Identifying the ubiquitination targets of E6AP by orthogonal ubiquitin transfer

Nat Commun. 2017 Dec 20;8(1):2232. doi: 10.1038/s41467-017-01974-7.


E3 ubiquitin (UB) ligases are the ending modules of the E1-E2-E3 cascades that transfer UB to cellular proteins and regulate their biological functions. Identifying the substrates of an E3 holds the key to elucidate its role in cell regulation. Here, we construct an orthogonal UB transfer (OUT) cascade to identify the substrates of E6AP, a HECT E3 also known as Ube3a that is implicated in cancer and neurodevelopmental disorders. We use yeast cell surface display to engineer E6AP to exclusively transfer an affinity-tagged UB variant (xUB) to its substrate proteins. Proteomic identification of xUB-conjugated proteins in HEK293 cells affords 130 potential E6AP targets. Among them, we verify that MAPK1, CDK1, CDK4, PRMT5, β-catenin, and UbxD8 are directly ubiquitinated by E6AP in vitro and in the cell. Our work establishes OUT as an efficient platform to profile E3 substrates and reveal the cellular circuits mediated by the E3 enzymes.

Publication types

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

MeSH terms

  • Blood Proteins / metabolism
  • CDC2 Protein Kinase / metabolism
  • Cyclin-Dependent Kinase 4 / metabolism
  • HEK293 Cells
  • Humans
  • Membrane Proteins / metabolism
  • Mitogen-Activated Protein Kinase 1 / metabolism
  • Protein-Arginine N-Methyltransferases / metabolism
  • Proteomics
  • Saccharomyces cerevisiae
  • Ubiquitin / metabolism*
  • Ubiquitin-Activating Enzymes
  • Ubiquitin-Protein Ligases / metabolism*
  • Ubiquitination*
  • beta Catenin / metabolism


  • Blood Proteins
  • FAF2 protein, human
  • Membrane Proteins
  • UBA1 protein, human
  • Ubiquitin
  • beta Catenin
  • PRMT5 protein, human
  • Protein-Arginine N-Methyltransferases
  • UBE3A protein, human
  • Ubiquitin-Protein Ligases
  • CDC2 Protein Kinase
  • CDK1 protein, human
  • CDK4 protein, human
  • Cyclin-Dependent Kinase 4
  • MAPK1 protein, human
  • Mitogen-Activated Protein Kinase 1
  • Ubiquitin-Activating Enzymes