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Abstract
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. We validated Dr-Trk ability to reversibly light-control several RTK pathways, calcium level, and demonstrated that their activation triggers canonical Trk signaling. Dr-TrkA induced apoptosis in neuroblastoma and glioblastoma, but not in other cell types. Absence of spectral crosstalk between Dr-Trks and blue-light-activatable LOV-domain-based translocation system enabled intracellular targeting of Dr-TrkA independently of its activation, additionally modulating Trk signaling. Dr-Trks have several superior characteristics that make them the opto-kinases of choice for regulation of RTK signaling: high activation range, fast and reversible photoswitching, and multiplexing with visible-light-controllable optogenetic tools.
Highlights
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions
The first optically regulated RTKs were developed by Chang et al.[3] by fusing catalytic kinase domains of tropomyosin receptor kinases (Trks) to the light-responsive photolyase homology region of cryptochrome 2 (CRY2)[3]
These findings suggest that RTK activation could be seen as merely a ligand-induced conformational rearrangement of the pre-existing dimers
Summary
Optical control over the activity of receptor tyrosine kinases (RTKs) provides an efficient way to reversibly and non-invasively map their functions. We combined catalytic domains of Trk (tropomyosin receptor kinase) family of RTKs, naturally activated by neurotrophins, with photosensory core module of DrBphP bacterial phytochrome to develop opto-kinases, termed Dr-TrkA and Dr-TrkB, reversibly switchable on and off with near-infrared and far-red light. Chemical inhibitors helped to dissect RTK signaling; they stalled on the specificity limitation: even most specific of them simultaneously inhibit several RTKs of the same family, making it hard to discern their biological effects Other chemical approaches, such as bump-and-hole strategy[1] and chemical dimerizers, played an essential role in RTK studies too, yet have a limited ability to control cell signaling with sufficient spatiotemporal precision. We hypothesized that the conformational changes accompanying ligand binding could be induced with the help of a light-sensitive dimeric protein fused to the cytoplasmic domains of an RTK, instead of its extracellular domains. Similar to other canonical bacterial phytochromes[7,8,9,10], DrBphP forms a head-to-head parallel dimer and consists of an N-terminal photosensory core module (PCM) and C-terminal histidine-kinase (HK) domain, connected by a long α-helix
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