\ A study of two protein kinases from Trypanosoma brucei Timothy Marcus Graham PhD Wellcome Unit of Molecular Parasitology Anderson College University of Glasgow May 1996 ProQuest Number: 11007874 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 11007874 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 lo53^ Qi i GLASGOW I j 'jN IV E E Sir? I ] ilflR&lT I Summary The protozoan parasite Trypanosoma brucei undergoes major differentiation events during its complex life cycle which involves the tsetse fly and mammal as obligate hosts. At the same time it alternates between proliferative and non-proliferative forms. In higher eukaryotes differentiation and the cell cycle are controlled by complex signalling networks many of which involve protein kinases as components and, by analogy, this would be expected to be the situation in T. brucei. However, very little is known about cellular signalling in this parasite and the work presented in this thesis is a study of two T. brucei protein kinases as an approach to the identification of pathways regulating differentiation and the cell cycle. Two approaches were followed: firstly, the characterisation and purification of a 60 kDa autophosphorylating protein kinase from T. brucei that was found to be expressed in a stage-specific manner (Hide et al., 1994). This protein kinase activity was found to localise in the 100 000 g supernatant of total extracts of bloodstream forms and this supernatant was used as the starting material for purification. Using anion-exchange chromatography as the first purification step, the autophosphorylating protein kinase was detected in two peaks of activity eluting at slightly different salt concentrations. A number of different chromatography matrices were then tested for their suitability in the further purification of the protein kinase and a further level of purification was achieved using a Sepharose to which the protein kinase inhibitor H-9 had been immobilised. In addition new assays for measuring activity were developed, one of which, based on autophosphorylation in solution was found to be robust and informative. The second approach taken was to isolate and sequence a full length cDNA corresponding to an amplified cDNA fragment (Hua and Wang, 1994) for which the predicted peptide sequence showed homology to catalytic domain sequences of a rat protein kinase C family member and the Drosophila protein kinase polo. Isolation and sequencing of genes encoding T. brucei protein kinase C family members would provide valuable evidence for cell signalling in this parasite and provide tools to complement earlier studies on protein kinase C like activities in T. brucei (Keith et al., 1990). A clone was isolated in a screen of a ^ g tll cDNA library using a probe homologous to the original amplified cDNA fragment. Partial sequencing of the insert has shown that it is a chimaera of cDNAs for a ribosomal protein S4 homologue and for a protein kinase (this part of the chimaera includes a section identical to the probe). Hybridisation of Southern blots of T. brucei genomic DNA to probes from the ribosomal protein and protein kinase coding regions, under high stringency conditions, shows that both sequences are of T. brucei origin but that the two sequences are not co-linear in the genome. Comparisons of the two predicted peptide sequences with protein sequences in the databases show that the open reading frames for both proteins are incomplete. The open reading frame for the protein kinase homologue is probably almost complete but there is no 3' stop codon. The 5' coding sequence could be complete but there could be an upstream start codon not contained within the clone isolated. The putative protein kinase has all the defining features of a member of the polo-like kinase family and is clearly not a protein kinase C. Polo-like kinases are implicated in regulation of mitotic spindle formation and therefore mitosis, and the T. brucei enzyme would be worthy of further study given that mitosis and spindle formation are very different in T. brucei compared to higher eukaryotes. Declaration This thesis and the results presented in it are entirely my own work except where otherwise stated. ) ^ ’I r L - C x iii Acknowledgements I would firstly like to thank my supervisors Dr G. Hide