Time-resolved FRET reports FGFR1 dimerization and formation of a complex with its effector PLCγ1

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.

Metformin influences drug sensitivity in pancreatic cancer cells

“…As KRAS has been frequently observed to be mutated in pancreatic cancer, attempts have been developed to generate effective RAS inhibitors as well as additional signaling molecules in this pathway. Attempts to inhibit the RAS/PI3K/PTEN/Akt/mTORC1/GSK-3 and other signaling pathways have been a central focus in many basic science laboratories and pharmaceutical companies for over three decades due to the involvement oncogenes in this pathway in human cancers (Yang et al, 2013;Fitzgerald et al, 2015;McCubrey et al, 2015;Anderson et al, 2016;Banfic et al, 2016;Cocco et al, 2016;Erneux et al, 2016;Falasca and Ferro, 2016;Fields et al, 2016;Geck and Toker, 2016;Maczis et al, 2016;Perdios et al, 2016;Pyne et al, 2016;Scarlata et al, 2016;Scoumanne et al, 2016;Tanaka et al, 2016;Ando et al, 2017;Carman and Han, 2017;Fujisawa, 2017;Geffken and Spiegel, 2017;Guo et al, 2017;Jang et al, 2017;Leonard and Johnson, 2017;Liu et al, 2017;Matsuzawa, 2017;McCubrey et al, 2017aMcCubrey et al, , 2017bMcCubrey et al, , 2017cMcCubrey et al, , 2017dObsil and Obsilova, 2017;Okazaki, 2017;Pyne et al, 2017;Ramos et al, 2017;Ratti et al, 2017;Rebello et al, 2017;Roth and Frohman, 2017;Rusnak and Fu, 2017;…”

Recent developments of genetically encoded optical sensors for cell biology

“…For monitoring protein–protein interactions of α‐synuclein, monomers of the protein were tagged with FlAsH (as a donor) and ReAsH (as acceptor), respectively (Roberti et al., ). Similarly, labelling the Src homology 2 domain of phospholipase Cγ with a FlAsH or ReAsH tag and fibroblast growth factor receptor with mTurquoise as a FRET donor permitted for time‐resolved FRET‐measurement of their interaction (Perdios et al., ).…”