Abstract
Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; thus, TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. However, mechanistic insights into TrKA signaling are lacking due to lack of site-dependent phosphorylation control. Here we engineer two light-sensitive tyrosine analogues, namely p-azido-L-phenylalanine (AzF) and the caged-tyrosine (ONB), through amber codon suppression to optically manipulate the phosphorylation state of individual intracellular tyrosines in TrkA. We identify TrkA-AzF and ONB mutants, which can activate the ERK pathway in the absence of NGF ligand binding through light control. Our results not only reveal how TrkA site-dependent phosphorylation controls the defined signaling process, but also extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy.
Highlights
Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer
Using the Y490 site as a reference, we studied the incorporation of AzF and ONB into TrkA at three different sites in the tyrosine kinase domain, Y670, Y674, and Y675, respectively (Fig. 3a)
The common concept of TrkA signaling activation is that the binding of ligand neuronal growth factor (NGF) from the extracellular domain promotes homo-oligomerization of TrkA on the cell membrane, which subsequently activates downstream signaling pathways
Summary
Tyrosine kinase A (TrkA) is a membrane receptor which, upon ligand binding, activates several pathways including MAPK/ERK signaling, implicated in a spectrum of human pathologies; TrkA is an emerging therapeutic target in treatment of neuronal diseases and cancer. Our results reveal how TrkA site-dependent phosphorylation controls the defined signaling process, and extend the genetic code expansion technology to enable regulation of receptor-type kinase activation by optical control at the precision of a single phosphorylation site. It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening in a site-directed phosphorylation pathway for targeted therapy. A specific pathway after phosphorylation for each site remains unaddressed
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