Direct characterization of E2-dependent target specificity and processivity using an artificial p27-linker-E2 ubiquitination system

BMB Rep. 2008 Dec 31;41(12):852-7. doi: 10.5483/bmbrep.2008.41.12.852.

Abstract

Little attention has been paid to the specificity between E2 and the target protein during ubiquitination, although RING-E3 induces a potential intra-molecular reaction by mediating the direct transfer of ubiquitin from E2 to the target protein. We have constructed artificial E2 fusion proteins in which a target protein (p27) is tethered to one of six E2s via a flexible linker. Interestingly, only three E2s (UbcH5b, hHR6b, and Cdc34) are able to ubiquitinate p27 via an intra-molecular reaction in this system. Although the first ubiquitination of p27 (p27-Ub) by Cdc34 is less efficient than that of UbcH5b and hHR6b, the additional ubiquitin attachment to p27-Ub by Cdc34 is highly efficient. The E2 core of Cdc34 provides specificity to p27, and the residues 184-196 are required for possessive ubiquitination by Cdc34. We demonstrate direct E2 specificity for p27 and also show that differential ubiquitin linkages can be dependent on E2 alone.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Anaphase-Promoting Complex-Cyclosome
  • Cyclin-Dependent Kinase Inhibitor p27
  • Humans
  • In Vitro Techniques
  • Intracellular Signaling Peptides and Proteins / chemistry
  • Intracellular Signaling Peptides and Proteins / genetics
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Lysine / chemistry
  • Models, Biological
  • Proliferating Cell Nuclear Antigen / chemistry
  • Proliferating Cell Nuclear Antigen / genetics
  • Proliferating Cell Nuclear Antigen / metabolism
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity
  • Ubiquitin-Conjugating Enzymes / chemistry
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitin-Protein Ligase Complexes / chemistry
  • Ubiquitin-Protein Ligase Complexes / genetics
  • Ubiquitin-Protein Ligase Complexes / metabolism
  • Ubiquitin-Protein Ligases / chemistry
  • Ubiquitin-Protein Ligases / genetics
  • Ubiquitin-Protein Ligases / metabolism
  • Ubiquitination / physiology*

Substances

  • CDKN1B protein, human
  • Intracellular Signaling Peptides and Proteins
  • Proliferating Cell Nuclear Antigen
  • Recombinant Fusion Proteins
  • Cyclin-Dependent Kinase Inhibitor p27
  • CDC34 protein, human
  • UBE2B protein, human
  • UBE2D2 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome
  • Ubiquitin-Protein Ligases
  • Lysine