Interactions of Phosphatidylinositol-4,5-bisphosphate (PIP2) and Phosphatidylserine (PS) with Epidermal Growth Factor Receptor Intracellular Domain

The epidermal growth factor receptor (EGFR) is a central regulator of cellular proliferation and survival, and its dysregulation drives the pathogenesis of non–small cell lung cancer (NSCLC). While structural and signaling mechanisms of EGFR activation have been extensively characterized, the influence of the lipid microenvironment on the intracellular kinase domain (EGFR-ICD) remains underexplored. In this work, we investigate how anionic phospholipids, specifically phosphatidylinositol-4,5-bisphosphate (PIP2) and phosphatidylserine (PS), modulate EGFR-ICD function using a liposome-based kinase bioassay. Unlike conventional solution-based systems, this membrane-mimetic approach recapitulates the electrostatic and spatial constraints of the plasma membrane, providing a more physiologically relevant framework to study membrane-bound enzyme kinetic activity and lipid–protein interactions. Kinetic analyses revealed that PIP2 acts as a reversible, predominantly uncompetitive inhibitor, lowering catalytic turnover suggesting a negative effect on the enzyme–substrate complexes, while PS exhibits concentration-dependent mixed inhibition, simultaneously altering substrate affinity and catalytic efficiency. Systematic determination of α and α′ parameters across lipid titrations (0.001–10 mol%) demonstrated that PIP2 and PS differentially reshape the Michaelis–Menten parameters (Km, Vmax). These effects are mediated by electrostatic interactions between negatively charged lipid headgroups and cationic residues within EGFR-ICD, influencing both structural stability and ATP occupancy in the catalytic cleft. Importantly, these inhibitory profiles were also observed in clinically relevant oncogenic mutants (Ex19del and L858R). Both PIP2 and PS inhibit the kinase activity of both mutant kinases indicating, that these negatively-charged phospholipids can modulate even the constitutively active form of EGFR. This lipid modulation offers new insight into how plasma membrane composition shapes receptor signaling output. Understanding how lipid interactions modulate mutant EGFR may help explain variable drug responses and resistance patterns and underscores the importance of considering the lipid milieu as a regulatory axis in receptor pharmacology. Collectively, this work advances a biophysical and clinical framework in which membrane lipids serve not merely as structural components but as active modulators of EGFR signaling, oncogenic potential, and drug susceptibility. Together, this work establishes a mechanistic and translational framework in which liposome-reconstituted kinase assays provide critical insights into the lipid-dependent regulation of EGFR, bridging structural biophysics and clinical oncology.