A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.
|A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells.
|Year of Publication
|Mukherjee A, Singh R, Udayan S, Biswas S, Reddy PPurushotha, Manmadhan S, George G, Kumar S, Das R, Rao BM, Gulyani A
|2020 Feb 04
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 () 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. 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.
|PubMed Central ID
|JRF-SRF Fellowship / / University Grants Commission /
DBT-RA Program in Biotechnology and Life Sciences / / Department of Biotechnology, Ministry of Science and Technology /
Grant BT/PR8071/MED/32/290/2013 / / Department of Biotechnology, Ministry of Science and Technology /
BT/IN/BMBF/03/AG/2015-16 / / Department of Biotechnology, Ministry of Science and Technology /
BT/IN/Denmark/07/RSM/2015-2016 / / Department of Biotechnology, Ministry of Science and Technology /