The ubiquitin conjugating enzyme, UbcM2, engages in novel interactions with components of cullin-3 based E3 ligases

Biochemistry. 2009 Apr 21;48(15):3527-37. doi: 10.1021/bi801971m.

Abstract

The class III ubiquitin conjugating enzymes (E2s) are distinguished from other E2s by the presence of unique N-terminal domains, and the utilization of importin-11 for transport into the nucleus in an activation dependent fashion. To begin determining the physiological roles of these enzymes, we carried out a yeast two-hybrid screen with the class III E2, UbcM2. This screen retrieved RCBTB1, a putative substrate adaptor for a cullin3 (CUL3) E3 ligase. We initially established through biochemical studies that RCBTB1 has the properties of a CUL3 substrate adaptor. Further analysis of the UbcM2-RCBTB1 complex led to the discovery and characterization of the following novel interactions: (i) UbcM2 binds an N-terminal domain of CUL3 requiring the first 57 amino acids, the same domain that binds to RCBTB1 and other substrate adaptors; (ii) UbcM2 does not bind mutants of CUL3 that are deficient in substrate adaptor recruitment; (iii) UbcM2 interacts with CUL3 independent of a bridging RING-finger protein; and (iv) can engage the neddylated (i.e., activated) form of CUL3. We also present evidence that UbcM2 can bind to the N-terminal halves of multiple cullins, implying that this E2 is a general cofactor for this class of ligases. Together, these studies represent the first evidence that UbcM2, in concert with substrate adaptors, engages activated CUL3 ligases, thus suggesting that class III E2s are novel regulators of cullin ligases.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Cell Line
  • Cullin Proteins / chemistry
  • Cullin Proteins / metabolism*
  • Guanine Nucleotide Exchange Factors / metabolism
  • HeLa Cells
  • Humans
  • Mice
  • Protein Binding
  • Substrate Specificity
  • Ubiquitin-Conjugating Enzymes / classification
  • Ubiquitin-Conjugating Enzymes / metabolism
  • Ubiquitin-Conjugating Enzymes / physiology*
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / metabolism*

Substances

  • Cul3 protein, mouse
  • Cullin Proteins
  • Guanine Nucleotide Exchange Factors
  • RCBTB1 protein, human
  • Ube2e1 protein, mouse
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligases