Identification of c-Cbl as a New Ligase for Insulin-like Growth Factor-I Receptor with Distinct Roles from Mdm2 in Receptor Ubiquitination and Endocytosis

Abstract

The insulin-like growth factor receptor (IGF-IR) plays several pivotal roles in cancer. Although most studies on the function of the IGF-IR have been attributed to kinase-dependent signaling, recent findings by our group and others have implicated biological roles mediated by ubiquitination of the receptor. As previously reported, the E3 ligases Mdm2 and Nedd4 mediate IGF-IR ubiquitination. Here we show that c-Cbl is a novel E3 ligase for IGF-IR. On ligand stimulation, both Mdm2 and c-Cbl associate with IGF-IR and mediate receptor polyubiquitination. Whereas Mdm2 catalyzed lysine 63 (K63) chain ubiquitination, c-Cbl modified IGF-IR through K48 chains. Mdm2-mediated ubiquitination occurred when cells were stimulated with a low concentration (5 ng/mL) of IGF-I, whereas c-Cbl required high concentrations (50-100 ng/mL). Mdm2-ubiquitinated IGF-IR was internalized through the clathrin endocytic pathway whereas c-Cbl-ubiquitinated receptors were endocytosed via the caveolin route. Taken together, our results show that c-Cbl constitutes a new ligase responsible for the ubiquitination of IGF-IR and that it complements the action of Mdm2 on ubiquitin lysine residue specificity, responsiveness to IGF-I, and type of endocytic pathway used. The actions and interactions of Mdm2 and c-Cbl in the ubiquitination and endocytosis of IGF-IR may have implications in cancer. In addition, identification and functional characterization of new E3 ligases are important in itself because therapeutic targeting of substrate-specific E3 ligases is likely to represent a critical strategy in future cancer treatment.

Polyubiquitination of Insulin-like Growth Factor I Receptor (IGF-IR) Activation Loop Promotes Antibody-induced Receptor Internalization and Down-regulation

“…3 and Ref. 30), whereas c-Cbl has been associated with caveolin-dependent endocytosis of IGF-IR (22). h10H5 treatment resulted not only in Lys-48-linked polyubiquitination, marking the receptor for proteasomal degradation similar to c-Cbl, but also in Lys-29-linked polyubiquitination.…”

“…Three E3 ligases, Nedd4, Mdm2, and Cbl, have been implicated in ligand-mediated ubiquitination (16,17,22). Nedd4-dependent ubiquitination was shown by the use of antibodies preferentially recognizing polyubiquitin chains over sin-gle ubiquitin molecules to induce multiubiquitination of IGF-IR (39).…”

“…Nedd4-dependent ubiquitination was shown by the use of antibodies preferentially recognizing polyubiquitin chains over sin-gle ubiquitin molecules to induce multiubiquitination of IGF-IR (39). In contrast, Mdm2 and c-Cbl conjugate polyubiquitin chains onto IGF-IR with distinct Lys-63 and Lys-48 linkages in an in vitro reconstitution system, respectively (22). The engagement of Mdm2 and c-Cbl correlates with low (5 ng/ml) and high (50 -100 ng/ml) IGF-I levels, respectively.…”

“…Three E3 ligases, Nedd4, Mdm2, and c-Cbl, have been reported to participate in ligand-mediated IGF-IR ubiquitination (16,17,22). Because A549 human lung cancer cells have been shown to undergo IGF-IR ubiquitination upon IGF-I treatment and are more readily transfected than SK-N-AS cells (18), we transfected them with siRNAs to the above E3 ligases.…”

“…Three E3 ligases Nedd4, Mdm2, and c-Cbl have been implicated in mediating IGF-IR ubiquitination (16,17,22). Although exactly how c-Cbl interacts with IGF-IR remains unknown, Nedd4 and Mdm2 have been demonstrated to exhibit their effects through adaptor molecules Grb10 and ␤-arrestin, respectively (16,17).…”