title: A FRET-Based Biosensor for Imaging SYK Activities in Living Cells.
abstract: Spleen tyrosine kinase (SYK) is crucial to cellular functions mediated by immunoreceptors and integrins. We have developed and characterized a new genetically-encoded Förster resonance energy transfer (FRET)-based biosensor for studying the dynamics of SYK activities in living cells at a subcellular level. It contains an N-terminal ECFP, SH2 domain, a peptide derived from a SYK substrate VAV2, and a C-terminal YPet. Upon the specific phosphorylation by SYKin vitro, the biosensor substrate peptide bound to the intramolecular SH2 domain to reduce the FRET efficiency. Transfection of the biosensor did not affect activation of the endogenous SYK in host cells. Phosphorylation of the biosensor followed the same kinetics as the endogenous VAV2. Using FRET imaging and ratiometric analysis with this SYK biosensor, we visualized and quantified the realtime activation of SYK in K562 cells upon IgG Fc engagement of Fcc receptor IIA and in mouse embryonic fibroblasts upon stimulation by the platelet derived growth factor. These results demonstrate our biosensor as a powerful tool for studying cellular signaling that involves SYK.
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA ; School of Life Sciences, SUN YAT-SEN University, Guangzhou 510275, China.
authors.forename: Xue
authors.initials: X
authors.lastname: Xiang
authors.affiliation: Department of Bioengineering, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA ; Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Jie
authors.initials: J
authors.lastname: Sun
authors.affiliation: School of Bioscience & Bioengineering, South China University of Technology, Guangzhou 510006, China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Wu
authors.affiliation: Centre d'Immunologie de Marseille-Luminy, INSERM-CNRS-Université de la Méditerranée, Case 906, 13288 Marseille Cedex 09, France.
authors.forename: Hai-Tao
authors.initials: HT
authors.lastname: He
authors.affiliation: Department of Bioengineering, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA ; Beckman Institute for Advanced Science and Technology, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Yingxiao
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.
authors.forename: Cheng
authors.initials: C
authors.lastname: Zhu
keywords: Biosensor; FRET; Immunoreceptor; SYK; Signaling