A quantitative microscopy method to probe RTK activation.

Abstract

Receptor tyrosine kinases (RTKs) are important for cell regulation, migration, differentiation, and proliferation. Activation of RTKs is characterized by phosphorylation at key tyrosine residues in the intracellular kinase domain. Phosphorylation is induced by ligand binding, which subsequently triggers multiple signaling cascades. We are seeking to develop a new method to quantify the activation of RTKs across the plasma membrane. While phosphorylation is typically characterized using western blotting, in this study we use a quantitative fluorescence technique to characterize the activation of EGFR and link ligand binding to the phosphorylation. EGFR plays an essential role in various biological responses such as differentiation and proliferation and is required in epithelization. One aim of this work is to investigate the activation of EGFR in direct response to ligand binding and the second aim is to investigate the effect of a mutation, known to cause a lung carcinoma, on EGFR activity.