Engineered Allosteric Regulation of Protein Kinases by Light
thesisposted 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.