Optical activation of TrkA signaling

Duan, Liting; Hope, Jen M.; Guo, Shunling; Ong, Qunxiang; François, Amaury; Kaplan, Luke; Scherrer, Grégory; Cui, Bianxiao

doi:10.1101/287409

Cited by 7 publications

(10 citation statements)

References 46 publications

(20 reference statements)

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“…By scanning through different phosphorylation sites, we managed to identify light-sensitive TrkA independent of ligand NGF. Compared to the recent demonstration of ligandindependent light-sensitive TrkA based on the fusion of lightsensitive protein Arabidopsis cryptochrome 2 [48][49][50] , our TrkA relies on the light activation of a specific phosphorylation site activating a specific signaling pathway using the two kinds of orthogonal tyrosyl-tRNA and pyrrolysyl-tRNA synthetase/ tRNA CUA pairs to incorporate light-sensitive Uaas. The introduction of Uaas to TrkA creates a much smaller change (10-100 Dalton) to the receptor than the light-sensitive proteins (~100 K Dalton); therefore, it is likely to maintain the function of the receptors, including ligand binding, protein-protein interactions and oligomerization.…”

Section: Discussionmentioning

confidence: 99%

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

Shu

Shi

et al. 2020

Commun Biol

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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.

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Section: Discussionmentioning

confidence: 99%

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

Shu

Shi

et al. 2020

Commun Biol

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“…So far, most opto-ERK-signaling tools activate signal transduction via receptor dimerization or translocation of receptorproximal pathway components to the plasma membrane (7)(8)(9)(10). These optogenetic systems mimic upstream activation steps and are therefore ideal for studies dissecting quantitative features of the downstream signaling cascade (11).…”

mentioning

confidence: 99%

Optimizing photoswitchable MEK

Patel

Yeung

McGuire

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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Optogenetic approaches are transforming quantitative studies of cell-signaling systems. A recently developed photoswitchable mitogen-activated protein kinase kinase 1 (MEK1) enzyme (psMEK) short-circuits the highly conserved Extracellular Signal-Regulated Kinase (ERK)-signaling cascade at the most proximal step of effector kinase activation. However, since this optogenetic tool relies on phosphorylation-mimicking substitutions in the activation loop of MEK, its catalytic activity is predicted to be substantially lower than that of wild-type MEK that has been phosphorylated at these residues. Here, we present evidence that psMEK indeed has suboptimal functionality in vivo and propose a strategy to circumvent this limitation by harnessing gain-of-function, destabilizing mutations in MEK. Specifically, we demonstrate that combining phosphomimetic mutations with additional mutations in MEK, chosen for their activating potential, restores maximal kinase activity in vitro. We establish that this modification can be tuned by the choice of the destabilizing mutation and does not interfere with reversible activation of psMEK in vivo in bothDrosophilaand zebrafish. To illustrate the types of perturbations enabled by optimized psMEK, we use it to deliver pulses of ERK activation during zebrafish embryogenesis, revealing rheostat-like responses of an ERK-dependent morphogenetic event.

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“…The general concept of TrkA signaling activation is that the ligand NGF from the extracellular domain promotes the homodimerization of TrkA at the cell membrane, which in turn activates downstream signaling pathways. However, because the TrkA signaling pathway is intricate and related to five intracellular phosphorylation sites (Y490, Y670, Y674, Y675, Y785) and their combinations, the mechanism has not yet been fully elucidated 19 .…”

Section: Discussionmentioning

confidence: 99%

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

Shu

Liu

2022

Preprint

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TrkA is a membrane receptor that upon ligand binding, induces autophosphorylation of tyrosine residues in the intracellular domain, and this process includes sites in the kinase activation loop (Y670, Y674, Y675) and two direct sites involved in downstream signaling pathways (Y490, Y785). At present, researchers cannot fully elucidate the regulatory mechanism of TrkA phosphorylation because TrkA signaling is a highly dynamic process, and a strategy with high temporal and spatial resolution will be beneficial to the mechanism research. Our previous study proposed a design scheme for photosensitive TrkA, which utilizes a new molecular light control technology to target TrkA-Y490 and three kinase domain sites (Y670, Y674, and Y675) through Genetic Code Expansion (GCE) technology combined with site-directed mutagenesis. We chose two light-controllable unnatural amino acids (UAAs) to introduce at the specific phosphorylation sites of the target protein TrkA. We focused on the regulation mechanism of these sites on the MAPK/ERK pathway downstream of TrkA. However, this method has not yet been validated for the TrkA-Y785 site. Therefore, this paper will continue to test the light-controlled method we established earlier in the Y785 site. We aim to improve further the experimental model of light-controlled phosphorylation of TrkA that we have established and finally lay the foundation for the comprehensive analysis of kinase-related pathways.

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Optical activation of TrkA signaling

Cited by 7 publications

References 46 publications

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

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

Optimizing photoswitchable MEK

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

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