Ligand-induced Ubiquitination of the Epidermal Growth Factor Receptor Involves the Interaction of the c-Cbl RING Finger and UbcH7

doi:10.1074/jbc.274.44.31707

Ligand-induced Ubiquitination of the Epidermal Growth Factor Receptor Involves the Interaction of the c-Cbl RING Finger and UbcH7*

From the Departments of ^‡Cell Biology,^§Orthopaedics, and ^‖Genetics, Yale University School of Medicine, New Haven, Connecticut 06510 and the ^¶Institute of Life Science, Kurume University, Aikawamachi 2432-3, Kurume 839-0861, Japan

Abstract

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an as yet undefined mechanism. We demonstrate here, using the yeast two-hybrid system and an in vitro binding assay, that the c-Cbl RING finger domain interacts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted with the wild-type c-Cbl RING finger domain but not with a RING finger domain that lacks the amino acids that are deleted in 70Z-Cbl, an oncogenic mutant of c-Cbl. The in vitro interaction was enhanced by sequences on both the N- and C-terminal sides of the RING finger. In vivo and in vitro experiments revealed that c-Cbl and UbcH7 synergistically promote the ligand-induced ubiquitination of the epidermal growth factor receptor (EGFR). In contrast, 70Z-Cbl markedly reduced the ligand-induced, UbcH7-mediated ubiquitination of the EGFR. MG132, a proteasome inhibitor, significantly prolonged the ligand-induced phosphorylation of both the EGFR and c-Cbl. Thus, c-Cbl plays an essential role in the ligand-induced ubiquitination of the EGFR by a mechanism that involves an interaction of the RING finger domain with UbcH7. This mechanism participates in the down-regulation of tyrosine kinase receptors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.

Footnotes

↵* This work was supported by the Japan Society for the Promotion of Science and by grants from the Nissan Science Foundation (to M. Y.), The Ministry of Education, Science, Sports and Culture, TORAY Research Foundation, Sumitomo Research Foundation (to A. Y.), and by National Institutes of Health Grant AR-42927 and Ariad Pharmaceuticals (to R. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
↵** To whom correspondence should be addressed: Depts. of Cell Biology and Orthopaedics, Yale University School of Medicine, 333 Cedar St., New Haven, CT 06510. Tel.: 203-785-4150; Fax: 203-785-2744; E-mail;roland.baron{at}yale.edu.
Abbreviations:

PTB

phosphotyrosine-binding domain

SH2

Src homology domain 2

EGFR

epidermal growth factor receptor

UbcH

human ubiquitin-conjugating enzyme

PCR

polymerase chain reaction

GST

glutathioneS-transferase

JAK

Janus kinase

SOCS

suppressor of cytokine signaling

STAT

signal transducers and activators of transcription
- Received March 16, 1999.
- Revision received August 5, 1999.
The American Society for Biochemistry and Molecular Biology, Inc.