A Study of Two Protein Kinases from Trypanosoma brucei

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 m 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 lambdagt 11 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