performed using His-SpaPK also apply to His-K42A-SpaPK.  3.2.3 Purification of recombinant His-SpaPK proteins from E.coli 3.2.3.1 Overexpression of wild-type His-SpaPK and catalytically inactive His-K42A-SpaPK from E.coli The recombinant plasmid was transformed into E. coli BL21(DE3), and individual colonies were picked and grown at 37 °C overnight in 1 ml of L B containing 120 pg/ml ampicillin. The next day, each saturated culture was diluted 1:50 into 10 ml of the same medium, and incubation was continued at 37 °C until the OD600 of the cultures reached 0.8. After addition of IPTG to a final concentration of 1 m M , the cultures were grown for another 3 hours. Cells were collected by centrifugation at 1,500X g, and the pellets were processed immediately or stored at -20 °C. The expression of recombinant wild-type or catalytically inactive His-SpaPK was verified by Western blotting. 3.2.3.2 Large-scale purification of recombinant His-SpaPK proteins from E. coli Typically, a culture of the appropriate strain was grown in 250 ml of L B containing 120 p.g/ml ampicillin. The cell pellet collected after induction was suspended in 50 ml of French Press Buffer (60 m M Tris-HCl [pH 7.6], 10 m M M g C l , 60 m M NH C1, 0.5 mM 2  4  EDTA) containing 5 m M (3-mercaptoethanol, centrifuged at 3,800X g for 5 minutes, resuspended  in 2X volumes (w/v) of French Press Buffer  containing 5 m M (J-  mercaptoethanol, 5% glycerol, 0.2 M PMSF, 2 ug/ml aprotonin, 0.8 ug/ml leupeptin, 0.8 pg/ml pepstatin A , and 20 |ag/ml DNase I. The cell suspension was passed twice through a French Press cell at 8,000 psi, and the lysate was centrifuged at 31,000X g at 4 °C for 45 minutes. The resulting pellet was washed three times with 10 ml of Buffer A (20 m M TrisHCl [pH 7.9], 500 m M NaCl, 10 % glycerol, 10 m M p-mecaptoethanol) containing 1% Triton® X-100 followed by centrifugation at 31,000X g at 4 °C for 20 minutes. The pellet  65  was dissolved in 5 ml of Buffer B (20 m M Tris-HCl [pH 7.9], 500 m M NaCl, 10 % glycerol, 10 mM p-mecaptoethanol, 6 M guanidine hydrochloride) containing 1 % Triton® X-100 at 4 °C for 1 hour, and centrifuged at 39,000X g at 4 "C for 20 minutes. The resulting supernatant containing denatured proteins was loaded onto a 5 ml Ni-column pre-equilibrated with 20X column volumes of Buffer B containing 1% Triton® X-100.  The unbound proteins were  washed off the column with 50 ml of Buffer B. The retained proteins were renatured on the column by washing with decreasing concentrations of guanidine hydrochloride: 30 ml of 1 to 1 ratio of Buffer A to Buffer B, 30 ml of 3 to 1 ratio of Buffer A to Buffer B, 30 ml of 7 to 1 ratio of Buffer A to Buffer B, and finally 30 ml of Buffer A . The contaminants were washed off with 20 ml of Buffer A containing 30 m M imidazole and 20 ml of Buffer A containing 60 m M imidazole. Recombinant His-SpaPK proteins were eluted with 20 ml of Buffer A containing 200 m M imidazole. A l l fractions were collected at 5 ml each. The purity of the fractions was monitored on 10% gels by SDS-PAGE.  Fractions containing  purified protein were pooled, concentrated to 3 ml using a Stirred Ultrafiltration Cell (Amicon 8200® or Amicon® 8050), and dialyzed against Buffer A at 4 °C overnight. Prior to loading onto SDS-PAGE gels, proteins in guanidine hydrochloride were precipitated with cold 95% ethanol at -20 °C for 10 minutes, centrifuged at 4 °C at 13,000X g for 10 minutes, washed with cold 90% EtOH and centrifuged as before.  Other protein  samples were precipitated with 5X volumes of acetone at -20 °C for 30 minutes, and centrifuged at 4 °C at 13,000X g for 10 minutes. 3.2.3.3 Circular dichroism spectroscopy Circular dichroism (CD) spectroscopy was used to determine the presence of secondary structure in the purified His-SpaPK recombinant proteins.  The measurements  were carried out in a 0.1 cm cell and protein solutions of approximately 0.25 to 0.5 mg/ml. All C D spectra were measured at 25 °C from 200 nm to 300 nm at 50 nm/min on a Jasco J500A recording spectrophotometer.  The recorded spectra were the average of 5 scans and  were corrected to background (buffer alone). To monitor the thermal unfolding of recombinant proteins, circular dichroism was recorded at 222 nm during the course of heating the solutions from 15 °C to 90 °C at a heating rate of 50 °C /hour.  66  3.2.4 Analysis of purified His-SpaPK 3.2.4.1 His-SpaPK kinase assay Autophosphorylatiori of His-SpaPK was carried out in a total volume of 20 pi of a kinase buffer (10 m M Tris H C l [pH 7.2], 10 mM M g C l , 3 m M M n C l ) containing 30 uCi of 2  2  32  [y- P] ATP and purified kinase. After incubation at room temperature for 30 minutes, the reaction was terminated by the addition of a SDS sample buffer, boiled for 5 minutes, and subjected to SDS-PAGE.  The gel was then stained with Coomassie brilliant blue R-250,  destained, and dried. The phosphorylated protein was visualized by autoradiography. 3.2.4.2 Phosphoaminoacid analysis Autophosphorylated His-SpaPK was subjected to SDS-PAGE and transferred to a polyvinylidene difluoride membrane (BioRad).  The phosphorylated SpaPK band was  localized by autoradiography, excised and subjected to phosphoamino acid analysis. The protein on the membrane was eluted and hydrolyzed in 100 ul of 6 N H C l at 110 °C for 1 hour. The sample was dried in a Speedvac evaporator and dissolved in 10 u.1 deionized water. The sample, together with 0.3 ug/ml each of phosphoserine, phosphothreonine, and phosphotyrosine standards, was subjected to two-dimensional thin layer electrophoresis in a H T L E 7000 apparatus (CBC Scientific). The first dimension was electrophoresed at 1.5 kV for 20 min in a pH 1.9 buffer (50 ml of 88% formic acid, 156 ml of glacial acetic acid, and 1794 ml of deionized water). The second dimension was electrophoresed at 1.3 kV for 16 min in a pH 3.5 buffer (100 ml of glacial acetic acid, 50 ml of pyridine, 0.5 m M E D T A and 1880 ml of deionized water).  32  P-labeled individual phosphoamino acids were identified by  an alignment of phosphoamino acid markers sprayed with 0.5% ninhydrin with the signals obtained by autoradiography. 3.2.4.3 Screening of peptide substrates for His-SpaPK phosphorylation Screening of peptide substrates for His-SpaPK phosphorylation was carried out in collaboration with Kinetek. Five ul of each peptide (Table 3.2) was plated in duplicate at 1 mg/ml onto a U-bottomed 96 well plate (Serocluster). Kinase assays were performed at room temperature for 15 minutes directly in each well, in the presence of 0.5 p.g of purified His-SpaPK, 5 ul of [y- P] ATP, and 10 pi of A D B buffer (25 m M p-glycerophosphate, 2 32  mM EDTA, 0.25 m M DTT, 20 m M M g C l , 20 m M MOPS, [pH=7.2]). Ten ul of each 2  67  kinase reaction were spotted onto a 96 well phosphocellulose P81 Multi Screen plate (Millipore), which was subsequently washed six times with 1% H P 0 for 15 minutes each, 3  4  blotted dry, quantitated for radioactivity using a scintillation counter.  3.2.5 Production of affinity purified anti-SpaPK polyclonal antibodies 3.2.5.1 Production of anti-SpaPK antiserum Anti-SpaPK polyclonal antibodies were raised in New Zealand female rabbits against purified His-SpaPK. For immunization, 500 ug of the purified His-SpaPK was emulsified with an equal volume of Freund's complete adjuvant and injected subcutaneously into rabbits, followed by three boosts with 500 ug of protein each in Freund's incomplete adjuvant at two week intervals.  Anti-SpaPK antisera were obtained 14 days after each  injection, and the specificity of the polyclonal antibodies was determined by immunoblot analysis. 3.2.5.2 Affinity purification of anti-SpaPK polyclonal antibodies SpaPK polyclonal antibodies were affinity-purified on a His-SpaPK-bound Ni-NTA column (Qiagen).  The crude immune serum was first precipitated with 26% (w/v)  ammonium sulphate, and the pellet was dissolved in TBS. The partially purified immune serum was then applied to the affinity column equilibrated with Buffer A (150 m M NaCI, 50 mM Tris-HCl, [pH 7.4]). After loading, the column was washed with 5X column volumes of Buffer A, and 5X column volumes of Buffer B (Tris-HCl [pH 7.4], 2 M NaCI). To obtain the purified antibodies, the column was incubated in 1 column volume of 4 M M g C l for 15 2  minutes, and antibodies were eluted with the same volume of 4 M M g C l . The eluate from 2  the first column volume was collected, dialyzed against deionized water at 4 °C for 30 minutes then against PBS at 4 °C overnight.  3.2.6 Extraction of protein from mammalian cells Tissue culture cells were trypsinized and washed twice in cold PBS. Cells were lysed in NP-40 lysis buffer (50 m M Tris-HCl [pH 7.4], 1% [v/v] NP-40, 150 m M NaCI, ImM EDTA) containing 1 m M PMSF, 1 pg/ml aprotonin, 1 pg/ml leupeptin, 1 pg/ml pepstatin A , 1 mM Na^VC^ and 1 m M NaF at 1 X 10 cells per pL lysis buffer. 4  The mixture was  incubated at 4 °C for 15 minutes on a Nutator mixer and then centrifuged at 13,000X g at 4  68  °C for 10 minutes. The protein concentration of the supernatant was determined by Bradford Assay (BioRad) according to manufacturer's instructions.  3.2.7 Western blot analysis Proteins were resolved on 8-12% gels by SDS-PAGE and transferred onto a nitrocellulose membrane (Life Technologies) at 140 mA at 4 °C for 1 hour in a transfer buffer (3.9 m M glycine, 48 m M Tris-HCl [pH 8.3], 0.037% SDS, 20% methanol), or transferred onto a PVDF membrane (Millipore) at 180 mA at room temperature for 1 hour in Towbin Buffer (25 m M Tris-HCl [pH 8.3], 192 m M glycine). The membranes were blocked with PBS containing 5% milk at room temperature for 1 hour or at 4 °C overnight. The blots were incubated with the appropriate primary antibody diluted in PBST (0.05% Tween 20 in PBS) at room temperature for 1 hour or at 4 °C overnight. The blots were then washed three times with PBST for 15 minutes each, incubated with the appropriate secondary antibody diluted in PBST at room temperature for 1 hour, and washed as before. The immunoreactive proteins were detected with ECL™ reagent and visualized on X - O M A T film. The following primary antibodies were used:  affinity-purified rabbit anti-SpaPK  antibodies, mouse anti-Express antibody (Invitrogen), mouse anti-HA antibody (kindly provided by Dr. Sadowski) at 1:1000, 1:5000, and 1:5000 respectively. The following secondary antibodies were used:  HRP-conjugated goat anti-rabbit  antibodies (Kirkegaard Perry Laboratories) and HRP-conjugated goat anti-mouse antibodies (Pierce) at 1:3000, and 1:5000 respectively.  69  3.3 RESULTS 3.3.1 Sequence analysis of SpaPK 3.3.1.1 Analysis of primary structure of SpaPK The open reading frame of SpaPK cDNA encodes a protein of 454 amino acids with a predicted molecular mass of 52.5 kDa and a pi of 6.6.  Analysis of the amino acid  composition of the SpaPK shows that the protein contains many charged residues: 12.3% glutamate, 4.6% aspartic acid, 9.5% arginine, and 7.0% lysine. The overall composition of negatively charged and positively charged residues are comparable to each other: 16.9% and 16.5%, respectively.  A closer examination of the protein sequence reveals that the  percentage of negatively charged (18.3%) and positively charged (21.1%) residues is higher in the carboxy terminus, from position 275 to 454.  Within these 180 amino acids, the  composition of glutamate, aspartic acid, arginine and lysine is 15.0%, 3.3%, 14.4%, and 6.7%, respectively. 3.3.1.2 Putative domain structures and motifs of SpaPK Sequence analysis of SpaPK revealed that the protein is organized into two putative domains, a protein kinase domain and a coiled-coil domain (Figure 3.1). The putative kinase domain, which spans 263 amino acids, is located at the amino terminus from position 13 to 275. It displays the classic composition of eleven subdomains that is characteristic of various protein kinase catalytic domains (Figure 3.1.A) (Hanks et al., 1988, Kennelly and Krebs, 1991). The sequence features that distinguish known serine/threonine kinases from tyrosine kinases lie in subdomains VI and VIII. Serine/threonine kinases contain the consensus AspLeu-Lys-Pro-Glu-Asn in subdomain VIb and Gly-Thr/Ser-X-X-Tyr/Phe-X-Ala-Pro-Glu in subdomain VIII, whereas tyrosine kinases contain either Asp-Leu-Arg-Ala-Ala-Asn or AspLeu-Ala-Ala-Arg-Asn in subdomain VIb and Pro-Ile/Val-Lys/Arg-Trp-Thr/Met-Als-Pro-Glu in subdomain VIII (Kennelly and Krebs, 1991). Therefore, the presence of amino acids AspLeu-Lys-Pro-Glu-Asn in subdomain VIb, and of Gly-Thr-Pro-Glu-Phe-Val-AIa-Pro-Glu in subdomain VIII strongly implicates that SpaPK as a serine/threonine-specific protein kinase. The carboxyl terminus of SpaPK contains a predicted coiled-coil domain, which functions in protein-protein interactions. A coiled coil is a helical bundle of 2 to 5 a-helices  70  A  I  II  MSTFRQEDVEDHYEMGEELGSGQFAIVRKCRQKGTGKEYAAKFIKKRR III 49:  IV  LSSSRRGVSREEIEREVNILREIRHPNIITLHDIFENKTDWLILELV 96 V  97:  48  Via  SGGELFDFLAEKESLTEDEATQFLKQILDGVHYLHSKRIAHFDLKPEN VIb  VII  144  VIII  145: IMLLDKNVPNPRIKLIDFGIAHKIEAGNEFKNIFGTPEFVAPEIVNYE 192 IX  X  193: PLGLEADMWSIGVITYILLSGASPFLGETKQETLTNISAVNYDFDEEY  240  XI 241: FSNTSELAKDFIRRLLVKDPKRRMTIAQSLEHSWIKAIRRRNVRG  71  285  B  U  0.8 0.6 0.4 0.2 0 101  201  301  401  454  Figure 3.1 Domain structures of SpaPK. (A) Kinase domain of SpaPK. The catalytic domain of SpaPK spans from amino acid 13 to 275. The eleven conserved subdomains known in various protein kinases are indicated by Roman numerals.  The protein  sequences are presented in single letter code. Amino acids are numbered on both left and right margins. (B) Coiled-coil domain of SpaPK. SpaPK sequence was analyzed using the COILS program (window 28). The predicted propensity of a sequence to form coiledcoils (Y axis) is plotted against the linear sequence of amino acids of SpaPK (X axis). A schematic view of the domain structure of SpaPK is shown at bottom, with the catalytic domain illustrated as a hatched region.  72  that have a distinctive packing of amino acid side chains at the core of the bundle (Lupas, 1996). The sequence of coiled coils shows heptad repeats of hydrophobic and hydrophilic amino acids at regular intervals. The seven structural positions of a heptad repeat are named a-g, where a and d are hydrophobic residues and form the helix interface core, and b, c, e, f and g are hydrophilic residues and form the solvent exposed part of the helical bundle. The heptad repeat of hydrophobic and hydrophilic residues has been used to detect regions of coiled coils in protein sequences. The coiled-coil domain, predicted by the COILS program, is located downstream of the catalytic domain, from amino acid 330 to 448 (Figure 3.1.B). In addition to these two putative domains, a putative bipartite nuclear targeting sequence is also found in SpaPK.  Nuclear proteins contain a targeting signal that specifies  their selective accumulation in the nucleus. The proposed general consensus of the nuclear targeting sequence is composed of two adjacent basic amino acids separated by a spacer region of any 10 residues and at least three basic residues in the five positions after the spacer region (Dingwall and Laskey, 1991; Garcia-Bustos et al., 1991).  The putative bipartite  nuclear targeting sequence, Arg-Arg-Asn-Val-Arg-Gly-Glu-Asp-Ser-Gly-Arg-Lys-Pro-GluArg-Arg-Arg, of SpaPK is located between the kinase domain and the coiled-coil domain, from amino acid 290 to 306 (Hofmann et al, 1999). 3.3.1.3 Putative phosphorylation and other post-translational modification sites of SpaPK Using ScanProsite (Hofmann et al., 1999) and PhosphoBase (Kreegipuu et al., 1999) search programs, multiple potential sites for post-translational modifications were identified on SpaPK. SpaPK sequence potentially contains 7 phosphorylation sites for protein kinase A (PKA), 7 for protein kinase C (PKC), 3 for protein kinase G (PKG), 4 for casein kinase I (CKI), 8 for casein kinase II (CKII), 6 for calmodulin-dependent protein kinase II (CaM kinase II), 2 for glycogen synthase kinase 3 (GSK3), 2 for protein kinase p70S6 (p70S6K), and 1 for tyrosine kinase, some of which are overlapping (Table 3.1). Among the total 43 potential phosphorylation sites, 27 are serine residue, 15 are threonine residue and 1 is a tyrosine residue. SpaPK can also be potentially modified by an attachment of an isoprenoid lipid to a cysteine residue. This modification requires a cysteine residue located three residues from the C-terminal extremity, as part of the CaaX box in which A represents aliphatic residues  73  Table 3.1 Predicted phosphorylation sites on SpaPK.  Phosphorylation site  Consensus site  Number of phosphorylation site  Protein Kinase A  [R]-X(2)-[S/T]-X  7  Protein Kinase C  [S/T]-X-[R/K]  7  Protein Kinase G  [R/K](2-3)-X-[S/T]-X  3  Casein Kinase I  [Sp/Tp/D/E]-X(2-3)-[S/T]-X  4  Casein Kinase II  [S/T]-X(2)-[Sp/Tp/D/E]  8  [R]-X-X-[S/T]-X  6  50, 57, 265, 299, 371,414  X-[S/T]-X-X-X-[Sp]  2  265,269  Calmodulindependent Protein Kinase II Glycogen Synthase Kinase-3  11  p70S6 Kinase  Coordinates ' 1  50, 57, 265, 299, 326, 371,414 35,51, 132,288, 299, 306, 345 50, 265,414 86, 97, 110, 1 17, 202,215,225, 227, 230, 245 35, 57, 78,97, 110, 221, 318, 414  2 [K/R]-X-[R]-X-X-[S/T]-X 50, 299 [R/K]-X(2)-[D/E]-X(3)-Y or Tyrosine kinase [R/K]-X(3)-[D/E]-X(2)-Y 1 133 a Site of phosphorylation is in bold type. b Amino acid numbered according to the SpaPK protein sequence. The potential translation initiation codon (Met) is defined as 1.  74  (Lowy and Willumsen, 1989). The sequence of the CaaX box on SpaPK is Cys-Gly-LeuArg, from position 451 to 454. 3.3.1.4 Comparison of amino acid sequence of SpaPK with other proteins Proteins highly similar to SpaPK are found in other mammalian species.  SpaPK  sequence shares 83.7%, 83.5% and 80.6% identity with its homologs from mouse (mZIPK), rat (rdlk), and rat smooth muscle (rsmZIPK), respectively (Figure 3.2). Compared to other homologs, the rsmZIPK contains a unique 16-residue extension at the N-terminal end. To determine the relationship of SpaPK to other kinases, B L A S T searches (default settings) of databases consisting of non-redundant (nr) protein sequences were performed using the kinase domain of SpaPK.  The catalytic domain of SpaPK showed the highest  similarity to human death associated protein kinase (DAP kinase) at 94%, and to a lesser extent, human myosin light chain kinase ( M Y L K ) at 66%, and human calcium/calmodulin dependent protein kinase II alpha, delta, and gamma (CaMK-a, -5, -y) subunits at 58%, 60% and 59%, respectively (Figure 3.3). Comparable similarities were also found between SpaPK and the aforementioned kinases from other species, including rat and mouse D A P kinases, bovine, chicken, rabbit, sheep and rat M Y L K s , and rat and mouse C a M K subunits. Compared to other model organisms, the kinase domain of SpaPK showed the highest similarity to a putative calcium-dependent protein kinase from Arabidopsis, an ankyrin and protein kinase from C.elegans, and a member of the Titin/Myosin light chain kinase family (Stretchin-MLCK) from Drosophda. Since SpaPK was identified in an attempt to find the human homolog of Rad53 of the budding yeast, Saccharomyces cerevisiae, sequence alignments were performed with Rad53 and its homolog Cdsl from fission yeast Schizosaccharomyces pombe. The alignments revealed that the catalytic domain of SpaPK shares 50% similarity with that of Rad53 (Figure 3.4). This similarity is the fifth highest scored amongst all S. cerevisiae proteins. In budding yeast, the proteins which display higher homology than Rad53 are calcium/calmodulindependent protein kinases. The kinase domain of SpaPK also shares 53% similarity with that of Cdsl protein (Figure 3.4). This homology is the sixth highest scored amongst all S. pombe proteins.  In fission yeast, the proteins which display higher homology than Cdsl are  calcium/calmodulin dependent protein kinases, carbon catabolite repressing protein kinase,  75  SpaPK mZIPK rdlk rsmcZIPK  SpaPK mZIPK rdlk rsmcZIPK  SpaPK mZIPK rdlk rsmcZIPK  SpaPK mZIPK rdlk rsmcZIPK  212 V T I Y T ~  L?  212 212 228  1  J A S P F L G E T K Q E ? L T H I S A VM Y D F DE E F  GASP- L G B " > * Q E T L T N I S A . V H V D F I J E E Y F 3 ST £ E L A F D F T P P L I  :YESt  76  VKnpt'PPvrnx • n c  S S T S S L A K D F I R R L L V K D P K R R X T I A Q S  269 269 269 285  Figure 3.2 Amino acid sequence comparison of SpaPK and its homologs in mouse and rat.  The deduced amino acid sequence of SpaPK is aligned with its homologs from  mouse (mZIPK), rat (rdlk), and rat smooth muscle (rsmZIPK).  The identity of each  protein is listed on the left. The numbers indicating the position of amino acid relative to the amino terminus of each protein is shown on the right of each line. Amino acids which are identical in all four proteins are highlighted in red. Amino acids which are identical between mZIPK, rdlk and rsmZIPK are colored in grey. Alignment of amino acids was performed using C L U S T A L .  77  A SpaPK hDAPK  LS lc o R R. S v 5 R E E  KE  TK  SpaPK hDAPK  SpaPK hDAPK  60 60  12 0 120  KQIt  •H  "I H L Q H H ^ ^ B ^ I H K  180 180  F  SpaPK hDAPK  SpaPK hDAPK  Q  275 275  !QD  B SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  I  -MB|FRQEDVE  |RQKGBGKE 1EKK|RK KVLAGQE  SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  FFK  AYBJ  IKIPIGQ  I|N—TKKLH  KKTSTQE  I|N—TKKLB  IFENKT AFEIKA  ^  FIKKRRLSSIR  SISlEG SISBEG SISlEG  53 48 51 52 52  106 100 103 104 104  160 152 155 156 15 6  78  SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  .GN-E|KNIF|  213  AG-SLKVLF|  205  EQQAJFGF;  209  GDQQAIFGF;  210  EQQA|FGF>  SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  210  1  TKQETLTN  S|VN|  1  DNETLAN  TSAT  QHRLYQQ K | G . QHRLYQQ K l G . QHKLYQQ K | G  PSPEJ^  : 2 64  SpaPK hMYLK hCaMKalpha hCaMKdelta hCaMKgamma  F i g u r e 3.3 Comparison of the amino acid sequence of SpaPK and other protein kinases. (A) Alignment of the catalytic domain of SpaPK with that of human death-associated protein kinase (hDAPK). Identical and similar amino acids are highlighted in red. (B) Alignment of the catalytic domain of SpaPK with that of human isoform I of myosin light chain kinase  (MYLC),  calcium/calmodulin hCaMKgamma).  and  dependent  -alpha,  -delta  protein  kinase  and II  -gamma  subunit  of human  (hCaMKalpha, hCaMKdelta,  Amino acids that are identical or similar in all six proteins are  highlighted in red. Amino acids that are identical or similar in five of the proteins are highlighted in gray and amino acids that are identical or similar in four of the proteins are highlighted in blue. The identity of each protein is listed on the left. The numbers indicating the position of amino acid relative to the amino terminus of each protein are shown on the right of each line.  Amino acid alignments were carried out using  CLUSTAL. Gaps (dashes) were used for optimization of the alignment.  79  A  SpaPK K RadS  : |s7F|QEDVElHYE|r|Kas|QHlHcRQ|GHE|AlF|KHsSSRRBE  :  59  3 : |VAN1TGIFK|FSI|O|V|Q|A|TKIE|THT|V1I|SH|IGNMDH- : 5 8 P  Rad53p  SpaPK Rad53p  EIER N•RE| Mr, AJNj EI,  IF!  'G|HMBKR|A|: A|K^HMG|S|:  : FLl : isl  |EKES1TBEATQ UIGABGHAGRE  U.J  E  SpaPK Rad53p R G K D T SVS  l ILDKNVPNPR^Bl EQDDP--VL^BT|  |HKIEA|NEF|NI K | VQGN|SFM|TF  •PLG- - -LE AMHHV|THHHASHL|E|: IKQ 1 Q P D E | E R N E Y S S L V i ^ H B c | v ^ ^ B H L | s | sI l D HL|  ETLT •SAW|DFDEEYFSNTBLHHRRHVK|KR|M|I|QS|E|SH •GRGS|HEGPLKDFRI|E^HDS|QV|NN|S|A|KA|N|P|  SpaPK Rad53p  : : QLYK  :118 :113  177 17 0  223 229  275 281  B  MSTFRQEDVED| GEEl IQI SQKNMIKSENS IRTI IT]  SpaPK Cdsl  SpaPK Cdsl  : :  MFQJ  SpaPK : Cdsl :  59 60  : 119 IQC^IHDDHF^HYIE^HM^HIANIG|D|Q|C: ICKPL : 1 2 0  |T|LH|KQG|T|F :  LAVEVNl  TL|HHKTHVMBIJSMHFHHAEKEEMTIDIATQF  IGIHYBSKRIAIE  SpaPK Cdsl  SpaPK Cdsl  |E|RI •S|H;  EIE  ' GBBEBA|F|KB-R|SBRRGVSR| IKCRQKNHHHI|I|N|KI|L|SEKRAT| 1  ILDKNVPNPRI TN  DFHI  -|PEB  |N|GG G|MG|^H^BKSKN|NHGGYDDK  Isl  IKIEAGNEIKNIIIG : 1 7 9 :V§HGTGT|LET|IC : 1 7 6  IHK IK  •LGEIKQETITNHA'V .L|K I2 3 2 LSI| A S S |QAK C|EK 236 'G  5AS  ClLl  N|DF|EEYFSNTMLAK|F|R«BVKD|KBB|IAQS|E|S|: I A|PI|PLLENEIHEGI|L|NHEIN|E^|ESEA|Q|P|IF  80  : 275 : 279  Figure 3.4 Comparison of the amino acid sequences of SpaPK, Rad53 and Cdsl. (A) Alignment of the catalytic domain of SpaPK with that of Rad53 from Saccharomyces cerevisiae.  (B) Alignment of the catalytic domain of SpaPK with that of Cdsl from  Schizosaccharomyces pombe. Identical and similar amino acids are highlighted in red. The identity of each protein is listed on the left. The numbers indicating the position of amino acid relative to the amino terminus of each protein is shown on the right of each line. Amino acids alignments were carried out using C L U S T A L . Gaps (dashes) were used for optimization of the alignment.  81  meiosis-specific serine/threonine protein kinase M e k l , and mitosis inducer protein kinase Cdrl. Database searches using the carboxyl terminal portion of SpaPK excluding the kinase domain, failed to reveal any proteins with significant similarity. Hence no insight into the function of the non-kinase portion of SpaPK was gained from using this method.  3.3.2 Construction of catalytically inactive SpaPK by site-directed mutagenesis Subdomain II of all protein kinase domains contains an invariant lysine residue (Hanks et al., 1988; Taylor et ah, 1993).  This lysine is directly involved in the  phosphotransfer reaction between the kinase and its substrate. In the presence of MgATP, the lysine residue interacts electrostatically with the non-transferable a- and P-phosphates of ATP, thereby providing stability to the complex (Hanks et al., 1988). Substitution of this universally conserved lysine residue in subdomain II interferes with the phosphotransfer reaction, rendering protein kinases inactive (Hanks and Quinn, 1991; Hanks et al., 1988). This universally conserved lysine residue is located at position 42 of SpaPK. Sitedirected mutagenesis was performed to replace lysine 42 with an alanine, using a pair of complementary  oligonucleotides (Section 3.2.3.1).  Sequencing data confirmed the  substitution (data not shown). The resulting protein product lacked protein kinase activity (see text below, Figure 3.7, Panel C). The inactive protein kinase form of SpaPK is designated as K42A-SpaPK.  3.3.3 Expression and purification of recombinant His-SpaPK proteins 3.3.3.1 Overexpression of His-SpaPK and His-K42A-SpaPK in E.coli To overexpress the wild-type or the catalytically inactive form of SpaPK in E.coli, the BamW 1/EcoR I fragment containing the corresponding open reading frame was inserted into the pRSETA bacterial expression vector (Section 3.2.3.2).  Thus the recombinant protein  includes an N-terminal hexahistidine tag (His-tag), and thrombin and enterokinase cleavage sites in its leader sequence. These modifications added 25 amino acids to the amino terminus of each protein, which is designated His-SpaPK for the wild-type form or His-K42A-SpaPK for the catalytically inactive form of SpaPK.  82  The recombinant plasmids were transformed individually into E.coli expression host strains and the 55.3 kDa recombinant proteins were successfully overexpressed by induction with 1 m M IPTG (Section 3.2.4.1) (Figure 3.5A). However, the recombinant proteins were not soluble. Varying expression conditions such as host strains, growth temperatures, IPTG concentrations, density of host cells and length of induction did not improve solubility. Since all of the overexpressed recombinant proteins formed inclusion bodies under all the above conditions, they were solubilized by denaturation prior to purification. 