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Regulatory Mechanisms of a Bacterial Multi-Kinase Network

Kowallis, Kimberly (2020) Regulatory Mechanisms of a Bacterial Multi-Kinase Network. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Cells sense and respond to their environment through signaling pathways which often require processing several signals prior to implementing a biological response. Bacterial signaling pathways are responsible for processes such as virulence, biofilm formation, survival, and symbiosis that are of research interest for medical, environmental, and industrial advancements. Emerging discoveries suggest that the systems that control these responses are more complex and intertwined than the previously understood two-component systems. Proteins such as scaffolds and pseudokinases regulate the localization, activity, and timing of the phosphotransfer reactions that dictate cellular decisions. This dissertation describes regulatory mechanisms of a multi-kinase network that controls asymmetric division in the model bacterium C. crescentus.
It has been proposed that the novel pseudokinase DivL reverses signal flow by exploiting conserved kinase conformational changes and protein-protein interactions. Chapter 2 describes the development and characterization of a series of DivL-based modulators to synthetically stimulate reverse signaling of the network in vivo. I propose that synthetic stimulation and sensor disruption provide strategies to define signaling circuit organization principles for the rational design and validation of synthetic pathways.
In Chapter 3, I further dissect the roles of each DivL domain on subcellular localization and downstream activity. While not catalytically active, pseudokinases have been repurposed to serve functions including complex signal recognition, integration, competition, and intermolecular allostery. I provide a refined model detailing how DivL plays each of these parts within its broader network.
The work in Chapter 3 also revealed multiple scaffolding interactions that orchestrate the multi-kinase network in time and space. In Chapter 4 I identify factors that lead to the accumulation of two biochemically distinct signaling hubs at opposite cell poles to provide the foundation for asymmetry. I also provide evidence that a scaffold not only recruits a key signaling protein to the correct location but mediates its switch between kinase and phosphatase activities that drives the cell cycle.
In each chapter, I discuss questions that remain and suggest future directions for study. Overall, this dissertation contributes strategies that can be used to interrogate other relevant multi-kinase networks in bacteria.


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Item Type: University of Pittsburgh ETD
Status: Unpublished
CreatorsEmailPitt UsernameORCID
Kowallis, Kimberlykas279@pitt.edukas279
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairChilders, W.
Committee MemberWeber, Stephen
Committee MemberIslam,
Committee MemberCooper, Vaughn
Date: 16 September 2020
Date Type: Publication
Defense Date: 9 July 2020
Approval Date: 16 September 2020
Submission Date: 12 May 2020
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Number of Pages: 210
Institution: University of Pittsburgh
Schools and Programs: Dietrich School of Arts and Sciences > Chemistry
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: bacterial signaling Caulobacter crescentus histidine kinase protein engineering scaffold
Date Deposited: 16 Sep 2020 14:18
Last Modified: 16 Sep 2020 14:18


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