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bioRxiv · 10.1101/227512

Single Molecule Pull-down for Quantification of Membrane Receptor Multisite Phosphorylation

Salazar-Cavazos, E.·Lidke, K. A.·Lidke, D. S.
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Abstract

The Epidermal Growth Factor Receptor (EGFR/ErbB1/HER1) plays an important role in both physiological and cancer-related processes. To study the factors that influence receptor phosphorylation, we have coupled Single Molecule Pull-down (SiMPull) measurements with rule-based modeling of EGFR signaling. Using SiMPull, we quantified the phosphorylation state of thousands of individual receptors. These measurements enabled the first direct detection of multisite phosphorylation on full-length EGFR and revealed that the extent of phosphorylation varies by tyrosine site and is dependent on the relative abundance of signaling partners that limit access by tyrosine phosphatases. We also evaluated the impact of oncogenic mutations and ligands with varying affinity on phosphorylation kinetics. Simulations highlight the importance of dimer lifetimes on EGFR phosphorylation and signaling output.

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Salazar-Cavazos, E., Lidke, K. A., Lidke, D. S.. 2017-12-01. Single Molecule Pull-down for Quantification of Membrane Receptor Multisite Phosphorylation. https://doi.org/10.1101/227512

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