Abstract 4441: Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d

Receptor tyrosine kinases (RTKs) have a demonstrated role as drivers of malignant transformation. Many RTKs (e.g., EGFR, MET, FLT3) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins a family of RING finger (RF) ubiquitin ligases (E3s). There are three mammalian family members Cbl, Cbl-b and Cbl-c. All Cbl proteins have a highly conserved N-terminal tyrosine kinase binding domain and a C3HC4 (RF) domain. Loss of Cbl protein function has be associated with malignant transformation drive by increased RTK activity. E3s such as the Cbl proteins confer specificity to the ubiquitination process and direct the conjugation of ubiquitin to one or more lysines on the specific target proteins in collaboration with ubiquitin-conjugating enzymes (E2s). The E2s bind to the E3 and together they mediate ubiquitination of specific substrates. There are approximately 30 human E2s. All of the E2s have a highly conserved catalytic domain (UBC domain). In this study, we characterized the interaction of Cbl with E2s. Using an in vitro E3 assay, we found that only the Ube2d family (Ube2d1, Ube2d2, and Ube2d3) of E2s mediates autoubiquitination of the three Cbl proteins. Subsequently, using the yeast two-hybrid system, we found that Ube2e1, Ube2e2, Ube2e3, Ube2L3, Ube2u, Ube2w and Ube2z can interact with Cbl even though they do not support autoubiquitination of Cbl in vitro E3 assay. Among these E2s, three Ube2e family members and Ube2w bind to the RF domain of Cbl as demonstrated by the loss of interaction when the RF domain is disrupted. Further, we demonstrated that Ube2e family members are involved in regulation of EGFR activity mediated by Cbl in HeLa cells. Knockdown of Ube2e increases down-regulation and ubiquitination of EGFR in HeLa cells. We further showed that three Ube2e inhibit autoubiquitination of Cbl mediated by Ube2d2 in vitro. Together these data suggest that RTK degradation can be modulated by competition between E2s for the Cbl E3s. Citation Format: Ke Ma, Stanley Lipkowitz, Rachel Klevit, Mariya Sergeyevna Liyasova. Ube2e inhibits the ubiquitination and degradation of EGFR mediated by Cbl and Ube2d. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4441. doi:10.1158/1538-7445.AM2014-4441