Ligand-induced ubiquitination of the epidermal growth factor receptor involves the interaction of the c-Cbl RING finger and UbcH7

c-Cbl plays a negative regulatory role in tyrosine kinase signaling by an a s 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 int eracts with UbcH7, a ubiquitin-conjugating enzyme (E2). UbcH7 interacted wi th 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 mutan t 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 experiment s 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 li gand-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 mec hanism that involves an interaction of the RING finger domain with UbcH7. T his mechanism participates in the down-regulation of tyrosine kinase recept ors and loss of this function, as occurs in the naturally occurring 70Z-Cbl isoform, probably contributes to oncogenic transformation.