Enigma prevents Cbl-c-mediated ubiquitination and degradation of RETMEN2A.

Therapeutic Approaches

Abstract

The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation.

Authors

Kales, Stephen; Nau, Marion M; Merchant, Anand S; Lipkowitz, Stanley;

Keywords

  • Adaptor Proteins, Signal Transducing/ metabolism
  • Blotting, Western
  • Breast Neoplasms/ metabolism
  • Breast Neoplasms/ mortality
  • Breast Neoplasms/ pathology
  • Cells, Cultured
  • Cytoskeletal Proteins/ metabolism
  • Female
  • HEK293 Cells
  • Humans
  • Immunoprecipitation
  • LIM Domain Proteins/ metabolism
  • Microarray Analysis
  • Multiple Endocrine Neoplasia Type 2a/ metabolism
  • Pancreatic Neoplasms/ metabolism
  • Pancreatic Neoplasms/ pathology
  • Proteolysis
  • Proto-Oncogene Proteins c-cbl/ metabolism
  • Proto-Oncogene Proteins c-ret/ metabolism
  • Two-Hybrid System Techniques
  • Ubiquitination

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