3.3.3.2 Renaturation of His-SpaPK and His-K42A-SpaPK on the nickel affinity column After unsuccessful attempts to renature the recombinant His-SpaPK proteins after purification under denaturing conditions, refolding was ultimately achieved by direct renaturation of recombinant His-SpaPK proteins on a nickel resin affinity column (Section 3.2.4.2). Figure 3.5.B shows the renaturation profile of wild-type His-SpaPK. The inclusion bodies containing the overexpressed His-SpaPK were washed with Triton X-100 to eliminate contaminants, solubilized in 6 M guanidine hydrochloride denaturant, then loaded onto a N i affinity column (lane 1). The binding of His-SpaPK was efficient since no target protein was detected from the unbound fraction (lane 2). Renaturation of His-SpaPK was carried out by washing with buffers containing decreasing concentrations of guanidine hydrochloride: 3 M (lane 3), 1.5 M (lane 4), 0.75 M (lane 5) and 0 M (lane 6). His-SpaPK remained bound to the column during the renaturation process.  Retention of His-SpaPK was confirmed by the  absence of signal on a Western blot probed with an anti-Express monoclonal antibody specific for the linker region of the fusion protein (data not shown). 3.3.3.3 Purification of His-SpaPK and His-K42A-SpaPK under native conditions After renaturation, recombinant His-SpaPK proteins were purified using nickel resin affinity chromatography.  The contaminants were washed off the column with buffers  containing 30 m M and 60 m M imidazole, after which recombinant protein was eluted with buffer containing 200 m M imidazole. A l l fractions containing purified recombinant protein were pooled, concentrated to 0.5 mg/ml and then dialyzed to remove imidazole.  The  purification achieved a relatively high homogeneity with a yield of approximately 5 mg per 200 ml of starting bacterial culture. The purified His-SpaPK has an apparent molecular mass of approximately 55 kDa, correlating well with its predicted molecular mass (Figure  83  S4  Figure 3.5  Overexpression, renaturation and purification of His-SpaPK.  Overexpression of His-SpaPK.  (A)  E.coli BL21(DE3) cultures harboring an empty  pRSETA vector (lane 1) or His-SpaPK construct (lane 2) were grown at 37 °C to early log phase and then induced for expression with 1 m M IPTG for 3 hours, as described in Section 3.2.3.1.  Cells from 1 ml of culture were collected by  centrifugation and boiled in 100 pi of SDS sample buffer. Proteins from 5 pi of each lysate were resolved by SDS-PAGE.  (B) Renaturation of His-SpaPK. Inclusion  bodies from 200 ml of bacterial culture harboring His-SpaPK were solubilized in 5 ml of a 6 M GdHCl denaturant and loaded onto a nickel resin affinity column. Proteins were renatured on the column by a gradual removal of GdHCl, as described in Section 3.2.3.2. Loadings on the SDS-PAGE are as follows. Lane 1, solubilized inclusion body (10 pi); lane 2, flow through (100 pi); lane 3, 3 M GdHCl wash through (100 pi); lane 4, 1.5 M GdHCl wash through (100 pi); lane5, 0.75 M GdHCl wash through (100 pi); lane 6, 0 M GdHCl wash through (100 pi). (C) Purification of His-SpaPK. His-SpaPK was purified using nickel resin affinity chromatography, as described Section 3.2.3.2.  Fractions containing the purified His-SpaPK were  pooled, concentrated and dialyzed. Lane 1 shows a sample of the purified HisSpaPK (5 pg). A l l SDS polyacrylamide gels, 10% (A), 10% (B) and 15% (C), are stained with Coomassie brilliant blue R-250. Molecular mass standards in kDa are shown on the left. The position of the recombinant His-SpaPK is indicated by an arrow.  85  3.5 C). The molecular mass of His-SpaPK was determined using unstained molecular mass standards, and correlated with the pre-stained molecular mass standards which were used throughout the thesis. Confirmation that the purified protein was His-SpaPK or His-K42ASpaPK was provided by Western blotting with an anti-Express monoclonal antibody and the anti-SpaPK polyclonal antibodies (data not shown).  3.3.4 Circular dichroism analysis of purified recombinant His-SpaPK proteins To determine whether His-SpaPK was refolded after renaturation and purification, the protein sample was subjected to circular dichroism (CD) analysis (Section 3.2.4.3). C D spectroscopy measures ellipticity as a function of wavelength, and is able to detect secondary structures of a protein. Ellipticity is defined as the difference in absorbance of right and left handed circularly polarized light by a substance. Minima in the ellipticity at 208 and 222 nm are characteristic of an tx-helical structure, while a minimum at 215 nm and 115 nm is typical of a p-sheet structure and random coils, respectively (website, 2). The C D spectrum of the purified His-SpaPK displays two minima centered at 208 and 222 nm (Figure 3.6.A). This is typical of a folded protein containing a considerable amount of ot-helical structure, suggesting that purified His-SpaPK was refolded. To further verify that purified His-SpaPK was refolded, it was subjected to thermal denaturation and analyzed by CD. Unfolding of the protein was monitored by the changes in its ellipticity at 222 nm during the course of heating the solution from 25 °C to 85 °C (Figure 3.6.B). Figure 3.6.B shows that His-SpaPK began to lose its cc-helical structure at 30 °C and was completely devoid of a-helices at 55 °C. The four transitions in the slope of the melting curve suggest the presence of three domains in the folded structure, two of which may be due to the small lobe and the large lobe subdomains within the catalytic domain of the kinase (Taylor et al, 1993).  Ellipticity as a function of wavelength showed that of the heat  denatured His-SpaPK lacked a-helical and P-sheet content (data not shown). The C D analysis suggests that refolding of His-SpaPK was successful since it contained a-helical secondary structure which was sensitive to heating. Similar C D spectra indicate that the His-K42A-SpaPK purified under the same conditions was also renatured (data not shown).  86  I 200  1  I 220  1  I 240  1  I 260  I 280  1  I 300  1  Wavelength (nm)  B  -10  o  \e  .&  -is  3  -20  -25 20  30  40  50  60  70  80  90  Temperature (°C) Figure 3.6  Secondary structure content of His-SpaPK.  (A) Circular dichroism  analysis of His-SpaPK. Purified His-SpaPK at 0.25 mg/ml was subjected to C D spectroscopy, as described in Section 3.3.4. (B) Thermal unfolding of His-SpaPK. The C D spectrum was measured at 222 nm while the protein solution was heated from 25 °C to 85 °C. The four transitions of the melting curve are indicated by arrows.  87  3.3.5 Autophosphorylation activities of wild-type His-SpaPK and catalytically inactive His-K42A-SpaPK The amino terminal portion of SpaPK is predicted to contain all of the conserved subdomains characteristic of protein kinases. To determine whether SpaPK possesses kinase activity, kinase assays were performed with the purified His-SpaPK and His-K42A-SpaPK (Section 3.2.5.1).  The products of the kinase reactions were subjected to SDS-PAGE,  electroblotted onto a P V D F membrane and visualized by autoradiography.  Figure 3.7.A  shows that His-SpaPK underwent autophosphorylation in the presence of [y- P]-ATP (lane 32  1). The activity of D A P kinase is stimulated by the presence of calcium and calmodulin. Since the kinase domain of SpaPK shows highest identity to that of D A P kinase, whether the activity of SpaPK also requires similar components was examined. The kinase activity of His-SpaPK is not stimulated by the addition of calcium chloride and calmodulin (lane 2), or DTT in the reaction buffer (lane 3). However, the requirement of calcium for SpaPK activity cannot be definitively stated since E D T A was lacking in assay mixtures. Panel C shows that substitution of the universally conserved lysine 42 residue with an alanine, a point mutation known to interfere with the phosphotransfer reaction in other kinases, abolished the kinase activity of this protein (lane 1). To monitor the amount of proteins transferred onto the PVDF membranes, Western blotting was performed using an anti-Express monoclonal antibody. Panel B and panel D show that the amount of protein is comparable in each lane. The kinase activity displayed by the purified His-SpaPK further supports the notion that the renaturation of the recombinant proteins during the purification process was successful.  3.3.6 Phosphoaminoacid analysis of autophosphorylated His-SpaPK To  identify the phosphorylated amino acid residues in the His-SpaPK, the  autophosphorylated recombinant protein was subject to phosphoaminoacid analysis (Section 3.2.5.2). Radiolabeled His-SpaPK was resolved using SDS-PAGE, transferred to a PVDF membrane, and hydrolyzed in the presence of HC1. The released amino acids were then separated and analyzed by two-dimensional thin-layer electrophoresis. Figure 3.8 shows that autophosphorylation of His-SpaPK occurs predominantly on serine residues, and to a lesser  88  D  Figure  3.7  Kinase  activities  of His-SpaPK  and  His-K42A-SpaPK.  (A)  Autophosphorylation activity of His-SpaPK. Kinase assays using 5 pg of purified His-SpaPK were performed, as described in Section 3.2.4.1 (lane 1), or in the presence of additional 10 m M calcium chloride and 25 p M calmodulin (lane 2), or 25 m M DTT (lane 3). (B) Western blot of (A) using an anti-Express monoclonal antibody. (C) Autophosphorylation activities of His-SpaPK and K42A-His-SpaPK. Kinase reactions were carried out using 0.5 pg of purified His-SpaPK (lane 1) or His-K42A-SpaPK (lane 2). monoclonal antibody.  (D) Western blot of (C) using an anti-Express  A l l products of kinase reactions were subjected to SDS-  P A G E , electroblotted onto a PVDF membrane and visualized by autoradiography. The position of His-SpaPK proteins is indicated by an arrow.  89  Figure 3.8  Phosphoaminoacid analysis of autophosphorylated  His-SpaPK.  Autophosphorylation of His-SpaPK was carried out as described in Section 3.2.5.1. After termination of the kinase reaction, P-labeled His-SpaPK was subjected to 32  SDS-PAGE, electroblotted onto a PVDF membrane and located by autoradiography. The position corresponding to radiolabeled His-SpaPK was excised and hydrolyzed in 6 M HC1, followed by two-dimensional thin-layer electrophoresis in the presence of internal phosphoamino acid standards, as described in Section 3.2.5.2.  The  dashed  and  circles  indicate  the  positions  of  inorganic  phosphate  (Pi)  phosphopeptides generated by partial hydrolysis (PP), and the locations of standards as visualized by ninhydrin staining for the phosphorylated forms of serine (S), threonine (T), and tyrosine (Y). The origin is indicated by a cross.  90  extent on threonine residues. No phospho-tyrosine was detected. These results support the hypothesis that His-SpaPK is a serine/threonine kinase, capable of phosphorylating serine and threonine residues found in its own amino acid sequence.  3.3.7 Identification of potential phosphorylation sequence of His-SpaPK To identify the preferred phosphorylation site in targets of SpaPK, His-SpaPK kinase reactions were performed using 48 peptides containing one or more serine and/or threonine phosphorylation sites. A list of the names of these substrates is presented in Table 3.2. The kinase reactions were carried out in duplicate in a 96 well plate, and transferred onto a phosphocellulose plate (Section 3.2.5.3). Scintillation counting of the radiolabelled products revealed two strong positives and several weaker positives from the screen. One of the positives, M B P , is a generic substrate of various protein kinases. The other, which gave a 3fold more intense signal than M B P , is the substrate of cAMP-dependent protein kinase. The potential phosphorylation target sequence of His-SpaPK was deduced by comparing sequences of the non-phosphorylated and phosphorylated peptides. The resulting predicted consensus phosphorylation sequence is (RZK)-X(not I/P/L/Q)-X(not L/I/G/Q)-(S/T), where the site of phosphorylation is in bold type. Four sites correlating to the deduced consensus phosphorylation sequence are found on SpaPK.  One of these sites is located within the  kinase domain, and three sites are located within the carboxy terminus of SpaPK. These are the sites which are most likely to be autophosphorylated by SpaPK.  3.3.8 Detection of endogenous SpaPK with anti-SpaPK polyclonal antibodies 3.3.8.1 Generation of anti-SpaPK polyclonal antibodies Purified His-SpaPK was used to immunize New Zealand White rabbits for a period of 8 weeks (Section 3.2.5). As shown in Figure 3.9, the resulting antiserum recognized a 55 kDa protein predicted for His-SpaPK in an E. coli extract harboring the construct (Panel A , lane2 and Panel B , lane2). This signal is not detected from a bacterial cell lysate harboring the empty vector (Panel A , lane 1 and Panel B, lane 1). The anti-SpaPK antiserum also failed to recognize a fragment  of His-tagged Drosophila  polyhomeotic protein (His-pH)  overexpressed in E.coli (Panel A , lane 3 and Panel B , lane 3) or a fragment of His-tagged murine Ets-1 (His-pt-L) purified from E.coli (Panel A , lane 4 and Panel B , lane 4). The  91  Table 3.2 Peptide sequences used for screening of potential phosphorylation sequences of H i s - S p a P K . Phosphorylation status  Name o f peptide  Sequence o f peptide  CaMKII-sub  MHRQETVDCLK  -  CDC2D  KIKKIEGEGTYGVVYKKK  -  cGPK-sub  RKISASEFDRPLR  -  M A P 2 R4  CGGPKKPTVPWKRLYGAADPKSLSCCDRILTFNP  -  M A P 2 R5  LPNKPTVPWKR  -  MBPK-C  CGGIDRLALFAHREDWTWNPSGY  - •  MIPK-CT  C G K E L T F Q L I Q A V R H Q SRR  -  MOS-III  ASQRSFWAELNIARLRHDNIVRVVAASTR  -  PAK1  MSNNGLDVQDKPC  -  PAK3-NT  MSD SLDNEEKPP AC  -  PDVII-NT  MLLSKINSLAHLRARACNDLHATKLAPGKEKEPLESGC  -  PKC-sub  CQKRPSQRSKYL  -  RAF-C2  PSLHRAAHTEDINAATLTTSPRLPVF  -  RSK2-PCT  CNRNQSPVLEPVGRS  -  WEE1-X  LTVVCAAGAEPLPRNGDQWHEIRQGRLPGGC  -  C a M K II  EETRVWHRRDGKWQNVHFHC  -  CDC3  PLFHGDSEEDQLFRIFRALGTPGGC  -  cPKC-III  KKDVVIQDDDVESTMVEKC  -  GSK-3p-XI  CSHSFFDELRDPNVK  -  M A P 2 R6  LYGAADPKSLSLLDRILTFNP  -  MEK5-PNT  AFEYEDEDGDRITVRSC  -  MLCK-1  KGTPKTPLPEK  -  NEM-N  VKRHKNTIGVWRLGKTLGTGGC  -  PAK3-NT  MSDSLDNEEKPPAC  -  PIK  CGYILGLGDRHPFRLTR  -  PKA  KKGSEQESVKEFLAKC  -  P ST A I R E  EGVPSTAIREISLLKEGGC  -  RAF-sub  IVQQFGFQRRASDDGRK  +  S6K-sub  CKRRRLASLR  -  cAPK-sub  CGRTGRRNSI  ++++  CDK8-NT  M D Y D F K V K L S SERERC  -  92  ERK5-PNT  SAEPPAREGRTRPHRC  -  HSP-27-sub  RRLNRQLSVA  -  MBP-NT  AAQKRPSQRTKYLA  +  MEKK-1  QDRPP SRELLKHP V F R  MLCK-3  ASGSSPTSPPNADK  -  NMA-III  KEREQLTAEFNILSSLRHPGGC  -  PAK1  MSNNGLDVQDKPC  -  PMI-III  VEKDRISDWGELPNGTRVPMEVGGC  -  PKB-sub  RPRAATF  RAC1-PH  FHVETPEEREEWTC  -  RAS-L2  E YDPTIED S Y R K Q C  -  SEK1-CT  CKILDQMP ATPS S P M Y V D  -  ++  Casein  -  HH1  -  MBP  +++  Phosvitin  -  Polyglutyr  -  Protamine  -  93  inability of the anti-SpaPK antiserum to identify other His-tagged recombinant proteins suggests that the antibodies recognize SpaPK rather than the His-tag. 3.3.8.2 Affinity purification of SpaPK antiserum To increase the titer and the purity of the anti-SpaPK antibodies, the rabbit antiserum was affinity-purified using purified His-SpaPK bound to a N i - N T A column (Section 3.2.6). Using an anti-Express monoclonal antibody, Western blotting was performed on all collected fractions to verify that His-SpaPK remained on the column during the purification process (data not shown). Western blotting using HRP conjugated anti-rabbit secondary antibodies was also performed to confirm the presence of antibodies in the eluted M g C I fractions (data 2  not shown). The resulting affinity-purified anti-SpaPK antibodies, free of the His-SpaPK, are designated ApSpaPK antibodies. 3.3.8.3 Detection of endogenous SpaPK from mammalian cell extracts In Western blots, ApSpaPK antibodies recognized the purified His-SpaPK (Figure 3.10, lane 3).  They also recognized a major immunoreactive band which migrated at  approximately 60 kDa from MCF-7 human breast carcinoma cell extract (Figure 3.10, lane 4), approximately 7.5 kDa larger than the predicted size of SpaPK. A protein of similar size was also detected from other mammalian cell lines, including MCF-7 mutant-p53, A549WT, A549 mutant-p53 and GM814 (data not shown). To confirm that the purified His-SpaPK and the 60 kDa protein from mammalian cell extracts are specifically recognized by the ApSpaPK antibodies, the same Western blotting was performed using pre-immune serum and ApSpaPK antibodies pre-incubated with the purified His-SpaPK. Figure 3.10 shows that the pre-immune serum did not recognize HisSpaPK (lane 1) or any proteins from MCF-7 cell extracts (lane 2).  Pre-incubation of  ApSpaPK antibodies with purified His-SpaPK before probing abolished the recognition of both His-SpaPK (lane 5) and the 60 kDa protein (lane 6). The ability of purified His-SpaPK to block antibody binding suggests that the recognition by ApSpaPK antibodies is specific, and that the 60 kDa protein is most likely the endogenous form of SpaPK.  94  B kDa 207 — 123 — 86 —  1 2  3  4  44 — 31 — * 18 —  Figure 3.9  Detection of His-SpaPK using anti-SpaPK antiserum. Duplicate 12%  SDS polyacrylamide gels were run simultaneously.  One was stained with  Coomassie brilliant blue R-250 (panel A). Proteins on the other gel were transferred onto a nitrocellulose membrane and subjected to Western blotting using an antiSpaPK antiserum (panel B). Lane 1: E. coli lysate containing the pRSETA empty vector; lane 2: E. coli lysate containing the overexpressed His-SpaPK; lane 3: E. coli lysate containing the overexpressed His-tagged Drosophila polyhomeotic protein fragment (His-pH); lane 4: purified His-tagged murine Est-1 fragment (His-pt-L). Molecular mass standards in kDa are shown on the left of each panel. The position of His-SpaPK, His-pH and His-pt-L are indicated by an arrow, an asterisk, and a leftright block arrow, respectively.  95  Pre^ " incubated immune ApSpaPK ApSpaPK serum antibodies antibodies r e  kDa  Figure 3.10 Detection of SpaPK from a MCF-7 cell extract using affinity purified anti-SpaPK antibodies. Triplicates of 1 pg of purified His-SpaPK (lane 1, 3, and 5) and MCF-7 cell lysate (lane 2, 4, and 6) were resolved on a 10% SDS polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and subjected to Western blotting using a pre-immnune serum (lane 1 and 2), ApSpaPK antibodies (lane 3 and 4), and ApSpaPK antibodies pre-incubated with 2.5 pg of purified HisSpaPK (lane 5 and 6). Molecular mass standards in kDa are shown on the left. The position of His-SpaPK and the endogenous SpaPK from cell lysate is indicated by an arrow and a left-right block arrow, respectively.  96  3.4 DISCUSSION 3.4.1 Evidence for an incomplete SpaPK  As detailed in Chapter 2, there are several lines of evidence suggesting that the cloned SpaPK cDNA is incomplete. Here, two additional observations supporting the hypothesis are discussed. 3.4.1.1 The size of endogenous SpaPK When detected on western blots using specific affinity purified anti-SpaPK polyclonal antibodies, the endogenous SpaPK from cell extracts migrates as an approximately 60 kDa band. This size is 7.5 kDa larger than the estimated molecular mass of SpaPK. Assuming that the average molecular mass of each amino acid is 110 Da, the endogenous protein contains about 68 additional amino acid residues than the sequence predicted for SpaPK. This means that there are over 200 nucleotides carrying coding information upstream of the initiation A T G codon which is located at position 144 to 146 on the SpaPK cDNA. Given that there are at least 87 missing nucleotides as indicated by the primer extension analysis, about 60 nucleotides of the missing sequence contain coding information and at least 25 nucleotides of the missing sequence are part of the 5' U T R of the SpaPK cDNA. Interestingly, SpaPK from rat smooth muscle contains an additional 16 amino acids at its N terminus in comparison to other homologs. Alternatively, proteins which carry a high overall negative charge or positive charge migrate slower or faster, respectively, than expected on a SDS polyacrylamide gel. SpaPK has a predicted pi of 6.6 which should not alter the rate of migration on a SDS denaturing gel. If amino acids encoded by the 5' U T R are included as part of SpaPK, then the predicted pi of the longer SpaPK is 8.4 which still would probably not change the rate of migration. Post-translational modifications including phosphorylation, glycosylation and methylation can also influence the overall molecular mass of a protein. The observed size difference between the detected endogenous and the predicted SpaPK is possibly due to an incomplete 5' end of the SpaPK c D N A or post-translational modifications or a combination of both. 3.4.1.2 Presence of a nuclear export signal in the 5' U T R of the SpaPK cDNA The localization of a protein from the nucleus to the cytoplasm is highly regulated. In addition to various factors, proteins that are exported from the nucleus contain a sequence known as the nuclear export signal (NES), which specifies selective translocation to the  97  cytoplasm (Fukuda et al, 1996). NES, a short leucine-rich motif, contains four hydrophobic residues at a conserved spacing (Wen et al, 1995). Many proteins have been reported to be spatially controlled by their NES, including protein kinase A inhibitor (PKI), M A P kinase kinase 1 (MKK1) (Fukuda et al, 1996; Tolwinski et al, 1999), cyclin B l (Toyoshima et al, 1998) , M A P K A P kinase-2 ( M A P K A P kinase-2) (Engel et al, 1998), p53 (Stommel et al, 1999) and HIV Rev protein (Fischer et al, 1995). Examination of the amino acid sequences encoded by the SpaPK cDNA revealed the presence of a N E S motif, as implied by its similarity to other known NESs (Figure 11). However, the nucleotide sequence encoding this motif is located within the 5' UTR, from 63 to 90 residues upstream of the predicted translational initiation codon.  Within the putative N E S of SpaPK are two potential  phosphorylation sites. Phosphorylation has been reported to be important in controlling the nuclear translocation of cyclin B l (Li et al, 1997a). The localization of endogenous SpaPK could be coordinately regulated by this putative nuclear export signal and the putative bipartite nuclear localization signal outside the kinase domain (Section 3.2.1.2 and Section 3.4.5.3). Therefore, the existence of such a potentially important motif within the 5' U T R suggests that the actual coding region extends into this part of the SpaPK sequence.  3.4.2 SpaPK is a serine/threonine-specific kinase The amino acid sequences from subdomain V I and VIII of SpaPK is consistent with a serine/threonine-specific kinase (Hanks et al, 1988). Phosphoaminoacid acid analysis of the autophosphorylated His-SpaPK confirms the prediction, showing the ability of the protein to phosphorylate serine and threonine residues and not tyrosine residues (Figure 3.8). However, these results do not entirely exclude the possibility that SpaPK is a tyrosine kinase. The analysis was performed using the renatured His-SpaPK. Although both C D spectra and the display of kinase activities indicate that the purified His-SpaPK is refolded, the extent of refolding was not monitored since there was no reference for comparison. The observed activities may be manifested by partially refolded protein or a mixture of partially refolded and native proteins.  98  SpaPK  YL  S L  E  ELALKLAGLDIN ALQKKLEELELD DLCQAFSDVILA EMT SALATMRVD MFRELNEALELK LQLPPLERLTLD  PKI MKK1  human cyclin B 1 M A P K A P kinase-2 human p53 HIV Rev  Figure 3.11  Q 7 A A V T AL  Alignment of the putative N E S sequence of SpaPK with that of other  proteins. The putative NES sequence of the SpaPK protein is presented on the top line. The NES sequences of protein kinase A inhibitor (PKI), M A P kinase kinase-1 ( M K K 1 ) , human cyclin B l , M A P kinase-activated protein kinase-2 ( M A P K A P kinase-2), human p53, and H I V Rev protein are aligned at the bottom. Important hydrophobic residues within the NES sequences are in bold-type.  99  Kinases which phosphorylate both tyrosine and serine/threonine residues on their target substrates are referred to as dual-specificity kinases. The yeast Rad53, a bona fide serine/threonine-specific protein kinase, has been reported to display an autophosphorylation activity towards tyrosine residues in immunoprecipitates (Zheng et al, 1993), although its physiological tyrosine-specific substrates have not been identified. Other dual-specificity kinases, such as Xenopus M y t l and members of the M A P kinase kinase (MEK) family, have been characterized in more detail. M y t l , a kinase important in regulation of the cell cycle, phosphorylates adjacent threonine and tyrosine residues of Cdkl complexed with cyclin B (Mueller et al., 1995). Members of the M E K family, which transduce signals of stress, damage and survival within a cell, simultaneously phosphorylate both threonine and tyrosine present in the conserved T - X - Y sequence motif of the activation loop of M A P kinases (Nakielny et al., 1992).  Another group of related protein kinases, D Y R K s which are  involved in cellular growth, have also been reported to have tyrosine phosphorylation activity. They appear to be activated through autophosphorylation of their T - X - Y activation loop, but otherwise function primarily as serine/threonine directed kinases (Becker et al., 1998; Himpel et al, 2000). Overall, dual-specificity kinases typically function in pivotal positions in signal transduction pathways. Further experiments such as testing the purified His-SpaPK against known tyrosine kinase substrates are required to verify whether the SpaPK kinase is a dual-specificity kinase.  3.4.3 Substrate specificity of SpaPK  Protein kinases selectively phosphorylate their targets in order to perform specific functions.  The selectivity is partly dependent on the nature of residues surrounding the  phosphorylated amino acid.  This type of selectivity, commonly referred to as sequence  specificity, is illustrated by the ability of various protein kinases to phosphorylate short peptides reproducing phosphoacceptor sites, with kinetics comparable to those of the intact substrate.  Based on the residues utilized for site recognition, serine/threonine-specific  protein kinases are classified roughly into three main groups (Pinna and Ruzzene, 1996). Basophilic protein kinases, such- as P K A , P K C and C a M kinases, use basic and often hydrophobic residues  as specificity determinants.  Proline-directed protein kinases,  represented by cyclin-dependent kinases and M A P kinases, require the presence of a proline  100  residue immediately C-terminal to the acceptor site.  And acidophilic/phosphate-directed  protein kinases, such as CKII and GSK3, utilize carboxylic and or phosphorylated side chains as specificity determinants. Determination of a reliable consensus sequence for phosphorylation by a given protein kinase is a laborious task. The success of the outcome is dependent on the amount and quality of data available. The potential consensus phosphorylation sequence for SpaPK, derived from 48 peptides, shows that the protein prefers an arginine residue located two residues N-terminal to the phosphoacceptor site, suggesting that SpaPK is a basophilic kinase. To further characterize the residues involved in recognition by SpaPK, one could utilize a SPOT assay in which each amino acid surrounding the site of phosphorylation is replaced.  These peptides are synthesized as individual spots on cellulose paper and  phosphorylated by a kinase of interest. In addition, a degenerate peptide library consisting of different amino acid residues at every position could also be used to examine the changes in phosphorylation efficiency.  There are several limitations in using these methods.  