Cellular and Molecular Bioengineering

, Volume 4, Issue 4, pp 670–677

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

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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 real-time activation of SYK in K562 cells upon IgG Fc engagement of Fcγ 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.

Keywords

SYK Immunoreceptor FRET Biosensor Signaling 

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Copyright information

© Biomedical Engineering Society 2011

Authors and Affiliations

  1. 1.Wallace H. Coulter Department of Biomedical EngineeringGeorgia Institute of Technology and Emory UniversityAtlantaUSA
  2. 2.School of Life SciencesSUN YAT-SEN UniversityGuangzhouChina
  3. 3.Department of BioengineeringUniversity of Illinois, Urbana-ChampaignUrbanaUSA
  4. 4.Beckman Institute for Advanced Science and TechnologyUniversity of Illinois, Urbana-ChampaignUrbanaUSA
  5. 5.School of Bioscience & BioengineeringSouth China University of TechnologyGuangzhouChina
  6. 6.Centre d’Immunologie de Marseille-LuminyINSERM-CNRS-Université de la MéditerranéeMarseille Cedex 09France

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