Many cellular proteins are post-translationally modified by the addition of a single ubiquitin or a polyubiquitin chain. Among these are receptor tyrosine kinases (RTKs), which undergo ligand-dependent ubiquitination. The ubiquitination of RTKs has become recognized as an important signal for their endocytosis and degradation in the lysosome; however, it is not clear whether ubiquitination itself is sufficient for this process or simply participates in its regulation. The issue is further complicated by the fact that RTKs are thought to be polyubiquitinated - a modification that is linked to protein degradation by the proteasome. By contrast, monoubiquitination has been associated with diverse proteasome-independent cellular functions including intracellular protein movement. Here we show that the epidermal growth factor and platelet-derived growth factor receptors are not polyubiquitinated but rather are monoubiquitinated at multiple sites after their ligand-induced activation. By using different biochemical and molecular genetics approaches, we show that a single ubiquitin is sufficient for both receptor internalization and degradation. Thus, monoubiquitination is the principal signal responsible for the movement of RTKs from the plasma membrane to the lysosome.

Cbl is a multi-adaptor protein involved in ligand-induced downregulation of receptor tyrosine kinases. It is thought that Cbl-mediated ubiquitination of active receptors is essential for receptor degradation and cessation of receptor-induced signal transduction. Here we demonstrate that Cbl additionally regulates epidermal growth factor (EGF) receptor endocytosis. Cbl rapidly recruits CIN85 (Cbl-interacting protein of 85K; ref. 6) and endophilins (regulatory components of clathrin-coated vesicles) to form a complex with activated EGF receptors, thus controlling receptor internalization. CIN85 was constitutively associated with endophilins, whereas CIN85 binding to the distal carboxy terminus of Cbl was increased on EGF stimulation. Inhibition of these interactions was sufficient to block EGF receptor internalization, delay receptor degradation and enhance EGF-induced gene transcription, without perturbing Cbl-directed receptor ubiquitination. Thus, the evolutionary divergent C terminus of Cbl uses a mechanism that is functionally separable from the ubiquitin ligase activity of Cbl to mediate ligand-dependent downregulation of receptor tyrosine kinases.

CIN85 is a multidomain adaptor protein implicated inCbl-mediated down-regulation of receptor tyrosine kinases. CIN85 binding to Cbl is increased after growth factor stimulation and is critical for targeting receptor tyrosine kinases to clathrin-mediated endocytosis. Here we report the identification of a novel polyproline-arginine motif (PXXXPR), specifically recognized by the SH3 domains of CIN85 and its homologue CMS/CD2AP. This motif was indispensable for CIN85 binding to Cbl/Cbl-b, to other CIN85 SH3 domains' effectors, and for mediating an intramolecular interaction between the SH3-A domain and the proline-rich region of CIN85. Individual SH3 domains of CIN85 bound to PXXXPR peptides of Cbl/Cbl-b with micromolar affinities, whereas an extended structure of two or three SH3 domains bound with higher stoichiometry and increased affinity to the same peptides. This enabled full size CIN85 to simultaneously interact with multiple Cbl molecules, promoting their clustering in mammalian cells. The ability of CIN85 to cluster Cbl was important for ligand-induced stabilization of CIN85⅐Cbl⅐epidermal growth factor receptor complexes, as well as for epidermal growth factor receptor degradation in the lysosome. Thus, specific interactions of CIN85 SH3 domains with the PXXXPR motif in Cbl play multiple roles in down-regulation of receptor tyrosine kinases.Growth factor binding to receptor tyrosine kinases (RTKs) 1 promotes receptor autophosphorylation, association of intracellular signaling proteins with receptors, and phosphorylation of multiple substrates (1). Subsequent changes in receptor substrates, including modulations of enzymatic activities and modifications of proteins and lipids, lead to the assembly of signaling networks that ultimately control cellular responses including cell proliferation, migration, or differentiation (2). Activated RTKs are also rapidly relocalized from the cell surface into the endosomal compartment, from where they can be recycled back to the plasma membrane or alternatively sorted to the lysosome for degradation. The processes of receptor internalization and endocytosis are regulated via a network of protein-protein and protein-lipid interactions, as well as protein post-translational modifications, such as phosphorylation or ubiquitination (3, 4). Several RTKs, including epidermal growth factor (EGF) receptors, are ubiquitinated and downregulated upon interactions with the Cbl family of ubiquitin ligases (5, 6). Cbl binding to EGF receptors occurs at the plasma membrane (7,8), and following receptor internalization they remain associated throughout the endocytic compartment (8, 9). Recent data (10) have indicated that attachment of a single ubiquitin, rather than polyubiquitin chains, to EGF and platelet-derived growth factor receptors is sufficient for receptor endocytosis and that multiple monoubiquitination events ensure proper receptor sorting and subsequent degradation in the lysosome.Cbl can also promote receptor endocytosis via a pathway that is functionally separable from its...

