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Vol. 19, Issue 8, 3454-3462, August 2008

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Ubc4/5 and c-Cbl Continue to Ubiquitinate EGF Receptor after Internalization to Facilitate Polyubiquitination and Degradation

Kyohei Umebayashi^*,, Harald Stenmark, and Tamotsu Yoshimori^*,

*Department of Cellular Regulation, Research Institute for Microbial Diseases, Osaka University, Osaka 565-0871, Japan; Centre for Cancer Biomedicine, University of Oslo and Department of Biochemistry, The Norwegian Radium Hospital, Montebello, N-0310 Oslo, Norway; and CREST, Japan Science and Technology Agency, Kawaguchi 332-0012, Japan

Submitted October 1, 2007; Revised May 12, 2008; Accepted May 16, 2008
Monitoring Editor: Sandra Schmid

c-Cbl is the E3 ubiquitin ligase that ubiquitinates the epidermal growth factor (EGF) receptor (EGFR). On the basis of localization, knockdown, and in vitro activity analyses, we have identified the E2 ubiquitin-conjugating enzyme that cooperates with c-Cbl as Ubc4/5. Upon EGF stimulation, both Ubc4/5 and c-Cbl were relocated to the plasma membrane and then to Hrs-positive endosomes, strongly suggesting that EGFR continues to be ubiquitinated after internalization. Our time-course experiment showed that EGFR undergoes polyubiquitination, which seemed to be facilitated during the transport to Hrs-positive endosomes. Use of a conjugation-defective ubiquitin mutant suggested that receptor polyubiquitination is required for efficient interaction with Hrs and subsequent sorting to lysosomes. Abrupt inhibition of the EGFR kinase activity resulted in dissociation of c-Cbl from EGFR. Concomitantly, EGFR was rapidly deubiquitinated and its degradation was delayed. We propose that sustained tyrosine phosphorylation of EGFR facilitates its polyubiquitination in endosomes and counteracts rapid deubiquitination, thereby ensuring Hrs-dependent lysosomal sorting.

Present address: Department of Biochemistry, University of Geneva, Sciences II, CH-1211 Geneva, Switzerland.

Address correspondence to: Kyohei Umebayashi (Kyohei.Umebayashi{at}biochem.unige.ch) or Tamotsu Yoshimori (tamyoshi{at}biken.osaka-u.ac.jp)

Abbreviations used: EGFR, epidermal growth factor receptor; ESCRT, endosomal sorting complex required for transport; MVB, multivesicular body; NEM, N-ethylmaleimide.

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