Ligand-induced down-regulation of two growth factor receptors, EGF receptor (ErbB-1) and ErbB-3, correlates with differential ability to recruit c-Cbl, whose invertebrate orthologs are negative regulators of ErbB. We report that ligand-induced degradation of internalized ErbB-1, but not ErbB-3, is mediated by transient mobilization of a minor fraction of c-Cbl into ErbB-1-containing endosomes. This recruitment depends on the receptor's tyrosine kinase activity and an intact carboxy-terminal region. The alternative fate is recycling of internalized ErbBs to the cell surface. Cbl-mediated receptor sorting involves covalent attachment of ubiquitin molecules, and subsequent lysosomal and proteasomal degradation. The oncogenic viral form of Cbl inhibits down-regulation by shunting endocytosed receptors to the recycling pathway. These results reveal an endosomal sorting machinery capable of controlling the fate, and, hence, signaling potency, of growth factor receptors.

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...

Two ErbB family members, ErbB-1 (also called the epidermal growth factor (EGF) 1 receptor) and ErbB-2 (also called Neu and HER2), are implicated in human cancer; genetic aberrations of the respective two genes cause their oncogenic activation (reviewed in Ref. 1). The neu/erbB2 oncogene was initially identified as an activated oncogene using a DNA transfection strategy (2). The human counterpart of the gene has been independently isolated using related human or viral erbB probes and termed HER2 (3) or c-erbB-2 (4 -6). The neu oncogene differs from c-neu/erbB-2 by a point mutation replacing a valine for a glutamate at position 664, which lies within the transmembrane region (7). The resulting oncogenic protein is constitutively active as a tyrosine kinase (8) due to its ligandindependent dimeric state (9). Although a similar mutation has not been reported in human cancers, oncogenic activation of Neu has been frequently used as a genetic model for breast cancer development in rodents (10 -14). Moreover, this protein is considered a major target for anti-tumor therapies that use kinase inhibitors or monoclonal antibodies (1). A humanized monoclonal antibody specific to ErbB-2 has been recently approved for treatment of metastatic breast cancers overexpressing ErbB-2 (15-17). Therapeutic anti-ErbB-2 antibodies are thought to enhance down-regulation and degradation of ErbB-2, but the underlying mechanism is still unknown (18,19).Activation of ErbB-2 through overexpression, mutagenesis, or stimulation of EGFR/ErbB-2 chimeras with EGF results in the recruitment of several substrates. The list of ErbB-2 substrates includes phospholipase C␥ (20, 21), phosphatidylinositol (PI) 3-kinase (22, 23), Shc (24), c-Src (25-27), the GTPaseactivating protein (20, 28), protein tyrosine phosphatase 1D (29), and GRB-7 (30). The status of one potential substrate of Neu/ErbB-2, namely c-Cbl, is currently unclear. This issue is important because recent studies have identified the c-Cbl adaptor protein as a negative regulator of activated receptor tyrosine kinases, including the receptors for EGF (31-34), the platelet-derived growth factor (35), and colony-stimulating growth factor-1 (36). Consistent with a negative regulatory role, c-Cbl-deficient mice exhibit hyperplasia of mammary ducts (37), a process known to be regulated by EGF-like growth factors. Recruitment of c-Cbl to the EGF receptor plays a key role in sorting the receptor to late endosomes, and it involves receptor ubiquitination and degradation (32,33). By using a cell-free ubiquitination assay, we and others (34,38,39) have shown that Cbl is a component of the ubiquitin conjugation machinery, and it qualifies as a ubiquitin-protein isopeptide ligase or a ligase ancillary protein.In view of the pathological significance of Neu/ErbB-2, identification of negative regulators of this oncogenic receptor is of clinical importance. We previously reported that the protooncogenic c-Cbl protein is specifically recruited by ErbB-1/EGF receptor but not by other ErbB proteins, including...

Colletotrichum trifolii is a fungal pathogen which is responsible for anthracnose disease of alfalfa. To initiate research on molecular communication in this fungus, a kinase-encoding gene (TB3) and the corresponding cDNA were cloned and sequenced. The deduced amino acid sequence of TB3 closely resembles that of a Neurospora crassa serine/threonine protein kinase, COT1, required for hyphal elongation and branching. The C-terminal catalytic domains of TB3 and COT1 are highly conserved but the N-terminal regions are divergent, particularly in the homopolymeric glutamine repeats of TB3. Northern analysis indicated that TB3 expression was highest 1 h after inducing conidial germination and 1 h before germ tubes were first observed. Expression of TB3 transcripts returned to constitutive levels by 4 h after induction of germination. TB3 complemented the cot-I mutant of Neurospora crassa, demonstrating the functional conservation of this kinase between a pathogenic and a saprophytic fungus.

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