Cbl-mediated Ubiquitinylation Is Required for Lysosomal Sorting of Epidermal Growth Factor Receptor but Is Dispensable for Endocytosis

• Published in: The Journal of biological chemistry 2003-08, Vol.278 (31), p.28950-28960
• Main Authors: Duan, Lei, Miura, Yuko, Dimri, Manjari, Majumder, Biswanath, Dodge, Ingrid L., Reddi, Alagarsamy L., Ghosh, Amiya, Fernandes, Norvin, Zhou, Pengcheng, Mullane-Robinson, Karen, Rao, Navin, Donoghue, Stephen, Rogers, Rick A., Bowtell, David, Naramura, Mayumi, Gu, Hua, Band, Vimla, Band, Hamid
• Format: Article
• Language: English
• Subjects: Animals; Blotting, Western; Cell Line; CHO Cells; Cricetinae; Down-Regulation; Embryo, Mammalian; Endocytosis; Endosomes - metabolism; Epidermal Growth Factor - pharmacology; ErbB Receptors - genetics; ErbB Receptors - metabolism; Fibroblasts - metabolism; Gene Expression; Humans; Lysosomes - metabolism; Mice; Mutation; Oncogene Protein v-cbl; Proto-Oncogene Proteins - deficiency; Proto-Oncogene Proteins - genetics; Proto-Oncogene Proteins - physiology; Proto-Oncogene Proteins c-cbl; Retroviridae Proteins, Oncogenic - deficiency; Retroviridae Proteins, Oncogenic - genetics; Retroviridae Proteins, Oncogenic - physiology; RNA, Messenger - analysis; Temperature; Transfection; Ubiquitin - metabolism; Ubiquitin-Protein Ligases
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M304474200

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.