Dissection of spatiotemporal intracellular signaling using engineered chemical and genetic tools
Dieter, Emily M
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Cellular signaling proteins are responsible for transmitting intracellular signals and enacting the appropriate response to received stimuli. These messages are propagated through networks that rely on allosteric regulation, post-translational modifications, or other mechanisms for modulating signaling protein activity. Scientists seeking to engineer systems that mimic endogenous pathways must be able to control and precisely tune the spatiotemporal activity of the proteins involved in these complex networks. This thesis describes the development of chemical genetic tools that specifically control individual signaling nodes in a cell using three distinct approaches. The first approach utilizes conformation-selective inhibitors that modulate the allosteric regulation of Src Family Kinases (SFKs). SFKs are multi-domain kinases that transmit cellular signals through their catalytic and non-catalytic activities. Although dysregulation of kinases is often implicated in human diseases, studying individual kinases is difficult due to their conserved catalytic domain. By combining genetics with small molecule probe development, we created potent and specific kinase inhibitors that are capable of allosterically controlling the intramolecular regulation of an individual kinase. We applied this strategy to the SFKs and studied the phenotypic effects that resulted from conformation-selective inhibition. The second approach employed a small molecule-controlled, genetically-encoded rheostatic switch for studying signaling mediated by localized pools of RAS. In the final approach, a second-generation chemically-disrupted proximity (CDP) system was developed. This optimized CDP system can be incorporated into numerous chemical genetic systems that rely on intramolecular and intermolecular regulation. Together, these three approaches represent a broad platform for interrogating and engineering diverse cellular processes.
- Chemistry