MOLECULAR CHARACTERIZATION OF THE ROLE OF CBL PROTEINS IN EGFR ENDOCYTOSIS

c-Cbl is the major E3 ligase involved in ubiquitination of Epidermal Growth Factor Receptor (EGFR). Ubiquitination by c-Cbl plays a critical role in EGFR endocytosis by targeting receptors to lysosomal degradation. Its involvement at early internalization steps is still debated, also due to the fact that multiple internalization pathways were described. Indeed, EGFR ubiquitination is required for non-clathrin mediated endocytosis (NCE), while it is not essential for clathrin endocytosis (CME). However, c-Cbl might still play a crucial function also in CME since, in addition to its role as an E3 ligase, it works also as an adaptor, by recruiting several proteins involved in the early phases of this process. 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, and are therefore predicted to affect E3 ligase activity. However, some mutations map outside this region, suggesting that they might impinge on the adaptor function without altering E3 ligase activity. None of these mutations was characterized in detail at the mechanistic level. 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 (KD) of c-Cbl (and its family members) on EGFR ubiquitination and endocytosis in two different cell systems, murine fibroblast and HeLa cells. From our data, we confirmed that c-Cbl is essential for NCE, by ubiquitinating the EGFR; however, it also plays a role in CME. Importantly, reconstitution experiments with RING finger mutants demonstrated that c-Cbl E3 ligase activity is also required for CME. Since EGFR ubiquitination is not essential for CME, we hypothesize that this activity is exerted not directly on the receptor but 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. Moreover, in order to dissect c-Cbl adaptor function vs E3 ligase activity, we plan to investigate the phenotype of c-Cbl mutations that map outside the E3 ligase domain in EGFR internalization and ubiquitination, exploiting both in vivo analyses (through reconstitution experiments in cell lines) and in vitro ubiquitination assay.