The  crucial determinants established with the consensus peptide sequence may be less important in the intact protein substrate (Ikebe et al, 1994). The relevance of an individual residue at a given position may depend on the rest of the sequence (Marin et al, 1992; Sarno et al., 1996; Srinivasan et al., 1995), as well as the vicinity of residues in three-dimensional space (Fiol et al, 1990). Furthermore, the length of the peptide excludes possible determinants acting at positions further upstream and/or downstream of the phosphoacceptor site. ZIP kinase, isolated from HeLa cells, phosphorylates the regulatory light chain of myosin II (MRLC) at Thr-18 and Ser-19 in vitro (Murata-Hori et al, 1999). Phosphorylation at Ser-19 leads to localization of M R L C to the midzones of separating chromosomes in dividing mammalian cells, suggesting that phosphorylation of this site is important for initiation of cytokinesis (Matsumura et al, 1998; Matsumura et al, 2001).  Examination of  amino acid sequence surrounding Ser-19 of M R L C revealed the presence of an arginine located two residues downstream of Ser-19.  This is in agreement with the deduced  phosphorylation sequence of SpaPK. However, sequences surrounding Thr-18 do not match the consensus motif.  Nevertheless, the correlation with the Ser-19 phosphorylation site  suggests that the deduced preferred phosphorylation sequence of SpaPK is valid. Searches of human protein databases using the consensus phosphorylation sequence revealed thousands  101  proteins containing potential SpaPK phosphorylation sites.  Such a large score was most  likely due to the fact that the query sequence was too short and ambiguous. A more stringent search using the amino acid sequence which gave the highest phosphorylation signal in the peptide screen, resulted in the identification of 56 human proteins.  Examples include  proteins involved in ion transport, transcription, translation, intracellular movement, and cell cycle.  3.4.4 Putative phosphorylation sites on SpaPK  SpaPK has the potential to be phosphorylated by multiple protein kinases, including P K A , P K C , P K G , C K I , CKII, C a M kinase II, GSK3, p70S6k and a tyrosine kinase. These phosphorylation sites were predicted by ScanProsite and PhosphoBase programs which carry consensus phosphorylation sites for 11 protein kinases. Although there are exceptions, the consensus sequence for phosphorylation by each of the above protein kinases is as follows. Substrate recognition by P K A requires the presence of a basic amino acid, particularly arginine, found one or two residues N-terminal to the phosphoacceptor site (Hofmann et al., 1999; Kennelly and Krebs, 1991). P K C has a preference for the phosphorylation of serine or threonine residues found close to a C-terminal basic residue (Hofmann et al., 1999; Kennelly and Krebs, 1991). residue  P K G targets sequences rich in basic residues which are located one  N-terminal to the  site  of phosphorylation (Kennelly  and Krebs, 1991).  Phosphorylation by C K I requires the presence of an acidic or a pre-phosphorylated serine or threonine located two to three residues from the phosphoacceptor group (Pinna and Ruzzene, 1996).  Substrates of CKII are characterized by an acidic residue or pre-phosphorylated  serine or threonine located three residues from the C-terminal side of the phosphorylated serine or threonine (Hofmann et al., 1999; Kennelly and Krebs, 1991).  Sequences  phosphorylated by C a M Kinase II contain an arginine which is three residues N-terminal of the site of phosphorylation (Kennelly and Krebs, 1991). GSK3 phosphorylates serine or threonine residues located four residues N-terminal to a pre-phosphorylated serine (Kennelly and Krebs, 1991). p70S6K phosphorylates sequences which contain an arginine and a basic residue three residues and five residues N-terminal to the site of phosphorylation, respectively (Pinna and Ruzzene, 1996). And finally, tyrosine-specific protein kinases, in general, phosphorylate tyrosine residue which lies seven residues to the C-terminal side of a  102  basic residue. A n acidic residue is often found at either three or four residues to the N terminal side of the tyrosine (Pinna and Ruzzene, 1996). The ability of the above kinases mentioned to phosphorylate SpaPK in vitro and in vivo remains unclear. A l l the protein kinases described above are pleiotropic regulators of many cellular processes, and therefore, potential phosphorylations by these kinases do not offer any insight into the function of SpaPK. However, further analysis of SpaPK sequence revealed that it also carries a phosphorylation site for Chkl protein kinase. C h k l is a critical regulator of the D N A damage checkpoint pathway. The consensus phosphorylation motif of Chkl is <E>-X-pX-X-(S/T), where O is a hydrophobic residue (M>I>L>V), p is a basic residue (R>K), X is any amino acid, and the site of phosphorylation is in bold type (Hutchins et al., 2000). This phosphorylation site is located close to the carboxyl terminus of SpaPK, from position 409 to 414.  Sequence specificity is not the only criterion ensuring the selectivity of proteins  kinases. Whether a protein kinase interacts with its substrate(s) also depends on subcellular compartmentalization and precise association often mediated by targeting elements outside the kinase domain.  3.4.5 Other post-translational modifications and motifs of SpaPK 3.4.5.1 The putative prenylation site of SpaPK Covalent modification by an attachment of an isoprenoid moiety, known as prenylation, occurs on a wide variety of cellular proteins. Known prenylated proteins include Ras superfamily G-proteins, nuclear lamins, and two protein phosphatase (Sinensky, 2000; Tamanoi et al., 2001a; Tamanoi et al., 2001b). Addition of the lipid groups to these proteins facilitates membrane localization and promotes protein-protein interaction. The carboxyl terminal residue of the CaaX motif determines which isoprenoid will be added to a protein. The cysteine residue of the CaaX-containing proteins is modified by a farnesyl group when X is usually a serine, cysteine, alanine, methionine or glutamine, or modified by a geranylgeranyl group when X is a leucine (Zhang and Casey, 1996). SpaPK contains a putative CaaX box where X is an arginine residue. The identity of this residue does not conform to the general consensus.  However, prenylated proteins  containing unusual CaaX motif have recently been identified.  CENP-E and CENP-F,  kinetochore-associated proteins, play critical roles during the M-phase of the cell cycle  103  (Abrieu et al, 2000). Both proteins carry an asparagine residue at position X within the CaaX box, and are farnesylated at the cysteine residue (Ashar et al, 2000). The sequences of the CaaX box are C K T Q for CENP-E and C K V Q for CENP-F. CENP-E, the microtubuleassociated motor protein, is required for the coupling of kinetochores and microtubules and the generation of motive force for chromosome movement during mitosis (Yao et al, 2000). Studies using inhibitors suggest that farnesylation of CENP-E does not alter its association with the kinetochore, but does promote its association with microtubules (Ashar et al, 2000). Prenylation of SpaPK on the putative CaaX box and the functional consequences remain to be tested. 3.4.5.2 The putative leucine zipper of SpaPK Leucine zipper motifs allow both homo- and hetero-dimerization of proteins. This protein-protein interaction motif was initially identified in families of transcription factors including C A A T / E B P , fos/jun, A T F (Landschulz et al, 1988) and myc/max (Blackwood et al, 1992). Recently, several kinases have been reported to contain putative or proven leucine zippers. These include P K N (Mukai and Ono, 1994), Zipper Protein Kinase (ZPK) (Reddy and Pleasure, 1994), Leucine zipper-bearing kinase (LZK) (Ikeda et al, 2001), Myosin light chain kinases (MLKs) (Hirai et al, 1997), Ataxia-telangiectasia mutated kinase (ATM) (Chen and Lee, 1996), DNA-dependent protein kinase (DNA-PK) (Hartley et al, 1995), I K B kinase (Regnier et al, 1997), and Dictyostelium NIMA-related kinase 2 (Nek2) (Graf, 2002). A leucine zipper motif is a special type of coiled coil, characterized by at least three heptad repeats where a and d of each repeat generally are leucine residues (Lupas, 1996). The carboxyl-terminus of SpaPK contains a series of leucine and valine residues from amino acid 426 to 441. Theoretically, this sequence does not fulfill the requirements of a leucine zipper, as ScanProsite did not predict its presence on SpaPK. However, Kawai et al. (1998) demonstrated that the homolog from mouse, mZIP kinase, does contain similar residues that are responsible for protein-protein interaction from amino acids 422 to 436.  A n intact  leucine zipper motif is required for dimerization since a C-terminal fragment of mZIP kinase containing a mutated leucine zipper failed to interact with itself or ATF4 transcription factor (Kawai et al, 1998). The authors also showed that the kinase activity of mZIP kinase is dependent on self-association since mutation of the leucine zipper in the full length protein abolished the phosphorylation activity (Kawai et al, 1998). The rat homolog of SpaPK, Dlk,  104  also interacts with other partners as revealed from two-hybrid screens.  The interaction  domain within Dlk is mapped to the leucine zipper when binding to CDC5 (Engemann et al., 2002) and apoptotic antagonistic transcription factor (AATF) (Page et al., 1999b), and to a C-terminal arginine-rich region when binding to prostate apoptosis response gene 4 (Par-4) (Page et al., 1999a). Par-4, a mediator of transcription containing a bona fide leucine zipper, can interact with the full length Dlk as well as a Dlk deletion mutant lacking the leucine zipper (Page et al., 1999a). These results imply that amino acid residues from position 426 to 441 on SpaPK are important for protein-protein association. However, additional features must also be important in determining interaction partner of SpaPK. 3.4.5.3 Nuclear localization signal SpaPK was predicted to carry a bipartite nuclear localization signal outside the kinase domain (Section 3.3.1.2). SpaPK may also carry an additional functional N L S , I K K R R L S , in the subdomain II of its kinase domain. A C-terminal deletion mutant of D A P kinase carrying a similar N L S , I K K R R L P , is localized to the nucleus rather than retained in the cytoplasm, suggesting that this N L S is inaccessible in the full length protein.  This  interpretation might apply to SpaPK.  3.4.6 Homology comparisons with other protein kinases  The kinase domain of SpaPK shows highest identity to that of death-associated protein kinase (DAP kinase). A l l DAP-related kinases have been implicated in promoting programmed cell death, but a direct role in this process has only been demonstrated for D A P kinase (Kogel et al., 2001). The catalytic domain of the human SpaPK has 81% of its amino acid sequence identical to that of the human D A P kinase. Particularly, subdomain IX is entirely identical. The high degree of sequence identity between SpaPK and D A P kinase suggests that SpaPK also functions as a positive regulator of apoptosis.  However, the  homology is limited to the kinase domain, while the sequences immediately C-terminal to the kinase domain differ completely. On one hand, the C-terminus of D A P kinase contains a calmodulin-binding domain which contributes to calcium and calmodulin dependent phosphorylation activity, a region of eight ankyrin repeats, a cytoskeleton binding domain, and a death domain which functions in the mediation of apoptosis (Cohen et al., 1997; Feinstein et al., 1995). On the other hand, the C-terminus of SpaPK does not carry any of  105  these motifs and does not show any significant homology to any other proteins in the database. The kinase domain of SpaPK also shows high similarity to calcium calmodulin dependent protein kinases and myosin light chain kinases from a variety of species. Both types of proteins phosphorylate their targets in a calcium calmodulin dependent manner. However, the activity of His-SpaPK is not influenced by the addition of calcium or calmodulin (Section 3.3.5), suggesting that SpaPK does not belong to the C a / C a M 2+  regulated protein, kinase subfamily.  Other proteins which display high similarity to the  catalytic domain of SpaPK are Rad53 and Cdsl. The sequence homology between SpaPK and other protein kinases may suggest possible functions, however, these assumptions need to be tested experimentally.  106  CHAPTER 4 CHARACTERIZATION OF THE FUNCTIONS OF SpaPK  4.1 INTRODUCTION The cell division phase, or the M phase, comprises mitosis when various stages of nuclear division occur and cytokinesis when cytoplasmic division occurs. The main purpose of M phase is to equally segregate parental cell contents, such that each daughter cell inherits one complete set of chromosomes, one centrosome, and an appropriate complement of cytoplasmic components.  The progression of M phase is dependent on the coordinated  activity of many elements, including microtubules, microtubule-associated motor proteins, centrosomes and signaling proteins.  4.1.1 Main events of mitosis and cytokinesis Mitosis is a term coined by Walther Flemming in 1882 from the Greek word for thread, reflecting the shape of mitotic chromosomes. Mitosis is sequentially divided into five stages: prophase, prometaphase, metaphase, anaphase and telophase. These stages are based on morphological features distinguished by light microscopy of living cells, and light and electron microscopy of fixed and stained cells.  During prophase, diffuse interphase  chromatin condenses into well-defined chromosomes. Each of the sister chromatid pair of chromosomes contains a specific D N A sequence called the centromere.  Meanwhile, the  duplicated centrosomes begin nucleating dynamic microtubules and migrating apart around the outside of the nucleus, thereby defining the poles of the bipolar spindle apparatus. Prometaphase begins with the breakdown of the nuclear envelope, allowing the spindle microtubules to interact with the chromosomes. A specialized proteinaceous complex, called the kinetochore, matures on each centromere, and captures the plus ends of some of the spindle microtubules. Chromosomes eventually congress to an equatorial plane in the cell during metaphase. Each chromosome is held in tension at the metaphase plate by the paired kinetochores and associated microtubules emanating from opposite poles of the spindle. After all chromosomes have undergone proper bipolar attachment, a sudden loss of tension between the paired kinetochores on sister chromatids triggers the onset of anaphase. Each chromosome is then pulled toward the spindle pole it faces in anaphase A , and the poles  107  themselves separate further apart in anaphase B. Once the chromosomes arrive at the spindle pole, a new nuclear envelope reforms around the separated daughter chromosomes and chromatin decondensation occurs in telophase. Finally, the cytoplasmic contents are equally divided by a process called cleavage during cytokinesis. Shortly after the onset of anaphase, a contractile ring of actin and myosin filaments begins to assemble beneath the plasma membrane midway between the two spindle poles perpendicular to the long axis of the mitotic spindle. The contractile ring is drawn inward to form a cleavage furrow. The cleavage furrow gradually deepens and encounters the spindle midzone composed of tightly packed remains of spindle microtubules. Furrow ingression continues further, such that a narrow neck, known as the midbody, connects the two daughter cells. The midbody finally breaks down at each end, resulting in two separate daughter cells. This brief description of M phase applies to animal cells.  Although numerous  variations exist between animals, plants and fungi, the basic strategy of cell division is remarkably similar among eukaryotic organisms.  4.1.2 Microtubules: a brief introduction Microtubules are involved in a wide variety of cellular functions, such as organelle transport, cell organization and shape, cell motility and division, and cell morphogenesis (Valiron et al, 2001). The building block of microtubules is an a/p-tubulin heterodimer. The a/p-tubulin heterodimers associate linearly to form protofilaments in which P tubulin in one dimer contacts a tubulin in the adjacent dimer. Typically in vivo, 13 protofilaments are joined through lateral interactions to form a 25 nm hollow cylindrical tube (Amos and Klug, 1974; Evans et al, 1985; Tilney et al, 1973). The alignment of tubulin dimers gives rise to structural and kinetic polarity within the microtubules. The P-tubulins are exposed at one terminus of the microtubules, known as the fast growing 'plus' end during polymerization. The a-tubulins are exposed at the other terminus of the microtubules, known as the slow growing 'minus' end during polymerization (Hunter and Wordeman, 2000). In a population of microtubules, most microtubules grow slowly at a steady state while a minority shrinks rapidly. Microtubules grow and shrink by addition or loss of tubulin heterodimers at the ends of microtubules (Kirschner and Mitchison, 1986). A n individual  108  microtubule alternates stochastically between phases of slow growth and rapid shrinkage, a behavior known as dynamic instability (Mitchison and Kirschner, 1984)]. The transition of a microtubule from growth to shrinkage is called catastrophe, and the converse transition is called rescue (Mitchison and Kirschner, 1984; Walker et al., 1988). Dynamic instability is characterized by the polymerization rate, depolymerization rate, catastrophe frequency and rescue frequency of the microtubule. Dynamic instability is thought to arise from hydrolysis of p-tubulin-bound GTP to GDP.  Incorporation of new tubulin heterodimers into a  microtubule stimulates hydrolysis of GTP bound to interior P-tubulins (Carlier, 1989; Stewart et al., 1990).  During rapid growth of microtubules, the rate of tubulin  polymerization is slightly faster than the rate of GTP hydrolysis, resulting in the generation of a GTP cap on the end of the microtubules.  Such a GTP cap stabilizes and promotes  continual growth of microtubules (Carlier, 1989; Drechsel and Kirschner, 1994). A decrease in the rate of polymerization leads to loss of the GTP cap and exposure of GDP-tubulins at the end of microtubules.  GDP-exposed microtubules change from the strained straight  conformation to a relaxed curved conformation (Hyman et al., 1995; Muller-Reichert et al., 1998). The release of energy accompanying the relaxation of the strained GDP-tubulin is thought to promote progressive shrinkage of the microtubule ends (Caplow and Shanks, 1996). Nocodazole, a microtubule depolymerizing drug, acts by sequestering tubulin dimers with which it forms inactive complexes.  This results in a decrease in free tubulin  concentration and eventual disassembly of microtubules (Xu and Luduena, 2002). Taxol, a tubulin-binding drug, appears to stabilize microtubules by inducing GDP-tubulin to adopt the straightened conformation (Arnal and Wade, 1995).  4.1.3 Microtubule-associated motor proteins Microtubule-based molecular motors are proteins which utilize the energy from ATP hydrolysis to perform their functions.  The roles of most of the microtubule-based motor  proteins are to directionally transport cargos along microtubules, or to crosslink and slide antiparallel microtubules past one another (Higuchi and Endow, 2002). The microtubulebased dynein and kinesin superfamilies are comprised of many related proteins which function in organelle transport, spindle and chromosome motility during mitosis, and cell  109  motility (Mountain and Compton, 2000).  A l l motor proteins are composed of one or more  motor domains which reversibly bind to microtubules and convert energy from A T P hydrolysis into force for translocation, and a region outside the motor domain(s) which interacts with the surface of its cargo (Karcher et al., 2002). The ability of motor proteins to transport a wide variety of cargos such as vesicles, organelles, protein complexes and nucleic acids and protein complexes, is partly due to the divergence of cargo-binding regions (Karcher et al., 2002).  The microtubule-based motor proteins relevant to mitosis are  described in further detail in the following sections (Table 4.1). 4.1.3.1 Dynein superfamily The dynein superfamily is grouped into three subfamilies according to their cellular functions (Gibbons, 1995). One of the subfamilies, the cytoplasmic dyneins, is found in all cell types and plays multiple roles in chromosome separation during mitosis. Cytoplasmic dynein hydrolyzes A T P to glide towards the minus ends along the microtubules (Wang et al., 1995). Cytoplasmic dynein is composed of two heavy chains, each of which containing a microtubule-binding site and four ATP-binding loops (King, 2000; Koonce, 1997), and several intermediate chains and light chains which participate in binding of cargo (Holzbaur and Vallee, 1994). Structurally, a short and complex stalk connects the motor domains of cytoplasmic dynein organized into two globular heads to a single base (Holzbaur and Vallee, 1994). This base is most likely composed of additional accessory proteins such as dynactin, which is a multi-subunit activator of cytoplasmic dynein (Gill et al., 1991). 4.1.3.2 Kinesin superfamily The kinesin superfamily is comprised of kinesin originally identified in squid axoplasm and a diverse set of kinesin-related motor proteins found in many different species (Goldstein, 2001). Each kinesin-related motor shares a conserved motor domain containing approximately 340 amino acids (Vale, 1996). According to the location of the motor domain within their amino acid sequences and their cellular functions, kinesins are categorized into four mains classes: K I N N , K I N C, K I N I and the Orphan group (Vale and Fletterick, 1997). The motor domain of the plus end-directed K I N N kinesins resides at the N terminus of the amino acid sequence. Within this class of proteins, there are several subgroups categorized according to their biochemical activities.  The subgroups involved in mitosis are the  multimeric bipolar kinesins which have motor domains at either end of the protein, and  110  Table 4.1 List of mitotic microtubule-associated motor proteins and their functions . 3  Class  Species"  Cytoplasmic dynein  K I N N Kinesin Bipolar  Chromokinesin  Centrosome separation and spindle orientation Microtubule minus end focusing at spindle poles Microtubule plus end attachment to kinetochores  bimC: A n Cln8: Sc K i p l : Sc Cut7: Sp Klp61F: Dm XlEg5: XI hsEg5: Hs klp38B: D m X k l p l : XI chromokinesin: Gg klf4: M m nod: D m kid: Hs  K I N C Kinesin  K I N I Kinesin  Orphan N-Orphan-kinetochore N-Orphan-midzone  a b  Properties  Direction of motility minus  Centrosome and spindle pole body separation Maintenance of spindle bipolarity Microtubule minus end focusing at spindle poles Microtubule crosslinking Opposition of K I N C kinesin activity  plus  Microtubule plus end attachment of chromatin Formation and maintenance of spindle bipolarity Chromosome segregation  plus  Microtubule plus end attachment of chromatin Chromosome segregation  KLP A: An K A R 3 : Sc P k l l : Sp KatA: At Ned: D m XCTK2/Xklp2: XI C H 0 2 : eg HSET: Hs X K C M 1 : XI M C A K : Hs  Microtubule minus end focusing at spindle poles Microtubule crosslinking Centrosome separation Opposition of bipolar K I N N kinesin activity  minus  Associate with microtubule plus ends Affect microtubule dynamics  none motile  CENP-E: Hs  Microtubule attachment at kinetochores Chromosome segregation Cytokinesis  plus/minus?  Pavarotii: Dm ZEN4: Ce C H O I : Cg M K L P 1 : Hs  plus  This table is adapted from (Mountain and Compton, 2 0 0 0 ) The following designations are used for each species: A n : Aspergillus nidulans; Sc: Saccharomyces cerevisiae; Sp: Schizosaccharomyces pombe; Dm: Drosophila melanogaster; XI: Xenopus laevis; Ce: Caenorhabditis elegans; Gg: Gallus gallus; Cg: Cricetulus griseus, M m : Mus musculus; Hs: Homo sapiens  111  chromosome-associated kinesins which have the additional capacity to bind chromatin through its DNA-binding domain. The motor domain of the minus end-directed K I N C kinesins lies at the C-terminus of the amino acid sequence. The motors of the K I N I class contain internally located motor domains.  These proteins all function as non-motile  monomers and modulate microtubule dynamics at the minus end of microtubules. The fourth class is the Orphan kinesin motors. The member of the N-Orphan kinetochore subgroup functions in microtubule attachment at the kinetochores.  The members of the N-Orphan  midzone subgroup are required for organization of the central spindle and the formation of a contractile ring during cytokinesis. These plus-end directed motor proteins contain an N terminus motor domain, but they are not related to any other members of the kinesin superfamily.  4.1.4 Centrosome The microtubule organization center (MTOC) is the organelle which directs the formation of a network of microtubules in cells. The centrosome is the major M T O C in most animal cells (Meraldi and Nigg, 2002). Vertebrate centrosomes are composed of a pair of barrel-shaped centrioles which are surrounded by an amorphous protein-dense matrix known as pericentriolar material (PCM) (Preble et al, 2000).  Centrioles, each consisting of nine triplets of microtubules, serve to  localize the pericentriolar material into a focal body (Marshall and Rosenbaum, 1999). The P M C functions to nucleate interphase microtubule arrays and essentially all mitotic spindle microtubules (Euteneuer and Mcintosh, 1981). To ensure proper mitotic division, the P M C also promotes the bipolarity of the spindle apparatus such that the minus ends of the mitotic microtubules converge at opposing centrosomes (Lange, 2002).  Although the molecular  organization of P M C is poorly defined, its biochemical properties and composition are characterized in some detail. There are at least four different classes of centrosomal proteins, some with overlapping functions (Lange, 2002). These classes are structural proteins which maintain stability of centrosomes and serve as a scaffold for the assembly of other centrosome-associated proteins, microtubule nucleator proteins such as y tubulin and the components of the y-tubulin ring complex (yTuRC), regulatory proteins such as kinases and  112  phosphatases, and anchoring proteins which physically link microtubule nucleator proteins and regulatory proteins. Based on morphological observations, the centrosome cycle has been divided into four stages, referred to as centrosome duplication during S phase, centrosome maturation at late G2, centrosome separation during the early stages of mitosis, and centriole disorientation at the end of mitosis (Meraldi and Nigg, 2002). This portion of the thesis focuses on the centrosome cycle in animal cells.  The properties of the spindle pole body, the yeast  equivalent of the centrosome, are discussed in (Helfant, 2002).  4.1.5 Mitotic kinases Several important protein kinases, including Cdkl/cyclin B , polo-like family kinases, aurora family kinases, N I M A family kinases, and checkpoint kinases, are involved in M phase progression (Table 4.2). The Cdkl/cyclin B , polo-like family kinases, aurora family kinases and N I M A family kinases are described below. Checkpoint kinases are described in Section 4.1.8. 