Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1.
File(s)
Published version
Author(s)
Type
Journal Article
Abstract
In vitro and in vivo imaging of protein tyrosine kinase activity requires minimally invasive, molecularly precise optical probes to provide spatiotemporal mechanistic information of dimerization and complex formation with downstream effectors. We present here a construct with genetically encoded, site-specifically incorporated, bioorthogonal reporter that can be selectively labelled with exogenous fluorogenic probes to monitor the structure and function of fibroblast growth factor receptor (FGFR). GyrB.FGFR1KD.TC contains a coumermycin-induced artificial dimerizer (GyrB), FGFR1 kinase domain (KD) and a tetracysteine (TC) motif that enables fluorescent labelling with biarsenical dyes FlAsH-EDT2 and ReAsH-EDT2. We generated bimolecular system for time-resolved FRET (TR-FRET) studies, which pairs FlAsH-tagged GyrB.FGFR1KD.TC and N-terminal Src homology 2 (nSH2) domain of phospholipase Cγ (PLCγ), a downstream effector of FGFR1, fused to mTurquoise fluorescent protein (mTFP). We demonstrated phosphorylation-dependent TR-FRET readout of complex formation between mTFP.nSH2 and GyrB.FGFR1KD.TC. By further application of TR-FRET, we also demonstrated formation of the GyrB.FGFR1KD.TC homodimer by coumermycin-induced dimerization. Herein, we present a spectroscopic FRET approach to facilitate and propagate studies that would provide structural and functional insights for FGFR and other tyrosine kinases.
Date Issued
2016-01
Date Acceptance
2015-09-22
Citation
Advances in Biological Regulation, 2016, 60, pp.6-13
ISSN
2212-4934
Publisher
Elsevier
Start Page
6
End Page
13
Journal / Book Title
Advances in Biological Regulation
Volume
60
Copyright Statement
© 2015 The Authors. Published by Elsevier Ltd. This is an open access article under the CC
BY license (http://creativecommons.org/licenses/by/4.0/).
BY license (http://creativecommons.org/licenses/by/4.0/).
License URL
Sponsor
Biotechnology and Biological Sciences Research Council (BBSRC)
Identifier
PII: S2212-4926(15)30015-4
Grant Number
BB/E000495/1
Subjects
Fibroblast growth factor receptor
Phospholipase Cγ
Time-resolved FRET
Publication Status
Published
Date Publish Online
2015-09-26