and Professor A. Tait for advice and encouragement throughout this project and for speedy correction of drafts of this thesis. I would also like to thank all those in WUMP, Vet. Parasitology and elsewhere who helped and encouraged me during this work. This work was supported by the Medical Research Council. CONTENTS Abstract i Declaration iii Acknowledgements iv List of contents V List of figures ix List of tables xii List of abbreviations xiii Page Number Chapter One : Introduction 1.1 Trypanosoma brucei 2 1.1.1 Life cycle 3 1.1.2 Genome and gene expression 5 1.1.3 Energy metabolism 8 1.1.4 Cytoskeleton and cell cycle, cell surface and flagellar pocket 9 1.1.5 Chemotherapy of African trypanosomiasis 12 1.1.6 Summary 15 1.2 Protein phosphorylation in eukaryotes 15 1.2.1 Protein kinase classification 17 1.2.2 Catalytic domains 21 1.3 Protein phosphorylation inT. brucei 24 1.3.1 Biochemical studies 24 1.3.2 Protein kinase and protein phosphatase genes 33 1.3.2.1 Nrk (Nekl and NimA related kinase) 34 1.3.2.2 cdc2-related protein kinases 36 1.3.2.3 A MAP kinase homologue 38 1.3.2.4 Partial cDNAs for T. brucei protein kinases 39 1.3.2.5 Protein phosphatase genes from T, brucei 40 1.3.3 Conclusions 41 1.4 Overview of thesis 43 v Chapter Two : Materials and Methods 2.1 Protein methods 53 2.1.1 T. brucei stocks and preparation of cell extracts 53 2.1.2 Isoelectric focusing and in situ protein kinase assay 53 2.1.3 Phosphoamino acid analysis 55 2.1.4 Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) 55 2.1.5 2-dimensional protein electrophoresis 57 2.1.6 Liquid chromatography 57 2.1.7 Slot blot kinase assay 58 2.1.8 Liquid kinase assay for column fractions 59 2.1.9 Preparation of H-9 Sepharose 59 2.1.10 Protein concentration/buffer exchange 60 2.2 Nucleic acid techniques 60 2.2.1 Growth and transformation ofE. coli 60 2.2.2 Bacterial vectors and preparation of vector DNA 60 2.2.3 Manipulations of plasmid DNA 60 2.2.4 Electrophoresis of DNA in agarose gels 61 2.2.5 Southern blotting of agarose gels 61 2.2.6 Labelling of oligonucleotide and DNA probes 62 2.2.7 Polymerase chain reaction 62 2.2.8 DNA sequencing 63 2.2.9 Isolation ofT. brucei genomic DNA and RNA, purification of poly(A)+ RNA and synthesis of 1st strand cDNA 63 2.2.10 Screening of a A, cDNA library 63 2.2.11 Isolation ofX DNA 64 Chapter Three : Identification of an autophosphorylating protein kinase from Trypanosoma brucei 3.1 Introduction 67 3.2 Analysis of autophosphorylating protein kinase activities in Trypanosoma brucei using the in situ IEF gel assay 68 vi 3.3 Phosphoamino acid analysis 69 3.4 Discussion 70 Chapter Four : Purification of the 60 kDa protein kinase 4.1 Introduction 79 4.1.1 Aims 79 4.1.2 Approaches to protein purification 80 4.1.2.1 General considerations 80 4.1.2.2 Ion-exchange chromatography 81 4.1.2.3 Size-exclusion chromatography 82 4.1.2.4 Reversed-phase and Hydrophobic interaction chromatography 83 4.1.2.5 Affinity chromatography 84 4.1.2.6 Purification approaches specific for protein kinases 86 4.1.2.7 Summary 90 4.2 Results 91 4.2.1 Development of anion-exchange chromatography 91 4.2.1.1 Initial anion-exchange separation 92 4.2.1.2 100 mM NaCl step elution 94 4.2.1.3 150 mM and 200 mM NaCl steps 96 4.2.1.4 100 mM-200 mM NaCl gradient elution 97 4.2.2 Hydrophobic interaction chromatography (HIC) 98 4.2.3 ATP affinity chromatography 100 4.2.4 H-9 affinity chromatography 101 4.2.4.1 Initial tests of protein kinase binding to H-9 Sepharose 102 4.2.4.2 ATP gradient elution of the 60 kDa protein kinase from an H-9 Sepharose column 104 4.2.5 Size-exclusion chromatography 106 4.3 Discussion 107 vii Chapter Five : Cloning of a Trypanosoma brucei cDNA encoding a protein kinase 5.1 Introduction 135 5.2 Results 136 5.2.1 Amplification of a cDNA fragment homologous to TbPK-A2 136 5.2.2 Amplification of the 5' cDNA sequence 137 5.2.3 Amplification of the 3' cDNA sequence 138 5.2.4 Isolation of a Xgtll clone containing the TbPK-A2 sequence 138 5.2.4.1 Subcloning and sequencing of pTbplkl 139 5.2.4.2 The putative protein kinase from pTbplkl is a member of the polo-like kinase family 142 5.2.4.3 The probable primary structure of TbPLK and alignments of the catalytic domain and C-terminal sequences with those of other polo-like kinases 143 5.2.4.4 Similarity of the partial T.