4.1.5.1 Cdkl/cyclin B Cdkl/cyclin B is a master orchestrator of the M phase events. The regulation of its activity is described in Section 1.3.2. Targets of Cdkl include both structural proteins and regulatory proteins which control the timing and execution of many mitotic events such as chromosome condensation, nuclear envelope breakdown, centrosome separation, spindle assembly, onset of anaphase and mitotic exit. 4.1.5.2 Polo-like kinases Polo-like kinases (Plks) are serine/threonine protein kinases named after the Drosophila polo gene, and are conserved in all eukaryotes (Glover et al, 1995). Whereas one Plk has been identified in S. cerevisiae, S. pombe, Drosophila and Xenopus, at least three Plks are found in mammals (Nigg, 2001). Of these mammalian Plks, P l k l functions during mitosis, and the function of the two other mammalian Plks is not well defined. They may play a role during M phase, as well as prior to mitosis (Donohue et al., 1995; L i et al., 1996; Ouymg etal., 1997). A l l known Plks contain an N-terminal catalytic domain, and a conserved sequence motif, known as the polo-box, in their carboxy-termini (Glover et al., 1998). The polo-box  113  has been proposed to function in targeting of Plks to subcellular compartments (Lee et al., 1998), in mediating interaction with other proteins (Kauselmann et al., 1999; Lee et al, 1998), and in autoregulation of kinase activity of Plks (Mundt et al, 1997).  Plks interact  transiently with several mitotic structures, including spindle poles, kinetochores, the central spindle midzone and the midbody (Arnaud et al., 1998; Glover et al., 1998; Golsteyn et al., 1995).  Also during M phase, Plks display highest kinase activity as a consequence of  phosphorylations (Golsteyn et al., 1995). Two candidate P l k l activating kinases are Xenopus xPlkkl and its structurally related human S L K (Ellinger-Ziegelbauer et al., 2000; Qian et al., 1998b). Their subcellular localization and the timing of their kinase activity suggest that Plks play multiple roles at the onset of mitosis and during M phase. These include regulation of G2/M transition (Section 1.3.2), centrosome maturation, exit from mitosis, and cytokinesis (Mayor et al., 1999). Shortly before mitosis, centrosomes recruit maturation markers such as ninein and cenexin/Odf2 as well as additional yTuRC, leading to a dramatic increase in microtubule nucleation activity of the centrosomes (Khodjakov and Rieder, 1999, Lange and Gull, 1995; Nakagawa et al., 2001; Piel et al., 2000). Genetic analysis and antibody injection have implicated Plks in centrosome maturation (Lane and Nigg, 1996; Sunkel and Glover, 1988).  One of the substrates identified is Drosophila microtubule-associated abnormal  spindle protein (Asp), which functions in microtubule nucleation (do Carmo Avides et al., 2001) or in formation of spindle poles and spindle midzone (Riparbelli et al., 2002; Wakefield et al, 2001).  Other studies have also implicated Plks as important upstream  regulators of the multiprotein complex, known as anaphase promoting complex (APC), which degrades mitotic cyclins (Descombes and Nigg, 1998; Kotani et al, 1998; Shirayama et ah, 1998). Degradation of cyclin B is required for exit from mitosis (Section 4.1.6.3). Dominant-negative approaches and immunodepletion-reconsititution experiments show that Xenopus P l x l is required for cyclin B degradation and M phase exit (Descombes and Nigg, 1998). In vitro, P l k l , pre-activated by Cdkl/cyclin B , can phosphorylate three subunits of APC, thereby enhancing its cyclin-ubiquitination activity (Kotani et al, 1998). In addition, Plks are important regulator of cytokinesis (Carmena et al., 1998; Lee and Erikson, 1997; Mundt et al., 1997). In this role, potential targets of Plks are the N-orphan midzone kinesins. Drosophila  pavarotti or mammalian M K L P 1  114  colocalizes to the midbody and co-  immunoprecipitates with Polo or P l k l , respectively (Adams et al., 1998; Lee et al., 1995). As described in Section 4.1.3.2, N-Orphan midzone kinesins are essential for cytokinesis. 4.1.4.3 Aurora family kinases Aurora from Drosophila melanogaster and increase in ploidy 1 protein (Ipllp) from S. cerevisiae are founding members of the aurora family kinases. Aurora, a protein essential for centrosome separation, was identified from Drosophila melanogaster during a screen designed to isolate mutants which display defects in the centrosome cycle (Glover et al, 1995). Ipllp, a protein involved in both chromosomal and spindle events during mitosis, was identified from S. cerevisiae during a search for mutants showing defects in chromosome segregation (Chan and Botstein, 1993).  Since then, the aurora family has grown to  encompass one member from budding yeast, two from Drosophila and C. elegans, and three from mouse and human (Giet and Prigent, 1999). A n unwanted consequence of this diversity is the confusing nomenclature.  For simplicity, the three aurora/Ipllp-related kinases are  referred to as Aurora-A, B and C (Nigg, 2001). Aurora/Ipllp-related kinases share a similar carboxy-terminal catalytic domain (Giet and Prigent, 1999), and their amino-termini vary in size and share little or no similarity. The kinase activity and subcellular localization of each type of Aurora kinases are cell cycle regulated. The activities of Aurora-A and Aurora-B are at maximal levels during mitosis, but Aurora-A activity reaches a maximum before that of Cdkl and Aurora-B reaches a maximum after that of Cdkl (Bischoff et al., 1998). A-type aurora kinases are involved in centrosome separation and/or mitotic spindle assembly (Giet et al., 1999b; Glover et al., 1995; Roghi et al., 1998). They localize to mitotic spindle poles throughout M phase (Bischoff et al., 1998). B-type aurora kinases play a role in chromosomal events and cytokinesis (Adams et al., 2000; Terada et al., 1998). They associate with the spindle midzone during anaphase and the midbody during telophase (Bischoff et al., 1998; Wheatley et al., 2001). The function of Aurora-C is unclear, but its localization to the centrosome only at anaphase implies a role after that of Aurora-A but before that of Aurora-B (Kimura et al., 1999). At present, little information is available about the substrates of aurora kinases. One of the targets of A-type aurora kinases is the bipolar kinesin Eg5, during centrosome separation (Giet et al., 1999a).  Xenopus A-type aurora kinase pEg2 and bipolar kinesin  XlEg5 colocalize first to an area between the duplicated centrosomes before centrosome  115  Table 4.2 List of mitotic kinases and their functions.  Class  Species  3  Localization  13  Properties  Putative substrates  Polo-like kinases Plkl  Cdc5p: Sc Plolp: Sp Polo: D m P l x l : XI Plkl: M m P l k l : Hs  Centrosome Kinetochore Spindle midzone Midbody  Centrosome maturation Establishment of bipolar spindle  Cdc 25C p-tubulin 85 kDa M A P Asp APC pavarotti pinl c  Plk2  Snk: M m Plk2/hSnk: Hs Fnk: M m Plk3/Prk: Hs  Plk3  Aurora-related kinases Aurora-A  aurora: Dm AIR-1: Ce E g 2 : XI ARKl/Aykl/IAKl: Mm Aurora2/Hs AIRK1 / A R K 1 / A i k / B T A K / S T K - 1 5 : Hs IAL: D m AIR-2: Ce AIRK2: XI AIM-l:Rn ARK2/STK1: M m Auroral/HsAIRK2/ARK2 /Aik2/AIM1/STK-12: Hs AIE1: M m Aurora3/HsAIRK3/ AIE2/Aik3/STK-13: Hs Ipllp: Sc P  Aurora-B  Aurora-C  Ipllp  d  Duplicated centrosomes Spindle pole  Centrosome separation  Kinetochore Spindle midzone Midbody  Chromosome movements Cytokinesis  Eg5 Cdc20  c  Histone H3 INCENPE Survivin C  c  Centrosome (anaphase) Spindle pole body  Histone H3 Ndc lOp  NIMA-like kinases NIMA  NIMA: An  Centrosome  Nek2  Nek2: Hs  Centrosome  Recruitment of C d k l / c y c l i n B to the nucleus and centrosome Chromosome condensation Centrosome separation Chromosome condensation  Histone H3?  C-Napl  a The following designations are used for each species: A n : Aspergillus nidulans; Sc: Saccharomyces cerevisiae; Sp: Schizocaccharomycespombe; Dm: Drosophila melanogaster; XI: Xenopus laevis; Ce: Caenorhabditis elegans; Gg: Gallus gallus; Cg: Cricetulus griseus, Rn: R norvegicus; M m : Mus musculus; Hs: Homo sapiens b Localization of Polo-like kinases and aurora-related kinases refers to those from mammalians. c Binding partners of corresponding kinases. d Yeast Ipllp does not belong to any particular subfamily of aurora-like kinases.  116  separation, then to the separated centrosomes and centrosome-associated spindles from prophase to metaphase. In telophase, pEg2 and XlEg5 remain on the centrosomes, but a pool of XlEg5 is also found at the spindle midzone (Giet et al, 1999a). pEg2 phosphorylates a serine residue in the stalk domain of XEg5, but the functional consequences of such phosphorylation have not been elucidated (Giet et al, 1999a).  Aurora-A has also been  detected in a complex with Cdc20 subunit of APC, suggesting that some function of APC is regulated through its association with Aurora-A (Farruggio et al, 1999). Phosphorylation of core histone H3 on serine 10 has been shown to correlate with chromosome condensation during mitosis. Genetic and biochemical data show that two of the H3 kinases are members of the aurora kinase family: Ipllp of S. cerevisiae and the B type aurora AIR-2 of C. elegans. Both kinases control the phosphorylation of histone H3 in opposition of PP1 (Hsu et al, 2000; Speliotes et al, 2000). In addition, Ipllp is proposed to function in the destabilization of kinetochore-microtubule interaction, by phosphorylating a kinetochore protein, NdclOp (Biggins et al, 1999). Recent findings further indicate that B type aurora kinases interact with two chromosomal passenger proteins, the inner centromeres protein (INCEP) and Survivin, where INCEP serves to target both B-type aurora and Survivin to the expected locations (Adams et al, 2000; Wheatley et al, 2001). A l l three proteins precisely colocalize to the kinetochore during metaphase, to the spindle midzone during anaphase and telophase, and to the midbody during cytokinesis in HeLa cells (Wheatley et al, 2001).  Interference with either Survivin or INCEP function causes  mislocalization of B-type aurora kinases in C. elegans and HeLa cells (Adams et al, 2000; Kaitna et al, 2000; Speliotes et al, 2000). Moreover, yeast two-hybrid and in vitro pulldown assays show that Survivin binds to both Aurora-B and INCEP (Wheatley et al, 2001). The interactions among B-type aurora kinases, INCENP and Survivin are important for mitotic progression, since inference with the function of either one leads to similar defects in mitosis and cytokinesis (Kaitna et al, 2000; Mackay et al, 1998; Speliotes et al, 2000). 4.1.5.4 N I M A family kinases NIMA-related kinases (Neks) are named after the never in mitosis (NIMA) gene product from the filamentous fungus Aspergillus nidulans (Schultz et al, 1994). N I M A , whose activity peaks at the G / M transition, is proposed to function in chromosome 2  condensation (Osmani et al, 1988). It is also required for localization of Cdkl/cyclin B to  117  the nucleus and to the spindle pole body, thereby directly cooperating with Cdkl to promote mitotic progression (Wu et al., 1998). Several structural relatives of N I M A have been found in yeasts, and seven Neks, named Nekl to Nek7, have been identified in mammals (Kandli et al., 2000; Krien et al., 1998).  However, the similarity between different N I M A family  members is mostly confined to the catalytic domain, suggesting diverse cellular functions for different family members. Of the NIMA-related kinases, the kinase domain of Nek2 and the non-catalytic coiled-coil domain are most similar to those of N I M A (Fry et al., 1999; Schultz et al., 1994). Localization and biochemical fractionation studies demonstrate that Nek2 is a core component of the centrosome (Fry et al., 1998b). The protein level and kinase activity of Nek2 are cell cycle-regulated, with peak levels in S/G2 and low activity during mitotic arrest (Schultz et al., 1994).  Recent evidence has implicated Nek2, together with the  components of the M A P K pathway, in chromosome condensation in mouse spermatocytes (Di Agostino et al., 2002). Another important function of Nek2 relates to the regulation of centrosome separation at the onset of mitosis (Fry et al., 1998b), as described in Section 4.1.6.1. Another NIMA-related kinase, Nek6, has recently been implicated in regulation of chromosome condensation due to the ability of recombinant kinase to phosphorylate histone HI and H3 but not casein in vitro (Hashimoto et al., 2002).  4.1.6 Spindle assembly and chromosome movements  Chromosome segregation is mediated by a transiently occurring, microtubule-based bipolar spindle apparatus.  There are three classes of spindle microtubules: astral  microtubules, which radiate from the spindle pole to the cell periphery outside the body of the spindle (Hyman, 1989); kinetochore microtubules, which emanate from the spindle pole to the kinetochores on the chromosomes (Rieder, 1981); and interpolar microtubules, which extend from one spindle pole into the central region and associate with antiparallel microtubules from the opposite pole, and to a lesser extent, parallel microtubules from the same spindle pole (Mastronarde et al, 1993). The spindle microtubules must be capable of coordinated growth and shrinkage to promote chromosome attachment, congregation to and alignment at the metaphase plate, and segregation during anaphase. The dynamic nature of spindle assembly and chromosome movements directly and indirectly involves a variety of proteins and enzymatic activities.  118  4.1.6.1 Spindle assembly The formation of a spindle apparatus requires two changes in microtubule organization and behavior at the onset of mitosis. One of the changes is the replacement of the population of stable interphase microtubules with short, highly dynamic mitotic microtubules. During mitosis, the catastrophe rate of mitotic microtubules markedly changes from minutes to seconds (Inoue and Salmon, 1995; McNally, 1996). The dramatic increase in the dynamic instability of the microtubules is driven by a variety of factors. Such factors include microtubule severing protein katanin (McNally and Vale, 1993), microtubule destabilization protein Op 18, whose activity is turned on by hyperphosphorylation during mitosis (Larsson et al, 1997), and kinetochore-associated K i n I kinesins, which bind to the plus ends of microtubules and promote microtubule catastrophe (Desai et al., 1999). Another change is the shift from one interphase centrosome to two mitotic centrosomes,  each  capable  of nucleating microtubules. During G2, the  duplicated  centrosomes remain closely paired and continue to function as a single M T O C .  Prior to  mitosis, centrosomes dramatically increase their microtubule nucleation activity while they mature (Palazzo et al., 2000). At the onset of mitosis, the centrosomes separate into two distinct MTOCs.  This process  dephosphorylation events.  appears to be regulated  by phosphorylation and  At an early step, an electron dense material connecting the  proximal ends of the two parental centrioles within the duplicated centrosomes is disrupted (Fry et al, 1998a; Paintrand et al., 1992). This process is dependent on the phosphorylation state of a centrosomal Nek2-associated protein (C-Napl), which may serve as a docking site for other centrosomal proteins at the cohesion structure (Fry et al., 1998a).  The  phosphorylation state of C-Napl is possibly regulated by the relative activities Nek2 and a type 1 phosphatase (PP1) (Fry et al, 1998a; Helps et al, 2000). Inactivation of PP1 at the onset of mitosis may contribute to cause an abrupt increase in C-Napl phosphorylation by Nek2, leading to the dissolution of the cohesion structure (Fry et al, 1998a; Helps et al, 2000). At a later step, cytoplasmic dynein and members of the bipolar K I N N and K I N C kinesins are required for centrosome separation (Endow and Komma, 1996; Robinson et al, 1999; Sawin et al, 1992; Vaisberg et al, 1993; Wilson et al, 1997). Prominent among these motors is Eg5, whose binding to the spindle at the poles depends on the phosphorylation of a highly conserved carboxy-terminal motif by Cdkl/cyclin B , and possibly by A-type aurora  119  kinase (Blangy et al, 1995; Giet et al, 1999a). The separation of the two centrosomes is thought to be maintained by the antagonistic actions of K I N N and K I N C kinesins which crosslink and slide overlapping antiparallel interpolar microtubules emanating from opposite spindle poles relative to one another (Gaglio et al, 1996; Mountain et al, 1999; O'Connell et al, 1993; Saunders et al, 1997; Sharp et al, 1999).  The two separating centrosomes  continue to enhance microtubule nucleation activity, leading to the formation of a radial array of microtubules, known as an aster, around each centrosome. These mitotic asters eventually establish the two poles of the bipolar spindle apparatus (Compton, 2000). The dissolution of the nuclear envelope permits access for the attachment of microtubules to the chromosomes at their kinetochores through a mechanism called search and capture (Kirschner and Mitchison, 1986). In this model, the microtubules, which are rapidly growing and shrinking from the two mitotic asters, come into proximity of and are then captured by a kinetochore.  This highly dynamic nature allows microtubules to  efficiently search the local cytoplasm for kinetochores (Holy and Leibler, 1994).  The  kinetochore microtubules, once captured, become preferentially stabilized, such that the turnover rate decreases to a level similar to that of interphase microtubules, and they rarely dissociate from kinetochores (Cassimeris et al, 1990; Mitchison et al, 1986). Eventually, the unattached kinetochore on the sister chromatid captures microtubules from the opposite aster. As a result, sister chromatids become connected by their kinetochores to a bundle of 10 to 40 microtubules which extend from opposite poles (McEwen et al, 1997). This ultimately leads to the bipolar attachment of chromosomes which is crucial for proper chromosome segregation. 4.1.6.2 Chromosome movements Movement of chromosomes is accompanied by the action of dynamic microtubules. Prometaphase  chromosomes oscillate back and forth, and ultimately congress to the  metaphase plate.  As the bipolar-oriented chromosomes move toward the center of the  spindle, the longer microtubules are shortened while the shorter microtubules are elongated. Chromosomes are maintained at the metaphase plate until all are properly aligned. Spindle microtubules at the metaphase plate display a poleward microtubule flux (Mitchison, 1989). The plus end undergoes a net addition of subunits and the minus end experiences a net loss, resulting in the poleward movement of tubulin subunits along these microtubules without net  120  change in overall length of the microtubules. The movement of the chromosomes toward the spindle pole during anaphase A is accompanied by the net loss of tubulin subunits at the kinetochore, leading to shortening of the kinetochore microtubules. Separation of spindle poles is accompanied by the net addition of tubulin subunits to the plus end of interpolar microtubules, leading to their elongation during anaphase B. Chromosome movements are unlikely generated by the growing and shrinking of dynamic microtubules alone (Houchmandzadeh et al, 1997). A balance of polar ejection forces and poleward forces is also important in propelling chromosome movement. Most likely, these forces are dependent on the coordination of multiple microtubule-associated motors located within the spindle. Polar ejection forces, which push chromosomes toward the metaphase plate, play a role in chromosome congression and alignment during prophase and metaphase. These forces are generated directly on the arms of chromosomes (Rieder and Salmon, 1994). Candidate motor proteins involved in this process are members of the plusend directed DNA-binding chromokinesins.  Immunodepletion and reconstitution studies  show that Xenopus xkid, associated with chromosomes during mitosis, is required for chromosome alignment in Xenopus egg extracts, xkid is thought to push chromosome arms away from the spindle pole and towards the equatorial plate from prophase until metaphase (Antonio et al., 2000; Funabiki and Murray, 2000). Other chromokinesins, Xenopus xklpl and Drosophila Nod, are also distributed along the chromosome arms. They function as docking proteins which maintain interaction between microtubules and chromatin, and are essential for spindle assembly (Afshar et al, 1995; Vernos et al, 1995). Poleward forces, which move chromosomes toward the spindle poles, are exerted by the kinetochores. These forces play a role in chromosome movement during prometaphase and become continuous during anaphase.  Three motors are candidates for generating  poleward forces either directly or indirectly. Cytoplasmic dynein appears to drive the first poleward chromosome motion that occurs immediately after initial capture of microtubule at kinetochores (Rieder and Alexander, 1990). The minus end-directed activity of cytoplasmic dynein is also required to drive chromosomes towards the spindle poles during anaphase (Savoian et al, 2000; Sharp et al, 2000). CENP-E, a member of the N-terminal Orphan subfamily of kinesins, is essential for chromosome congression to and alignment at the metaphase plate, and may also contribute to poleward movement during anaphase A (Schaar  121  et al, 1997; Wood et al, 1997; Yao et al, 2000). In vitro experiments show that CENP-E functions to maintain attachment of the kinetochore to the highly dynamic plus ends of microtubules during poleward motion (Yao et al, 2000). The third motor, the K I N I kinesin, promote depolymerization of kinetocnore microtubules during chromosome poleward movement, and may be essential during anaphase to power chromosome migration toward the spindle poles (Desai et al, 1999; Maney et al, 1998).  4.1.7 Anaphase onset and mitotic exit  A multimeric ubiquitin ligase, known as anaphase promoting complex (APC) or cyclosome, plays a pivotal role in M-phase progression. A P C ubiquitinates and targets key regulatory proteins for degradation by the 26S proteasome (Zachariae and Nasmyth, 1999). To ensure the correct order of mitotic events, the activity of A P C is temporally regulated. Two forms of A P C are sequentially activated during the metaphase to anaphase transition and the anaphase to G i transition, by association with Cdc20 and Cdc20 homolog 1 (Cdhl), respectively (Morgan, 1999). A P C complex containing one or the other of these subunits is designated A P C  c  or A P C  . The activity of A P C is also dependent on phosphorylation.  Phosphorylation of core subunits and perhaps Cdc20 is required for activation of A P C  Cdc2  °  (Kramer et al, 2000). On the one hand, kinases such as C d k l , P l k l , BubRl and possibly Aurora-A have been implicated in the activation of A P C  C d c 2 0  (Chan et al, 1999; Descombes  and Nigg, 1998; Farruggio et al, 1999; Kotani et al, 1998; Kramer et al, 2000; Shirayama et al, 1998) and P K A has been described as a negative regulator (Kramer et al, 2000). On the other hand, phosphorylation of Cdhl retains A P C 2000). Phosphorylation of A P C  C d h l  C d h l  in an inactive state (Kramer et al,  is accomplished by Cdk cyclin complexes (Kramer et  al, 2000), and the activating dephosphorylation of A P C  c h l  is accomplished by Cdcl4p in S.  cerevisiae (Jaspersen et al, 1999; Visintin et al, 1998). The two sister chromatids of mitotic chromosomes are primarily connected to each other at their centromeres by a multiprotein complex called cohesin. The onset of anaphase begins with the separation of sister chromatids, resulting from the loss of sister-chromatid cohesion at the centromeres.  The loss is dependent on the degradation of an anaphase  inhibitor, termed Securin, by A P C  Cdc2  ° mediated ubiquitin-dependent proteolysis (Cohen-Fix  et al, 1996; Funabiki et al, 1996; Nasmyth et al, 2000; Yanagida, 2000). This degradation  122  leads to the liberation of a protease, termed Separin (Uhlmann et al, 2000), which in turn allows separation of sister chromatids by cleaving the cohesion subunit Sccl (Uhlmann et al., 1999) . Exit from mitosis requires ubiquitination and degradation of cyclin B (Sigrist et al, 1995; Yeong et al., 2000). Evidence has shown that degradation of a subpopulation of cyclin B, localized to the chromosomes and spindle poles, occurs at the metaphase to anaphase transition in mammalian cells (Clute and Pines, 1999). Budding yeast A P C  C d c 2 0  can also  target a pool of clb2 for degradation at the onset of anaphase (Yeong et al., 2000). However, the majority of cyclin B is degraded by activated A P C  C d h l  after anaphase, thereby promoting  mitotic exit and driving cells through cytokinesis (Shirayama et al, 1999; Yeong et al, 2000) . In yeast, regulation of A P C  C d h l  requires additional proteins from the mitotic exit  network (MEN) (Bardin and Amon, 2001).  4.1.8 Spindle assembly checkpoint Two different mitotic checkpoints monitor passage of cells through M phase. The spindle assembly checkpoint functions to inhibit anaphase onset until all chromosomes are properly aligned on the spindle and achieve bipolar attachment at their kinetochores (Amon, 1999). A second checkpoint in budding; yeast, links proper orientation of the mitotic spindle to mitotic exit (Bardin and Amon, 2001). This spindle positioning checkpoint is dependent on Bub2p, and inhibits the mitotic exit network until the completion of chromosome separation. Structural homologs have been found in higher eukaryotes, but little information is known about their functional relevance. This checkpoint is not described further in this chapter. 4.1.8.1 Spindle assembly checkpoint proteins The spindle assembly checkpoint is activated by improperly attached kinetochores, leading to inhibition of A P C activity required to drive cells into anaphase (Amon, 1999). The resulting metaphase arrest increases the time available for all chromosomes to attach to the mitotic spindle and align at the metaphase plate. A similar arrest is induced upon treating cells with microtubule poisons such as nocodazole or taxol. Yeast checkpoint proteins were identified in screens for mutants which failed to cell cycle arrest when treated with microtubule poisons.  Six checkpoint proteins, budding uninhibited by benomyl 1 and 3  123  (Bubl and Bub3) (Hoyt et al., 1991), mitotic arrest deficiency 1, 2 and 3 (Madl, Mad2 and I  Mad3) (Li and Murray, 1991) and monopolar spindle 1 (Mpsl) (Weiss and Winey, 1996), are essential for the execution of spindle assembly checkpoint in S. cerevisiae.  Since then,  homologs for most of these checkpoint proteins have been identified from higher eukaryotes. The N-terminus of Bubl and Mad3 are structurally related, containing a Cdc20-binding domain and a Bub3-binding domain (Hardwick et al., 2000). However, Bublp contains an additional C-terminal serine/threonine protein kinase domain. A l l animal cells carry two isotypes of Bubl, both containing a carboxy-terminal kinase domain. The kinase with the Mad3-like amino-terminus is named BubRl (Cahill et al., 1998).  Functional studies of  human and mouse Bubl (Cahill et al., 1998; Taylor and McKeon, 1997), human BubRl (Chan et al., 1999), human and Xenopus Mad2 (Chen et al., 1996; L i and Benezra, 1996; Michel et al., 2001), human and Xenopus M p s l (Abrieu et al., 2001; Stucke et al., 2002) show that they are all required for cells to arrest in mitosis in the presence of microtubule inhibitor-induced spindle defects.  