title: Epidermal growth factor-stimulated Akt phosphorylation requires clathrin or ErbB2 but not receptor endocytosis.
abstract: Epidermal growth factor (EGF) binding to its receptor (EGFR) activates several signaling intermediates, including Akt, leading to control of cell survival and metabolism. Concomitantly, ligand-bound EGFR is incorporated into clathrin-coated pits--membrane structures containing clathrin and other proteins--eventually leading to receptor internalization. Whether clathrin might regulate EGFR signaling at the plasma membrane before vesicle scission is poorly understood. We compared the effect of clathrin perturbation (preventing formation of, or receptor recruitment to, clathrin structures) to that of dynamin2 (allowing formation of clathrin structures but preventing EGFR internalization) under conditions in which EGFR endocytosis is clathrin dependent. Clathrin perturbation by siRNA gene silencing, with the clathrin inhibitor pitstop2, or knocksideways silencing inhibited EGF-simulated Gab1 and Akt phosphorylation in ARPE-19 cells. In contrast, perturbation of dynamin2 with inhibitors or by siRNA gene silencing did not affect EGF-stimulated Gab1 or Akt phosphorylation. EGF stimulation enriched Gab1 and phospho-Gab1 within clathrin structures. ARPE-19 cells have low ErbB2 expression, and overexpression and knockdown experiments revealed that robust ErbB2 expression bypassed the requirement for clathrin for EGF-stimulated Akt phosphorylation. Thus clathrin scaffolds may represent unique plasma membrane signaling microdomains required for signaling by certain receptors, a function that can be separated from vesicle formation.
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Camilo
authors.initials: C
authors.lastname: Garay
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Gurjeet
authors.initials: G
authors.lastname: Judge
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Lucarelli
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Stephen
authors.initials: S
authors.lastname: Bautista
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Rohan
authors.initials: R
authors.lastname: Pandey
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada.
authors.forename: Tanveer
authors.initials: T
authors.lastname: Singh
authors.affiliation: Department of Chemistry and Biology, Ryerson University, Toronto, ON M5B 2K3, Canada cantonescu@ryerson.ca.
authors.forename: Costin N
authors.initials: CN
authors.lastname: Antonescu
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D002966:Clathrin / Q000037:antagonists & inhibitors / Q000378:metabolism*; D022163:Clathrin-Coated Vesicles / Q000378:metabolism*; D034285:Dynamin II / Q000378:metabolism; D004705:Endocytosis / Q000502:physiology; D004815:Epidermal Growth Factor / Q000378:metabolism / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D021962:Membrane Microdomains / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013449:Sulfonamides / Q000494:pharmacology; D053778:Thiazolidines / Q000494:pharmacology
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D002966:Clathrin; D034285:Dynamin II; D004815:Epidermal Growth Factor; D051057:Proto-Oncogene Proteins c-akt; D018719:Erb-b2 Receptor Tyrosine Kinases; D013449:Sulfonamides; D053778:Thiazolidines; C098587:GAB1 protein, human; C578512:pitstop 2; C508053:ERBB2 protein, human