Abstract 1220: Enigma blocks Cbl-c-mediated RET ubiquitination and degradation

Abstract

Abstract RET (rearranged during transfection) is an RTK that is activated by glial cell-derived neurotrophic factors (GDNF) and is critical to neuronal and kidney development. RET mutations causing loss of function are associated with Hirschsprung's disease, while gain of function mutations are associated with thyroid carcinomas and mutiple endocrine neoplasias (MEN2A&B). Cbl proteins are a family of ubiquitin protein ligases that mediate RTK degradation and thereby serve as negative regulators of RTK signaling. Cbl-c (a.k.a. Cbl-3), the most recently identified of the human Cbl proteins, is restricted to epithelial cells and most resembles the Cbl proteins found in lower organisms. Cbl-c can ubiquitinate and downregulate RTKs including activated RET and EGFR. Through yeast two-hybrid screening, we identified the LIM protein Enigma (a.k.a. PDLIM7) as a Cbl-c interacting protein. Enigma is an adapter protein for kinase anchoring to the cytoskeleton and is required for the mitogenic signaling of the RET/PTC2 oncogene. The interaction was confirmed in mammalian cells through co-immunoprecipitation of transfected and endogenous Cbl-c and Enigma. Enigma did not co-immunoprecipitate with c-Cbl or Cbl-b indicating that this interaction is specific to Cbl-c. Cbl-c can ubiquitinate and downregulate an activated form of RET, RET-MEN2A and we show that Enigma prevents Cbl-c-mediated RET ubiquitination and downregulation. This effect is specific to RET as EGFR ubiquitination was unaffected by the presence of Enigma. An Enigma mutant, lacking the RET binding site, does not block Cbl-c recruitment and subsequent ubiquitination and downregulation of RET. Our data suggest that Enigma blocks Cbl-c binding to the RET receptor and prevents its ubiquitination. This is further supported by microscopy data which demonstrates maintained surface expression of RET in the presence of Enigma. Together these data demonstrate a novel interaction between a positive and negative regulator or RET signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1220. doi:10.1158/1538-7445.AM2011-1220