A Fyn biosensor reveals pulsatile, spatially localized kinase activity and signaling crosstalk in live mammalian cells

Elife. 2020 Feb 4;9:e50571. doi: 10.7554/eLife.50571.

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.

Keywords: E. coli; FRET; Fyn kinase; S. cerevisiae; biochemistry; biosensor; cell biology; chemical biology; compartmentalized; human; mouse; pulsatile; src kinases; virus.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques*
  • Cell Line
  • Cell Physiological Phenomena / genetics
  • Fluorescence Resonance Energy Transfer
  • Fluorescent Dyes / metabolism
  • HEK293 Cells
  • Humans
  • Magnetic Resonance Spectroscopy
  • Mice
  • Phosphorylation / genetics
  • Proto-Oncogene Proteins c-fyn* / chemistry
  • Proto-Oncogene Proteins c-fyn* / genetics
  • Proto-Oncogene Proteins c-fyn* / metabolism
  • Signal Transduction / genetics*
  • Yeasts / genetics

Substances

  • Fluorescent Dyes
  • Proto-Oncogene Proteins c-fyn

Associated data

  • PDB/5ZAU