Studies in animal cells also indicate that the spindle  assembly checkpoint functions in the timing of anaphase initiation in unperturbed cell division, since unattached kinetochores are present in every prophase and prometaphase cells during normal mitosis (Chan et al., 1999; Rieder et al., 1994; Taylor and McKeon, 1997). Localization experiments have placed all six mammalian checkpoint proteins, Bubl, BubRl, Bub3, M a d l , Mad2 and M p s l , at kinetochores during mitosis (Abrieu et al., 2001; Chen et al., 1996; Jablonski et al., 1998; Jin et al., 1998; L i and Benezra, 1996; Stucke et al., 2002; Taylor et al., 1998; Taylor and McKeon, 1997). Whereas human M a d l , human and Xenopus Mad2, and mouse Bubl are not detectable on kinetochores after proper alignment at the metaphase plate (Campbell et al., 2001; Chen et al., 1996; L i and Benezra, 1996; Taylor and McKeon, 1997), human B u b l , BubRl and Mpsl associate with kinetochores until late anaphase (Jablonski et al., 1998; Stucke et al., 2002). Human Bubl and BubRl also relocate to the midbody during cytokinesis (Jablonski et al., 1998). 4.1.8.2 Sensors of spindle assembly checkpoint The spindle assembly checkpoint senses signals generated by improperly attached kinetochores, either through unattached kinetochores and/or absence of bipolar tension at kinetochores (Nicklas, 1997; Shah and Cleveland, 2000; Skoufias et al., 2001; Waters et al., 1998; Zhou et al., 2002). Experimental data indicate that the motor protein CENP-E is not  124  only involved in kinetochore attachment to microtubules and or tension generation, but is also implicated in the control of spindle checkpoint function. Depending on the experimental system, CENP-E is required either for[ the activation of checkpoint response or for the silencing of a BubRl-dependent checkpoint arrest (Abrieu et al, 2000; Chan et al, 1999; Yao et al, 2000).  Localization of CENP-E to the kinetochores is dependent on its  kinetochore-binding domain located C-terminal of its microtubule-binding domain (Chan et al, 1998). The assembly of checkpoint proteins at the kinetochore is an ordered process. Localization of BubRl to the kinetochores occurs after that of B u b l , and before that of CENP-E (Jablonski et al, 1998). A n association with Bub3 is required for the recruitment of Bubl and BubRl to the unattached kinetochores (Taylor et al, 1998). 4.1.8.3 Interaction between spindle checkpoint proteins Spindle assembly checkpoint proteins form complexes that regulate orderly chromosome segregation and nuclear division. Details of the timing, location and identities of these interactions remain to be fully understood.  The combined data from yeast two  hybrid, co-immunoprecipitation, co-transfection and co-localization experiments show that CENP-E interacts with BubRl (Chan et al, 1998; Jablonski et al, 1998; Yao et al, 2000). BubRl is also capable of directly binding to and inhibit Cdc20, thereby inhibiting the activity of A P C independently of its kinase activity (Chan et al, 1999; Fang, 2002; Tang et al, 2001; Yao et al, 2000).  There are other known associations among kinetochore-associated  checkpoint proteins in higher eukaryotes. Madl recruits Mad2 to unattached kinetochores, forming an association essential for Mad2 function (Chen et al, 1998; Luo et al, 2002; Sudakin et al, 2001; Tang et al, 2001): Mad2 functions to bind Cdc20 and inhibit A P C activity upon activation of spindle assembly checkpoint (Fang et al, 1998; Kallio et al, 1998; L i et al, 1997b). Mad2 undergoes structural changes upon binding to Madl or Cdc20, and the change is important for its function (Luo et al, 2002). These data show that A P C  cdc2  °  is negatively regulated by both BubRl and Mad2 checkpoint proteins. Recently, two protein complexes, containing B u b R l , Bub3 and Cdc20 with or without Mad2, were independently isolated from mitotic human cells (Sudakin et al, 2001; Tang et al, 2001). The protein complex containing Mad2, known as the mitotic checkpoint complex (MCC) is 3000 fold more efficient than Mad2 in inhibiting A P C  c d c 2 0  . In vitro experiments show that Cdc20 can  form two separate inactive complexes, a higher affinity complex with BubRl and a lower  125  affinity complex with Mad2. Binding and ubiquitination assays show that BubRl and Mad2 act synergistically in enhancing binding to Cdc20 and inhibiting A P C , thereby cooperatively exerting their checkpoint effects. 4.1.8.4 Current model of spindle assembly checkpoint According to the current model of spindle assembly checkpoint, CENP-E interacts with BubRl and Bub3 at the kinetochore. The interaction of BubRl and CENP-E might be crucial for the detection of appropriate or inappropriate microtubule and kinetochore attachment, and leads to phosphorylation of as yet unidentified substrates. These events are believed to regulate the recruitment of the Madl-Mad2 complex to unattached kinetochores. The unattached kinetochores are thought to function as sites of continuous assembly and release of conformationally altered active Mad2. Together with B u b R l and Bub3, Mad2 blocks the activating interaction between Cdc20 and APC.  Upon attachment of the last  kinetochore, the checkpoint proteins are displaced from the kinetochore, allowing Cdc20 to dissociate from the complex and to activate APC, resulting in the degradation of Securin and progression of anaphase. The model presented here is a simple and linear pathway and does not take into account many other factors.  The function of M p s l is unclear.  Available  evidence suggest that M p s l may function upstream of Bub and Mad gene products to generate an anaphase inhibitory signal (Abrieu et al, 2001; Hardwick et al., 1996). Mpsl is also required for the efficient recruitment of CENP-E to the kinetochores (Abrieu et al., 2001). Bubl has been suggested as a possible regulator of Madl-Mad2 complex, since Bubl can phosphorylate Madl in vitro and Bubl-Bub3 complex can bind Madl-Mad2 (Seeley et al., 1999). In addition, Xenopus BubRl is hyperphosphorylated at kinetochores, and this process is dependent on Bubl and Madl but not Mad2. In these cells, BubRl is required for kinetochore association of B u b l , Bub3, M a d l , Mad2 and C E N P - E (Chen, 2002). Moreover, M A P K , Plk, and Aurora-B have also been detected at kinetochores, suggesting that these enzymes may function in checkpoint signaling (Adams et al., 2000; Arnaud et al., 1998; Zecevic etal, 1998).  In this chapter, we describe the characterization of SpaPK.  We were originally  interested in studying the Rad53-like cell cycle checkpoint functions of SpaPK. Instead, we discovered that SpaPK plays a role in mitosis. Using specific affinity purified anti-SpaPK  126  polyclonal antibodies, the cell cycle dependence of gene expression, protein expression and subcellular localization of endogenous SpaPK. were investigated.  Dependence of SpaPK  localization on inhibitors of signaling proteins and on microtubule polymerization was also studied. In addition, the effect of recombinant SpaPK proteins on cell cycle progression was examined. Finally, attempts were made to identify binding partners of SpaPK.  127  4.2 MATERIALS AND METHODS 4.2.1 Chemicals, enzymes, plasmids and bacterial strains Same as in Section 2.2.1 and 2.2.2 unless specified otherwise.  4.2.2 Yeast strains Yeast strains were kindly provided by Dr. Philippe Szankasi from the Fred Hutchinson Cancer Research Center.  Table 4.3 List of yeast strains. Strain  Genotype  MEC2  MATa ade2, ade3, leu2, trpl, ura3 cyh2 SCR::URA3  Mec2-I  MATa mec2-l ade2, ade3, leu2, trpl, ura3 cyh2 SCR::URA3  4.2.3 Molecular biological methods 4.2.3.1 Construction of a HA-SpaPK for overexpression in yeast The open reading frame of SpaPK was amplified by PCR with oligonucleotides XC3 ( 5 ' - C G G j \ A T T C A T G T C C A C G T T C A G G C A G G A G G - 3 ' ) and X C 4 ( 5 ' - C C C C T C G A G C T A C T A G C G C A G C C C G C A C T C C A C - 3 ' ) using SpaPK cDNA as a template. X C 3 contains an EcoR I restriction site (underlined) and XC4 contains a Xho I restriction site (underlined) at the 5'-ends. The amplified PCR fragment was digested with EcoR I and Xho I restriction enzymes, and cloned into the EcoR l/XJio I sites of the yeast expression vector BKK244. The resulting construct encoded an H A tag at the N-terminus of SpaPK. The nucleotide sequence encoding HA-tagged SpaPK was subsequently cloned into the BamW MXho I sites of two yeast expression vectors, p414 GAL1 and p424 G A L 1 , kindly provided by Dr. Phil Hieter. The nucleotide sequences of all constructs were confirmed by D N A sequencing.  The  recombinant plasmids were individually transformed into MEC2 wild-type and mec2-l mutant yeast strains and used in complementation assays.  128  4.2.3.2 Construction of recombinant wild-type and catalytically inactive His-SpaPK proteins for overexpression in mammalian cells The open reading frame of SpaPK was amplified by P C R with oligonucleotides XC3 and XC4 (Section 4.2.3.1) using SpaPK cDNA as a template. The amplified PCR fragment was digested with EcoR I and Xho I restriction enzymes and cloned into EcoR \IXho I sites of mammalian expression vector pCDNA3.1/HisC (Invitrogen).  The open reading frame of  K42A-SpaPK was amplified by PCR with oligonucleotides XC1 and X C 2 (Section 3.2.3.2) using K42A-SpaPK c D N A as a template. The amplified catalytically inactive PCR fragment was digested with BamH I and EcoR I restriction enzymes, and cloned into the same mammalian expression vector digested with BamH I and EcoR I. The nucleotide sequences of all constructs were confirmed by D N A sequencing. The resulting constructs, designated hHis-SpaPK and hHis-K42A-SpaPK, encoded the wild-type and the catalytically inactive SpaPK, respectively, downstream of a six histidine tag. 4.2.3.3 Transformation of yeast Competent yeast cells were prepared using the lithium sorbitol method. A n overnight culture of yeast cells was diluted 1:5, and grown at 30 °C with shaking for 4 to 5 hours. The cells were collected by centrifugation at 1,800X g for 2 minutes in a sterile Falcon tube and suspended in 1 ml of Li-TE-Sorbitol (100 mM LiOAc in TE-Sorbitol) and 1 ml of sterile 40 % glycerol. Plasmid D N A (1 to 3 ul) and denatured salmon sperm D N A (8 pi at 5 mg/ml) were mixed with 100 ul of heat-shock competent yeast cells and 100 ul of 70% PEG. The mixture was incubated at 30 °C for 30 minutes, and heat shocked at 42 °C for 15 minutes. The cells were pelleted at 13,000X g for 1 minute, and resuspended in 400 ul of Y E P D medium (10 g/1 yeast extract, 20 g/1 Bacto Peptone, 0.2% glucose). After incubation at 30 °C for 30 minutes, 300 ul of TE-Sorbitol (10 m M Tris-HCl [pH 7.5], 1 m M E D T A , 1M Sorbitol) were added to the cells. The cells were then plated and grown on selective medium for 2 to 3 days.  4.2.4 Culturing of yeast cells Wild-type MEC2 and mec2-l mutant cells were grown in Y E P R medium (10 g/1 yeast extract, 20 g/1 Bacto Peptone, 2% raffinose), and transformed yeasts were grown in  129  tryptophan-deficient SR selective medium (6.7 g/1 yeast base, 1.5 g/1 dropout medium lacking tryptophan, 2% raffinose).  4.2.5 Extraction of proteins from yeast Exponentially growing yeast cells were pelleted at 1,800X g at 4 °C for 2 minutes, washed once in distilled water and resuspended in 100 pi of 2X SDS sample buffer. The cell suspension was vortexed twice in a mixture of approximately 100 pi of glass beads for 30 seconds, boiled for 5 minutes, incubated on ice for 5 minutes and vortexed for 10 seconds. The sample was centrifuged at 13,000X g for 10 minutes. Five to ten pi of the supernatant was loaded onto SDS-PAGE gels for analysis.  4.2.6 Yeast complementation test Hydroxyurea  (HU) sensitivity  assays  were  performed  in  liquid  medium.  Logarithmically growing cultures were obtained by inoculation of 20 ml of YEPR or tryptophan-deficient SR medium with an aliquot of a stationary phase culture and grown at 30 °C overnight. The next morning, when OD oo of the culture reached 0.6, 2% galactose 6  was added for induction of overexpression of HA-SpaPK. After incubation at 30 °C for 2 hours, cultures were collected and suspended in 1 ml of water. One hundred pi of each cell suspension were used to inoculate 1 ml of Y E P R or tryptophan-deficient SR medium containing 2% galactose in the absence or presence of HU at 3 m M , 8 m M , 10 mM, 30 mM or 80 mM. The cultures were further incubated at 30 °C for 2 hours, and then washed twice with water to remove H U . To determine cell viability, an equal volume of cells was diluted, plated onto Y E P D plates and incubated at 30 °C for 48 hours. Colonies were scored, and percent survival was expressed as a ratio of colonies formed by HU-treated samples compared to untreated samples.  4.2.7 In vitro phosphorylation of Xenopus Cdc25C fragments by His-SpaPK Phosphorylation of 1 pg of GST-tagged Xenopus CDC25C fragment and its variants was performed using 30 ng of purified His-SpaPK or GST-tagged human Chkl kinase (hChkl) (Hutchins et al., 2000). Kinase reactions using His-SpaPK were carried out under the same reaction conditions as in Section 3.2.5.1. Kinase reactions using GST-hChkl were  130  carried out in 20 ul of a kinase buffer (50 mM HEPES [pH 7.5], 10 m M M g C l , 1 mM DTT) 2  containing 30 uCi of [y- P]-ATP. Substrates used were fragments of wild-type Cdc25C and 32  Cdc25C containing a point mutation from serine to alanine corresponding to amino acid position 285 or 287.  4.2.8 Mammalian cell culture MCF-7 cells and A549 cells, MCF-7 mutant-p53 cells overexpressing a dominantnegative mutant p53, and A549 mutant-p53 cells expressing the human papillomavirus E6 protein that stimulates p53 degradation were cultured in D M E M (Life Technologies) medium supplemented with 0.37% (w/v) sodium bicarbonate,  1% penicillin-streptomycin (Life  Technologies), 1% M E M non essential amino acids (Life Technologies), 1% L-glutamine (Life Technologies), 1% sodium pyruvate (Life Technologies), 5 p.g/ml bovine insulin (Life Technologies), 1 jig/ml hydrocortisone (Sigma), 1 ng/ml hEGF (Life Technologies), 1 ng/ml P-estradiol (Sigma), and 10% fetal calf serum (Life Technologies). HeLa cells were cultured in D M E M medium supplemented with 1% penicillin-streptomycin and 5% fetal calf serum. All tissue culture cells were maintained at 37 °C in a humidified 5% C 0 incubator. 2  4.2.9 Drug treatments of mammalian cells MCF-7 wild-type cells were treated with the following inhibitors of protein kinases or protein  phosphatases:  staurosporine  (Sigma)  at  100  n M ; 5,6-Dirchloro-l-P-D-  ribofuranosylbenzimidazole (DRB) (Calbiochem) at 6 u M , 15 u M or 150 u M ; H89 (Calbiochem) at 50 u M or 140 u M ; A3 (Calbiochem) at 10 u M or 80 u M ; olomucine (Sigma) at 100 u M ; bisindolymaleimide 1 (Calbiochem) at 1 u M or 5 u M ; M L - 7 (Calbiochem)  at  1 uM  or  50  u M ; wortmannin  at  2  uM  or  50  u M ; and  debromohymenialdisine (DBH) at 40 u M .  4.2.10 Mitotic spreads of mammalian cells Cells were collected by trypsinization and centrifugation at 300X g. Cells were then suspended in 1 ml of 75 m M K G and subsequently in 0.5 ml of Carnoy's fixative mix (1:3 [v/v] acetic acid: methanol). Cells were incubated in each solution at room temperature for  131  10 minutes, and collected by centrifugation as before.  The resulting cell pellet was  suspended in 50 pi of the supernatant. Approximately 10 to 20 pi of the suspended cells were spotted on a glass slide, air dried, and stained with 1 pg/ml Hoechst 33342 at room temperature for 7 minutes. Finally, each sample was mounted with a coverslip and 20 pi of Flouromount (0.2 M n-propyl gallate and 90% glycerol in PBS), and viewed under a microscope.  4.2.11 Cell cycle synchronization of mammalian cells HeLa cells were synchronized at Gi/S by a double thymidine-aphidicholin block. Exponentially growing cells were seeded at a density of 5 x 10 cells per 10 cm culture plate. 5  The next day, cells were treated with 2 mM thymidine and incubated for 14 hours. The plates were then washed twice with PBS and released into standard growth medium (Section 4.2.7). Following 14 hours of incubation, cells were treated with 1 pg/ml aphidocholin, and incubated for an additional 14 hours. Cells were then washed twice with PBS followed by the addition of normal growth medium. This time point was designated zero. At regular intervals, cells were collected by trypsinization and subjected to flow cytometric analysis (Section 4.2.13), Northern blot analysis (Section 2.2.3.10) and Western blot analysis (Section 3.2.5.8). Synchronization of MCF-7 cells and MCF-7 mutant-p53 cells was as follows. For arresting cells in Go, cells were incubated in serum-free medium for 24 hours.  For  synchronization of cells in G], cells were synchronized in G , and then released into complete 0  medium for eight hours. Synchronization in S phase was established by treatment of cells with 1 pg/ml aphidocholin for 24 hours. Arresting cells in mitosis was accomplished by treatment of cells with either 1 pg/ml nocodazole for 20 hours or I p M taxol for 24 hours.  4.2.12 Immunofluorescence microscopy Cells were seeded on poly-L-lysine coated cover-slips at a density of 1.5 x 10 cells 5  per 35 mm culture dish. The next day, cells were fixed with 3.7% formaldehyde in TBS at room temperature for 10 minutes, and permeabilized with a blocking buffer (1% Triton® X 100 and 1% B S A in TBS) at room temperature for 30 minutes. Cells were incubated with antibody of interest for 1 hour at room temperature, washed twice in T B S for 5 minutes each,  132  followed by incubation with the appropriate secondary antibody. Cells were also incubated with 20 ng/ml Hoechst 33342 for 2 minutes to visualize D N A . Coverslips were mounted on glass slides with Flouromount and sealed with clear nail polish. Samples were examined using a Nikon Eclipse E400 epifluorescence microscope equipped with a 60X objective. Photographs were taken with a Microimager II Programmable Digital Camera and analyzed using Photoshop 6. The primary antibodies, affinity purified rabbit anti-SpaPK antibodies, mouse antiExpress antibody (Invitrogen), and FITC-conjugated mouse anti-p-tubulin antibody (Sigma), were used at 1:200, 1:500, and 1:30, respectively. The secondary antibodies, CY3-conjuated sheep anti-rabbit antibodies (Sigma), CY3-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch), and Alexa 488-conjugated  goat anti-mouse antibodies (Molecular  Probes), were used at 1:4000, 1:3000, and 1:1000, respectively.  4.2.13 Transient transfection of mammalian cells MCF-7 cells were seeded at a density of 3 x 10 cells per 35 mm culture dish 20 hours 5  before transfection. Transfection was performed using lipofectin (Life Technologies) based on the manufacturer's instructions. Cells were transfected with 5 pi of lipofectin and 2 pg of pCDNA3.1/HisC expression vector containing cDNAs encoding wild-type SpaPK or catalytically inactive SpaPK.  Control cells were mock-transfected with the appropriate  amount of expression vector containing no insert. Cells were allowed to incorporate the cDNA constructs for 6 hours at 37 °C in serum-free and antibiotic-free medium, washed and incubated for further 18 hours in complete medium. Atttempts at making stable cell lines harbouring the recombinant SpaPK were not successful.  4.2.14 Flow cytometry To label D N A only, tissue culture cells were collected by trypsinization, washed twice in cold PBS, and suspended in 0.5 ml of PBS. 5 X 10 cells were fixed with 10X cell 5  volumes of ice-cold 70% EtOFI at 4 °C for at least 30 minutes.  After centrifugation at  1,500X g at 4 °C, the pellet was resuspended in 250 pi of PBS and an equal volume of Vindelov's PI solution (10 m M Tris-HCl [pH 8.0], 10 m M NaCI, 0.1% NP-40, 10 pg/ml RNase A, 100 pg/ml PI). Samples were kept at 4 °C for at least overnight in darkness.  133  For dual labeling, tissue culture cells were collected by typsinization, washed in 10 ml of Standard Azide Buffer (SAB: 1% fetal calf serum and 0.1% N a N  3  in PBS), and  suspended at 6 x 10 cells per ml in the same buffer. Cells were fixed with 10X cell volumes 6  of ice cold 70% ethanol, and stored at 4 °C for at least 1 hour or overnight. The fixed cells were washed twice with 10 ml of Tw-SAB (SAB containing 0.5% Tween®-20), incubated in the same buffer for 30 minutes to permeabilize the cells and block nonspecific binding sites, and then suspended at 5 x 10 cells per ml in Tw-SAB. Approximately 1 x 10 cells were 6  6  labeled for each sample with the appropriate primary antibody diluted in Tw-SAB containing 5% fetal calf serum in a final volume of 100 pi. After 1 hour of incubation on ice, the cells were washed twice in 1 ml of Tw-SAB, and incubated with the appropriate secondary antibody diluted in the same solution for 30 minutes on ice in darkness. Cells were further washed twice in 1 ml of Tw-SAB, and treated with 250 ul of RNase A (Roche Diagnostics, 500 U/ml in 4 mM sodium citrate buffer, pH 8.4) at 37 °C for 30 minutes. To label D N A , an equal volume of propidium iodide (50 p.g/ml in sodium citrate buffer, p H 8.4) was added and incubated for a further 20 minutes. Cells were finally suspended in 1 ml of propidium iodide (25 u.g/ml in sodium citrate buffer, pH 8.4), and stored at 4 °C overnight in darkness. Stained samples were analyzed on a Becton-Dickinson FACScan with standard laser and filter configurations.  Data were collected for a minimum of 10,000 events at a low flow rate  setting, saved in listmode format and analyzed using WinMDI freeware. The primary antibody, mouse anti-Express antibody (Invitrogen) was used at 1:500. The secondary antibody, Alexa 488-conjugated goat anti-mouse antibody (Molecular Probes), was used at 1:1000.  4.2.15 Activation of Cdc2 in interphase Xenopus extract by recombinant cyclin AB A low speed (10,000X g) supernatant of an interphasic Xenopus egg extract containing cyclohexidmide was prepared as described by Clarke (Clarke, 1995). The extract was then supplemented with 5% (v/v) glycerol and 10 p.g/ml aprotonin before freezing and storage in aliquots (100 ul) in liquid nitrogen. The activation of Cdc2 was initiated by the addition of cyclin AB in 1 ul of a buffer A (50 mM Tris-HCl [pH 7.8], 100 m M KC1, 5 m M MgCb, 1 m M DTT and 10 mg/ml leupeptin) to give a final concentration of 200 n M (Clarke et al., 1995). At times shown, samples were removed, diluted in Extraction Buffer (80 mM 134  (3-glycerophosphate [pH 7.3], 20 mM EGTA, 15 m M M g C l , 1 m M N a V 0 , 1 m M DTT, 25 2  3  4  pg/ml aprotinin, 1 m M benzamidine, 0.5 mM phenymethylsulphonyl fluoride) and assayed for Cdc2/cyclin B activity. Cdc2/cyclin B activity was assayed using 1 pg/ml histone HI in diluted Extraction Buffer (1:3) containing 0.05 pCi (0.5-1 x 10 cpm/nmole) of [y- P]-ATP 6  32  and 0.3 m M ATP. The addition of purfied His-SpaPK and His-K42A-SpaPK in buffer (20 mM Tris-HCl [pH 7.9], 500 m M NaCI, 10 % glycerol, 10 m M p-mecaptoethanol) was made immediately before cyclin addition. Buffer only was substituted in the absence of the wildtype or the catalytically inactive His-SpaPK.  4.2.16 Farwestern blot Tissue culture cells were collected by trypsinization and washed twice in cold PBS. Cells were lysed in NP-40 lysis buffer, as described in Section 3.2.6. The resulting pellet was then incubated at 4 °C for 15 minutes on a Nutator mixer in a Cytoskeletal Extraction Buffer (20 m M Tris-HCl [pH 7.4], 0.6 M KC1, 10 m M M g C l , 50 U/ml micrococcal 2  nuclease, 1% Triton® X-100) containing 1 m M PMSF, 1 pg/ml aprotonin, 1 pg/ml leupeptin, 1 pg/ml pepstatin A , 1 m M Na3V0 and 1 m M NaF. 4  Protein samples (50 pg) were resolved by SDS-PAGE and transferred onto a membrane as described in Section 3.2.5.8. After transfer, the blot was rinsed twice in PBS to remove SDS, rinsed in A C Buffer (20 m M Tris [pH 7.6], 10% glycerol, 100 m M NaCI, 0.5 mM EDTA, 0.1% Tween®-20) to remove PBS, and then blocked at 4 °C overnight in Blocking Solution (AC buffer containing 2% milk). The next day, a [y- P] ATP-labeled 32  His-SpaPK probe was prepared as described in Section 3.2.5.1. The probe was then diluted using A C Buffer to a final volume of 100 pi, and loaded onto a 0.8 ml Sephadex G25 (Pharmacia) column to remove unincorporated nucleotides.  The purified probe was pre-  incubated with 10 ml of Blocking Solution containing 1 m M D T T at 4 °C for 20 minutes. The membrane was then incubated in the above probe mixture at 4 °C for 3 hours. After probing, the blot was rinsed twice in A C Buffer, and washed in Blocking Solution containing 1 m M DTT at 4 °C for 15 minutes. Finally, the blot was rinsed in A C Buffer, followed by four twenty minutes washes in A C Buffer at 4 °C. autoradiography.  135  The signals were detected by  4.3 RESULTS 4.3.1 Complementation of mec2-l yeast mutant Complementation has often been used to demonstrate biological activity for mammalian homologs of yeast proteins. mec2-l is a Rad53 mutant and displays sensitivity to HU treatment. H U blocks D N A replication by inhibiting ribonucleotide reductase which is responsible for the de novo synthesis of deoxyribonucleotides. Treatment with HU causes wild-type cells to arrest cell division in S phase and remain viable. However, HU-treated mec2-I mutants fail to cell cycle arrest and die rapidly. The effects of overexpressed SpaPK on the growth of yeast cells and its ability to rescue the H U sensitivity phenotype of mec2-l were examined. 4.3.1.1 Overexpression of HA-SpaPK in 5. cerevisiae To overexpress SpaPK in S. cerevisiae, the EcoR. VXho I fragment containing the open reading frame of the SpaPK gene was first inserted into a BKK244 vector containing a hemagglutinin (HA) tag.  Subsequently, the nucleotide sequence carrying the HA tagged  SpaPK was cloned into a galactose-inducible p414GAL yeast expression plasmid, and the resulting recombinant protein was designated HA-SpaPK. HA-SpaPK recombinant protein consists of an H A tag at the TN-terminus, and has a predicted molecular mass of 54 kDa. The empty p414Gall vector or the p414Gall vector carrying the HA-SpaPK gene under the control of G A L promoter were individually introduced into wild-type MEC2 or mec.2-1 mutant.  Transformants were grown in non-inducing tryptophan-deficient SR media and  induced for expression with the addition of 2% galactose, as described in Section 4.2.7. As shown in Figure 4.1, HA-SpaPK is absent from MEC2 (lane 1) and mec2-l strains (lane 3) growing in non-inducing raffinose medium, and was successfully expressed at comparable levels in MEC2 (lane 2) and mec2-l strains (lane 4) under induction. 4.3.1.2 Complementation assay To determine whether SpaPK is a functional homolog of Rad53 checkpoint kinase, the ability of HA-SpaPK overproduction to suppress the HU sensitivity phenotype of mec2-l was examined. Complementation assays in liquid cultures were performed, as described in Section 4.2.7.  Logarithmically growing wild-type MEC2 and mutant mec2-l, as well as  corresponding strains carrying an empty vector or HA-SpaPK were induced with galactose for expression. Yeast strains were then treated with or without H U for varying amounts of  136  time or incubated for 2 hours with different concentrations of H U , and plated to score for the number of colonies. The percent survival relative to non-treated sample for each strain was determined.  A day-to-day variability in the absolute but not the relative level of colony  formation for all strains was observed. Since the relative sensitivities of strains were highly reproducible, the data shown are from experiments performed on the same day and treated for an equivalent period of time at each concentration. The results from a typical set of experiment are shown in Figure 4.2. Panel A shows the percent survival of yeast strains treated with 10 mM of H U for up to 6 hours. Not surprisingly, mec2-l mutant displayed sensitivity after 4 hours of H U treatment, while wildtype MEC2 did not show significant changes during the period of treatment. The relative viability of MEC2 was not affected by overproduction of HA-SpaPK.  