A FRET-Based Biosensor for Imaging SYK Activities in Living Cells

• Type of Material: Article
• Authors: Xue Xiang, Georgia Institute of Technology; Jie Sun, University of Illinois; Jianhua Wu, South China University of Technology; Hai-Tao He, INSERM-CNRS-Université de la Méditerranée; Yingxiao Wang, University of Illinois; Cheng Zhu, Emory University
• Language: English
• Date: 2011-12-01
• Publisher: Springer (part of Springer Nature): Springer Open Choice Hybrid Journals
• Publication Version: Author Accepted Manuscript (After Peer Review)
• Copyright Statement: © 2011 Biomedical Engineering Society.
• Final Published Version (URL): http://dx.doi.org/10.1007/s12195-011-0211-x
• Title of Journal or Parent Work: Cellular and Molecular Bioengineering
• ISSN: 1865-5025
• Volume: 4
• Issue: 4
• Start Page: 670
• End Page: 677
• Keywords: Immunoreceptor; IMMUNORECEPTOR; SYK; ROLES; Engineering; ACTIVATION; Biosensor; SRC; Cell Biology; Science & Technology; Life Sciences & Biomedicine; PHAGOCYTOSIS; ANTIGEN RECEPTOR; Engineering, Biomedical; FRET; PROTEIN-TYROSINE KINASE; CONTACT-AREA FRAP; Biophysics; ASSOCIATION; ZAP-70; Signaling; Cell & Tissue Engineering; Technology
• Research Categories: Engineering, Biomedical; Biology, Cell

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 SYK in 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.