Fig. 1

Design and initial screening of DrBphP-PCM kinase fusions. a Activation of receptor tyrosine kinases (RTKs) by dimerization upon binding of a growth factor ligand. b Schematically depicted structures of the full-length TrkB, DrBphP, and developed for initial screening DrBphP-PCM-cyto-Trk fusion constructs. c Scheme of luciferase assay for kinase activity. The system consists of the reporter plasmid, pFR-Luc, where firefly luciferase expression is controlled with the synthetic promoter, containing 5× tandem repeats of the yeast UAS GAL4 binding sites, and the transactivator plasmid pFA-Elk-1. In the transactivator plasmid, the activation domain of the Elk-1 is fused with the yeast GAL4 DNA binding domain (DBD). Under 780 nm light, DrBphP-PCM-cyto-Trk is active, which results in the activation of the MAPK/ERK pathway. The phosphorylated Elk-1-GAL4-DBD fusion dimerizes, binds to 5× UAS, and activates transcription of firefly luciferase. Under 660 nm light, DrBphP-PCM-cyto-Trk is inactive, MAPK/ERK pathway (mitogen-activated protein kinase/extracellular signal-regulated kinase) is inhibited, and luciferase expression is switched OFF. d Luciferase assay of initial DrBphP-PCM-cyto-Trk constructs in PC6-3 cells. PC6-3 cells were co-transfected with the pCMVd2-DrBphP-PCM-cyto-Trk, pFR-Luc, and pFA-Elk-1 plasmid mixture (1:100:5), and 6 h after transfection, culture medium was replaced with serum-starving one. Cells were grown for additional 30 h under 780 nm or 660 nm light (both 0.5 mW cm⁻²), lysed, and analyzed for luciferase activity. Error bars represent s.d., n = 3 experiments