Overexpression of  HA-SpaPK in mec2-l, compared to a non-transformed mutant, appears to suppress the rate of killing by H U . However, compared to a mutant carrying the empty p414Gall plasmid, the extent of suppression is minor after 2 hours and insignificant after 3 hours. The ability of HA-SpaPK to rescue mec2-l mutant from treatments with different concentrations of H U was also tested (Panel B). The percent survival of different yeast strains was tested using 3 mM, 8 mM, 10 mM, 30 mM, 50 mM and 80 mM of H U for 2 hours. Similar to that observed in Panel A, overexpression of the HA-SpaPK had no effect on the relative viability of MEC2 at all H U concentrations tested, and had a partial effect in mec2-l compare to the negative controls. Moreover, a complementation assay using an empty high copy p424Gall yeast expression plasmid also had an effect that was not very different from overexpressing the kinase (data not shown). Therefore, overexpression of HA-SpaPK led to, at best, a weak complementation of the mutant in the presence of H U , suggesting that SpaPK is not a functional homolog of Rad53.  4.3.2 Cell cycle profile of SpaPK expression 4.3.2.1 Synchronization of HeLa cells and MCF-7 cells HeLa cells have been used as a suitable cell line to analyze biological events during cell cycle progression, since the procedures for cell cycle synchronization and mitotic index determinations are well established (Maity et al, 1996). HeLa cells were synchronized at the  137  MEC2  kDa 207 —  U  mec2-l  I  U  I  123 — 86 —  44 —  Figure 4.1 Overexpression of HA-SpaPK in yeast strains. Overexpression of H A SpaPK was carried as described in Section 4.2.7. mec2-l  Wild-type MEC2  and mutant  were transformed with p414Gall plasmid or p414Gall plasmid containing  HA-SpaPK.  The transformants were induced for protein expression with 2%  galactose for 2 hours. Protein extracts from MEC2 and 4) before  (U) and after  (lane 1 and 2) and mec2-l (lane 3  induction (I) were resolved on a 10% SDS  polyacrylamide gel, electroblotted onto nitrocellulose membrane, and probed with anti-HA monoclonal antibody. Molecular mass standards in kDa are shown on the left of each panel. The position of HA-SpaPK is indicated by an arrow.  138  139  Figure 4.2 Suppression of HU sensitivity of mec2-l mutants by overexpression of HA-SpaPK.  Complementation assays using MEC2 (closed diamonds), mec2-l(open  diamonds), MEC2 overexpressing HA-SpaPK (closed triangles), mec2-l carrying p414Gall plasmid (open circles), and mec2-l overexpressing HA-SpaPK (open triangles) were performed as described in Section 4.2.7. (A) H U lethality at varying incubation periods.  Log phase cells were treated with or without 10 m M H U .  Aliquots were removed at timed intervals and plated onto Y E P D plates to score for viable colony-forming units. (B) HU lethality at varying concentrations. Log phase cells were treated with H U for 2 hours at the indicated concentrations and plated onto Y P E D plates.  Percent survival relative to non-treated controls for each strain  was plotted.  140  G,/S transition with double thymidine/aphidocholin block as described in Section 4.2.9, and followed through the completion of mitosis.  After release into complete medium, cell  samples were taken at different time points for the analysis of mRNA expression and protein expression of SpaPK (Section 4.3.2.2 and Section 4.3.2.3). D N A content at each time point was analyzed by flow cytometry (Figure 4.3.A). Flow cytometry analysis shows that the synchronization of HeLa cells at the G|/S boundary was successful. After the final release from drug treatment, HeLa cells progressed to S phase after 4 hours, entered G / M phase 2  after 8 hours, and re-entered G, at 12 hours after release. The mitotic index of HeLa cells peaked at 10 to 11 hours (data not shown), suggesting that most of the HeLa cells were in mitosis from the tenth to the eleventh hours after release. M C F - 7 cells and M C F - 7 mutant-p53 cells were synchronized in G , G i , S and M 0  phases of the cell cycle, as described in Section 4.2.9.  Aliquots of each sample were  subjected to Western blotting to monitor the protein expression level of SpaPK (Section 4.3.3.3).  Analysis by flow cytometry indicated that the synchronization of M C F - 7 was  successful (data not shown). 4.3.2.2 Gene expression of SpaPK during the cell cycle To examine the cell cycle dependence of expression of the SpaPK gene, Northern blotting analysis of HeLa cells synchronized at and released from G\/S transition was performed. Exponentially growing HeLa cells were analyzed in parallel (Figure 4.3.B). A probe corresponding to the first 865 nucleotides of the SpaPK c D N A hybridized to a 2.4 kb transcript from total RNAs extracted from the synchronized HeLa cells. The bottom panel is an ethidium bromide stained gel showing the level of 28S rRNA in each lane. 28S rRNA acted as a control for loading. Loading of total RNA isolated 7 hours after G[/S release is higher than other lanes, which corresponded to higher hybridization signal.  After  normalization, the level of detected SpaPK transcript remained constant throughout the cell cycle. 4.3.2.3 Protein expression of SpaPK during the cell cycle Cell extracts prepared from synchronized HeLa cells were resolved by SDS-PAGE, and the protein level of SpaPK was also analyzed by Western blotting using ApSpaPK antibodies.  Figure 4.3.C shows that SpaPK levels displayed minor variations throughout  141  Hours after release Expo  0  2  4  6  8  10  11  12  G, G / M G, G / M G, G / M G, G / M G, G / M G, G / M G, G / M G, G / M G, G,/M • • H TT TT • T M TT TT ? f 2  2  2  2  2  2  2  2  A  Hours after release Expo  0  4  5  7  8  9  10  12 SpaPK m R N A  28S r R N A  Hours after release 0  4  5  7  8  9  10  12 SpaPK  Expo  G  0  G,  S  M SpaPK  142  Figure 4.3 Cell cycle analysis of gene expression and protein expression of SpaPK. (A) FACS analysis of synchronized HeLa cells. HeLa cells were arrested at G,/S using thymidine/aphidicolin and then released, as described in Section 4.2.9. FACS analysis was performed on exponentially growing HeLa cells (Expo) and cells harvested at 0, 2, 4, 6, 8, 10, 11 and 12 hours after release from G,/S block, as described in Section 4.2.12. The positions of G, and G / M phases are indicated an 2  arrows.  (B) Northern blot analysis of synchronized HeLa cells.  32  P labeled  randomly primed SpaPK cDNA was used in a Northern blot of total RNAs extracted from exponentially growing FleLa cells (Expo), as well as cells harvested at 0, 4, 5, 7, 8, 9, 10, and 12 hours after release from G,/S block (Top panel). The amounts of total R N A loaded in each lane were monitored by ethidium bromide staining of 28S rRNA (Bottom panel).  (C) Western blot analysis of synchronized HeLa cells.  Proteins from exponentially growing HeLa cells (Expo) and cells harvested at 0, 4, 5, 7, 8, 9, 10, and 12 hours after release from G,/S block were resolved on a 10% SDS polyacrylamide gel, electroblotted onto a nitrocellulose membrane, subjected to Western blotting using ApSpaPK antibodies. cellular protein (50 pg) were loaded in each lane.  and  Equal amounts of total (D)  MCF-7 cells were  synchronized at G , G , , S and M as described in Section 4.2.9. 0  Proteins from  exponentially growing MCF-7 cells and synchronized cells were resolved on a 10% SDS polyacrylamide gel, electroblotted onto a nitrocellulose membrane, subjected to Western blotting using using ApSpaPK antibodies.  143  and  the cell cycle, and no evidence was obtained for a mobility shift which might be indicative of cell cycle-dependent posttranslational modifications. The level of SpaPK from synchronized MCF-7 cells was also analyzed by Western blotting using the ApSpaPK antibodies (Figure 4.3.D).  Protein extracts from cells  synchronized in Go (lane 2), in G | (lane 3), at S (lane 4), and in M phase by nocodazole treatment (lane 5) were resolved using SDS-PAGE.  Exponentially growing cells were  analyzed in parallel (lane 1). Cell extracts were normalized for protein content (50 pg) before loading. Protein expression of SpaPK, as revealed by Western blotting, shows that SpaPK is expressed at all stages of the cell cycle, but to a much lesser extent in serumstarved cells arrested in Go.  In proliferating cells, the amount of SpaPK is similar in  exponentially growing cells and synchronized G i , S and M cells. However, SpaPK in mitotic cells showed reduction in mobility of approximately 5 kDa during SDS-PAGE (lane M) when compared to others, suggesting that SpaPK is post-translationally modified, perhaps by phosphorylation, during mitosis.  A similar protein expression profile of SpaPK was  observed for MCF-7 mutant-p53 cells, although the level of SpaPK is much lower in nonproliferating Go arrested cells (data not shown).  The difference in protein expression of  SpaPK in HeLa cells and MCF-7 cells may be attributed to differences in cell lines.  4.3.3 Subcellular localization of SpaPK 4.3.3.1 Subcellular localization of SpaPK during mitosis The subcellular localization of endogenous SpaPK was determined by indirect immunofluorescence microscopy.  Exponentially growing MCF-7 cells were fixed with  formaldehyde and probed with FITC-conjugated anti-P-tubulin monoclonal antibody and anti-ApSpaPK polyclonal antibodies (Section 4.2.10). Figure 4.4 shows that the subcellular localization pattern of SpaPK is cell cycle dependent.  In interphase cells, SpaPK is  expressed at slightly higher levels in the cytoplasm than in the nucleus, and seems to be concentrated to small granular aggregates in the cytoplasm.  SpaPK undergoes dramatic  redistribution as the cells progress through mitosis. In prophase, SpaPK becomes associated with duplicated centrosomes after they are separated and during their migration around the nucleus. From prometaphase to late anaphase, SpaPK remains associated with centrosomes and is also found on spindle pole-proximal microtubules. However, during late anaphase, a  144  pool of SpaPK distributes to a region corresponding to the spindle midzone. During early telophase, staining for SpaPK disappears from spindle poles, and instead becomes all concentrated at the midbody.  SpaPK dissociates from mitotic structures during late  telophase, and becomes diffusely distributed throughout the cells. Counterstaining of cells with an anti-P-tubulin monoclonal antibody that recognizes microtubules demonstrated that the ApSpaPK polyclonal antibodies were indeed binding to the centrosomes and mitotic spindles.  The staining pattern of SpaPK was observed  independently of p-tubulin staining and was not the result of a strong FITC signal bleeding through into the CY3 channel (data not shown). A similar pattern of subcellular localization of SpaPK was observed in MCF-7 mutant-p53, A549 and A549 mutant-p53 cell lines. The localization to mitotic structures suggests that SpaPK may play an important role in spindle function during chromosome segregation in mammalian cells To determine the specificity of ApSpaPK polyclonal antibodies in immunostaining for endogenous SpaPK, indirect immunofluorescence microscopy was performed using exponentially growing MCF-7 cells. Cells were triple labeled with Hoechst 33342 for DNA, FITC-conjugated anti-P-tubulin monoclonal antibody for microtubules, and ApSpaPK polyclonal antibodies pre-incubated with buffer only or with purified His-SpaPK. As shown in Figure 4.5, mock competition using only buffer had no effect (Row A), whereas preincubation of ApSpaPK antibodies with purified His-SpaPK completely abolished staining of centrosomes and centrosome-associated spindle microtubules (Row B).  The ability of  purified His-SpaPK to compete with immunostaining demonstrates that the observed cell cycle dependent localization pattern was due to specific recognition of endogenous SpaPK by the ApSpaPK antibodies. 4.3.3.2 Effects of mitotic blockers on the localization of SpaPK To determine whether mitosis-specific association of SpaPK with centrosomes is dependent on microtubules, localization of SpaPK was analyzed in exponentially growing MCF-7 cells treated with microtubule poisons. The distribution of P-tubulin and SpaPK in these cells was examined by indirect immunofluorescence microscopy, using a FITC-  145  DNA  p-Tubulin  Interphase  Prophase  Metaphase  Anaphase  Late Anaphase  Telophase  Late Telophase  146  SpaPK  Co-localization  Figure 4.4 Subcellular localization of SpaPK during the cell cycle. Localization of SpaPK during the cell cycle was examined using indirect  immunofluorescence  microscopy, as described in Section 4.2.9. Dividing MCF-7 cells were triple stained with Hoechst 33342 for D N A , with a FITC-conjugated anti-P-tubulin monoclonal antibody for microtubules and with ApSpaPK polyclonal antibodies for SpaPK. Colocalization of green fluorescence from p-tubulin and red fluorescence from SpaPK produces a yellow signal. The cell cycle stages of MCF-7 cells are shown on the left.  147  Figure 4.5 Specificity of polyclonal ApSpaPK antibodies in immunostaining. Exponentially growing MCF-7 cells were subjected  to triple labeling using  immunofluorescence microscopy, as described in section 4.2.9. staining with Hoechst  In addition to  33342 for DNA and FITC-conjugated anti-P-tubulin  antibodies, cells were stained with either ApSpaPK polyclonal antibodies preincubated with buffer only (Row A) or with 5 pg of purified His-SpaPK (Row B). Cells presented in this figure are mitotic cells cycling through metaphase.  148  conjugated  anti-p-tubulin monoclonal antibody and ApSpaPK polyclonal antibodies,  respectively. 4.3.3.2. A Localization of SpaPK in MCF-7 cells treated with nocodazole or taxol Microtubule poisons, nocodazole and taxol, cause cells to arrest and accumulate at a metaphase-like stage.  Treatment with nocodazole leads to depolymerization of the  cytoplasmic microtubule network, while the centrosomes are still intact (Alieva and Vorobjev, 1997; Fry et al, 1998b). In nocodazole-treated mitotic and interphase MCF-7 cells, diffused P-tubulin staining was observed throughout the cell. Staining of SpaPK in interphase cells was similar to that of non-treated interphase cells. In metaphase-arrested cells, SpaPK was distributed throughout the cells and did not localize to any recognizable structures (Figure 4.6 Rows A and B). The inability of SpaPK to localize to centrosomes in nocodazole-arrested mitotic cells suggests that the association of SpaPK with the mitotic centrosomes is dependent on microtubule polymerization. Treatment with taxol leads to stabilization of microtubules.  In taxol-treated  interphase cells, the microtubules appear as long dense bundles and are detached from centrosomes (De Brabander et al., 1986). In such cells, SpaPK was not associated with microtubule bundles or centrosomes (Figure 4.6 Row C). When taxol-treated cells enter mitosis, the long microtubule bundles are replaced by the formation of multiple MTOCs, also referred to as mitotic asters. Two of these mitotic asters contain centrosomes while others do not.  In these MCF-7 cells, each of these mitotic asters was also stained for SpaPK, as  visualized by anti-P-tubulin staining (Figure 4.6 Row D). The association of SpaPK with mitotic asters is specific since the signal can be competed with ApSpaPK antibodies preincubated with purified His-SpaPK (data not shown). These data suggest that localization of SpaPK to the mitotic spindle poles is not dependent on the presence of centrosomes, but derived from interactions with microtubules associated with centrosomal and acentrosomal MTOCs. 4.3.3.2.B Localization of SpaPK in MCF-7 cells released from nocodazole arrest Localization of SpaPK was also examined in MCF-7 cells recovering from nocodazole treatment upon removal of the drug (Figure 4.7). Seven minutes after release, interphase cells consistently showed a microtubule nucleation center from which many long  149  DNA  p-Tubulin  SpaPK  Co-localization  Figure 4.6 Subcellular localization of SpaPK in nocodazole and taxol treated M C F 7 cells.  Localization of SpaPK in nocodazole-treated  interphase (Row A) and  mitotic cell (Row B), and taxol-treated interphase (Row C) and mitotic cell (Row D) were examined using indirect immunofluorescence microscopy, as described in section 4.2.9. Exponentially growing MCF-7 cells were incubated with 1 ug/ml nocodazole or 1 u M taxol for 24 hours, and then triple labeled with Hoechst 33342 for D N A , with an FITC-conjugated anti-P-tubulin monoclonal antibody microtubules and with anti-ApSpaPK polyclonal antibodies  for SpaPK.  for Co-  localization of green fluorescence from P-tubulin and red fluorescence from HisSpaPK produces a yellow signal.  150  151  152  Figure 4.7  Subcellular localization of SpaPK in M C F - 7 cells released from a  nocodazole block. Exponentially growing M C F - 7 cells were incubated with 1 ug/ml nocodazole for 24 hours, washed twice and incubated in fresh medium for various times. removal  Localization of SpaPK in interphase cells (A) and mitotic cells (B) after of nocodazole  was  examined  microscopy, as described in Section 4.2.9.  using  indirect  immuno-fluorescence  M C F - 7 cells were triple stained with  Hoechst 3 3 3 4 2 for D N A , with an FITC-conjugated anti-(3-tubulin monoclonal antibody for microtubules and with ApSpaPK polyclonal antibodies for SpaPK. Colocalization of green fluorescence from P-tubulin and red fluorescence from SpaPK produces a yellow signal. The times after release from nocodazole block are shown on the left.  153  thin microtubules emanated.  This highly focused MTOC, presumably the centrosome,  remained in interphase cells until 15 minutes after release, by which time cells displayed an organized microtubule network in the cytoplasm (Panel A). The staining of SpaPK in these interphase cells remained similar to that of untreated interphase cells, and did not coincide with the M T O C .  In metaphase-arrested cells (Panel B), the microtubules were still  depolymerized at seven minutes after release from the nocodazole block.  However,  prolonged recovery periods allowed regrowth of microtubules from multiple MTOCs, presumably the centrosomes and other sites within the cytoplasm.  The appearance of  multiple mitotic asters was first observed 15 minutes after release and persisted until at least 45 minutes after release. Under these conditions, SpaPK was found on each of the mitotic asters and proximal microtubules. These results further suggest that SpaPK is a component of the centrosomal and acentrosomal microtubule organization center during mitosis. 4.3.3.3 Effects of protein kinase inhibitors on the localization of SpaPK To determine the involvement of protein phosphorylation and of proteins involved in the regulation of the subcellular localization of SpaPK, indirect  immunofluorescence  microscopy using FITC-conjugated anti-P-tubulin monoclonal antibody and ApSpaPK polyclonal antibodies was performed on MCF-7 cells treated with a variety of protein kinase inhibitors (Section 4.2.9). A list of these inhibitors and their targets is presented in Table 4.4. Table 4.5 presents a summary of the effects of these inhibitors on the distribution of SpaPK during prophase, metaphase and telophase. The pattern of localization of SpaPK is most sensitive to treatment with staurosporine at 100 n M (Figure 4.8 Rows A and B) and H-89 at 140 p M , which inhibited staining of SpaPK to the spindle poles and the spindle apparatus during M phase. Staurosporine also inhibited localization of SpaPK at the taxol-induced mitotic asters (Figure 4.8 Row C). Instead, the staining for SpaPK is diffused throughout the cell. In addition, localization of SpaPK to the duplicated centrosomes during prophase is inhibited by H89 at 50 p M , DRB at 150 p M , A3 at 10 p M , M L - 7 at 50 p M , wortmannin at 50 nM and 2 p M and Na3V04 at 100 p M .  Localization to the spindle poles during  metaphase is inhibited by D B H at 40 p M , and localization to the midbody during telophase is inhibited by M L - 7 at 50 p M , D B H at 40 p M . The inhibition of localization by different  154  Table 4.4 Inhibitors of protein kinases.  Drugs  Staurosporine  H-89  DRB  Potency (IC )  a  50  M L C K = 0.0013 u.M P K A = 0.007 u.M PKC - 0.0007 uM PKG = 0.0085 uM CaM kinase = 0.02 uM P K A = 0.048 uM P K G = 0.48 uM M L C K = 28.3 uM PKC = 3 1.7 uM CKI = 38.3 uM CKII = 137 uM CKII = 6 uM  Concentration used  100 uM  50 yiM 140 u M  6 |.iM 15 u.M 150 u M  PKG = 3.8 uM P K A = 4.3 JLIM  CKII = 5.1 uM A3 M L C K = 7.4 uM PKC = 47 uM CKI = 80 uM CDC2 = 7 uM Olomucine p44 M A P kinase = 25 uM CDK4 > 1 mM CDK6 > 250 uM PKC= KlnM Bisindolymaleimide 1 PKA = 2 uM PI 3-kinase = 50 nM Wortmannin M L C K = 0.2 uM M L C K = 300 nM ML-7, hydrochloride P K A = 21 yiM PKC = 42 uM Chkl =3 u M DBH Chk2 = 3.5 pM a IC_so taken from Calbiochem and Sigma catalogues.  155  10 p M 80 p M  100 uM 1 LiM 5 uM 50 n M 2 LIM 1 uM 50 p.M 40 nM  Table 4.5 Effects o f Inhibitors of protein kinases and phosphatases on the subcellula localization o f the SpaPK protein in mitotic cells. SpaPK localization and drug effect Drug  Concentration used  Untreated  Prophase  Metaphase  Telophase  Duplicated centrosomes  Spindle poles  Midbody  Staurosporine  100 n M  Inhibit  Inhibit  Inhibit  H-89  50 p M 140 p M  n/a Inhibit  No effect Inhibit  N o effect Inhibit  6 pM 150 p M  No effect No effect Inhibit  N o effect N o effect N o effect  N o effect N o effect N o effect  A3  10 p M 80 p M  Inhibit n/a  No effect N o effect  N o effect N o effect  Olomucinc  100 p M  n/a  No effect  No effect  Bisindolymaleimide I  1 pM 5 pM  No effect n/a  No effect No effect  N o effect n/a  Wortmannin  50 n M 2 pM  Inhibit Inhibit  No effect No effect  N o effect No effect  1 pM  No effect No effect  n/a Inhibit  Inhibit  Inhibit  DRB  15 p M  ML-7  50 p M  n/a Inhibit  DBH  40 p M  n/a  n/a  not observed.  156  A  B  C  Figure 4.8 Subcellular localization of SpaPK in staurosporine-treated MCF-7 cells. Localization of SpaPK in staurosporine treated metaphase (Row A), anaphase (Row B), and taxol-arrested  MCF-7 cells (Row C) was examined using indirect  immunofluorescence microscopy, as described in section 4.2.9.  Exponentially  growing or taxol arrested MCF-7 cells were incubated with 100 nM staurosporine for 1 hour, and then triple labeled with Hoechst 33342 for D N A . with an 1TTCconjugated anti-P-tubulin monoclonal antibody for microtubules and with ApSpaPK polyclonal antibodies for SpaPK.  157  protein kinase inhibitors clearly shows that the localization of SpaPK is controlled by protein phosphorylation, and also suggests that the localization is possibly differentially regulated by a number of upstream kinases.  4.3.4 Subcellular localization of ectopically expressed recombinant SpaPK proteins in mammalian cells 4.3.4.1 Overexpression of hHis-SpaPK and hHis-K42A-SpaPK in mammalian cells To overexpress the wild-type and catalytically inactive SpaPK in mammalian cells, the EcoR \IXho I fragment containing the open reading frame of SpaPK and the BamH MEcoR I fragment containing the open reading frame of K42A-SpaPK were individually cloned into the pCDNA3.1/His C mammalian expression vector. The recombinant proteins included codons for an N-terminal His-tag in their leader sequences. The modification added 35 amino acids to the N-terminus of the wild-type SpaPK which is designated hHis-SpaPK, and extended 40 amino acids to the N-terminus of the catalytically inactive SpaPK which is designated hHis-K42A-SpaPK. The recombinant plasmids were introduced into mammalian cells by transient transfection. The expression of the 55.7 kDa hHis-SpaPK and the 56.3 kDa hHis-K42A-SpaPK were monitored by an anti-Express monoclonal antibody. 4.3.4.2 Localization of ectopically expressed hHis-SpaPK and hHis-K42A-SpaPK To gain insight into the possible function of SpaPK at mitosis, the wild-type or catalytically inactive hHis-SpaPK were expressed in MCF-7 cells and their localization was determined. Exponentially growing MCF-7 cells were transiently transfected with an empty vector, the hHis-SpaPK construct or the hHis-K42A-SpaPK construct (Section 4.2.13). After 24 hours, transfected cells were identified by immunostaining with an anti-Express monoclonal antibody and microtubules were monitored by staining with a FITC-conjugated monoclonal anti-(3-tubulin antibody (Section 4.2.12). In interphase cells, the localization of both recombinant hHis-SpaPK proteins was different from that of endogenous SpaPK, and varied amongst transfected MCF-7 cells. As shown in Figure 4.9, hHis-SpaPK was found in the nucleus and the cytoplasm in similar  158  DNA  p-Tubulin  hHis-SpaPK  Co-localization  Figure 4.9 Subcellular localization of hHis-SpaPK. Exponentially growing MCF-7 cells were transiently transfected with hHis-SpaPK construct, as described in section 4.3.13.  Localization of hHis-SpaPK was examined using indirect immuno-  fluorescence microscopy, as described in section 4.2.12. MCF-7 cells were triple stained with Hoechst 33342 for D N A . with a FITC-conjugated anti-P-tubulin monoclonal antibody for microtubules and an anti-Express monoclonal antibody for hHis-SpaPK.  Co-localization of green fluorescence from P-tubulin and red  fluorescence from His-SpaPK produces a yellow signal.  159  amounts, or was excluded from the nucleus.  hHis-SpaPK was also associated, albeit  infrequently, with aggregates in the cytoplasm, some of which coincided with p-tubulin staining. The immunostaining pattern for hHis-K42A-SpaPK was also varied, displaying similarity to that of the wild-type recombinant protein, with a majority of the signal excluded from the nucleus (data not shown). Microtubules in transfected interphase cells, for the most part, formed an organized network as in non-expressing neighboring interphase cells. Interestingly, none of the observed transfected cells were cycling through mitosis. Taken together, these data suggest that overexpression of either of the recombinant hHis-SpaPK proteins possibly inhibits cell cycle progression. 4.3.4.3 Localization of ectopically expressed hHis-SpaPK and hHis-K42A-SpaPK in MCF-7 cells treated with nocodazole or taxol Following transient transfection with the wild-type or the catalytically inactive hHisSpaPK construct, MCF-7 cells were treated with nocodazole or taxol and subjected to indirect immunofluorescence using an anti-Express antibody and FITC-conjugated anti-Ptubulin monoclonal antibody (Sections 4.2.13, 4.2.11 and 4.2.12). The staining pattern for the recombinant hHis-SpaPK proteins was comparable to that of the non-treated transfected cells.  In these cells, the staining pattern for p-tubulin appeared similar to that of non-  transfected interphase cells, with diffuse tubulin staining in nocodazole-treated cells and with microtubule bundles in taxol-treated cells (data not shown). As observed in Section 4.3.4.3, none of the transfected cells were in mitosis upon treatment with mitotic inducers nocodazole or taxol. This observation further suggests that cells expressing recombinant hHis-SpaPK proteins were arrested at a cell cycle stage prior to mitosis.  4.3.5 Effects of recombinant SpaPK proteins on cell cycle progression 4.3.5.1 Cell cycle distribution of MCF-7 cells overexpressing hHis-SpaPK or hHis-K42ASpaPK Immunostaining of transfected MCF-7 cells, in which mitotic cells were not observed, indicated that overexpression of recombinant hHis-SpaPK proteins affects cell cycle progression. To further characterize this effect, the cell cycle distribution of MCF-7 cells transiently transfected with hHis-SpaPK or hHis-K42A-SpaPK construct was analyzed 24 hours, 48 hours and 72 hours post-transfection. The MCF-7 cells expressing recombinant  160  hHis-SpaPK proteins were distinguished from non-expressing cells in each mixture of transfected cell populations using an anti-Express monoclonal antibody.  The percent  distribution of non-expressing cells and expressing cells in G , , S and G / M phases was 2  determined by flow cytometry (Section 4.2.14), and in M phase was examined by indirect immunfluorescence microscopy using an additional FITC-conjugated P-tubulin monoclonal antibody (Section 4.2.13). Figure 4.10 and Figure 4.11 represent typical data from the same experiment. Figure 4.10 compares the cell cycle distribution of non-expressing and SpaPK-expressing MCF-7 cells after transient transfection with hHis-SpaPK (Column A) or hHis-K42A-SpaPK (Column B) for 24 hours, 48 hours and 72 hours. Parental MCF-7 cells (data not shown), vector-transfected control cells and transfected but non-expressing cells exhibited similar DNA profiles at each time point, with approximately 55% of cells in G i phase and 30% of cells in G / M phase. Compared to the non-expressing cell population, the distribution of 2  hHis-SpaPK and hHis-K42A-SpaPK-expressing cells was lower in S phase and slightly higher in Gi and G / M at 24 hours and 48 hours post-transfection. 2  And after 72 hours of  transfection, an approximately 15% increase in G / M distribution was observed in hHis2  SpaPK and hHis-K42A-SpaPK-expressing MCF-7 cells, accompanied by a decrease in Gi cell population. Similar changes were observed from experiment to experiment. As shown in Figure 4.11, the change in G / M distribution is due to an accumulation of hHis-SpaPK2  expressing cells (Panel A ) and hHis-K42A-SpaPK expressing cells (Panel B) in G / M after 2  72 hours of transfection, while the cell cycle distribution of SpaPK-expressing MCF-7 cells in S phase remained similar during the same time period. The cell cycle distribution of MCF-7 cells overexpressing wild-type recombinant SpaPK and catalytically inactive recombinant SpaPK are similar, suggesting that the activity of the kinase is not required for accumulation of cells in the G / M phase. 2  Indirect  immunfluorescence microscopy showed that none of the wild-type-expressing cells and mutant-expressing cells were cycling in mitosis when observed at 24 hours, 48 hours and 72 hours post-transfection, whereas the percent distribution of mitotic cells in non-expressing cell population was normal. The combined flow cytometry data and immunostaining data indicate that overexpression of wild-type or catalytically inactive recombinant SpaPK  161  A  B 100  100  90  90  80  80  G,  S  G /M 2  T = 24 hr  T = 24 hr  100  100  90  90  80  80  G,  S  G /M 2  T = 48 hr  ID  -S c  W o u 0) EL,  100 90 80 70 60 | 50 j 40 j 30 20 10 0  <u  & c  s  G /M ?  T = 72 hr  100 90 80 70 60 50 40 30 20 10 0  I 1  s T = 72 hr  162  WL GJM  Figure 4.10 Comparison of cell cycle distribution of MCF-7 cells expressing wildtype hHis-SpaPK or catalytically inactive hHis-K42A-SpaPK with that of transfected but non-expressing cells 24, 48 and 72 hours post-transfection. MCF-7 cells were transiently transfected with hHis-SpaPK (Column A) or hHis-K42A-SpaPK (Column B), as described in Section 4.2.13. The SpaPK-expressing cells were detected from each transiently transfected cell population using an anti-express monoclonal antibody. The transfected cells were then subjected to flow cytometry to determine the percentage of non-expressing cells (diagonal stripe) and SpaPK-expressing cells (dots) in G,, S and G / M , as described in Section 4.2.14. The cell cycle distribution 2  of MCF-7 cells transiently transfected with the empty vector (vertical stripe) was used as an internal control at each cell cycle stage for each time point.  163  A  Figure 4.11 Cell cycle distribution of SpaPK-expressing MCF-7 cells 24, 48 and 72 hours post-transfection.  MCF-7 cells were transiently transfected with wild-type  hHis-SpaPK (Panel A) or catalytically inactive hHis-K42A-SpaPK (Panel B), as described in Section 4.2.13.  The SpaPK-expressing cells were detected using an  anti-Express monoclonal antibody, and the percentage of transfected cells in G , , S and G / M was determined using flow cytometry, as described in Section 4.2.14. The 2  cell distribution of transfected cells 24 hours (dark), 48 hours (grey) and 72 hours (light) post-transfection was plotted for G,, S and G / M cell cycle stage. 2  164  inhibits cell cycle progression, and prolonged overexpression causes cells to accumulate at G . 2  4.3.5.2  Effects of purified His-SpaPK and His-K42A-SpaPK on the onset of mitosis in  Xenopus egg extract The observation that SpaPK-expressing cells accumulate in G phase and do not 2  undergo mitosis raises the possibility that SpaPK prevents cells from entering mitosis. To address this question in a cell-free system, the purified His-SpaPK and His-K42A-SpaPK were supplied to Dr. James Hutchins from the research group of Dr. Paul Clarke at University of Dundee. The effects of the purified proteins on the onset of mitosis were tested using interphasic Xenopus egg extract. Their results are discussed here with permission. Xenopus extracts, made from early embryos, have provided a useful cell-free system for studying cell cycle processes (Clarke, 1995). The cell cycle of Xenopus early embryos lacks G i and G  2  phases, and oscillates rapidly in succession between mitosis and interphase  when D N A replication occurs. Xenopus oocytes can be stimulated to mature by progesterone treatment within the female frog. Maturation consists of progression through meiosis I, and then arrest in metaphase of meiosis II with high Cdc2/cyclin B activity. When a laid mature egg is fertilized, the activated egg is released from metaphase (meiosis II) arrest and Cdc2/cyclin B activity declines to an interphase state. This is followed by eleven rapidly dividing mitosis-interphase cell cycles. Interphasic egg extracts are prepared from mature eggs activated with an electric pulse or calcium ionophore treatment to release them from metaphase arrest and to degrade mitotic cyclins, followed by incubation in cycloheximide to inhibit re-synthesis of cyclins. Therefore, all required components for the cell cycle, other than mitotic cyclins, are present in an interphasic Xenopus egg extract. Typically, the onset of mitosis is studied using cycloheximide-treated interphasic extracts by the addition of recombinant mitotic cyclins to activate Cdc2, which can occur after a lag phase varying from a few minutes to a few hours. The activation of Cdc2 in Xenopus extract was monitored by histone HI phosphorylation activity, every 15 minutes for a period of 2 hours (Section 4.2.15). The assays were performed with the addition of cyclin AB in the presence of buffer only, 0.5 nM of purified His-SpaPK, or 0.5 nM of purified His-K42A-SpaPK.  Cyclin A B is a non-  degradable form of full-length cyclin B, due to an N-terminal truncation of its destruction  165  25 .3.  |  20  'o  B OH  15  03  10  C  X c o  0  20  40  60  80  100  120  140  Incubation time after cyclin addition (min)  Figure 4.12  Time course of Cdc2/cyclin B activation in an Xenopus extract.  Activation of Cdc2 in an Xenopus extract was performed as described in Section 4.2.15. The assays were performed without any addition (closed diamond), with the addition of cyclin A B in the presence of buffer alone (closed square), 0.5 n M of purified His-SpaPK (closed triangle) or 0.5 nM of purified His-K42A-SpaPK (closed circle). Aliquots of extract were taken at every 15 minutes for a period of 2 hours, and measured for Cdc2 dependent histone H1 phosphorylation activity.  166  box domain. As shown in Figure 4.12, the Xenopus egg extract, lacking cyclin B, did not show activation of histone HI kinase activity. The addition of cyclin AB triggered activation of Cdc2/cyclin B after 15 minutes of incubation. This lag period before Cdc2/cyclin B activation is lengthened by 30 minutes upon additional incubation with purified His-K42ASpaPK, and by 60 minutes upon additional incubation with purified His-SpaPK. Both HisSpaPK and His-K42A-SpaPK were able to consistently delay the activation of Cdc2, although  the  relative  timing of Cdc2  activation  varies  among  different  trials.  Supplementation of Xenopus extract with the wild-type or the catalytically inactive HisSpaPK delayed the onset of mitosis by up to 60 minutes, suggesting that SpaPK exerts its effect on Cdc2 activation independently of its kinase activity. 4.3.5.3 His-SpaPK phosphorylation of Xenopus Cdc25C fragment and variants Cdc2 histone HI kinase activity is an indirect measurement of the activity of Cdc25C phosphatase.  Cdc25C  triggers  activation  of Cdc2  by  removing the inhibitory  phosphorylation on Thr-14 and Tyr-15 in the ATP-binding site of the kinase. The function of Cdc25C is also regulated by phosphorylation. Additional experiments examined the ability of His-SpaPK to phosphorylate Cdc25C protein phosphatase using a GST-tagged Xenopus Cdc25C fragment and its variants as substrates were performed by Dr. James Hutchins (Section 4.2.7) and are discussed here with permission. The wild-type Cdc25C fragment is derived from residues 271 to 316 of Cdc25C protein. It includes Ser-285 and Ser-287 whose phosphorylation can activate and inhibit the function of Cdc25C, respectively. Chkl has been shown to phosphorylate Ser-287 in vitro (Kumagai et al., 1998). As shown in Figure 4.13, purified GST-tagged human C h k l  (GST-hChkl)  predictably phosphorylated the wild-type Cdc25C fragment (SPS) (lane 5), the Cdc25C fragment carrying a Ser-285 to alanine substitution (APS) (lane 6), but not the Cdc25C fragment carrying a Ser-287 to alanine substitution (SPA) (lane 7).  The observed  phosphorylations were not due to the presence of the GST tag since addition of GST alone did not generate any signal (lane 8). As shown in Figure 4.13, purified His-SpaPK was also able to phosphorylate SPS (lane 1), but to a much lower level than GST-hChkl. APS was phosphorylated to a similar extent compared to SPS (lane 2), indicating that Ser-285 is not required for recognition by His-SpaPK. In contrast, SPA was not phosphorylated (lane 3), suggesting that Ser-287 is the  167  Kinase: Substrate:  His-SpaPK  GST-hChkl  SPS  APS  SPA  GST  1  2  3  4  SPS A P S SPA GST  5  6  Figure 4.13 His-SpaPK phosphorylation of an Xenopus  7  8  Cdc25C fragment and its  variants. Kinase reactions were carried out using 30 ng of purified protein kinases and 1 ug of GST-tagged fragment of Xenopus  Cdc25C and its variants as the  substrates, as described in materials and methods. The protein kinases used were purified His-SpaPK (lane 1 to 4), and GST-hChkl (lane 5 to 8). The substrates used were SPS, wild-type Cdc25C (lane 1 and 5; APS, Cdc25C containing a serine to alanine mutation at position 285 (lane 2 and 6); SPA, Cdc25C containing a serine to alanine mutation at position 287 (lane 3 and 7); and GST (lane 4 and 8). The products of the kinase reactions were resolved by SDS-PAGE and located by autoradiography. Molecular mass standards and their sizes in kDa are shown on the left. The position of hChkl, His-SpaPK, Cdc25C and GST is indicated by an arrow, an asterisk, a left-right block arrow, and a left-right arrow, respectively.  168  site of phosphorylation. As with GST-hChkl, the observed signals generated by His-SpaPK were not due to phosphorylation of the GST tag (lane 4). The ability to phosphorylate APS but not SPA suggests that His-SpaPK targets Ser-287 in Xenopus Cdc25C.  4.3.6 Farwestern blotting of M C F - 7 cell extracts  Farwestern overlay blotting was used in an attempt to identify potential interaction partners of SpaPK (Section 4.2.16). A n initial experiment using the non-ionic NP-40 lysis buffer resulted in the detection of three positive signals in the pellet fraction (data not shown). Further extraction of the pellet with the cytoskeletal extraction buffer resulted in the solubilization of these three proteins. Figure 4.14 shows a Farwestern blot of an NP-40 soluble fraction of MCF-7 cells (lane 6), a cytoskeletal soluble fraction of MCF-7 cells (lane 5) and a cytoskeletal insoluble fraction, of MCF-7 cells (lane 4) probed with purified His-SpaPK.  3 2  P labeled  The blot reveals two strong signals corresponding to a doublet of  approximately 30 kDa, and a weaker signal of approximately 50 kDa. The sizes of the positive signals raised the possibility that the 50 kDa band is tubulin or endogenous SpaPK, and that the doublet is histones. However, the failure of the radiolabeled probe to bind to tubulin (lane 2), histones (lane 3) and purified His-SpaPK (lane 4) imply that they are not binding partners of SpaPK. Nocodazole or taxol-treated mitotic MCF-7 cells and MCF-7 cells overexpressing hHis-SpaPK showed same pattern of bands (data not shown). overexpressed hHis-SpaPK was also not recognized by radiolabeled probe.  The  Farwestern  blotting shows that His-SpaPK can bind to three components of the MCF-7 cytoskeleton during interphase and mitosis.  169  kDa 207 — 123 _ 86 —  1  2  3  4  5  6  44 — 31 —  Figure 4.14 Detection of binding partners of His-SpaPK. Twenty pg of purified tubulin (lane 1), 40 pg of purified histones (lane 2), and 1 pg of purified His-SpaPK (lane 3),  50 pg of each of cytoskeletal insoluble fraction (lane 4), cytoskeletal  soluble fraction (lane 5) and NP-40 soluble fraction (lane 6) of MCF-7 cells were resolved on a 10% SDS polyacrylamide gel, electroblotted onto a nitrocellulose membrane, and subjected to Farwestern blotting probed using a  3 2  P labeled His-  SpaPK, as described in Section 4.2.16. Molecular mass standards in kDa are shown on the left.  170  4.4 DISCUSSION 4.4.1 SpaPK is not a functional homolog of Rad53 The amino acid sequence within the kinase domain of SpaPK shows high similarity to that of S. cerevisiae Rad53, suggesting that these two proteins may be functionally related. However, complementation assays performed in liquid media show that the recombinant HASpaPK is unable to rescue the H U sensitivity of mecl-1 yeast mutant (Section 4.3.1). Furthermore, mecl-1 transformants overexpressing HA-SpaPK failed to grow on H U containing solid media (not shown). The complementation assays indicate that SpaPK is not a functional homolog of Rad53. During the course of this study, the human homolog of S. cerevisiae Rad53 and S. pombe Cdsl checkpoint kinases, designated Chk2, was independently cloned by two research groups (Blasina et al., 1999; Matsuoka et al., 1998). Rad53 and Cdsl protein kinases are required for checkpoint responses to D N A replication interference or D N A damage. Blasina et al. (1999) and Matsuoka et al. (1998) identified CHK.2 using database screening for human EST sequences homologous to Rad53/Cdsl and subsequent cloning. similar to that described in this thesis.  This approach is  However, they had access to ESTs from the  proprietary LifeSeq® database or additional updated ESTs which were not available from the GeneBank™ database at the beginning of this project.  C H K 2 is 26% identical and 37%  similar to RAD53, and 26% identical and 34% similar to C D S l . Compared to Rad53, the overall structures of Chk2 and Cdsl lack the long carboxyl terminal extension.  Sequence  alignment of these homologs shows the conservation of a forkhead-associated (FHA) domain outside the kinase domain. The F H A domain has been shown to be important in signaling pathways mediated by Rad53 and Chk2, and its function in Cdsl is unclear (Durocher and Jackson, 2002; Lee and Chung, 2001; Sun et al., 1998). This motif, however, is not found in SpaPK.  In contrast to the SpaPK gene, Matsuoka et al. (1998) showed that CHK2 is a  functional homolog of RAD53 since CHK2 was able to rescue the lethality of a RAD53 deletion and increase resistance to replication interference by HU of rad53 mutants.  171  4.4.2 Possible biological functions of SpaPK The broad expression profiles of the SpaPK gene and the high degree of conservation between SpaPK and its homologs from mouse and rat suggest that this protein kinase serves an important function. Several possible biological functions for SpaPK are discussed below. 4.4.2.1 A role in apoptosis The similarity between the catalytic domains of SpaPK and D A P kinase suggests that like D A P kinase, SpaPK may play a role in apoptosis.  Loss of apoptotic response and  increased apoptotic rate are implicated in a wide variety of diseases and pathologies. The decision to undergo apoptosis is regulated by a balance of cell death and cell survival signals, and apoptotic and anti-apoptotic proteins. DAP kinase family is a group of pro-apoptotic serine/threonine kinases (Kogel et al, 2001) (Figure 4.15). The founding member, death-associated protein kinase (DAP kinase), was identified as a protein whose expression, when reduced by antisense mRNA, led to the protection of HeLa cells from interferon y-induced programmed cell death (Deiss et al, 1995). Subsequently, D A P kinase was shown to play a critical role in mediating apoptosis induced by tumor necrosis factor-ct (TNF-a), activated Fas receptors, and detachment from the extracellular matrix (Cohen et al., 1999; Inbal et al, 1997). The cell death promoting activity of D A P kinase is dependent on a number of factors.  The correct intracellular  localization to the microfilament network mediated by its cytoskeleton-binding domain is critical for the disruption of stress fibers, a hallmark of apoptosis (Cohen et al, 1997). The presence of the C-terminal death domain is essential for the induction of apoptosis (Cohen et al, 1999). A functional kinase activity is also required for mediation of apoptosis. The kinase activity of DAP kinase is negatively regulated by the calmodulin-binding domain adjacent the kinase domain. The autoinhibitory effect is relieved by the binding of the calcium activated calmodulin (Cohen et al, 1997). D A P kinase may therefore respond to changes in cytosolic calcium concentration known to occur during apoptosis (Baffy et al, 1993; Pinton et al, 2000). A serine-rich C-terminal tail, located within the last 17 amino acids of the protein, also exerts an autoinhibitory function, presumably by modulating interactions between D A P kinase and its substrate(s) (Raveh et al, 2000). In addition to its function as an apoptotic protein, D A P kinase is also suggested to function as a tumor  172  Figure 4.15  Schematic representation of structural organization of D A P kinase  family members:  death associated protein kinase (DAP kinase), death-associated  protein kinase related protein 1/death-associated protein kinase death-associated (DRAK1), (DRAK2),  2  (DRP-1/DAPK2),  protein kinase related apoptosis inducing protein kinases 1  death-associated protein kinase related apoptosis inducing protein kinase and death-associated  protein like kinase from rat (rDlk), zipper  interacting protein kinase from mouse (mZIP kinase), and SpaPK.  The kinase  domain, calcium calmodulin-binding domain (CAM), cytoskeleton-binding region (CYT), death domain (DD), and leucine zipper motif are indicated in r e d l , p i n k l , b l u e l , b l a c k l , and yellowD, respectively.  173  suppressor since the expression of DAP kinase is lost in several human tumor-derived cell lines (Inbal etal, 1997; Kissil etal,  1997; Raveh and Kimchi, 2001).  All D A P kinase family members are ubiquitously expressed in various tissues, and are capable of inducing apoptosis upon overexpression in cultured cells (Inbal et al, 2000; Kawai et al, 1999; Sanjo et al, 1998). Examples of other members of the D A P kinase family include death-associated protein kinase related protein 1/death-associated protein kinase 2 (DRP-1/DAPK2) (Inbal et al, 2000; Kawai et al, 1999), and death-associated protein kinase related apoptosis inducing protein kinases 1 and 2 (DRAK1 and DRAK2) (Sanjo et al, 1998). D A P kinase family members exhibit high degree of sequence similarity in their kinase domains, but differ in their non-catalytic C-terminal regions. DRP-1/DAPK2, a soluble cytoplasmic protein, is the only member other than D A P kinase to possess a calmodulin-binding domain (Inbal et al, 2000; Kawai et al, 1999). D R A K 1 and D R A K 2 are nuclear proteins whose C-terminal domains do not reveal any known structural motifs or significant homology to other proteins.  They are more distantly related to D A P kinase  (Sanjo et al, 1998). As discussed in Section 2.4.1, the primary sequence of SpaPK is identical to that of placenta and HeLa ZIP kinase. ZIP kinase is another member of the D A P kinase family and carries an additional leucine zipper motif compared to other members of the D A P kinase family (Kogel et al, 2001). Various authors have investigated the functions of ZIP kinase homologs from mouse (mZIP kinase) and rat (Dlk).  Although mZIP kinase and Dlk are  99.8% identical, they differ in their subcellular localizations and proposed functions. Recombinant mZIP kinase is localized to the nucleus, and possibly functions as a positive regulator of apoptosis (Kawai et al, 1998). Overexpression of mZIP kinase, and not the kinase inactive mutant, caused the morphological changes typical of apoptosis in 45% of transfected NIH 3T3 cells, suggesting that the kinase activity of ZIP kinase triggers apoptosis (Kawai et al, 1998). Recombinant Dlk exhibits a diffused as well as a speckled distribution in the nucleus (Kogel et al, 1998). Immunostaining showed that the nuclear speckles of Dlk overlap considerably with promyelocytic leukemia (PML) bodies (Kogel et al, 1999). P M L bodies are composed of 20 or more different proteins, including the death-inducing proteins bax and p27 which are recruited to P M L bodies by the P M L protein (Guo et al, 2000). Recently, the P M L gene has been implicated in the control of cell death and cell survival  174  (Salomoni and Pandolfi, 2002). Therefore, some evidence suggested that mZIP kinase and Dlk play a positive role in inducing apoptosis.  However, other studies of Dlk presented  contradictory data on their death-inducing properties. Dlk was identified using cDNAs from SV40-transfromed rat cells, in search for novel kinases which play a role in growth control or large T-mediated transformation (Kogel et al, 1998). Overexpression of recombinant Dlk in Sf9 insect cells and in rat fibroblast REF52 cells did not cause apoptotic-like morphological alterations (Kogel et al, 1998). Moreover, U V irradiation of REF52, Rati, SV52, and p53negative BT1 cells at doses that induce apoptosis led to reduction of Dlk transcript levels, arguing against a positive role in apoptosis (Kogel et al, 1998). Further characterization of Dlk suggests that the protein may act as a molecular switch between apoptotic and antiapoptotic pathways.  The activation of apoptotic activity of Dlk was achieved by  truncation of the most C-terminal nuclear localization signal (Kogel et al., 1999).  The  deletion mutant, targeted to the actin cytoskeleton in the cytoplasm, was a potent inducer of apoptosis (Kogel et al., 1999). Regulation of apoptotic activity of Dlk can also occur through interaction with other proteins. One of the binding partners of Dlk is Par-4 (Page et al, 1999a).  Par-4 was identified as an immediate early response gene whose expression is  upregulated upon induction of apoptosis in prostate carcinoma cells (Sells et al., 1994). Overexpression of Dlk or Par-4 on its own did not induce apoptosis (Page et al., 1999a). However, coexpression of Dlk and Par-4 resulted in cytoplasmic retention of Dlk to the actin filaments and efficient induction of apoptosis (Page et al., 1999a). Recently, another binding partner of Dlk, named apoptotic antagonistic transcription factor (AATF), was identified (Page et al., 1999b). Interestingly, additional coexpression of A A T F with Dlk and Par-4 or with Dlk lacking the functional nuclear localization signal resulted in reduction of apoptosis (Page et al., 1999b).  Thus, under certain conditions, the apoptotic function of Dlk is  dependent on its subcellular localization from the nucleus to cytoplasm and association with the actin filament system, and on the identity of its interaction partner. As detailed in Chapter 3, SpaPK is 83.7% and 83.5% similar to mZIP kinase and Dlk, respectively.  The high sequence similarities suggest that SpaPK may play a role in  apoptosis; however, this was not observed in MCF-7 cells. The recombinant hHis-SpaPK proteins do not localize to the same subcellular compartments as the overexpressed mZIPK or Dlk. Preliminary F A C S analysis revealed that compared to mock plasmid-transfected  175  control, no significant apoptosis was observed in MCF-7 cells expressing wild-type or catalytically inactive hHis-SpaPK even after 72 hours of post-transfection (data not shown). 4.4.2.2 A role in transcription A function in regulation of transcription and/or splicing has been proposed for nuclear Dlk.  Disperse staining of recombinant Dlk in the nucleus may represent chromatin-  associated Dlk. Consistent with this assumption is the ability of Dlk to phosphorylate core histones H 2 A and H4 as exogenous substrates and histone H3 as an endogenous substrate from nuclear extracts (Kogel et al., 1998). Localization to nuclear speckles suggests that Dlk is associated with transcription or splicing centers. Dlk is found to interact with other leucine zipper containing transcription factors such as ATF-4 (Kawai et al., 1998), Par-4 (Page et al., 1999a) and A A T F (Page et al., 1999b).  ATF-4 and Par-4 are negative regulators of  transcription by interfering with ATF1/2 or WT-1 mediated transcription, respectively (Page et al., 1999b). Another interaction partner of Dlk is the rat homolog of S. pombe CDC5 (Engemann et ah, 2002). CDC5 is a putative transcription and splicing factor involved in regulation of mitotic entry (Bernstein and Coughlin, 1998). Both Dlk and CDC5 colocalize perfectly to speckle-like structures (Engemann et al., 2002). However, they do not colocalize with splicing factors SC35 or U5-116 which are markers for nuclear speckles representing spliceosomes. Rather, SC35 was partially displaced from the nuclear speckles (Engemann et al., 2002). Alteration in subcellular localization of SC35 by coexpression of Dlk and CDC5 suggests a role in alternative splicing. Additionally, partial colocalization of Dlk with P M L bodies, which are also associated with C B P and nascent R N A , further suggests a role in transcription (Kogel et al., 1999).  Thus, Dlk may serve a general function in the  transcription of a set of genes, perhaps by mediating interaction between transcription factors and splicing factors, and by remodeling the chromatin structure during transcription. As a homolog of Dlk, SpaPK may also play a role in linking transcription and splicing during cell cycle. This is certainly a valid area to be explored. 4.4.2.3 A role in mitosis 4.4.2.3.A Formation of spindle poles The level of gene expression and protein expression of endogenous SpaPK remain relatively constant during cell cycle progression. However, the subcellular localization of SpaPK appears to be tightly regulated.  During mitosis, it is first found on duplicated  176  centrosomes and it remains on the centrosomes and centrosome-associated spindles until late anaphase, after which it redistributes to the spindle midzone and midbody until late telophase.  