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Analysis of the CRK3 Kinase of Leishmania Mexicana

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Analysis of the CRK3 Kinase of Leishmania Mexicana Analysis of the CRK3 kinase of Leishmania mexicana Paul Hassan Wellcome Unit of Molecular Parasitology Anderson College University of Glasgow Glasgow G11 6NU This thesis is presented in submission for the degree of Doctor of Philosophy in the Faculty of Veterinary Medicine March 1999 ■■ J k , 1 ProQuest Number: 13815606 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest ProQuest 13815606 Published by ProQuest LLC(2018). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC. ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106- 1346 GLASGOW UNIVERSITY LIBRARY ABSTRACT Control of cell cycle progression in eukaryotes is governed by members of a highly conserved family of serine threonine protein kinases, the cyclin-dependent kinases (CDKs), which associate with a regulatory cyclin partner protein to attain full activity. The CRK3 gene product ofLeishmania mexicana is one of two putative CDKs so far identified in this organism. The possible function of CRK3 and the requirement of the CRK3 gene for Leishmania survival was investigated using molecular genetic techniques. Attempts to create null mutants lacking an intact CRK3 locus failed repeatedly and instead a series of transgenic cell lines were generated that had undergone changes in ploidy or genomic rearrangements. However, both alleles could be successfully disrupted when extra copies of CRK3 were introduced on an episome into a heterozygote mutant, prior to disruption of the second chromosomal CRK3 allele. Together these results provide evidence that CRK3 is an essential gene in the promastigote form of Leishmania mexicana. CDKs are regulated not only by cyclin binding, but also by inhibitory and activatory phosphorylation events. Inhibition of Leishmania mexicana phosphotyrosine phosphatase activity by bpV(phen) resulted in inhibition of cell cycle progression, and led to an accumulation of cells in the G1 and S-phases of the cell cycle. Cells treated with bpV(phen) had a low level of CRK3 activity and upon release from inhibition CRK3 activity increased. Inhibition of a phosphotyrosine phosphatase activity could be involved in regulation of CRK3 activity, either directly or indirectly. The CDK inhibitor flavopiridol is a potent inhibitor of mammalian cdkl, cdk2 and cdk4, enzymes, which all have a role in cell cycle progression. Flavopiridol was found to be a potent inhibitor of L. mexicana CRK3 activity and inhibits purified CRK3 with an IC50 value of 100 nM. Flavopiridol inhibited growth of L. mexicana promastigotes in liquid culture, with 50% inhibition of growth achieved with a concentration of 250 nM. Inhibition of growth is due to inhibition of cell cycle progression, as cells were found to accumulate in the G2 phase of the cell cycle, probably due to inhibition of CRK3 activity. Flavopiridol-induced growth inhibition is reversible up to 24 hours after addition of the dmg. Release from flavopiridol inhibition resulted in a partially synchronous re-entry into the cell cycle. This method may be used to obtain cell samples enriched for cells in particular phases of the cell cycle. In contrast to several plant and animal CDKs, CRK3 failed to complement for loss of function of CDC28 activity in the Saccharomyces cerevisiae cdc28-lNt\ cdc28-4ts and cdc28-13ts mutants, suggesting that mechanisms of cell cycle control inLeishmania may be less conserved than those of other eukaryotes. The findings that CRK3 is an essential gene in Leishmania, that CRK3 is inhibited by specific inhibitors and that CRK3 has features that distinguish it from mammalian homologues, make CRK3 a novel drug target of some promise. TABLE OF CONTENTS Page TITLE PAGE i ABSTRACT ii TABLE OF CONTENTS iv LIST OF FIGURES iix LIST OF TABLES X ABBREVIATIONS xi NOTE ON GENETIC NOMENCLATURE xiii DECLARATION xiv ACKNOWLEDGEMENTS XV CHAPTER 1 INTRODUCTION 1.1 The trypanosomatids 1 1.1.2 The single organelles of trypanosomes andLeishmania 4 1.1.3 Avoidance of host immune responses 5 1.1.4 Molecular genetic analysis of trypanosomatid gene function 7 1.2 Control of the Eukaryotic Cell Cycle 18 1.3 Cell cycle control in fission yeast 18 1.4 Cell cycle control in budding yeast 22 1.5 Vertebrate cell cycle control 29 1.6 Cyclin dependent kinase inhibitors (CKIs) 38 1.7 Chemical inhibitors of cyclin-dependent kinases 40 1.8 Cell division and cell