Clathrin and TOM1L1 regulate Epidermal Growth Factor Receptor Signaling at the Plasma Membrane

Abstract

Epidermal growth factor (EGF) receptor (EGFR) controls many aspects of cell physiology via the activation of intracellular signaling pathways. Ligand‐bound EGFR elicits phosphorylation of Gab1, which activates phosphatidylinositol‐3‐kinase (PI3K), which in turn leads to Akt phosphorylation and activation, controlling cell survival and metabolism. Rapidly upon binding EGF, the receptor is recruited to clathrin‐coated pits (CCPs), eventually leading to EGFR endocytosis. As such, receptor‐proximal EGFR signaling occurs while the receptor is enriched with clathrin‐coated plasma membrane microdomains. Whether and how clathrin microdomains may control EGFR signaling at the plasma membrane leading to Gab1 and Akt phosphorylation remains poorly understood. Using several specific molecular perturbations, we found that clathrin is required for EGF‐stimulated phosphorylation of Akt. Similar perturbations of dynamin were without effect on EGF‐stimulated Akt phosphorylation. This indicates that clathrin microdomains regulate EGFR signaling at the plasma membrane.Clathrin interacts with and recruits numerous proteins to the plasma membrane including TOM1L1. We have examined the role of TOM1L1, which interacts with clathrin, EGFR and Src‐family kinases (SFKs). SiRNA gene silencing of TOM1L1 impairs EGF‐stimulated Akt phosphorylation. Using fluorescent labeling of clathrin, TOM1L1 and other signaling intermediates coupled to total internal reflection fluorescence microscopy, we have examined the hierarchy of the recruitment of EGFR signals to clathrin‐coated plasma membrane microdomains. Our findings suggest that TOM1L1 has a clathrin‐localized regulatory role in EGF‐stimulated Akt phosphorylation.