Analysis of cyclin dependent kinases in Leishmania

The results obtained from the experiments presented in this study aimed to further explore the role of cyclin dependent kinases and cyclins in the protozoan parasite Leishmania major. Cdks in kinetoplastids, CRKs, are the key regulators that allow cells to progress through different cell cycle phases and promote parasite proliferation during infection. In chapter 3 of this study, the results presented showed that L. major CYCA is capable of activating CRK3 in an in vitro kinase assay using histone H1 as substrate. The CRK3/CYCA active complex was then used to analyse the effect of the phosphorylation at the CRK3 activation threonine using a kinase activating kinase (yeast CAK or Civ-1). Phosphorylated CRK3 activity was compared to non-phosphorylated CRK3 and it was found that the phosphorylation promotes a 5-fold increase in kinase activity of the complex. The accessory protein Cks1 was assayed in vitro with the active CRK3/CYCA complex and it was shown that Cks1 might have an inhibitory effect when histone H1 substrate is used. The IC50 for two different kinase inhibitors (Flavopiridol and Indirubin) was determined for the in vitro CRK3/CYCA complex and compared with the values found for the in vivo purified CRK3. Similar values were obtained suggesting that the in vivo complex is indeed represented by the recombinant complex. In the following chapter 4, yeast Civ-1 purified from E. coli, was used to try to phosphorylate, in a similar manner, the activation of threonine/serine residues from other L. major CRKs. The kinases assessed were CRK1, CRK2, CRK4, CRK6 and CRK7. None of these were phosphorylated by Civ-1 suggesting that the only CRK under this type of regulation is CRK3. L. major CRK1-4 and CRK6-8 were tested in kinase assays by mixing under described conditions with L. major CYC9 and kinase activities towards three different substrates were assessed. L. major CYC9 was not able to activate the above kinases and the kinase subunit that interacts with this cyclin could not be identified. In chapter 5, the L. major CYCA was used to elucidate the characteristics of this cyclin in vivo. A gene disruption strategy aimed to replace the two genomic alleles of this protein gene by homologous recombination. Plasmids were developed with flanking regions of this gene placed in association with two different drug resistance genes, one for each of the allele’s disruption. These constructs were not able to produce the first allele knock out suggesting that not only this gene might be essential but the levels of expression may also be important. Tagging L. major CYCA was also attempted in vivo using two different strategies (i.e. two different tagging systems). The first tag employed was the TAP tag syste. Although drug resistant transfected cell lines were obtained, no tag detection could be observed by western blot using different tag-specific antibodies (α-protein-A and α-calmodulin antibodies). The second tag employed was HA, the 9-amino acid sequence YPYDVPDYA, derived from the human influenza hemagglutinin (HA) protein. Plasmids that contained C and N-terminal HA tagged L. major CYCA were used to transfect WT cells and cells extracts of resistant cell lines analysed by western blot. Both C and N-terminal HA tagged CYCA were detected by the α-HA antibody. Following the confirmation of the presence of the tagged CYCA in the cell extracts an affinity purification using an HA affinity matrix was attempted and the matrix binding material was used in in vitro kinase assays. The presence of kinase activity towards Histone H1 confirmed that CYCA was being succesfully immunoprecipitated in complex with a kinase partner. The identity of the co-eluted CRK could be confirmed using specific α-CRK3 antibody that detected CRK3 in the eluted material.