Photosensitive tyrosine analogues unravel site-dependent phosphorylation in TrkA initiated MAPK/ERK signaling

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.

Light-controlled phosphorylation in the TrkA-Y785 site by photosensitive UAAs activates the MAPK/ERK signaling pathway

“…treatment, AzF typically generates a nitrogen radical, which may subsequently be reduced to an amine or form a covalent bond with an adjacent atom within a 3-4 Å distance. Unlike the previous results in the kinase domain region 14 , the Y785 site did not show an apparent photo-controllable activation level of p-ERK after the introduction of AzF. However, in the absence of NGF stimulation, there was no significant difference in p-ERK expression levels with or without light in two mutants (figure 3b).…”

“…To sum up, we supplemented light control results of the TrkA-Y785 site based on our previous research and further expanded the application of genetic code expansion technology in kinase receptors; Combined with the previous research results 14 , we propose that these five phosphorylation sites may have different contributions to the activation of the MAPK pathway, and site-dependent phosphorylation in TrkA may have polymorphic control over the signaling process. Through precise optical control [28][29][30][31][32] , the activity of kinase receptors can be regulated at a single phosphorylation site to achieve the effect of site-directed light-controlled phosphorylation.…”

“…This is reasonable because Y785 has been reported in previous studies to activate ERK in a PLCγ-PKC signaling-dependent manner, and cell differentiation was significantly reduced after simultaneous inhibition of both Y490 and Y785 phosphorylation, suggesting that Y785 makes important contributions in regulating the ERK pathway. Our light-controlled approach offers advantages in explaining the phenotypic roles of the Y785 site and other single tyrosine sites 14 . We expect that through separation and combination light-control studies of these critical sites, it will provide a powerful approach to resolve the polymorphism of tyrosine receptor phosphorylation and the diversity of its functions.…”

“…Compared to HEK293T cells, which were able to efficiently transfect and successfully express TrkA-ONB, TrkA-AzF mutants, the successful application of this technology to neurons required extensive experimental optimization. We have tested light-sensitive TrkA mutants (Y490, Y670, Y674, Y675) in SH-SY5Y cells in our previous study to establish a light-controlled method for neurite outgrowth 14 . Therefore, using the same strategy, we introduced AzF and ONB at the Y785 site and carried out TrkA-AzF and TrkA-ONB fluorescence expression detection in SH-SY5Y and PC12 cells.…”

Activation of neurotrophin signalling with light‑inducible receptor tyrosine kinases

“…Techniques that rely on the genetic code expansion ( 33 ), which allows the site-specific incorporation of unnatural amino acids into proteins, can potentially enable smaller modifications of the protein. Recently, we have successfully demonstrated the feasibility of controlling TrkA activity using genetic code expansion technique ( 34 ). Another important alternative strategy to reengineering kinase activity is through chemical genetics.…”