Engineering Protein Kinases for Selective Control of Cellular Pathways

Persistent Link:
http://hdl.handle.net/10150/297791
Title:
Engineering Protein Kinases for Selective Control of Cellular Pathways
Author:
Tye, Blake Wells
Issue Date:
2013
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Protein 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.
Type:
text; Electronic Thesis
Degree Name:
B.S.
Degree Level:
bachelors
Degree Program:
Honors College; Biochemistry
Degree Grantor:
University of Arizona
Advisor:
Ghosh, Indraneel

Full metadata record

DC FieldValue Language
dc.language.isoenen_US
dc.titleEngineering Protein Kinases for Selective Control of Cellular Pathwaysen_US
dc.creatorTye, Blake Wellsen_US
dc.contributor.authorTye, Blake Wellsen_US
dc.date.issued2013-
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.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.en_US
dc.typetexten_US
dc.typeElectronic Thesisen_US
thesis.degree.nameB.S.en_US
thesis.degree.levelbachelorsen_US
thesis.degree.disciplineHonors Collegeen_US
thesis.degree.disciplineBiochemistryen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorGhosh, Indraneel-
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