Cbl-mediated ubiquitinylation is required for lysosomal sorting of epidermal growth factor receptor but is dispensable for endocytosis.
Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identified Cbl-mediated ubiquitinylation of EGFR as a mechanism of ligand-induced EGFR down-regulation. However, the role of endogenous Cbl in EGFR down-regulation and the precise step in the endocytic pathway regulated by Cbl remain unclear. Using Cbl-/- mouse embryonic fibroblast cell lines, we demonstrate that endogenous Cbl is essential for ligand-induced ubiquitinylation and efficient degradation of EGFR. Further analyses using Chinese hamster ovary cells with a temperature-sensitive defect in ubiquitinylation confirm a crucial role of the ubiquitin machinery in Cbl-mediated EGFR degradation. However, internalization into early endosomes did not require Cbl function or an intact ubiquitin pathway. Confocal immunolocalization studies indicated that Cbl-dependent ubiquitinylation plays a critical role at the early endosome to late endosome/lysosome sorting step of EGFR down-regulation. These findings establish Cbl as the major endogenous ubiquitin ligase responsible for EGFR degradation, and show that the critical role of Cbl-mediated ubiquitinylation is at the level of endosomal sorting, rather than at the level of internalization.
Duan
Lei
L
Division of Rheumatology, Immunology and Allergy, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusetts 02115, USA.
Miura
Yuko
Y
Dimri
Manjari
M
Majumder
Biswanath
B
Dodge
Ingrid L
IL
Reddi
Alagarsamy L
AL
Ghosh
Amiya
A
Fernandes
Norvin
N
Zhou
Pengcheng
P
Mullane-Robinson
Karen
K
Rao
Navin
N
Donoghue
Stephen
S
Rogers
Rick A
RA
Bowtell
David
D
Naramura
Mayumi
M
Gu
Hua
H
Band
Vimla
V
Band
Hamid
H
D000818:Animals; D015153:Blotting, Western; D016466:CHO Cells; D002460:Cell Line; D006224:Cricetinae; D015536:Down-Regulation; D004622:Embryo, Mammalian; D004705:Endocytosis; D011992:Endosomes / Q000378:metabolism; D004815:Epidermal Growth Factor / Q000494:pharmacology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism*; D005347:Fibroblasts / Q000378:metabolism; D015870:Gene Expression; D006801:Humans; D008247:Lysosomes / Q000378:metabolism*; D051379:Mice; D009154:Mutation; D050722:Oncogene Protein v-cbl; D011518:Proto-Oncogene Proteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D050721:Proto-Oncogene Proteins c-cbl; D012333:RNA, Messenger / Q000032:analysis; D015682:Retroviridae Proteins, Oncogenic / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D013696:Temperature; D014162:Transfection; D025801:Ubiquitin / Q000378:metabolism*; D044767:Ubiquitin-Protein Ligases
D050722:Oncogene Protein v-cbl; D011518:Proto-Oncogene Proteins; D012333:RNA, Messenger; D015682:Retroviridae Proteins, Oncogenic; D025801:Ubiquitin; D004815:Epidermal Growth Factor; D050721:Proto-Oncogene Proteins c-cbl; D044767:Ubiquitin-Protein Ligases; D066246:ErbB Receptors; C067419:CBL protein, human; C493849:Cbl protein, mouse