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

Abstract

Ligand-induced down-regulation controls the signaling potency of the epidermal growth factor receptor (EGFR/ErbB1). Overexpression studies have identifiedCbl-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. Growth factor receptor tyrosine kinases (RTKs)1 play crucial roles in cellular proliferation, survival, migration, and differentiation. Epidermal growth factor receptor (EGFR/ErbB1) is a member of the ErbB family (ErbB1-4) of RTKs, which play crucial homeostatic roles and are implicated in oncogenesis. Ligand-induced activation of RTKs leads to the assembly of signaling protein complexes and subsequent activation of downstream signaling pathways. The ligand-activated RTKs also undergo rapid endocytosis (1). The endocytosed receptors then undergo a sorting process, which determines receptor fate and signal intensity. The receptors can be targeted to the lysosome for degradation, which terminates receptor signals. Alternatively, the internalized receptors can be recycled back to the cell surface for continued ligand binding and signaling (2-5). The relative efficiency of lysosomal sorting versus recycling is a key determinant of the signaling potency of RTKs (6). For example, EGFR is predominantly delivered to lysosomes when activated by EGF. In contrast, heregulin-activated ErbB2 is primarily recycled. The greater efficiency of the recycling process is thought to be a major determinant of the signaling superiority of ErbB2 over EGFR (7-9).Despite a critical role of endocytic sorting as a determinant of ErbB receptor down-regulation, the biochemical mechanisms that regulate this process have only recently begun to be elucidated. We, and others, have identified Cbl as one such regulator (10 -12). Cbl is recruited to the activated EGFR through both direct and indirect binding. Direct Cbl-EGFR interaction is mediated through the N-terminal tyrosine kinase-binding domain of Cbl, which binds to phosphorylated Tyr-1045 on EGFR (13). Indirect Cbl-E...

Reduced Cbl phosphorylation and degradation of the ζ‐chain of the T‐cell receptor/CD3 complex in T cells with low Lck levels

“…Surprisingly, our results indicate that neither full Lck activity nor full c-Cbl phosphorylation is necessary for TCR/CD3 internalization, but that they are important for lysosomal targeting and degradation of z-chain. Data along these lines have been reported for the EGF receptor [29,30]. However, this is not in complete agreement with previous reports on Cbl knockout mice, where TCR internalization was reduced in both c-Cbl À/À , Cbl-b À/À and Cbl double knockout T cells [26], suggesting that altered activity may result in a different phenotype than complete removal of a protein.…”

EGFRvIII undergoes activation-dependent downregulation mediated by the Cbl proteins

“…The ability of all three members of the Cbl family of E3s (Cbl, Cbl-b, and Cbl-c) to ubiquitinate and downregulate the EGFR following stimulation with EGF is well-characterized (Ettenberg et al, 1999b(Ettenberg et al, , 2001Levkowitz et al, 1999;Waterman et al, 1999a;Yokouchi et al, 1999;Duan et al, 2003). In this study, we establish that the Cbl proteins can downregulate the constitutively active mutant of the EGFR known as the EGFRvIII.…”

“…These internalized antibodies become localized to vesicles in the perinuclear Golgi region and are rapidly catabolized, suggesting that the internalized EGFRvIII:monoclonal antibody complex is trafficked to the lysosome. The Cbl proteins are critical regulators of the trafficking of the WT EGFR to the lysosome (Duan et al, 2003) and this study has established that they regulate the constitutively active EGFRvIII. Furthermore, the inhibition of the TK activity of the EGFRvIII prevents its downregulation by the Cbl proteins and decreases the amount of EGFRvIII located in intracellular vesicles (Figure 2).…”

“…The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are negative regulators of WT EGFR signaling (Ettenberg et al, 1999a(Ettenberg et al, , b, 2001Keane et al, 1999;Levkowitz et al, 1999;Waterman et al, 1999a;Yokouchi et al, 1999;Duan et al, 2003). The Cbl proteins all contain an aminoterminal TK-binding (TKB) domain, a RING finger domain, and a region of proline-rich sequences in their carboxy-terminus (Nau and Lipkowitz, 2003).…”

“…They are recruited to the activated EGFR either by the direct binding of their TKB domain to the phosphotyrosine residue at position 1045 in the EGFR (Levkowitz et al, 1999) or by an indirect mechanism mediated by Grb2 -the SH3 domains of Grb2 bind the proline-rich region of the Cbl proteins and the SH2 domain of Grb2 binds the phosphorylated EGFR (Waterman et al, 2002;Jiang et al, 2003). The RING finger domain of the Cbl proteins allows them to function as ubiquitin ligases (E3s) and so to target the EGFR signaling complex for internalization and subsequent degradation in the lysosome (Ettenberg et al, 1999b(Ettenberg et al, , 2001Levkowitz et al, 1999;Waterman et al, 1999a;Yokouchi et al, 1999;Duan et al, 2003). Thus, the Cbl proteins mediate the downregulation of the EGFR following stimulation with EGF.…”