Specialized Clarthrin-Enriched Signaling Nanodomains Control Epidermal Growth Factor Receptor Signaling At The Plasma Membrane

Abstract

The Epidermal Growth Factor (EGF) Receptor (EGFR) is a key receptor tyrosine kinase that controls many facets of cell physiology, including proliferation, survival and migration. EGF binding to EGFR elicits receptor phosphorylation and membrane recruitment and phosphorylation of Gab1 followed by activation of phosphatidylinositol-3-kinase (PI3K), leading to Akt phosphorylation and activation. Concomitantly upon ligand binding, EGFR is simultaneously is recruited to clathrin-coated pits (CCPs), eventually leading to receptor endocytosis. We previously uncovered that perturbation of clathrin, but not of receptor endocytosis, impairs EGF-stimulated activation of Akt signaling. Moreover, we found that some key EGFR signaling intermediates such as phosphorylated Gab1 are enriched with a subset of CCPs. We thus proposed that in addition to their well- known function as endocytic portals, some CCPs have a direct role in controlling EGFR signaling at the plasma membrane prior to receptor internalization. How CCPs and clathrin may directly control EGFR signaling at the plasma membrane prior to receptor endocytosis had remained poorly understood, which I have now examined here. I uncovered that perturbations of TOM1L1 (a clathrin-binding regulator of Src-family kinases) and the TOM1L1-binding Src-family kinase Fyn impaired EGF-stimulated Akt phosphorylation, similarly to the effects of clathrin perturbations. A similar requirement for an additional cytosolic kinase Ack1 was also found. Using total internal reflection fluorescence microscopy and automated image analysis, we found that TOM1L1, Fyn, and Ack1 are enriched within a subset of CCPs, and that those that house EGFR are also those that preferentially contain Fyn or Ack1. Perturbation of TOM1L1 prevented Fyn recruitment to CCPs, and mutants of TOM1L1 defective in Fyn binding or clathrin binding abrogated Akt phosphorylation downstream of EGF stimulation. Importantly, TOM1L1- or Fyn-positive CCPs exhibit unique properties, such as distinct lifetimes, size, and recruitment of endocytic accessory proteins. Thus, these results indicate that a subset of CCPs become specialized signaling-capable structures by enrichment with key signaling regulators to control EGFR signaling, a phenomenon required for subsequent Akt activation. Given the importance of EGFR in driving growth and survival of certain types of cancer, obtaining a better understanding of how CCPs organize key EGFR signals in space and time within the PM may lead to the development of novel anti-cancer treatments.