Moreover, SpaPK is associated with microtubule organization centers in cells  recovering from nocodazole. These data suggest that SpaPK may play a role in spindle function during chromosome segregation. SpaPK is also found to associate with MTOCs in taxol-induced mitotic cells. Instead of two discrete centrosome-containing  MTOCs, taxol induces formation of multiple  microtubule nucleation centers throughout the cytosol of mitotic cells. The composition of taxol-induced MTOCs is similar to that of spindle poles (Verde et al., 1991). Several lines of evidence have shown that centrosomes alone are not sufficient to act as functional spindle poles. First, specialized cells, such as plant and some mammalian oocytes, lack conventional centrosomes but can still form focused spindle poles (Heald et al., 1996; M c K i m and Hawley, 1995; Smirnova and Bajer, 1992; Steffen et al., 1986). Second, when the bipolar spindle is well established, the centrosomes are dispensable for the organization and proper function of spindle poles (Murray et al., 1996; Nicklas et al., 1989).  Third, electron  microscopy data has shown that many spindle microtubules are not embedded in the centrosome and consequently may be bound by other factors (Mastronarde et al., 1993; McDonald et al, 1992; McEwen et al., 1997). Thus, spindle poles are defined as the regions of the mitotic spindle where microtubule minus ends are focused, and as the sites of convergence of sister chromatids upon segregation during anaphase (Compton, 1998; Mountain and Compton, 2000). The taxol-induced microtubule asters are formed by the action of non-centrosomal proteins independent of the presence of centrosomes.  These non-centrosomal structural  proteins, which normally assemble around the centrosomes, have been found to be essential for the cohesion of microtubule minus ends at the spindle pole and for the tethering of centrosomes to the body of the spindle. Three of these important factors are cytoplasmic dynein, dynactin and nuclear matrix A (NuMA). Cytoplasmic dynein is capable of focusing microtubule minus ends (Gaglio et al., 1997; Gaglio et al,  1996; Gaglio et al., 1995;  Walczak et al, 1998), and also of transporting N u M A to the spindle poles. N u M A then becomes concentrated at an insoluble matrix which probably clamps microtubule minus ends into a tightly focused bundle independent of centrosomes (Dionne et al., 1999; Gaglio et al.,  177  1997; Gaglio et al., 1996; Merdes et al, 1996). Disruption of activity of any one of the proteins results in the disorganization of microtubule minus ends at spindle poles despite the presence of centrosomes (Gaglio et al., 1997; Gaglio et al., 1996; Gaglio et al., 1995; Palazzo et al., 1999; Quintyne et al., 1999). In addition, two kinesin related proteins, the minus end-directed K I N C kinesin HSET and the plus end-directed bipolar kinesin Eg5, are also required for the efficient formation of spindle poles. They both functions to promote and maintain spindle pole formation through their motor activities, when the plus enddirected microtubule movement is counter balanced by the activities of minus end-directed motors during spindle pole organization (Endow, 1999; Gaglio et al., 1996; Merdes et al., 1996; Mountain et al, 1999). The association of SpaPK with taxol-induced MTOCs suggests that SpaPK plays a role in spindle function, which would affect centrosome separation and chromosome segregation during mitosis. However, immunostaining of MCF-7 cells treated with different kinase inhibitors show that the presence of SpaPK is not required for the formation or the maintenance of duplicated centrosomes, spindle poles, cleavage furrow or mitotic asters, since these structures lacking SpaPK are still intact (Section 4.3.3.3). 4.4.2.3.B G /M transition 2  Several lines of evidence suggest that SpaPK may function at the G / M transition. 2  Ectopic expression of wild-type hHis-SpaPK or catalytically inactive hHis-SpaPK lead to alteration of cell cycle progression with an accumulation in G (Section 4.3.5.1). 2  The  accumulation of these cells in G may be due to the ability of SpaPK to delay the onset of 2  mitosis, as was observed in Xenopus extract assays (Section 4.3.5.2). Due to the sensitivity of the assay, the delaying effect caused by addition of the purified wild-type His-SpaPK was at times longer or shorter than by the catalytically inactive His-K42A-SpaPK. Nevertheless, both recombinant His-SpaPK proteins were able to inhibit the activation of Cdc2 histone H1 kinase activity, thereby delaying the onset of mitosis. One of the targets of SpaPK-mediated inhibition of mitosis is possibly Cdc25C. Cdc25C is a protein phosphatase which controls the entry into mitosis by dephosphorylating Cdc2 at Thr-14 and Tyr-15. Cdc25C is localized in the cytoplasm during interphase and translocates to the nucleus prior to the onset of mitosis (Dalai et al., 1999). The cytoplasmic retention of Cdc25C has been shown to require a 58 amino acid region, in which a serine at  178  position 216 mediates the interaction of Cdc25C with a family of 14-3-3 proteins (Dalai et al, 1999; Peng et al., 1997). Ser-216 is the major phosphorylation site of Cdc25C during interphase,  and its dephosphorylation coincides with mitotic entry, suggesting that  dephosphorylation of Ser-216 accompanies Cdc25C activation (Ogg et al., 1994). Three kinases, C h k l , Chk2 and Cdc25-associated protein kinase 1 (C-TAK1), have been shown to phosphorylate Ser-216 of Cdc25C (Blasina et al., 1999; Peng et al., 1998; Sanchez et al., 1997). In vitro kinase assays in this study suggests that SpaPK could also phosphorylate Ser216 of Cdc25C. Purified His-SpaPK was able to target Ser-287 for phosphorylation within a fragment of Xenopus Cdc25C, although the level of phosphorylation is significantly less than that obtained by purified recombinant Chkl (Section 4.3.5.3). Ser-287 of Xenopus Cdc25C is analogous to Ser-216 of human Cdc25C. Immunostaining showed that a pool of SpaPK is located in the cytoplasm during interphase. SpaPK could promote association with  Phosphorylation of cytoplasmic Cdc25C by 14-3-3 proteins, thereby preventing nuclear  translocation of Cdc25C and consequential activation of Cdc2 required for the onset of mitosis. The catalytically inactive His-K42A-SpaPK also had a delaying effect on Cdc2 activation, suggesting that the activity of the kinase is not required for inhibition in the experimental system used.  The mechanism of inhibition by both the wild-type and the  catalytically inactive kinase is possibly through binding and inhibiting an activator of mitosis, such as Cdc25C, at a region outside the kinase domain of SpaPK. However, this does not exclude the importance of the kinase activity in vivo. Further experiments are required to characterize the effect of SpaPK on the activity of Cdc2 and Cdc25C at the G2/M transition. During mitosis, Cdc2 is not phosphorylated at Thr-14 and Tyr-15, and exists as single form instead of multiple phosphorylated forms.  Also during mitosis, Cdc25C is  hyperphosphorylated but is not phosphorylated at Ser-287 (Izumi et al., 1992; Kumagai and Dunphy, 1992).  The phosphorylation status of Cdc2 and Cdc25C are currently under  investigation in Xenopus extracts incubated with either of the recombinant His-SpaPK proteins, using specific antibodies. The potential interaction between recombinant proteins and Cdc25C can be investigated with immunoprecipation and subsequent Western blotting using specific antibodies.  179  Studies performed with the rat homolog Dlk, also support a role at the G2/M transition. REF52.2 cells expressing exogenous Dlk showed no signs of mitosis, suggesting that overexpression of Dlk may lead to growth arrest (Kogel et al., 1998). This effect is probably accomplished by the interaction of Dlk with Cdc5. Cdc5, a putative transcription factor or splicing factor, is a positive regulator of G2 progression and mitotic entry (Bernstein and Coughlin, 1998).  Overexpression of Cdc5 reduced the length of G2 phase, and a  dominant-negative Cdc5 mutant delayed entry into mitosis (Bernstein and Coughlin, 1998). Dlk may sequester Cdc5, thereby inhibiting Cdc5-dependent transcription or splicing of G2/M-specific genes involved in mitotic entry. 4.4.2.4 Factors contributing to discrepancies in the results Endogenous SpaPK displays different subcellular localization during the cell cycle; however, staining for the overexpressed recombinant SpaPK shows varied localization patterns which are different from that of the endogenous protein. This may be due to the possibility that SpaPK functions during mitosis as well as prior to mitosis.  Therefore,  recombinant proteins can only exert their effect at an earlier stage of the cell cycle and consequently, the effect during mitosis could not be observed. Alternatively, the proposed missing 5' end sequence, lacking in the recombinant proteins, may possibly be required for targeting the proteins to the mitotic structures. Finally, overexpression of a protein may alter its physiological function due to the abundance of the protein, therefore, not always truly reflecting the roles of endogenous protein. There are also differences in behaviour among ectopically expressed hHis-SpaPK proteins and mZIPK and Dlk. Potential explanations are quantitative differences in the expression levels of recombinant proteins, and qualitative difference between the cell lines used. Another possibility is that the role of SpaPK is different from those of its structural mouse and rat homologs.  4.4.3 Potential regulators of SpaPK  4.4.3.1 Protein kinases associated with the centrosome and the mitotic spindle A number of signaling proteins have been found on the centrosome and the spindle during mitosis. These are kinases including Cdkl/cyclin B (Bailly et al., 1989), casein kinase I-a (Brockman et al., 1992), casein kinase II (Krek et al., 1992), calcium calmodulin  180  dependent protein kinase II (Ohta et al., 1990), P K A (Nigg et al., 1985), PKC-x (Passalacqua et al., 1999), phosphoinositide-3 kinase (Kapeller et al., 1993), Plkl (Golsteyn et al., 1995), Aurora-A (Gopalan et at., 1997) and Aurora-C (Kimura et al., 1999), and protein phosphatases such as protein phosphatase 4 (Brewis et al., 1993) and protein phosphatase 1a (Andreassen et al., 1998).  Because of its protein kinase activity and the timing of its  association with the centrosome and the mitotic spindle, SpaPK could be involved in the regulation of phosphorylation changes on these structures during mitosis. Interestingly, the subcellular localization of SpaPK during mitosis is strikingly similar to that described for Aurora-A and the bipolar kinesin Eg5, suggesting that they may interact and control each other's function. 4.4.3.2 Candidate regulators of SpaPK identified from drug inhibition studies To identify potential regulators of SpaPK, the ability of different protein kinase inhibitors to change the subcellular localization of SpaPK was examined.  This study  unambiguously indicates the importance of phosphorylation in controlling the localization of SpaPK, since different protein kinase inhibitors were found to inhibit its localization at certain or all stages during mitosis. However, a definitive assignment of inhibition to a particular protein kinase is difficult from drug inhibition studies alone, for a number of reasons. Concentrations used were based on the IC50 of each drug for the respective kinases determined from in vitro experiments. The effective dose of a drug in vivo is often higher than that in vitro. The discrepancy could be related to the permeability of the compound in cell membranes, or the inability of the compound to out compete the high concentration of intracellular ATP. In addition, a drug can often act on many different targets, including those yet to be identified. The inhibitory ability of a drug may also be limited due to the different intracellular distribution of the drug and the potential target. Furthermore, these compounds could potentially directly inhibit the activity of SpaPK. This possibility was not tested in vitro with the purified His-SpaPK. Nonetheless, the drug experiments do provide clues on the potential regulators of the localization of SpaPK as discussed below. The localization of SpaPK is most sensitive to staurosporine and H-89 treatments. Staurosporine, a general kinase inhibitor, prevented localization of SpaPK throughout mitosis. H-89, a high affinity compound for P K A (Chijiwa et al., 1990), was also able to inhibit the distribution of SpaPK at all stages of mitosis. However, the changes observed  181  were at a concentration at which H-89 is a general, non-specific inhibitor. To narrow the possibile candidate regulators of SpaPK, a number of other inhibitors were used. At low concentrations, DRB has been shown to be a potent and specific inhibitor of CKI1 (Andreassen et ah, 1998). D R B did cause change at prophase, but only at a concentration which also has an inhibitory effect on a number of other protein kinases. Other compounds effective at prophase are A3, wortmannin and ML-7. In addition to staurosporine and H-89, the localization of the SpaPK was affected by D B H at metaphase, and by D B H and ML-7 at telophase.  Olomucine, an inhibitor of C D K 1 , and bisindolymaleimides I, an inhibitor of  PKC, appeared to have no effect. Collectively, these data suggest that P K A , CKII, CDK1 and P K C are not involved in the regulation of localization of SpaPK. The localization of SpaPK to mitotic structures is controlled by staurosporine-sensitive kinase(s) and H-89sensitive kinase(s). Additional possible regulators of its localization at prophase are P K G , myosin light chain kinase ( M L C K ) and phosphoinositide 3-kinase (PI 3-kinase), at metaphase are Chkl and Chk2, and at telophase are C h k l , Chk2 and M L C K . These data provided several candidate regulators involved in controlling the localization of SpaPK.  PI 3-kinase is a lipid kinase which has been implicated in the  regulation of various cellular processes, such as proliferation, growth, apoptosis and cytoskeletal rearrangement.  Immunofluorescence studies using antibodies against the p85  subunit of PI 3-kinase have shown that p85 is localized to microtubules through direct interaction with a/p heterodimmers (Kapeller et ah, 1993; Kapeller et ah, 1995). PI 3-kinase is also localized to centrosomes possibly through association with y-tubulin (Kapeller et ah, 1995). Moreover, PI 3-kinase has been proposed to be involved in the progression of the first mitotic cell cycle of fertilized sea urchin egg (De Nadai et ah, 1998). Inhibition of PI 3kinase activity led to prometaphase-like arrest, with drastic decrease of Cdc2 activity and perturbation of bipolar mitotic spindle formation.  Mammalian PI 3-kinase localized to  centrosomes may also control various events activated at the time of mitosis, and may be involved in the recruitment of SpaPK to the spindle poles. The possible involvement of Chkl and Chk2 in SpaPK signaling was unexpected. Chkl and Chk2 are activated and required for cell cycle arrest in response to D N A damage and/or D N A replication blocks. D N A damage was not introduced during the course of the experiment where Chkl and Chk2 were inactive. Possibly, D B H may acts on target(s) other  182  than Chkl and Chk2. Indeed, D B H can target the multifunctional protein kinase GSK-3 (unpublished) and cyclin-dependent kinases (Meijer, 2000).  Interestingly, two putative  GSK-3 phosphorylation sites are located in the non-catalytic domain of SpaPK. D B H was also tested on other protein kinases and showed no effect on their phosphorylation status associated with activation or inhibition. These protein kinases are CaMK4, Cdk2, C K l a , C K 2 a , E r k l , Erk2, p38 Hog mitogen-activated protein kinase, M e k l , Mek2, Mek3, Mek4, Mek5, Mekk3, P i m l , P K C a , PKCB, P K C ^ Rskl, p70S6 kinase, p46 SAPK, p54 SAPK, and Takl (Curman et al, 2001). Alternatively, Chkl and Chk2 may function at unperturbed G2/M transition. Chkl is reported to phosphorylate Cdc25C at Ser-216 in the absence of D N A damage at the S to M phase of the cell cycle (Kaneko et al., 1999). Recently, studies in G2-arrested Xenopus oocytes showed that basal levels of C h k l inhibit Cdc25C, suggesting that Chkl may function as an ordinary regulator of Cdc25C and contribute to G2 arrest of immature oocytes independent of D N A damage (Oe et al., 2001). The presence of a putative Chkl phosphorylation site on SpaPK further provides the possibility of involvement of Chkl in SpaPK-mediated signaling. Studies with kinase inhibitors also suggest that M L C K is involved in the regulation of localization of SpaPK. M L C K exists as two isotypes, with the high molecular mass M L C K as the predominant form in cultured non-muscle cells (Gallagher et al., 1995). The high molecular mass M L C K localizes to stress fibers during interphase and to the cleavage furrow during cytokinesis (Poperechnaya et al, 2000). The activity of high molecular mass M L C K is regulated in a cell cycle-dependent manner, where the activity is low during early mitosis and is two-fold higher during cytokinesis and interphase (Poperechnaya et ah, 2000). M L C K phosphorylates Ser-19 and to a lesser extent Thr-18 of the regulatory light chain of myosin II (MRLC) (Sellers et al, 1981). The level of phosphorylation of M R L C at Ser-19 plays an important role in the activation of myosin II motor activity and the initiation of cytokinesis in non-muscle cells of higher eukaryotes (Matsumura et al, 1998; Poperechnaya et al, 2000; Yamakita et al, 1994). M R L C , phosphorylated at Ser-19, is detected at spindle poles from prophase to metaphase.  During late anaphase, M R L C with increased level of Ser-19  phosphorylation is found at the spindle midzone, and appears to reach beyond the mitotic spindle to the peripheral region where the cleavages furrow would later form. At telophase, the immnunostaining is concentrated at the cleavage furrow until the end of cytokinesis  183  (Matsumura et al., 1998). Interestingly, SpaPK isolated from HeLa cells phosphorylates Ser19 as well as Thr-18 of M R L C (Murata-Hori et al., 1999). The distribution of M L C K to the spindle midzone during late anaphase may be a pre-requisite for localization of SpaPK during late stages of mitosis, where both kinases can function cooperatively.  4.4.4 Potential binding partners of SpaPK Farwestern blotting revealed the presence of three potential interaction partners for His-SpaPK from the cytoskeletal fraction of MCF-7 cells. Extraction of cells with nonionic detergent in physiologic ionic strength buffers leaves elements of the cytoskeleton in the particulate fraction.  These cytoskeletal components include a pool of microtubules and  microtubule-associated proteins, actin, myosin, intermediate filaments and their associated proteins, nuclear lamins, histones and other nuclear matrix-associated proteins.  Further  extraction with a high ionic strength buffer containing 0.6 M KC1 and DNase solubilizes some of the cytoskeletal proteins.  These three proteins are not tubulin, or histones, or  endogenous SpaPK. These three protein bands and ectopically expressed hHis-SpaPK were also detected in the cytoskeletal fraction of transfected MCF-7 cells, by Farwestern blotting and Western blotting, respectively. Therefore, pull-down of the ectopically expressed protein from the transfected MCF-7 cells using a specific antibody may be useful to further characterize the three cytoskeleton-associated  proteins.  Alternatively, Farwestern blotting of further  fractionations of the cytoskeleton components may also aid in the identification of these potential binding partners of SpaPK. The leucine zipper of mZIPK was reported to be required for the dimerization and the activity of the kinase (Kawai et al., 1998). Recently, the leucine zipper of the kinesin-like protein X C T K 2 was found to be required for localization to the spindle poles in Xenopus (Wittmann et al., 1998). Therefore, the putative leucine zipper located outside the kinase domain of SpaPK may be important for protein-protein interaction as well as for localization. Surprisingly, hHis-SpaPK from cytoskeletal cell extracts and purified His-SpaPK were not recognized by the radiolabeled His-SpaPK probe on Farwestern blot. This suggests that SpaPK does not cany a functional leucine zipper motif, or the experimental system is not  184  sufficiently sensitive or optimized for detecting such an interaction, or that the blotted protein dimerized during renaturation.  In conclusion, characterization of the functions of SpaPK was performed with both endogenous SpaPK and ectopically expressed His-SpaPK. Endogenous SpaPK is uniquely associated with centrosomes and the spindle apparatus during mitosis, and is not detected on other structures in the mitotic cells or interphase cells. This interaction of SpaPK with the spindle poles is independent of the presence of centrosomes. Overexpression of recombinant SpaPK led to inhibition of mitosis in mammalian cultured cells and delay of mitosis in Xenopus extracts.  This delaying effect may be due to the ability of His-SpaPK to  phosphorylate and inactivate Cdc25C, leading to negative regulation of Cdc2/cyclin B activity. Taken together, these data suggest that SpaPK may serve as a regulator of the spindle organization and chromosome segregation during mitosis, or as a regulator of the G2/M transition through the action of Cdc25C.  185  CHAPTER 5 CONCLUSIONS AND PERSPECTIVES  The human genome carries an estimated 40,000 genes encoding numerous types of proteins. Many are signaling protein kinases whose phosphorylation activities coordinate most cellular processes. Their importance is evident in their significant representation in the genome of many eukaryotic organisms: an estimated 113/6144 genes in the yeast S. cerevisiae, 411/18266 genes in the worm C. elegans, and 250/13338 in the fly D. melanogaster (Hunter and Plowman, 1997; Morrison et al, 2000; Plowman et al., 1999). Remarkably, the genome of the plant Arabidopsis thai carries genetic codes for 1005 putative protein kinases, representing about 4% of its total genes (website, 1). Finally, there appears to be more than 500 protein kinases encoded by the human genome (Kostich et al., 2002; Morrison et al., 2000). One of the important cellular processes involving protein kinases is the cell cycle. In an attempt to identify the human homolog of an essential yeast cell cycle checkpoint protein kinase Rad53, we have isolated a gene encoding, at the time, a novel protein kinase, named SpaPK for spindle pole associated protein kinase. Several lines of evidence suggest that the cloned SpaPK c D N A is missing a short upstream exon. The size of the SpaPK mRNA is longer than that of the isolated clone. A n in-frame stop codon is lacking from the supposed 5' UTR of the SpaPK cDNA.  The supposed 5' U T R of SpaPK c D N A is interrupted by  genomic sequences. The sequence surrounding the putative initiation A T G is not an optimal Kozak consensus. The size of endogenous SpaPK detected from cell extract is larger than that of the estimated molecular mass for the recombinant SpaPK. And a putative nuclear export signal is located within the supposed 5' UTR of the SpaPK cDNA. Primer extension analysis indicates that there are at least 87 nucleotides missing from the 5' extremity of the cloned SpaPK cDNA.  Extensive effort was devoted to identifying the missing sequences  using 5 ' - R A C E and Northern blotting, unfortunately, the identity of only 16 additional nucleotides were found. However, the missing sequences may not cause dramatic changes in the enzymatic function of SpaPK.  Therefore, experiments were performed on endogenous  SpaPK or using the cloned SpaPK cDNA.  186  SpaPK is a basophilic serine/threonine protein kinase which prefers an arginine or a lysine residue located at two positions N-terminal of its phosphorylation site. One of its in vitro substrates is Xenopus Cdc25C, which is phosphorylated at the site L-Y-R-S-P-S-M-P-E where the phosphorylated serine is in bold type. This sequence conforms to the deduced phosphorylation sequence of SpaPK. SpaPK is expressed in various tissues and cell lines, as well as at all stages of the cell cycle.  However, yeast complementation studies showed that SpaPK is not a functional  homolog of Rad53. Instead, it is a regulator of mitotic functions. SpaPK functions in mitotic cells possibly by controlling the organization of the mitotic spindle and the movement of chromosomes. prophase,  It is localized to duplicated centrosomes after their separation during  and remains associated with the centrosomes  and  centrosome-associated  microtubules until late anaphase, when it redistributes to the spindle midzone and finally to the midbody during early telophase. Endogenous SpaPK is also associated with MTOCs, independent of centrosomes, in cells released from nocodazole block and in taxol-induced mitotic cells.  SpaPK may also function at the G / M transition. The overexpression of 2  recombinant SpaPK proteins causes accumulation of cells in G , and the addition of purified 2  recombinant SpaPK proteins causes delay of mitosis in Xenopus extract. Many aspects of the signaling pathways controlling the onset of mitosis are still not well characterized.  A  simplistic view of SpaPK signaling at the G / M transition is presented in Figure 5.1. 2  A difficult task in signal transduction research is to properly map a signaling protein in a functional pathway. There are several candidate upstream kinases, such as PI 3-kinase, M L C K and C h k l , involved in the regulation of activity of SpaPK. Potential downstream targets of SpaPK include M R L C and Cdc25C. SpaPK specifically phosphorylates Ser-287 of Xenopus Cdc25C. Phosphorylation of Ser-287 is known to create a binding site for 14-3-3 proteins. The formation of the Cdc25C-14-3-3 complex results in the cytoplasmic retention of Cdc25C, thereby preventing the activation of nuclear pool of Cdkl/cyclin B required for the onset of mitosis. In addition, the three unidentified proteins from the cytoskeletal fraction of cell extract may represent both upstream regulators and/or downstream effectors of SpaPK.  187  Cytoplasm  Nucleus  F i g u r e 5.1  A proposed model for SpaPK signaling at the G / M transition. SpaPK 2  phosphorylates Ser-287 of Xenopus Cdc25C, which is analogous to Ser-216 of human Cdc25C. This phosphorylation creates a binding site for 14-3-3 proteins, thereby preventing nuclear translocation of Cdc25C and consequential activation of Cdkl/cyclin B required for the onset of mitosis.  188  Transfection  and  in vitro experiments  with wild-type or catalytic  inactive  recombinant SpaPK show that the activity of the kinase is not required to exert its effect. Although these observations may not apply to endogenous SpaPK, they do suggest that the non-catalytic domain of the kinase is important for the regulation of activity, subcellular targeting, and substrate recognition. The coiled-coil domain outside the kinase domain of SpaPK may serve as a scaffold for the assembly of multi-protein complexes and the colocalization of specific signaling proteins and their substrates, at the spindle poles, the spindle midzone and the midbody. Precise assembly and function of the mitotic spindle during cell division is essential for the accurate partitioning of the replicated chromosomes to daughter cells. Many of these key events during mitosis are controlled by phosphorylation.  Aberrant regulation of  phosphorylation activity may lead to chromosomal instability and cancer. mitotic kinases have been implicated in tumorigenesis.  Indeed, several  P l k l and aurora kinases are  frequently overexpressed in tumors, and are able to transform cultured cells (Bischoff et al., 1998; Knecht et al, 1999; Smith et al., 1997). In addition, inactivated Bubl was found in some tumor cell lines (Cahill et al., 1998). The functions of SpaPK during mitosis await further exploration.  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