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Published online 25 February 2002. doi:10.1083/jcb.200106056
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© The Rockefeller University Press, 0021-9525/2002/3/843 $5.00
The Journal of Cell Biology, Volume 156, Number 5, March 4, 2002 843-854

Article

Ubiquitination and proteasomal activity is required for transport of the EGF receptor to inner membranes of multivesicular bodies



Karianne E. Longva, Frøydis D. Blystad, Espen Stang, Astrid M. Larsen, Lene E. Johannessen and Inger H. Madshus

Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway

Address correspondence to Inger H. Madshus, Institute of Pathology, The University of Oslo, Rikshospitalet, N-0027 Oslo, Norway. Tel.: 47-23-073-536. Fax: 47-23-071-511. E-mail: i.h.madshus{at}labmed.uio.no

EGF, but not TGF{alpha}, efficiently induces degradation of the EGF receptor (EGFR). We show that EGFR was initially polyubiquitinated to the same extent upon incubation with EGF and TGF{alpha}, whereas the ubiquitination was more sustained by incubation with EGF than with TGF{alpha}. Consistently, the ubiquitin ligase c-Cbl was recruited to the plasma membrane upon activation of the EGFR with EGF and TGF{alpha}, but localized to endosomes only upon activation with EGF. EGF remains bound to the EGFR upon endocytosis, whereas TGF{alpha} dissociates from the EGFR. Therefore, the sustained polyubiquitination is explained by EGF securing the kinase activity of endocytosed EGFR. Overexpression of the dominant negative N-Cbl inhibited ubiquitination of the EGFR and degradation of EGF and EGFR. This demonstrates that EGF-induced ubiquitination of the EGFR as such is important for lysosomal sorting. Both lysosomal and proteasomal inhibitors blocked degradation of EGF and EGFR, and proteasomal inhibitors inhibited translocation of activated EGFR from the outer limiting membrane to inner membranes of multivesicular bodies (MVBs). Therefore, lysosomal sorting of kinase active EGFR is regulated by proteasomal activity. Immuno-EM showed the localization of intact EGFR on internal membranes of MVBs. This demonstrates that the EGFR as such is not the proteasomal target.

Key Words: EGF; c-Cbl; ubiquitination; proteasome; multivesicular bodies


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