cycle control in trypanosomatids 42 CHAPTER 2 MATERIALS AND METHODS 2.1 Molecular methods 56 2.1.1 Bacterial strains 56 2.1.2 Glycerol stocks 56 2.1.3 Bacterial culture 56 2.1.4 Preparation of competent cells 57 2.1.5 Plasmid preparation 57 2.1.6 Large scale plasmid preparation 58 2.1.7 Polymerase chain reaction (PCR) amplification of DNA fragments 59 2.1.8 Restriction digestion 60 2.1.9 Gel electrophoresis 60 2.1.10 DNA extraction from agarose gel 60 2.1.11 Phosphatase treatment 61 2.1.12 DNA Ligation 61 2.1.13 Bacterial transformation 62 2.1.14 DNA sequencing 62 2.2 Generation of constructs described in this thesis 62 iv 2.2.1 pGL89 63 2.2.2 pGL97 (CRK3::BLE knockout construct) 63 2.2.3 pGL105 (CRK3::HYG knockout construct) 63 2.2.4 pGL96 (pXCftOhis) 64 2.2.5 pGLlOO (pTEXCRO) 64 2.2.6 pGL310 (pTEXCROhis) 64 2.2.7 pGL120 (pRS416METCRK3) 64 2.2.8 pGL332 (pRS314METC7C7) 65 2.2.9 pGL292 (pRS314METCYC2) 65 2.2.10 pGL293 (pRS314METC7C3) 65 2.3 Leishmania mexicana methods 66 2.3.1 Leishmania mexicana cell line 66 2.3.2 Leishmania mexicana cell culture 66 2.3.3 Determination of cell density 66 2.3.4 Preparation ofL. mexicana promastigote cell pellets 67 2.3.5 Preparation of stabilates 67 2.3.6 Electroporation procedure 67 2.3.7 Preparation of Leishmania mexicana genomic DNA. Large scale 68 prep 2.3.8 Preparation of genomic DNA. Mini-prep method 69 2.3.9 Southern blot transfer 69 2.3.10 Nucleic acid hybridisation 70 2.3.11 bpV(phen) block and release 70 2.3.12 Flavopiridol block and release 71 2.3.13 DNA content analysis by flow cytometry 71 2.3.14 SDS-PAGE 72 2.3.15 Preparation of pl3sucl beads 72 2.3.16 pl3sucl Binding Kinase Assay 72 2.3.17 Nickel NT A agarose selection and flavopiridol IC50 determination 73 2.3.18 DAPI staining and microscopy 73 2.4 Yeast methods 74 2.4.1 Saccharomyces cerevisiae strains 74 2.4.2 Saccharomyces cerevisiae transformation 74 2.4.3 S. cerevisiae cell lysates 75 2.4.4 Western blot transfer 76 2.4.5 Antibody hybridisation 76 2.5 Buffers and reagents 77 CHAPTER 3 TARGETED GENE DISRUPTION OF Leishmania mexicana CRK3 3.1 Introduction 84 3.2 Results 85 3.2.1 Gene disruption of first and second alleles ofCRK3 85 3.2.2 Introduction of an episomal copy ofCRK3 followed by gene 90 disruption 3.2.3 Co-transfection of the CRK3 episome with the second targeting 92 V construct 3.2.4 Episomal expression ofCRK3 followed by second round gene 96 disruption in the absence of Geneticin selection 3.2.5 Expression of an inactive CRK3 kinase followed by gene disruption 97 results in changes in ploidy 3.2.6 Expression of a modified CRK3his gene and generation of null 99 mutants using a pX-based construct 3.2.7 Co-transfection of an episomal vector expressing CRK3his with the 101 CRK3: :BLE targeting fragment 3.3 Discussion 102 CHAPTER 4 THE EFFECTS OF DIRECT AND INDIRECT INHIBITION OF CRK3 ON CELL CYCLE PROGRESSION IN Leishmania mexicana 4.1 Introduction 131 4.2 Inhibition ofLeishmania growth by the protein tyrosine 134 phosphatase inhibitor bpV(phen) 4.2.1 Incubation of Leishmania promastigotes with bpV(phen) 135 results in a reduction of CRK3 activity 4.2.2 Restoration of CRK3 activity and re-entry into the cell cycle 137 upon release from bpV(phen) induced growth arrest 4.3 Inhibition of CDKs by chemical inhibitors 139 4.3.1 Inhibition of Leishmania Promastigote Growth by the CDK 140 Inhibitor Flavopiridol 4.3.2 Flavopiridol inhibits CRK3 with an IC50 value of 100 nM 141 4.3.3 Flavopiridol blocks promastigote growth by causing cells to arrest 142 in G2 phase of the cell cycle 4.3.4 Release from flavopiridol induced cell cycle arrest 144 4.3.5 Flavopiridol inhibits the growth of bloodstream and procyclic 146 forms of T. brucei 4.4 Discussion 147 CHAPTER 5 COMPLEMENTATION EXPERIMENTS WITH SACCHAROMYCES CEREVISIAE cdc28« MUTANTS 5.1 Introduction 168 5.2 Results 169 5.2.1 CRK3 fails to complement S. cerevisiae cdc28-lNts, cdc28-4ts 169 and cdc28-13ts 5.2.2 Co-expression of T. brucei cyclins withL. mexicana CRK3 172 fails to complement for loss ofS. cerevisiae CDC28 activity 5.3 Discussion 173 CHAPTER 6 GENERAL DISCUSSION 6.1 Gene Disruption of CRK3 185 6.2 Indirect inhibition of CRK3 189 6.3 Direct inhibition of CRK3 by the cdk inhibitor flavopiridol 191 6.4 CRK3 fails to complement Saccharomyces cerevisiae cdc28ts 193 mutants 6.5 Future directions 197 REFERENCES 202 vii LIST OF FIGURES FIGURE Page Fig. 1.1 The life cycle ofLeishmania mexicana 48 Fig. 1.2 Activation and inhibition of cyclin-dependent kinases 49 Fig.
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