Includes bibliographical references (p. 104-119).

Thesis (Ph. D.)--Hong Kong University of Science and Technology, 2003. / Includes bibliographical references (leaves 130-138). Also available in electronic version. Access restricted to campus users.

Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). HTLV-1 Tax is the major viral transactivator and transforming protein centrally involved in the proviral transcription, transformation and proliferation of infected T-cells as well as progression of diseases caused by HTLV-1 infection. Salt-inducible kinases (SIKs) are serine/threonine protein kinases belonging to the AMPK-related kinase (AMPK-RK) family. SIK subfamily consists of three isoforms named SIK1, SIK2 and SIK3 respectively. We have previously demonstrated the negative regulatory role of SIK1 in Tax-mediated activation of proviral transcription from long terminal repeats (LTR). In this study, we reported that both SIK2 and SIK3 exhibited a kinase-dependent suppressive effect on Tax-activated LTR transcription. We also found that SIK1, SIK2 and SIK3 act additively to suppress Tax activation of LTR. We further demonstrated that the SIK2- and SIK3-mediated suppression on LTR transcription was achieved through phosphorylation of TORC1, an essential transcriptional coactivator of CREB required for Tax-mediated transcriptional activation of LTR. Our findings revealed a new function of SIK2 and SIK3 in host restriction to HTLV-1 transcription. Pharmaceutical activation of SIKs or upstream kinase such as LKB1 may provide a new strategy for anti-HTLV-1 therapy. / published_or_final_version / Biochemistry / Master / Master of Medical Sciences

Background and objectives: Endothelial dysfunction and cancer are two of the important aspects of obesity-related medical complications, the prevalence of which is reaching an epidemic level worldwide. The protein kinase LKB1 has been shown to play opposite roles in these two metabolic diseases by promoting cellular senescence and inhibiting cell proliferation through regulating a series of its downstream targets. However, the molecular mechanisms wherebyLKB1 itself is regulated by its upstream molecules remains poorly understood. The major objectives of this study are to identify novel upstream regulators of LKB1 and to investigate how these upstream regulators modulate the subcellular localization and physiological functions of LKB1 by post-translational modifications. Key findings: 1. Our proteomic analysis demonstrated that LKB1 was modified by both acetylation and phosphorylation. The acetylation sites of mouseLKB1 include Lys48, Lys64and Lys312. The phosphorylation sites of mouseLKB1 include: Ser31, Thr32,Tyr36, Ser69, Thr71, Ser334and Thr336. 2. In both human embryonic kidney 293 (HEK293)cells and primary porcine aortic endothelial cells (PAECs), the nicotinamide adenine dinucleotide (NAD+)-dependent protein deacetylase SirT1 attenuated the acetylation levels of LKB1,which consequently resulted in enhancedLKB1ubiquitination, thereby leading to the proteasome-mediated degradation of LKB1. 3. In primary PAECs, overexpression of SirT1 protected cells from cell cycle arrest and cellular senescence, whereas overexpression of LKB1 exhibited the opposite effects.SirT1 antagonizedLKB1-induced G1 phase arrest and cellular senescence by promoting the deacetylation and protein degradation of LKB1. 4. The in vitro phosphorylation assay and mass spectrometry analysis demonstrated that LKB1 could be phosphorylated by the Akt kinase at Ser334which was critical for the interaction between LKB1 and 14-3-3. The enhanced association between LKB1 and 14-3-3 subsequently attenuated the interaction between LKB1 and Ste20 related adaptor α(STRADα), which further promoted the nuclear accumulation of LKB1. 5. The cell proliferation and cell cycle distribution analysis of the stably-transfected MDA-MB-231 breast cancer cells demonstrated that overexpression of the LKB1 mutant S334D, which mimicked Ser334 phosphorylation and localized exclusively in the nucleus, completely lost its anti-tumor activities. On the other hand, the S334A mutation enhanced the tumor suppressive functions of LKB1. 6. Nude mice inoculated with the LKB1 S334A stably-transfected MDA-MB-231 cells exhibited delayed tumor onset, decreased tumor growth rate and tumor weight. By contrast, inoculation of nude mice with the MDA-MB-231 cells overexpressing LKB1 S334D mutation showed the opposite effects on these parameters. Conclusions: These results collectively suggest that the deacetylase SirT1 and the protein kinase Aktare the two important upstream regulators of LKB1. SirT1 prevents LKB1-induced cellular senescence and protect endothelial ageing by promoting proteasome-mediated degradation of LKB1. Akt inhibits the tumor-suppressive activity of LKB1 by enhancing the phosphorylation-dependent nuclear translocation. Further investigations on the precise mechanisms whereby SirT1 and Akt regulate LKB1 functions may help to design novel therapeutic strategies for treating obesity-related diseases, such as diabetes, cardiovascular disease and cancer. / published_or_final_version / Medicine / Doctoral / Doctor of Philosophy

abstract / Anatomy / Doctoral / Doctor of Philosophy

abstract / Anaesthesiology / Doctoral / Doctor of Philosophy

No description available.