No abstract

Addition of ubiquitin or ubiquitin chains to target proteins leads to their mono-or polyubiquitination, respectively. Whereas polyubiquitination targets proteins for degradation, monoubiquitination is thought to regulate receptor internalization and endosomal sorting. Cbl proteins are major ubiquitin ligases that promote ligand-dependent polyubiquitination and degradation of receptor tyrosine kinases. They also recruit CIN85-endophilin in the complex with activated receptors, thus controlling receptor endocytosis. Here we show that the adaptor protein CIN85 and its homologue CMS are monoubiquitinated by Cbl͞Cbl-b after epidermal growth factor (EGF) stimulation. Monoubiquitination of CIN85 required direct interactions between CIN85 and Cbl, the intact RING finger domain of Cbl and a ubiquitin acceptor site present in the carboxyl terminus of CIN85. Cbl-b and monoubiquitinated CIN85 are found in the complex with polyubiquitinated EGF receptors during prolonged EGF stimulation and are degraded together in the lysosome. Dominant interfering forms of CIN85, which have been shown previously to delay EGF receptor degradation, were also impaired in their monoubiquitination. Thus, our data demonstrate that Cbl͞Cbl-b can mediate polyubiquitination of cargo as well as monoubiquitination of CIN85 to control endosomal sorting and degradation of receptor tyrosine kinases.

The mechanisms leading to the ubiquitination and degradation of the activated c-Abl kinase have not yet been identified. We found that the multi-adaptor protein ArgBP2 links c-Abl to the ubiquitin ligase Cbl. Phosphorylation of Cbl and ArgBP2 by c-Abl resulted in the stabilization of their interactions, thus facilitating Cbl-induced ubiquitination and subsequent degradation of c-Abl and ArgBP2.

Cbl proteins control multiple cellular processes by acting as ubiquitin ligases and multifunctional adaptor molecules. They are involved in the control of cell proliferation, differentiation and cell morphology, as well as in pathologies such as autoimmune diseases, inflammation and cancer. Here we review recent advances in understanding the role of Cbl and the importance of a growing repertoire of Cbl-interacting proteins in the regulation of signaling pathways triggered by growth factors, antigens, cell adhesion, cytokines and hormones. We also address key issues of the nature of proteins that bind Cbl in particular cells, where they are located, and how they are altered or traffic within cells upon stimulation. It is becoming obvious that temporal and spatial changes in Cbl signaling networks are essential for the control of physiological processes in a variety of cells and organs and that their deregulation can result in the development of human diseases.

The Pix/Cool proteins are involved in the regulation of cell morphology by binding to small Rho GTPases and kinases of the Pak family. Recently, it has been shown that bPix/Cool-1 associates with the ubiquitin ligase Cbl, which appears to be a critical step in Cdc42-mediated inhibition of epidermal-growth-factor-receptor (EGFR) ubiquitylation and downregulation. Here we show that the SH3 domain of bPix specifically interacts with a prolinearginine motif (PxxxPR) present within the ubiquitin ligase Cbl and Pak1 kinase. Owing to targeting of the same sequence, Cbl and Pak1 compete for binding to bPix. In this complex, Cbl mediates ubiquitylation and subsequent degradation of bPix. Our findings reveal a double feedback loop in which the Cdc42/bPix complex blocks Cbl's ability to downregulate EGFR, while Cbl in turn promotes degradation of bPix in order to escape this inhibition. Such a relationship provides a mechanism to fine-tune the kinetics of RTK endocytosis and degradation depending on the pool of active Cdc42 and the duration of EGFR signaling.