Photosensitive UAAs Optically Control Site-dependent Phosphorylation In TrkA Activated MAPK/ERK Signaling

Tropomyosin receptor kinase A(TrkA)is a membrane receptor which upon ligand binding autophosphorylates and activates several pathways including MAPK/ERK,PI3K/AKT,PLCγ signaling.The sites and characteristics of TrkA phosphorylation control cell proliferation,differentiation,growth,and apoptosis,while the NGF/TrkA signaling pathway is implicated in a spectrum of human pathologies such as Alzheimer’s disease,chronic pain,inflammation,and cancer,thus,it is an emerging therapeutic target in treatment of neuronal diseases and cancer.However,the downstream pathways of TrkA are complicated and related to the phosphorylation sites and their combinations in the intracellular segment.Therefore,the phosphorylation of TrkA has not been fully elucidated so far.One important reason is that TrkA signaling is a highly dynamic process,and current research methods lack separate control for site-dependent phosphorylation in the receptorTherefore,in this paper,we proposed a design scheme for photosensitive TrkA,which used a new molecular light control technology.Light-controllable unnatural amino acids(UAAs)were introduced at the specific phosphorylation sites of the target protein TrkA through genetic code expansion(GCE)and site-directed mutagenesis,aiming to study the regulation mechanism of different phosphorylation sites of TrkA.We used the amber stop codon for identifying unnatural amino acids.By mutating the TrkA tyrosine phosphorylation sites into the stop codon,we introduced the corresponding tRNA and protein synthetase(RS),using two pairs of engineered and specific orthogonal tRNA/RS to inhibit the expression of the amber stop codon.Two photosensitive tyrosine analogs,namely AzF(p-azido-L-phenylalanine)and ONB(caged-tyrosine),were introduced at the five phosphate sites of TrkA(Y490,Y670,Y674,Y675,Y785).These two types of UAAs provide different photochemical reaction mechanisms.ONB acts as a "light-controlled switch" to control the phosphorylation state of kinases,while AzF is a potential photo-crosslinking molecule.The purpose of our research was to optically manipulate the phosphorylation state of individual intracellular tyrosine in TrkA and observe the activation of downstream signaling pathwaysOur content was mainly divided into two parts.The first part was the design and exploration of appropriate light control methods,that was,the second and third chapters of the paper.There were three main conclusions:1)First,we tested the orthogonality of tRNA/RS pairs in mammalian cell HEK293T using two fluorescence reporting systems,EGFP-amber and EGFP-amber-mCherry,and then introduced AzF and ONB at the TrkA-Y490 site to establish a feasible method for encoding photosensitive UAAs.2)We found that HEK293T cells could be used to specifically respond to p-ERK activity activated by exogenous TrkA;3)Two genetic code expansion systems were introduced into three cell models,including PC12,HEK293T and SH-SY5Y cells.We found that AzF and ONB could be introduced at the five phosphorylation sites of TrkA and there were no errors in reading through.The HEK293T cell was a suitable cell model for expressing exogenous TrkA-AzF,TrkA-ONB mutants due to its higher expression efficiency,while SH-SY5Y cells could serve as a neural cell model for testing light-controlled TrkA mutantsThe second part was the detection of light-controlled TrkA mutants on downstream cell signaling pathways and cell differentiation regulation,which was the fourth chapter of the paper.The main conclusions were:1)We first used HEK293T cells to verify the feasibility of activating ERK signaling in TrkA-Y490ONB under light control.The phosphorylation of Y490 activates the MAPK/ERK pathway and is related to cell proliferation and differentiation,which has been previously reported.Secondly,MAPK/ERK activation was also detected through light-controlled TrkA-AzF,TrkA-ONB mutants at other phosphorylation sites(Y670,Y674,Y675,Y785),especially at the three sites of the kinase active region whose function is not clear.Interestingly,by introducing AzF and ONB to Y674 and Y675 sites,we found that light could directly activate the MAPK/ERK pathway without binding to ligand NGF,which provided an optical control model using light simulating NGF to activate downstream signaling pathways.Our results show that in addition to the two main sites Y490 and Y785,the three sites in the kinase circuit also play important roles in the regulation of the MAPK/ERK pathway.2)Due to the small interference of endogenous TrkA in SH-SY5Y cells,we combined the detection of ERK pathway in light-sensitive TrkA mutants(microscopic)with neuronal differentiation(cell phenotype),finding that the mutants which can activate p-ERK by light control can also show a significant degree of cell differentiation before and after light exposure,resulting in good consistencyIn conclusion,our research not only further extend the application of the genetic code expansion technology in kinase receptors,but also reveal how site-dependent phosphorylation in TrkA controls defined signaling process and initially establish an experimental model of light-controlled phosphorylation in kinase receptor.Through precise optical control,the activity of kinase receptors can be regulated at a single phosphorylation site,achieving the effect of site-directed light-controlled phosphorylation.It paves the way for comprehensive analysis of kinase-associated pathways as well as screening of compounds intervening a site-directed phosphorylation pathway for targeted therapy.