title: Proteasome inhibition blocks ligand-induced dynamic processing and internalization of epidermal growth factor receptor via altered receptor ubiquitination and phosphorylation.
abstract: Epidermal growth factor (EGF) receptor (EGFR), a member of the EGF superfamily of receptor tyrosine kinases, is a critical regulator of cell growth and an important target for single agent and combination anticancer therapeutics. To further investigate the dynamics of ligand-induced EGFR processing and regulation noninvasively, we developed a chimeric EGFR-firefly luciferase (FLuc) fusion reporter to directly monitor processing of EGFR in real-time. In a stable HeLa cell line expressing the reporter at physiologically relevant levels, bioluminescence imaging continuously monitored reporter dynamics, correlating with the ligand-induced response of endogenous EGFR as determined by Western blot, subcellular localization of an EGFR-green fluorescent protein (GFP) fusion protein, and validated pharmacologic responses. The signaling competency of the reporter was confirmed by gene rescue experiments in EGFR-null cells. Bioluminescence analysis further showed that proteasome inhibition with bortezomib or MG132 attenuated overall ligand-induced degradation of EGFR. In cells expressing EGFR-GFP, pretreatment with proteasome inhibitors trapped essentially all of the receptor at the cell membrane both before and after ligand-induced activation with EGF. Furthermore, proteasome inhibition enhanced receptor ubiquitination in both the basal and ligand-activated states as well as delayed the processing of ligand-activated phosphorylation of the receptor, kinetically correlating with attenuated receptor degradation. These observations point to a potential mechanism for the synergistic therapeutic effects of combination EGFR- and proteasome-targeted therapies.
authors.affiliation: Molecular Imaging Center, Mallinckrodt Institute of Radiology, Washington University School of Medicine, St Louis, Missouri 63110, USA.
authors.forename: Aparna H
authors.initials: AH
authors.lastname: Kesarwala
authors.forename: Mustapha M
authors.initials: MM
authors.lastname: Samrakandi
authors.forename: David
authors.initials: D
authors.lastname: Piwnica-Worms
mesh_terms: D001897:Boronic Acids / Q000494:pharmacology; D000069286:Bortezomib; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D006367:HeLa Cells; D006801:Humans; D049409:Luciferases, Firefly / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence / Q000379:methods; D010766:Phosphorylation; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex / Q000378:metabolism; D061988:Proteasome Inhibitors; D011719:Pyrazines / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection; D054875:Ubiquitination
chemical_list: D001897:Boronic Acids; D011480:Protease Inhibitors; D061988:Proteasome Inhibitors; D011719:Pyrazines; D011993:Recombinant Fusion Proteins; D004815:Epidermal Growth Factor; D000069286:Bortezomib; D049409:Luciferases, Firefly; D066246:ErbB Receptors; D046988:Proteasome Endopeptidase Complex