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Author Notes:

Yingxiao Wang, Department of Bioengineering, University of Illinois, Urbana-Champaign, Urbana, IL 61801, USA;

Cheng Zhu, Wallace H. Coulter Department of Biomedical Engineering, Georgia Institute of Technology and Emory University, Atlanta, GA 30332, USA.

X. Xiang and J. Sun contributed equally; Associate Editor John Shyy and Yingxiao Wang oversaw the review of this article.

Subjects:

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Technology
  • Cell & Tissue Engineering
  • Biophysics
  • Cell Biology
  • Engineering, Biomedical
  • Engineering
  • SYK
  • Immunoreceptor
  • FRET
  • Biosensor
  • Signaling
  • PROTEIN-TYROSINE KINASE
  • CONTACT-AREA FRAP
  • ANTIGEN RECEPTOR
  • ACTIVATION
  • ZAP-70
  • ROLES
  • SRC
  • IMMUNORECEPTOR
  • PHAGOCYTOSIS
  • ASSOCIATION

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

Tools:

Journal Title:

Cellular and Molecular Bioengineering

Volume:

Volume 4, Number 4

Publisher:

, Pages 670-677

Type of Work:

Article | Post-print: After Peer Review

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.

Copyright information:

© 2011 Biomedical Engineering Society.

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