Easy as pY(tag): visualizing receptor tyrosine kinase activity in embryos

Summary by Cat Rogers: Ho, E.K., Kim-Yip, R.P., Simpkins, A.G., Farahani, P.E., Oatman, H.R., Posfai, E., Shvartsman, S.Y., and Toettcher, J.E. (2025). In vivo measurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient. bioRxiv. 10.1101/2025.01.06.631605.

Image credit: Wikimedia Commons (Peggy Greb)

Signal transduction is a biological process that allows remarkably few inputs to modulate many outputs. In receptor tyrosine kinase (RTK) signaling, a signal (ligand) binds to a receptor extracellularly leading to receptor phosphorylation intracellularly followed by phosphorylation of a signaling effector. The phosphorylated effector “passes the message along” ultimately changing a cell’s gene expression and identity. However, this stepwise system presents a challenge for connecting input-output relationships of signaling: it requires identifying pathway components and how they interconnect. Researchers have observed and perturbed ligand and often use phosphorylated effectors as a signaling activity readout. But there is a missing link between ligand and phosphorylated effector: the phosphorylated receptor.

Ho et. al. presents “pYtag”, a tool which repurposes a naturally-occurring phosphotyrosine-binding domain interaction to visualize phosphorylated receptors [1]. T-cell receptors have an ITAM (immunoreceptor tyrosine-based activation motif) which in its phosphorylated state recruits and binds a tandem SH2 domain of ZAP70 (ZtSH2). pYtag consists of an ITAM appended to an RTK of interest and fluorescently labelled ZtSH2 dispersed in the cytosol. When the modified RTKs cross phosphorylate upon ligand binding, the appended ITAM is also phosphorylated recruiting the fluorescent ZtSH2. Since the fluorescent ZtSH2 goes from dispersed to localized, this can be visualized on a fluorescence scope in real time. To use this tool Ho et. al. appended the ITAM sequence and ZtSH2 to RTKs of interest in Drosophila embryos.

They tested the efficacy of pYtag using three well-studied RTKs including Torso, the RTK upstream of the ERK effector. Using pYtag, they observe the formation of the doubly phosphorylated ERK (ppERK) gradient comparing the domain of RTK phosphorylation with that of ppERK in embryos at the same developmental stages. Reproducing results from a previous study [2], Ho et. al. found that ppERK initially spans 40% of the embryo length in a shallow gradient, which then retracts to the poles and steepens over this developmental window. However, Torso pYtag reports receptor activity that has a narrower domain and uniformly decreases in amplitude over time. This begs the question; how are the activity domains and amplitudes of these receptors and effectors so different?

Ho et. al. have a two-part hypothesis for this discrepancy in receptor- and effector-level activity. Firstly, it may be due to ppERK feedback inhibition where phosphatases are targets of ERK phosphorylation and feed back to regulate receptor activity. They test the possibility of a feedback loop by inhibiting ERK activation downstream of the Torso RTK. They find that loss of ERK activation results in loss of the decreased Torso activity amplitude over time. Secondly, they test and support the idea that this discrepancy may also be due to ppERK diffusion away from the source in the syncytial embryo.

Altogether this study shows that while effector activity is a useful readout of signaling activity, researchers may be missing a wealth of information from an unsuspecting regulatory node in signaling pathways. Ho et. al. have presented a modular tool to explore this missing link in myriad RTK mediated signaling pathways, maybe even in other species.

1. Ho, E. K. et al. In vivo measurements of receptor tyrosine kinase activity reveal feedback regulation of a developmental gradient. bioRxiv, doi:10.1101/2025.01.06.631605 (2025).

2.  Coppey, M., Boettiger, A. N., Berezhkovskii, A. M. & Shvartsman, S. Y. Nuclear trapping shapes the terminal gradient in the Drosophila embryo. Curr Biol 18, 915-919, doi:10.1016/j.cub.2008.05.034 (2008).