Proteasome Inhibition Blocks Ligand-Induced Dynamic Processing and Internalization of Epidermal Growth Factor Receptor via Altered Receptor Ubiquitination and Phosphorylation

A. H. Kesarwala, M. M. Samrakandi, D. Piwnica-Worms
2009 Cancer Research  
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 EGFRfirefly luciferase (FLuc) fusion reporter to directly monitor processing of EGFR in real-time. In a stable HeLa cell line expressing the
more » ... 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 ligandinduced 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. [Cancer Res 2009;69(3):976-83] Note: A.H. Kesarwala and M.M. Samrakandi contributed equally. Requests for reprints:
doi:10.1158/0008-5472.can-08-2938 pmid:19176375 fatcat:wvybhybk2bcgjnvjthebckggv4