|Institution:||University of British Columbia|
|Full text PDF:||http://hdl.handle.net/2429/52854|
The eukaryotic protein kinases (ePKs) constitute one of the largest families of enzymes encoded by eukaryotic genomes. They regulate all cellular processes by transducing inter- and intracellular signals via the phosphorylation of specific protein substrates. The primary sequences of ePK catalytic domains are highly conserved, indicating a common ancestry. They all share a conserved catalytic core for their phosphotransferase function and often a common activation mechanism by phosphorylation of a variable segment known as the activation T-loop. Starting from a manually aligned and annotated map of 492 typical human protein kinase domains, I first explored the origin of ePKs using a modified BLAST method in various species. Comparisons of primary, secondary and tertiary structures supported the hypothesis that protein kinases and choline kinases evolved from an ancient aminoacyl-tRNA ligase. Secondly, I studied the functional roles of two extremely conserved phosphosites that exist ubiquitously in the activation T-loops of most protein-serine/threonine kinases. The extensively examined extracellular signal-regulated kinases (ERKs) 1/2 from the mitogen-activated protein kinase family were used as a model in this study. I discovered that both Thr-207 and Tyr-210 from human ERK1 were essential for regulation of its phosphotransferase activity. Autophosphorylation of Thr-207 played an inhibitory role to control ERK1 activity after the initial phosphorylation and activation by MEK1. This may serve as a general mechanism for kinase autoinhibition. I also examined the substrate specificities of more than 200 human protein kinases with peptide microarrays populated with semi-optimal substrate sequences. The resultant data were used to expand the training datasets for development of next generation protein kinase substrate predictive algorithms. Additionally, I describe a novel method to produce a more unbiased polyclonal generic phosphotyrosine antibody than the monoclonal antibodies that are commonly used to enrich and track tyrosine-phosphorylated proteins. The tools and knowledge resulting from this research should enable improvements in the characterization of protein kinases and establishing their linkages to a variety of human diseases for the development of better diagnostic tests and therapeutic drugs. This work also provides basic insights into the evolution of life and the specific architecture of protein kinase-based signalling systems.