BioID-based Identification of Skp Cullin F-box (SCF)β-TrCP1/2 E3 Ligase Substrates

Mol Cell Proteomics. 2015 Jul;14(7):1781-95. doi: 10.1074/mcp.M114.045658. Epub 2015 Apr 21.


The identification of ubiquitin E3 ligase substrates has been challenging, due in part to low-affinity, transient interactions, the rapid degradation of targets and the inability to identify proteins from poorly soluble cellular compartments. SCF(β-TrCP1) and SCF(β-TrCP2) are well-studied ubiquitin E3 ligases that target substrates for proteasomal degradation, and play important roles in Wnt, Hippo, and NFκB signaling. Combining 26S proteasome inhibitor (MG132) treatment with proximity-dependent biotin labeling (BioID) and semiquantitative mass spectrometry, here we identify SCF(β-TrCP1/2) interacting partners. Based on their enrichment in the presence of MG132, our data identify over 50 new putative SCF(β-TrCP1/2) substrates. We validate 12 of these new substrates and reveal previously unsuspected roles for β-TrCP in the maintenance of nuclear membrane integrity, processing (P)-body turnover and translational control. Together, our data suggest that β-TrCP is an important hub in the cellular stress response. The technique presented here represents a complementary approach to more standard IP-MS methods and should be broadly applicable for the identification of substrates for many ubiquitin E3 ligases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Biotin / metabolism*
  • Eukaryotic Initiation Factor-2 / metabolism
  • Gene Knockdown Techniques
  • HeLa Cells
  • Humans
  • Intracellular Signaling Peptides and Proteins / metabolism
  • Membrane Proteins / metabolism
  • Multiprotein Complexes / metabolism
  • Nuclear Envelope / metabolism
  • Phosphorylation
  • Protein Phosphatase 1 / metabolism*
  • Protein Stability
  • Reproducibility of Results
  • SKP Cullin F-Box Protein Ligases / metabolism*
  • Substrate Specificity
  • Ubiquitin / metabolism
  • Ubiquitin-Protein Ligases / metabolism*
  • beta-Transducin Repeat-Containing Proteins / metabolism*


  • Eukaryotic Initiation Factor-2
  • Intracellular Signaling Peptides and Proteins
  • Membrane Proteins
  • Multiprotein Complexes
  • SUN2 protein, human
  • Ubiquitin
  • beta-Transducin Repeat-Containing Proteins
  • Biotin
  • SKP Cullin F-Box Protein Ligases
  • Ubiquitin-Protein Ligases
  • Protein Phosphatase 1