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Probing the phosphoproteome with direct activity sensors: Applications in liver signaling and disease
Cellular signal transduction is an essential function of all life. All organisms rely on a multitude of signaling strategies in order to respond to their environment, communicate with other cells, grow, move or divide and maintain homeostasis. Dysregulation of signaling pathways has been implicated in most disorders and diseases. This dysregulation has been a major focus of study in modern biochemistry and molecular biology since the middle of the last century. Protein phosphorylation is one of the most widely utilized signal transduction strategies across all kingdoms of life. Protein kinases and protein phosphatases work in harmony to maintain the integrity of the phosphoproteiome. Protein kinases catalyze the transfer of a phosphate group to specific amino acid residues on protein substrates. Conversely, protein phosphatases catalyze the removal of phosphate groups from amino acid residues and thus act in coordinated opposition to protein kinases. In order to study this signal transduction mechanism a number of creative and useful technologies have been developed over the past half century. These technologies range from the earliest broadly applicable radiometric assays to more current and elegant genetically encodable FRET-based assays. While there are many examples of technologies that are useful and, indeed, highly utilized there remain improvements to be made in terms of selectivity, costs, dynamic range, real-time measurements and applicability to protein kinases as well as protein phosphatases. Herein the phosphorylation sensitive fluorophore known as Sox is utilized to develop a fluorescence-based, real-time activity sensor for Focal Adhesion Kinase (FAK). The Sox fluorophore is then applied for the first time in order to develop probes for both protein tyrosine phosphatases (PTPs) as well as protein serine/threonine phosphatase (PSPs). These new phosphatase probes are coupled with the existing battery of Sox based kinase sensors in order to investigate signaling dynamics in hepatocytes as well as perturbations in disease-related phosphorylation signaling in liver tissue. The new sensors described in this work expand the chemical biology toolbox and should be useful in discovery based applications involving the dysregulation of protein phosphorylation signaling.
Beck, Jon Robert, "Probing the phosphoproteome with direct activity sensors: Applications in liver signaling and disease" (2016). ETD collection for University of Nebraska - Lincoln. AAI10247496.