title: Detection of focal adhesion kinase activation at membrane microdomains by fluorescence resonance energy transfer.
abstract: Proper subcellular localization of focal adhesion kinase (FAK) is crucial for many cellular processes. It remains, however, unclear how FAK activity is regulated at subcellular compartments. To visualize the FAK activity at different membrane microdomains, we develop a fluorescence resonance energy transfer (FRET)-based FAK biosensor, and target it into or outside of detergent-resistant membrane (DRM) regions at the plasma membrane. Here we show that, on cell adhesion to extracellular matrix proteins or stimulation by platelet-derived growth factor (PDGF), the FRET responses of DRM-targeting FAK biosensor are stronger than that at non-DRM regions, suggesting that FAK activation can occur at DRM microdomains. Further experiments reveal that the PDGF-induced FAK activation is mediated and maintained by Src activity, whereas FAK activation on cell adhesion is independent of, and in fact essential for the Src activation. Therefore, FAK is activated at membrane microdomains with distinct activation mechanisms in response to different physiological stimuli.
authors.affiliation: Neuroscience Program, University of Illinois, Urbana-Champaign, Urbana, Illinois 61801, USA.
authors.forename: Jihye
authors.initials: J
authors.lastname: Seong
authors.forename: Mingxing
authors.initials: M
authors.lastname: Ouyang
authors.forename: Taejin
authors.initials: T
authors.lastname: Kim
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.forename: Po-Chao
authors.initials: PC
authors.lastname: Wen
authors.forename: Shaoying
authors.initials: S
authors.lastname: Lu
authors.forename: Yue
authors.initials: Y
authors.lastname: Zhuo
authors.forename: Nicholas M
authors.initials: NM
authors.lastname: Llewellyn
authors.forename: David D
authors.initials: DD
authors.lastname: Schlaepfer
authors.forename: Jun-Lin
authors.initials: JL
authors.lastname: Guan
authors.forename: Shu
authors.initials: S
authors.lastname: Chien
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
mesh_terms: D000818:Animals; D002448:Cell Adhesion; D002462:Cell Membrane / Q000737:chemistry* / Q000201:enzymology* / Q000235:genetics; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D051416:Focal Adhesion Protein-Tyrosine Kinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D010982:Platelet-Derived Growth Factor / Q000378:metabolism; D017434:Protein Structure, Tertiary
chemical_list: D010982:Platelet-Derived Growth Factor; D051416:Focal Adhesion Protein-Tyrosine Kinases