Growth factors stimulate specific receptor tyrosine kinases, but subsequent receptor endocytosis terminates signaling. The ubiquitin ligase c-Cbl targets epidermal growth factor receptors (EGFRs) to endocytosis by tagging them with multiple ubiquitin molecules. However, the type of ubiquitylation is unknown; whereas polyubiquitin chains signal proteasomal degradation, ubiquitin monomers control other processes. We report that in isolation c-Cbl mediates monoubiquitylation rather than polyubiquitylation of EGFRs. Consistent with the sufficiency of monoubiquitylation, when fused to the tail of EGFR, a single ubiquitin induces receptor endocytosis and degradation in cells. By using receptor and ubiquitin mutants, we infer that c-Cbl attaches a founder monoubiquitin to the kinase domain of EGFR and this is complemented by the conjugation of additional monoubiquitins. Hence, receptor tyrosine kinases are desensitized through conjugation of multiple monoubiquitins, which is distinct from polyubiquitin-dependent proteasomal degradation.Protein ubiquitylation has emerged as a versatile regulatory strategy (reviewed in Ref. 1). In its best characterized role as a signal for proteasomal degradation, productive recognition of ubiquitylated substrates is shown to minimally require a tetraubiquitin chain (2). Alternatively, studies in yeast attribute to monoubiquitylation an intrinsic capacity to target substrates both for internalization at the plasma membrane and sorting at multivesicular bodies toward destruction in the vacuole (reviewed in Ref. 3). With subsequent identification of ubiquitin binding activities, such as the UIM, 1 a rationale for ubiquitindependent recognition of substrates has materialized (4 -7). In higher eukaryotic systems, ubiquitylation of cell-surface receptors, likewise, correlates with their down-regulation via orthologous trafficking pathways that employ counterparts conserved from yeast (8 -11). Ligand-activated ubiquitylation of EGFR, as well as other RTKs, is mediated by c-Cbl (12-14).Whether or not EGFR ubiquitylation is sufficient for its internalization remains an open question. Likewise, although it is clear that each endocytosed receptor is conjugated to several molecules of ubiquitin, it is currently unknown to which extent branching of the EGFR-conjugated ubiquitins occurs in living cells. Here we present evidence indicating that the action of c-Cbl is limited to the addition of monomeric ubiquitins, and these are sufficient for receptor endocytosis and degradation. EXPERIMENTAL PROCEDURESReagents and Antibodies-Unless indicated, reagents were purchased from Sigma. E1 was from Affiniti (Mamhead, Exeter, UK), and rabbit reticulocyte lysate from Promega (Madison, WI). The 528-IgG antibody was isolated from hybridomas and a Fab fragment prepared and labeled with Cy3. An antibody to EGFR was from Alexis (San Diego, CA). Anti-EEA1 mouse antibody was from Transduction Laboratories (Lexington, KY). Fluorescently labeled antibodies were purchased from Jackson ImmunoResearch (West Grove, PA).Co...

Ligand‐induced desensitization of the epidermal growth factor receptor (EGFR) is controlled by c‐Cbl, a ubiquitin ligase that binds multiple signaling proteins, including the Grb2 adaptor. Consistent with a negative role for c‐Cbl, here we report that defective Tyr1045 of EGFR, an inducible c‐Cbl docking site, enhances the mitogenic response to EGF. Signaling potentiation is due to accelerated recycling of the mutant receptor and a concomitant defect in ligand‐induced ubiquitylation and endocytosis of EGFR. Kinetic as well as morphological analyses of the internalization‐defective mutant receptor imply that c‐Cbl‐mediated ubiquitylation sorts EGFR to endocytosis and to subsequent degradation in lysosomes. Unexpectedly, however, the mutant receptor displayed significant residual ligand‐induced ubiquitylation, especially in the presence of an overexpressed c‐Cbl. The underlying mechanism seems to involve recruitment of a Grb2 c‐Cbl complex to Grb2‐specific docking sites of EGFR, and concurrent acceleration of receptor ubiquitylation and desensitization. Thus, in addition to its well‐characterized role in mediating positive signals, Grb2 can terminate signal transduction by accelerating c‐Cbl‐dependent sorting of active tyrosine kinases to destruction.

The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.[Keywords: Endocytosis; growth factor; HIV; ubiquitin; signal transduction] Supplemental material is available at http://www.genesdev.org.

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.Growth factors and their transmembrane receptors, harboring intrinsic tyrosine kinase activity, play essential roles in cell fate determination. Whereas the process leading to growth factor-induced activation of signaling pathways is relatively well understood, mechanisms that initiate signal desensitization are only beginning to be unraveled (reviewed in Ref. 1). In the case of the epidermal growth factor receptor (EGFR) 2 and related receptor tyrosine kinases, the major signal attenuation process involves ligand-induced internalization of activated receptors and their sorting to degradation in lysosomes (reviewed in Ref.2). In analogy to the pivotal role played by phosphotyrosine and respective binding domains (e.g. Src homology domain 2) in positive signaling pathways, ubiquitin and ubiquitin-binding domains (e.g. ubiquitin-interacting motifs (UIMs)) regulate receptor endocytosis and sorting for lysosomal degradation (3). For example, recruitment of an E3 ubiquitin ligase, called c-Cbl (4 -7), enables subsequent conjugation of ubiquitin to multiple lysines of EGFR (8 -10). The appended ubiquitins are thought to recruit a set of endocytic proteins (e.g. Eps15) through their UIMs (reviewed in Ref. 11).In contrast to the well understood cellular functions of protein ubiquitylation, the roles played by ubiquitin-like proteins such as Nedd8 and SUMO are less characterized (12). Nedd8 is the closest kin of ubiquitin and it is linked to other proteins by an amide bond linking the carboxyl-terminal carboxylate to lysine residues. Neddylation is initiated by a heterodimeric complex comprising Uba3 and the amyloid precursor proteinbinding protein (APP-BP1). Ubc12, an E2-like component, then mediates conjugation of Nedd8 (13), but the identity and function of Nedd8-specific E3 ligases are less understood. Modification of Cullins, the first discovered neddylation substrate, is promoted by Roc1/Rbx, a RING finger protein (14, 15), which recruits Ubc12 (16, 17). Cullins are shared subunits of SCF (Skp1-Cdc53/CUL-F-box) complexes, assembled E3 ubiquitin ligases that regulate ubiquitylation of proteins involved primarily in cell cycle control. Neddylation of Cullin1 and Cullin2 activates their E3 ubiquitin ligase activity toward subs...

Ligand-dependent endocytosis of the epidermal growth factor receptor (EGFR) involves recruitment of a ubiquitin ligase, and sorting of ubiquitylated receptors to lysosomal degradation. By studying Hgs, a mammalian homolog of a yeast vacuolar-sorting adaptor, we provide information on the less understood, ligand-independent pathway of receptor endocytosis and degradation. Constitutive endocytosis involves receptor ubiquitylation and translocation to Hgs-containing endosomes. Whereas the lipid-binding motif of Hgs is necessary for receptor endocytosis, the ubiquitin-interacting motif negatively regulates receptor degradation. We demonstrate that the ubiquitin-interacting motif is endowed with two functions: it binds ubiquitylated proteins and it targets self-ubiquitylation by recruiting Nedd4, an ubiquitin ligase previously implicated in endocytosis. Based upon the dual function of the ubiquitin-interacting motif and its wide occurrence in endocytic adaptors, we propose a ubiquitin-interacting motif network that relays ubiquitylated membrane receptors to lysosomal degradation through successive budding events.