SHAAYA-DISSERTATION-2021.pdf (5.97 MB)

Engineered Allosteric Regulation of Protein Kinases by Light

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posted on 01.05.2021, 00:00 by Mark Shaaya
Key biological processes in mammalian cells are regulated by protein kinases 1. A single kinase can elicit distinct effects depending on its temporal activity dynamics as well as its subcellular localization 2-5. This complexity of kinase signaling necessitates the development of tools enabling spatial and temporal control of their activity, which otherwise cannot be achieved with pharmacological and genetic methods. Here, I describe the development of an optogenetic tool, Light-Regulated (LightR) switch domain, that we engineered and utilized to achieve allosteric control of kinase activity. Using tyrosine kinase Src as a model, we developed LightR-Src. We demonstrate an efficient regulation of the kinase with fast on-kinetics and we define signaling responses within seconds and minutes after LightR-Src activation. We show that the off-kinetics of LightR switch can be tuned by modulating its photoconversion cycle. Therefore, a fast cycling LightR-Src variant enables stimulation of transient pulses of Src activation in living cells. Local activation of LightR-Src in a cell induces local protrusions and cell polarization towards the light. Interestingly, continuous local Src activity stimulates recurring waves of protrusions mediated by Rho-associated protein kinase and myosin light-chain kinase. We demonstrate the broad applicability of this tool by achieving light-mediated regulation of Abl and bRaf kinases, as well as a different type of enzyme, Cre recombinase. Overall, we developed an optogenetic method that allows us to mimic dynamic protein activity at subcellular levels.

History

Advisor

Karginov, Andrei

Chair

Karginov, Andrei

Department

Pharmacology

Degree Grantor

University of Illinois at Chicago

Degree Level

Doctoral

Degree name

PhD, Doctor of Philosophy

Committee Member

Natarajan, Viswanathan Komarova, Julia Minshall, Richard Kazlauskas, Andrius

Submitted date

May 2021

Thesis type

application/pdf

Language

en

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