Cbl and its role in the ubiquitylation and endocytosis of tyrosine kinase receptors

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Cbl was first identified as the cellular homolog of v-Cbl, a protein expressed by murine retroviruses that potently induces B-cells lymphomas [149]. In mammals, the Cbl family comprises three members,  c- Cbl, Cbl -b and Cbl -c (also named Cbl3), of which c- Cbl is the most studied. c- Cbl, Cbl -b are ubiquitous proteins which exhibit enhanced expression in hematopoïetic tissues, whereas Cbl-c is mostly expressed in epithelial cells. The N-terminus of Cbl proteins contains a TKB (Tyrosine Kinase Binding) domain consisting of a four-helical domain followed by a EF-Hand domain (which link Ca++) and a non classical Src homology 2 (SH2) domain. The variable C-terminal half of Cbl carries a long proline-rich domain, several phosphorylation sites, and a UBA (ubiquitin associated) domain except for Cbl-c. A centrally located RING finger domain separates these two parts of the protein (Fig. 1). Extensive studies in the past years have highlighted the key role of Cbl family members in downregulation of several tyrosine-kinase receptors (RTK).

(RTKs) are downregulated following ligand binding to surface receptors. As RTKs play a determinant role as regulators of cell growth, proliferation and differentiation, extensive investigations have been carried out by many groups with a view to elucidating the process of ligand-induced RTKs downregulation. One prototype RTK is the epidermal growth factor receptor, EGFR. Upon ligand binding, monomeric receptors rapidly dimerize and catalyse auto-phosphorylation on several C-terminal Tyr residues that recruit signaling molecules at the cell surface. Efficient internalization then occurs by means of clathrin-coated pits that invaginate to form coated vesicles. The first clue to the process underlying the internalization of EGFR came from studies of vulva development in worms. Genetic screening in C. elegans identified the SLI-1 protein as an inhibitor of the EGFR-induced differentiation of vulva precursor cells [150]. SLI-1 is a worm ortholog of the mammalian proto-oncogene Cbl  [151].

Extensive mutagenesis in the genes encoding both EGFR andc-Cbl, as well as the possibility to reproduce EGFR ubiquitylation in vitro showed the sequence of events to be as follows. Binding of EGF to EGFR which stimulates the EGFR tyrosine kinase domain, results in an increase in the phosphorylation of the C-terminal Tyr residues, including a specific Tyr that then serves as a docking site for the TKB domain of Cbl. This interaction triggers the phosphorylation of c-Cbl, resulting in recruitment of an E2 and the activation of c-Cbl, which promotes EGFR ubiquitylation [15]. Thus, c-Cbl acts as an E3, and its RING finger domain is required for this activity [152]. Cbl mutants lacking a functional RING finger domain are unable to mediate receptor ubiquitylation and downregulation. Such mutants include oncogenic variants of Cbl, such as N-Cbl [153].

It was initially suggested that ubiquitylation of EGFR occured in endosomes [154]. However, it was then reported that polyubiquitylated forms of the EGFR receptor accumulated upon addition of EGF to HeLa cell overproducing a mutant form of dynamin that blocks the internalization step of endocytosis. Thus, conjugaison with ubiquitin must occur at the plasma membrane before recruitment to clathrin-coated pits [155, 156]. It was further shown that Cbl and EGFR associate at the plasma membrane, and remain associated throughout the endocytic pathway [155]. Whether cell surface EGFR ubiquitylation is the key element of receptor internalization, or merely required for later steps of endocytosis is still a matter of controversy in the literature. Overproduction of Cbl accelerates degradation of EGFR, while overproduction of v-Cbl, a dominant negative form of Cbl inhibiting ubiquitylation does not prevent internalization, but favors recycling of internalized EGFR at the cell surface [15, 154]. This led to the suggestion that EGFR ubiquitylation, despite its occurrence at the plasma membrane, is probably required for late events in the endocytic pathway, such as sorting to the internal vesicles of multivesicular bodies (MVB), an ubiquitin-dependent process [3]. This conclusion was further supported by experiments performed with a cell line carrying a thermosensitive E1 enzyme, which inhibited receptor degradation and not internalization [157]. Contrary to initial conclusions that growth factor receptor are polyubiquitylated, experiments performed with antibodies specific against mono- or polyubiquitin, and overproduction of mutant forms of ubiquitin unable to form polyubiquitin chains demonstrated that EGFR is monoubiquitylated on multiples cytoplasmic tail Lys residues (multi-monoubiquitylation) [158, 159]. Fusion of ubiquitin to a truncated form of EGFR lacking its cytoplasmic tail triggers the ability to internalize radio-labeled EGF in constitutive way, but with lower rate than that observed with wild type EGFR [158]. This observation was interpreted as an additional argument in favor of ubiquitylation acting as an internalization signal, and underlined the requirement of multiple monoubiquitins for efficient internalization. A possible way to reconcile the various data and models would be to consider that EGFR can be internalized by redundant internalization pathways [160, 161], one of these being the CIN85/endophilin pathway. In addition to its role in the ubiquitylation of EGFR and various other RTKs (Table II), c-Cbl was indeed reported to play a critical role in the internalization of RTKs by recruiting various components of the endocytic machinery ([162, 163]. Soubeyran and coworkers searched for partners of c-Cbl; they identified CIN85, an adaptor containing three Src-homology 3 (SH3) domains and a proline-rich region (Fig. 1). The binding of CIN85 to Cbl is increased by EGF stimulation. Furthermore, CIN85 constitutively associates with endophilins via its proline-rich domain and thus recruits endophilins to the complex with activated EGFR receptors [162]. In EGF-stimulated cells, EGFR internalization requires association with an intact Cbl-CIN85-endophilin complex. These data convincingly demonstrate the dual role of c-Cbl in the internalization step of endocytosis. In addition to its action as an E3, c-Cbl recruits endophilin via CIN85, possibly facilitating the invagination of membrane pits [164]. Furthermore, the Cbl-CIN85-endophilin complex remains associated with EGFR along the endocytic pathway