Engineering Protein Kinases for Selective Control of Cellular Pathways
PublisherThe University of Arizona.
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AbstractProtein kinases form a large family of enzymes that perform a simple chemical modification: addition of a phosphate group to a residue on a target protein. It turns out that this modification plays an integral part in the function of cognate proteins, making kinases key players in the complex orchestra of cellular pathways. Much work has been put into studying and targeting kinases, but to date the picture of how these highly similar proteins achieve such elegant specificity in a properly functioning cell remains incomplete. Here we report the development of an assay for the binding of small molecules by tyrosine kinases based off of our previously reported split luciferase assay. We show the process of creating the new chemical inducer of dimerization from the known inhibitor dasatinib and optimization for use in the tyrosine kinase group, and show that it can effectively be used as a competitive inhibition assay in a robust and rapid fashion against a panel of potential inhibitors. We then explore the structural and sequence characteristics of the ligand binding pocket of kinases in a kinome-wide manner. These results lead us to the apparent feature that the ligand and substrate binding clefts are confined to the N- and C-terminal lobes, respectively. We attempt the construction of chimeric kinases based on the PKA C-lobe and the N-lobe of various tyrosine kinases in hopes of making a chimera with native PKA substrate specificity, but active towards a tyrosine residue. We found that the kinases were inactive towards our predicted substrates when translated and assayed in vitro. Finally, we explore possible causes of the inactivity and propose our current approach to engineering kinases with our desired specificity characteristics in hopes of creating a tool for systematically controlling and dissecting cellular pathways involving kinases.
Degree ProgramHonors College