Roberta Pascolutti

c-Cbl is the major E3 ligase involved in ubiquitination of Epidermal Growth Factor Receptor (EGFR). Ubiquitination by c-Cbl targets EGFR to lysosomal degradation. Its involvement at early internalization steps is still debated, due to the fact that multiple internalization pathways were described. Moreover, in addition to its role as an E3 ligase, c-Cbl works also as an adaptor, by recruiting several proteins involved in the early phases of clathrin-mediated endocytosis (CME). Importantly, c-Cbl has been found mutated in different disorders, from myeloproliferative disease to Noonan syndrome and non-small cell lung cancer (NSCLC). Most of these mutations are located within the Ring finger domain and in the regulatory linker region, therefore predicted to affect E3 ligase activity. In order to draw a more precise molecular picture of c-Cbl activity in EGFR ubiquitination and endocytosis, we investigated the effects of different set of cancer-relevant mutations, combining two distinct approaches: 1) RNA interference-based functional assays and 2) in vitro ubiquitination assays.
1) First, we characterized the effect of the knockdown of Cbl proteins on EGFR ubiquitination and endocytosis, confirming its essential role in NCE, by ubiquitinating the EGFR. Importantly, reconstitution experiments with RING finger mutants demonstrated that c-Cbl E3 ligase activity is also required for CME and is possibly exerted on endocytic adaptors. In agreement, Eps15 monoubiquitination is impaired upon c-Cbl KD.
2) We were able to reconstitute the EGFR ubiquitination reaction in vitro, and now we can use this tool to study the molecular details of c-Cbl catalysis.