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Cancer Cell death Cell cycle Cytoskeleton Exo/endocytosis Differentiation Division Organelles Signalling Stem cells Trafficking
Cell Biology International (2006) 30, 31–43 (Printed in Great Britain)
Two different stages of epidermal growth factor (EGF) receptor endocytosis are sensitive to free ubiquitin depletion produced by proteasome inhibitor MG132
Maria S. Melikova, Kirill A. Kondratov and Elena S. Kornilova*
Institute of Cytology of Russian Academy of Sciences, Tikhoretsky Ave, 4, 194064, Sankt Petersburg, Russia


Abstract

Numerous studies implicate proteasomes in the regulation of EGF receptor (EGFR) endocytosis on the basis of the ability of inhibitors to decrease EGFR degradation, but the exact mechanisms remain obscure. We demonstrated that EGFR itself is not a direct target for proteasome, since it is delivered to lysosomes intact. Evidence is presented that the inhibitory effect of MG132 on EGF degradation is due mostly to free ubiquitin depletion resultant from the suppression of proteasomal functioning by MG132. By subcellular fractionation, we show two MG132-sensitive steps in the EGFR degradation pathway: sorting from early (EE) to late (LE) endosomes, and late stage of LE maturation. MG132 treatment resulted in stabilization of EGFR tyrosine phosphorylation and its association with c-Cbl. Nevertheless, ubiquitination of EGFR at late stages of endocytosis was significantly lower than that in control cells. Highly ubiquitinated forms of EGFR demonstrated more sensitivity to MG132 treatment.


Key words: EGF receptor, Endocytosis, Degradation, Endosomes, Ubiquitin, c-Cbl, MG132.

*Corresponding author. Tel.: +7 812 247 4596; fax: +7 812 247 0341.


Received 22 April 2005/22 August 2005; accepted 1 September 2005

doi:10.1016/j.cellbi.2005.09.003


ISSN Print: 1065-6995
ISSN Electronic: 1095-8355
Published by Portland Press Limited on behalf of the International Federation for Cell Biology (IFCB)