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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.

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand-and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.

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...