A Liposome-Reconstitution Assay Reveals Lipid-Dependent Modulation of EGFR Kinase Activity

Abstract

The epidermal growth factor receptor (EGFR) is a receptor tyrosine kinase whose activity is regulated by ligand binding and dimerization but also by its membrane environment. Although anionic lipids such as phosphatidylinositol 4,5-bisphosphate (PIP2) and phosphatidylserine (PS) have been implicated in modulating EGFR activation, direct biochemical evidence for lipid-dependent effects on EGFR catalytic function has not yet been reported. Here, we reconstitute the catalytically active intracellular domain (ICD) of EGFR (residues 669–1210) on synthetic liposomes to probe the influence of lipid composition on kinase activity. We find that both PIP2 and PS suppress the apparent catalytic output of membrane-tethered EGFR-ICD and produce distinct changes in ATP-dependent kinetic parameters. PIP2 produced stronger suppression than PS under matched assay conditions, consistent with electrostatic modulation of EGFR-ICD catalytic output. These results show that defined anionic lipid compositions can modulate EGFR-ICD catalytic activity and establish a platform for quantifying membrane-dependent regulation of receptor kinase activity.