Extended Data Fig. 2: Engineering and characterization of opto-EGFR and opto-HER2 prototypes.
From: A general approach for engineering RTKs optically controlled with far-red light
(a) Left: Ligand binding causes RTK autophosphorylation, interaction with Grb2 and SOS, and results in activation of ERK1/2 pathway consisting of RAS, RAF, MEK, and ERK1/2 kinases. Consequently, ERK1/2 activation leads to immediate early gene (IEG) expression driven by transcription factor Elk-1. Right: Likewise, activation of chimeric opto-RTK with far-red light leads to activation of ERK1/2 pathway and induction of Elk-1 dependent IEG expression. (b) Scheme of luciferase reporter assay. Top: ERK1/2 is inactive, and Elk-1 fused to Gal4DBD is monomeric and inactive. Bottom: ERK1/2 is active, and activated ERK1/2 phosphorylates Elk-1. Phosphorylated Elk-1-Gal4DBD fusion dimerizes, binds to 5x UAS sequence, and drives Firefly luciferase (FLuc) reporter expression. (c, d) Light-induced activation of Elk-1-dependent Firefly luciferase reporter expression by opto-EGFR (c) and opto-HER2 (d) prototypes in PC6-3 cells. Renilla luciferase (RLuc) was co-transfected as a normalization control. In the darkness, Firefly luciferase expression is suppressed. 660 nm light activates eDrRTKs and upregulates Firefly luciferase expression. Expression of Firefly and Renilla luciferases was analyzed after 24 h of illumination. Relative luciferase expression activity (RLA) is shown as a ratio of the Firefly luciferase expression relative bioluminescence units to the Renilla luciferase expression relative bioluminescence units (FLuc/RLuc). 25 µM BV was added to the culture medium in all experiments. Data are presented as mean values, error bars represent standard deviation (s.d.), (n≥3, transfection experiments).
