3. Electronic Theses and Dissertations (ETDs) - All submissions
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Item Reversible phosphorylation of proteins in proliferating and differentiating cells : cyclic variations and the effect of growth regulators / Gracinda Maria Nunes Ferreira.(1994) Ferreira, Gracinda Maria NunesLiving cells are highly auto-dynamic entities which means that the underlying biochemistry is equally dynamic, a reality which is ignored by most researchers. Theoretical studies indicate that such a state must be due to the existence of oscillatory variations in the levels and activities of key components in the cell. In this study, the dynamic behaviour of four major, interrelated areas of cell biochemistry (phosphorylation, dephosphorylation, the terminal reaction of glycolysis and the amount of soluble protein) were examined and all systems found to oscillate in murine erythroleukaemic cells (MEL) and, where examined, also in the human HL-60 leukaemic cell line. (Abbreviation abstract)Item Activation of JNK1B1 by phosphorylation: implications for its function, stability and dynamics(2015-01-29) Owen, Gavin RayThe c-Jun N-terminal kinases (JNKs) are mitogen-activated protein kinases (MAPKs) that are activated by the dual phosphorylation of a canonical threonine and tyrosine residue. While it is well known that the activation of JNK mediates many important cellular processes such as differentiation, proliferation, and apoptosis, the mechanisms by which phosphorylation induces its activation are not known. An understanding of the structural and biophysical basis for the activation of JNK is highly desirable however, as dysregulation of the kinase has been implicated in numerous prominent diseases. Aiming first to improve upon the previously reported inadequacies in acquiring active JNK, this work describes a novel method for the purification of large yields of pure and phosphorylated JNK1β1, the most abundant JNK isoform. Using codon harmonization as a precautionary measure toward increasing the soluble overexpression of the kinase raised unique questions about the role of translation kinetics in both the heterologous and natural co-translational modification of kinases. After purifying the upstream activating kinases of JNK, phosphorylation of JNK1β1 was achieved by reconstituting the MEKK1 → MKK4 → JNK MAPK activation cascade in vitro. Activated JNK1β1 was thereafter able to phosphorylate its substrate, ATF2, with high catalytic efficiency. Characterising the nature of JNK1β1 modification by MKK4, mass spectrometry revealed that the latter kinase phosphorylates JNK1β1 not only at its activation residues (T183 and Y185), but also at a recognised yet uncharacterised phospho-site (S377) as well as two novel phospho-residues (T228 and S284) whose phosphorylation appear to have functional significance. Unfolding studies and amide hydrogen-deuterium exchange (HX) mass spectrometry (MS) were then used to investigate the changes to the stability and structure/conformational dynamics of JNK1β1 induced by phosphorylation and nucleotide substrate binding. Increased flexibility detected at the hinge between the N- and C-terminal domains upon phosphorylation suggested that activation may require interdomain closure. Patterns of solvent protection by the ATP analogue, AMP-PNP, reflected a novel mode of nucleotide binding to the C-terminal domain of a destabilised and open domain conformation of inactive JNK1β1. HX protection at both domains following AMP-PNP binding to active JNK1β1 revealed that the domains close around nucleotide upon phosphorylation, simultaneously stabilising the kinase. This reveals that phosphorylation activates JNK1β1 in part by enhancing the flexibility of the hinge to enable interdomain closure and the formation of a functional active site. This work thus offers novel insight into the unique molecular mechanisms by which JNK1β1 is regulated by nucleotide binding and phosphorylation by MKK4, and by the complex interplay that exists between them.Item Characterization of a plasmodium falciparum protein kinase(2014-02-07) Roets, SashaMalaria is caused by Plasmodium parasites and is the world’s most devastating tropical infectious disease. The need for identifying novel drug targets is fuelled by an increased resistance of these parasites against available drugs. The human host red cell membrane plays an important role during invasion and subsequent development of the parasite within the red cell and undergoes several structural, functional and biochemical changes triggered by various protein-protein interactions between the parasite and the host cells. These interactions form a fundamental part of malaria research, since the parasite spends the pathogenic stage of its life cycle in the human erythrocyte. The Plasmodium kinome is complex and the exact role of protein phosphorylation in malaria parasites is not yet fully understood. This study aims to characterise the kinase domain of Plasmodium falciparum (3D7) Protein Kinase 8 (PfPK8), described as a putative protein on the Plasmodium falciparum database. PfPK8 is encoded by the PfB0150c gene (recently renamed as PF3D7_0203100) situated on chromosome 2 of the parasite genome. A 1 507bp section of the PfB0150c gene, containing a 822bp centrally located kinase domain was cloned into a pTriEx-3 expression vector. A soluble recombinant octa-histidine-tagged PfPK8 was expressed in Escherichia coli Rosetta 2 (DE3) cells, but with relatively low yield and purity.To improve the expression, a recombinant PfB0150c-baculovirus infected Spodoptera frugiperda (Sf9) insect cell system was attempted, but without success. A different tag was employed and glutathione-S-transferase-PfPK8 was successfully expressed in Escherichia coli Rosetta 2 (DE3) cells, with a higher yield and purity. Recombinant GST-PfPK8 was used in non-radioactive coupled spectrophotometric kinase assays in the presence of known kinase substrates casein, MBP and H1 to determine kinetic parameters of the enzyme. It phosphorylated all three substrates at a temperature of 37ºC and pH of 7.4. Recombinant GST-PfPK8 was inactive at a pH below 6 and most active at pH 7.4. The relative activity of the enzyme was highest at a temperature synonymous to a fever spike in a Plasmodium falciparum infected individual. Secondary structural analysis of PfPK8 revealed the position of a conserved substrate binding domain containing an ATP-binding site and binding loop within the kinase domain. The kinase domain of rPfPK8 was modelled using available crystal structures of its identified homologues. The gene is expressed throughout the intraerythrocytic stages of the parasite life cycle, as well as in gametocytes. Protein-protein binding studies revealed that host-parasite protein-protein interactions exist between rPfPK8 and erythrocyte membrane protein, band 3. Plasmodium falciparum PK8 could therefore play a role during invasion of host erythrocytes and during the intraerythrocytic development of the parasite, by phosphorylating red blood cell membrane proteins. This study provides the groundwork for future X-ray crystallographic studies to elucidate the structure of the enzyme, and for additional gene manipulation experiments to ascertain whether it is essential for parasite survival in all the intraerythrocytic stages and therefore a potential new drug target candidate.