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 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.
Yokouchi
M
M
Department of Cell Biology, Yale University School of Medicine, New Haven, Connecticut 06510, USA.
Kondo
T
T
Houghton
A
A
Bartkiewicz
M
M
Horne
W C
WC
Zhang
H
H
Yoshimura
A
A
Baron
R
R
D003546:Cysteine Endopeptidases / Q000187:drug effects / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism*; D007976:Leupeptins / Q000494:pharmacology; D008024:Ligands; D008025:Ligases / Q000378:metabolism*; D008954:Models, Biological; D009097:Multienzyme Complexes / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D046988:Proteasome Endopeptidase Complex; D011485:Protein Binding; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D050721:Proto-Oncogene Proteins c-cbl; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D020798:Two-Hybrid System Techniques; D014443:Tyrosine / Q000378:metabolism; D044763:Ubiquitin-Conjugating Enzymes; D044767:Ubiquitin-Protein Ligases; D014452:Ubiquitins / Q000378:metabolism*; D016335:Zinc Fingers
D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D008024:Ligands; D009097:Multienzyme Complexes; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014452:Ubiquitins; D014443:Tyrosine; D004815:Epidermal Growth Factor; C079437:UBE2L3 protein, human; D044763:Ubiquitin-Conjugating Enzymes; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; D008025:Ligases; C067419:CBL protein, human; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde