A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

Mukherjee, A.; Singh, R.; Udayan, S.; Biswas, S.; Pothula, P.; Manmadhan, S.; George, G.; Dilip Kumar, S.; Das, R.; Rao, B. M.; Gulyani, A.

doi:10.1101/726133

bioRxiv · 10.1101/726133

A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk

Mukherjee, A.·Singh, R.·Udayan, S.·Biswas, S.·Pothula, P.·Manmadhan, S.·George, G.·Dilip Kumar, S.·Das, R.·Rao, B. M.·Gulyani, A.

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Abstract

Cell behavior is controlled through spatio-temporally localized protein activity. Despite unique and often contradictory roles played by Src-family-kinases (SFKs) in regulating cell physiology, activity patterns of individual SFKs have remained elusive. Here, we report a biosensor for specifically visualizing active conformation of SFK-Fyn in live cells. We deployed combinatorial library screening to isolate a binding-protein (F29) targeting activated Fyn. Nuclear-magnetic-resonance (NMR) analysis provides the structural basis of F29 specificity for Fyn over homologous SFKs. Using F29, we engineered a sensitive, minimally-perturbing fluorescence-resonance-energy-transfer (FRET) biosensor (FynSensor) that reveals cellular Fyn activity to be spatially localized, pulsatile and sensitive to adhesion/integrin signaling. Strikingly, growth factor stimulation further enhanced Fyn activity in pre-activated intracellular zones. However, inhibition of focal-adhesion-kinase activity not only attenuates Fyn activity, but abolishes growth-factor modulation. FynSensor imaging uncovers spatially-organized, sensitized signaling clusters, direct crosstalk between integrin and growth-factor-signaling, and clarifies how compartmentalized Src-kinase activity may drive cell fate.

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Mukherjee, A., Singh, R., Udayan, S., Biswas, S., Pothula, P., Manmadhan, S., George, G., Dilip Kumar, S., Das, R., Rao, B. M., Gulyani, A.. 2019-08-05. A Fyn - specific biosensor reveals localized, pulsatile kinase activity and spatially regulated signaling crosstalk. https://doi.org/10.1101/726133

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