published_or_final_version / Biochemistry / Master / Master of Philosophy

published_or_final_version / Physiology / Master / Master of Philosophy

published_or_final_version / Zoology / Master / Master of Philosophy

No description available.

RdoA is a bacterial protein kinase from Salmonella enterica serovar Typhimurium first noted for its regulation of dsbA expression in this organism. The crystal structure of RdoA’s homologue, YihE from Escherichia coli, revealed a basic bi-lobal kinase domain that is a hallmark of the eukaryotic Ser/Thr, Tyr protein kinase superfamily. YihE however, bears the greatest structural similarity to choline kinase and aminoglycoside 3’-phosphotransferase [APH(3’)]-IIIa which are both atypical kinases. RdoA and YihE have demonstrated the capacity for autophosphorylation in vitro and the ability to phosphorylate myelin basic protein, however, the native kinase target protein has not been identified. Based on structural alignment with APH(3’)-IIIa, predictions were made of key residues involved in ATP binding and catalysis and five YihE mutants were generated. Both the wildtype and YihE mutants were cloned for expression as N-terminal histidine-tagged proteins. In the work presented here, these proteins have been overexpressed and purified for further study. Mutational analyses revealed that four of the five mutants had decreased kinase activity in comparison to the wildtype protein, thereby establishing the mutated residues as important for enzymatic activity. Several attempts were made to elucidate the substrate of RdoA/YihE, however, it remains unknown. Further investigation is necessary to identify its substrate(s) and to pinpoint its physiological significance. RdoA is a member of the Cpx regulon and its absence stimulates Cpx activation. Since the Cpx system is involved in regulating expression of cell surface appendages and is one of three envelope stress response systems, it is hypothesized that RdoA serves to relay Cpx activation signals. This is supported by studies on the effect of pH on Cpx activity in wildtype and rdoA- cells presented here. RdoA homologues are present in at least 85 different genera. This level of conservation is indicative of an important biological role for this previously uncharacterized bacterial protein kinase. / Thesis (Master, Microbiology & Immunology) -- Queen's University, 2007-12-04 18:19:29.574

Fission yeast Srk1 kinase is essential for cellular responses to extracellular stimuli. It is activated downstream of the MAPK Spc1 and participates in controlling mitotic entry by directly phosphorylating and inhibiting Cdc25 phosphatase during the normal cell cycle and also in response to osmotic stress. Following phosphorylation, Cdc25 is exported out of the nucleus. Heat stress caused by a temperature shift from 25°C to 36°C, which is within the normal temperature tolerance of fission yeast, temporarily inhibits nuclear division in wildtype cells. The same response is seen in cells deleted for the Pyp1 and Pyp2 phosphatases which normally serve to down regulate the Spc1 stress response. I have shown that the transient block in nuclear division caused by temperature shifts within the physiological range does not occur in srk1- cells but instead there is a stimulation of mitosis and cell division in response to the heat stress. This pattern of mitotic stimulation is phenocopied in cdc25-9a cells where nine putative Srk1 phosphorylation sites on Cdc25 are changed to alanine. Cells lacking srk1, however, display the same cell cycle progression pattern as the wildtype cells in response to osmotic stress. This experiment clearly distinguishes separate pathways for these two stress responses. Also, Cdc25 is found to be phosphorylated after a mild heat stress and seems to be exported out of the nucleus. These data indicate that Srk1 kinase plays the central role in regulating mitotic entry in response to mild heat stress by negatively regulating the Cdc25 tyrosine phosphatase. Thus my work highlights the role of Srk1 kinase in cell cycle regulation and is consistent with the Spc1 MAPK cascade linking the G2/M transition to extracellular stress. This response to mild heat stress is important because temperature sensitive mutants are frequently used in fission yeast research and this work shows that a temperature shift from 25°C to 36°C, which is within the physiological growth range, can trigger a stress response. / Thesis (Master, Biology) -- Queen's University, 2011-07-28 09:34:52.164

Fer is a ubiquitously expressed non-receptor protein-tyrosine kinase that regulates normal physiology through signaling in a variety of cell types. Fer signals downstream of growth factor receptors frequently activated or amplified in human cancers and Fer has been identified as a positive regulator of cancer progression in the prostate and liver. Epidermal growth factor (EGF) receptor (EGFR) is frequently activated due to gene amplification or gain-of-function mutations in non-small cell lung carcinomas (NSCLC) leading to aggressive tumours that frequently metastasize. Since EGFR activates Fer, I tested whether Fer participates in EGFR-driven NSCLC cell migration, tumour progression and metastasis. Here, I show that Fer is expressed in cell lines derived from both normal lung epithelia and NSCLC and is activated following EGF treatment of NSCLC cells. To probe Fer function we used a lentiviral shRNA system to achieve stable knock-down (KD) of Fer in H1299 cells. Compared to control cells, Fer KD cells displayed a significant reduction in EGF-induced cell migration and invasion which correlated with reduced phosphorylation of the guanine nucleotide exchange factor Vav2. Consistent with Vav2 phosphorylation promoting Rac activation, we observed reduced localization of active, GTP- bound Rac1 to the leading edge of Fer KD cells treated with EGF. Tumour xenograft experiments were performed to test the role of Fer in NSCLC tumour progression and metastasis in immune compromised mice. Growth of primary tumours was normal, despite efficient Fer silencing in vivo. Interestingly, fewer spontaneous lung metastases were observed from subcutaneous Fer KD tumours compared to control. However, no differences were observed in lung seeding efficiency in experimental metastasis assays, suggesting that Fer may play a role in early stages of metastasis. Together, this study identifies Fer as a potential new therapeutic target for the treatment of EGFR-driven lung cancer metastasis. / Thesis (Master, Biochemistry) -- Queen's University, 2012-08-31 12:29:43.856

The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.

No description available.

The non-receptor tyrosine kinase activity of fusion gene BCR-ABL derived oncoproteins is the key factor responsible for development and progress of Philadelphia positive (Ph+) chronic myeloid leukemia (CML). In the search for a superior and novel peptide-based inhibitor of Bcr-Abl, here I investigated a naturally occurring molecule, called apoptin. Apoptin is a 13.6 kDa protein derived from chicken anemia virus (CAV) and known to induce apoptosis in a wide range of transformed but not in primary cells. Apoptin is a protein without any reported structural and/or functional homolog and is an interesting candidate to initiate protein-protein interactions and subsequent downstream effects. Initially by an array-based analysis I found that apoptin interacts with the SH3 domain of Abl. By high stringency pull-down and co-immunoprecipitation assays the apoptin and Bcr-Abl interaction was further confirmed. Subsequently, a set of apoptin and Bcr-Abl deletion mutants were used to map this interaction precisely that mainly occurred between a proline rich domain of apoptin and the SH3 domain of Bcr-Abl. I further investigated the role of apoptin on Bcr-Abl. Apoptin was able to modify the phosphorylation of a series of targets (e.g. CrkL, STAT5, c-Myc) downstream of Bcr-Abl kinase. In addition, I used computational algorhythms for protein modeling to study the 3D structure of apoptin and it’s docking with Bcr-Abl at the molecular level. In controlled studies using the 2-pheny-laminopyrimidine derived specific tyrosine kinase inhibitor Imatinib® I found that apoptin has comparable effects on CML cells, suggesting that the interacting segment of the apoptin molecule acts as an adaptor and negatively regulates the Bcr-Abl kinase by deactivating many cell proliferation and anti-apoptotic pathways in CML cells. Briefly, this work provides important insights towards the development of peptide based tyrosine kinase inhibitors as new anti-cancer agents.