Phosphorylation/dephosphorylation is likely the most crucial chemical reaction taking place in all living organisms. It is the basis for the regulatory control of many diverse biological events triggered by extracellular effectors. Moreover, it is a ubiquitous element of intracellular signal transduction pathways that regulates a wide range of processes. While protein phosphorylation has been extensively characterized in eukaryotes, far less is known about its emerging counterparts in prokaryotes. This study involved determination of the crystal structures and functional characterization of two protein kinases, YihE and AceK (also a protein phosphatase), and two nucleotide pyrophosphatases, YjjX and YhdE. X-ray crystallographic structure determination combined with bioinformatics analyses, mutageneses and biochemical experiments, both in vitro and in vivo, were utilized for the functional characterization of each protein. YihE was found to be a previously unknown kinase component of a new type of bacterial phospho-relay mechanism, thus adding kinase activity as another response to the Cpx sensing system that functions to maintain cellular homeostasis. AceK, which possesses both kinase and phosphatase activities, modifies isocitrate dehydrogenase (ICDH) to regulate the flux of isocitrate into the glyoxylate cycle. The structures of Acek alone and in complex with its substrate, ICDH, provided us with information to explain the mechanisms underlying its bifunctionality and its molecular switch. Through structural comparison and, particularly, functional characterization, we revealed that YjjX is a novel ITPase/XTPase responsible for the removal of non-canonical nucleotides from the cell during oxidative stress in Escherichia coli. YhdE, identified as a novel dTTPase, was observed to retard cell growth and form a filamentous phenotype when overexpressed in the cell, suggesting that YhdE is involved in the control of cell growth and division by regulating the cell nucleotide pool for DNA synthesis. In summary, this research has made a substantial ii contribution to the investigation of bacterial phosphorylation and dephophorylation systems that respond to various environmental conditions. / Thesis (Ph.D, Biochemistry) -- Queen's University, 2009-05-29 11:41:41.832

Using a novel purification scheme and a new assay for detection of DNA kinase activity, a Polymin P-precipitable DNA kinase has been identified and characterized from calf thymus extracts. The DNA kinase activity was able to phosphorylate RNA as well as single-stranded and double-stranded DNA, therefore it has been termed Polymin P-precipitable polynucleotide kinase (PP-PNK). The enzyme had a neutral to alkaline, broad pH optimum that distinguished it from the previously described mammalian DNA kinases that have an acidic pH optimum. The sedimentation coefficient of the enzyme was 3.4-3.8 S, indicating a molecular weight of about 50 kDa. Estimates for the K$ sb{ rm M}$ for ATP were 52 $ mu$M and for the oligonucleotide substrate 8 $ mu$M. The activity was inhibited by pyrophosphate anions and to a lesser extent by sulfate anions. These results differentiate PP-PNK from other mammalian polynucleotide kinases.

Whole cell extracts from fresh calf thymus glands were subjected to Polymin P fractionation and Q Sepharose chromatography. Three peaks of DNA kinase activity, designated SNQI, SNQII and SNQIII, were found in the supernatant fraction. Studies of SNQI have revealed an estimated molecular mass of 50-90 kDa by Superose 12 chromatography, and activity gel analysis following SDS-PAGE identified an active polypeptide of approximately 55 kDa. This enzyme preparation, purified 10,000-fold, phosphorylated 5$ sp prime$-OH-terminated oligodeoxyribonucleotides and double stranded DNA, yet was inactive on an oligoriboadenosine ladder. SNQI functions optimally at an acidic pH in 10 mM MgCl$ sb2$, but is inhibited by both sulfate and pyrophosphate anions. The estimated K$ sb{ rm M}$ values were 2.3 $ mu$M for the oligonucleotide substrate and 11.8 $ mu$M for ATP. Similar to an enzymatic activity previously isolated from rat liver, SNQI is the first bovine preparation to display both 5$ sp prime$ kinase and 3$ sp prime$ phosphatase activities. / Partial purification and characterization of SNQII revealed similarities to SNQI, such as an acidic pH optimum and the presence of 3$ sp prime$ phosphatase activity. DNA kinase activity was also demonstrated in two mammalian cell lines.