Cbl interacts with multiple E2s in vitro and in cells

PLoS One. 2019 May 23;14(5):e0216967. doi: 10.1371/journal.pone.0216967. eCollection 2019.

Abstract

Many receptor tyrosine kinases (RTKs, such as EGFR, MET) are negatively regulated by ubiquitination and degradation mediated by Cbl proteins, a family of RING finger (RF) ubiquitin ligases (E3s). Loss of Cbl protein function is associated with malignant transformation driven by increased RTK activity. RF E3s, such as the Cbl proteins, interact with a ubiquitin-conjugating enzyme (E2) to confer specificity to the ubiquitination process and direct the transfer of ubiquitin from the E2 to one or more lysines on the target proteins. Using in vitro E3 assays and yeast two-hybrid screens, we found that Ube2d, Ube2e families, Ube2n/2v1, and Ube2w catalyze autoubiquitination of the Cbl protein and Ube2d2, Ube2e1, and Ube 2n/2v1 catalyze Cbl-mediated substrate ubiquitination of the EGFR and SYK. Phosphorylation of the Cbl protein by by Src resulted in increased E3 activity compared to unphosphorylated cbl or Cbl containing a phosphomimetic Y371E mutation. Ubiquitin chain formation depended on the E2 tested with Cbl with Ube2d2 forming both K48 and K63 linked chains, Ube2n/2v1 forming only K63 linked chains, and Ube2w inducing monoubiquitination. In cells, the Ube2d family, Ube2e family, and Ube2n/2v1 contributed to EGFR ubiquitination. Our data suggest that multiple E2s can interact with Cbl and modulate its E3 activity in vitro and in cells.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • ErbB Receptors / metabolism
  • Gene Expression Regulation
  • Gene Silencing
  • HEK293 Cells
  • HeLa Cells
  • Humans
  • Mutation
  • Phosphorylation
  • Protein Binding
  • Proto-Oncogene Proteins c-cbl / genetics
  • Proto-Oncogene Proteins c-cbl / metabolism*
  • Two-Hybrid System Techniques
  • Ubiquitin / metabolism
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitination

Substances

  • Ubiquitin
  • UBE2D2 protein, human
  • UBE2E1 protein, human
  • UBE2E3 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Proto-Oncogene Proteins c-cbl
  • EGFR protein, human
  • ErbB Receptors
  • CBL protein, human