Priming and extending: a UbcH5/Cdc34 E2 handoff mechanism for polyubiquitination on a SCF substrate

Mol Cell. 2010 Mar 26;37(6):784-96. doi: 10.1016/j.molcel.2010.02.025.

Abstract

We describe a mechanistic model of polyubiquitination by the SCF(beta TrCP2) E3 ubiquitin (Ub) ligase using human I kappaB alpha as a substrate. Biochemical reconstitution experiments revealed that the polyubiquitination of I kappaB alpha began with the action of the UbcH5 E2 Ub-conjugating enzyme, transferring a single Ub to I kappaB alpha K21/K22 rapidly and efficiently. Subsequently, the Cdc34 E2 functioned in the formation of polyubiquitin chains. It was determined that a Ub fused at I kappaB alpha K21 acts as a receptor, directing Cdc34 for rapid and efficient K48-linked Ub chain synthesis that depends on SCF(beta TrCP2) and the substrate's N terminus. The I kappaB alpha-linked fusion Ub appears to mediate direct contacts with Cdc34 and the SCF's RING subcomplex. Taken together, these results suggest a role for the multifaceted interactions between the I kappaB alpha K21/K22-linked receptor Ub, the SCF's RING complex, and Cdc34 approximately S approximately Ub in establishing the optimal orientation of the receptor Ub to drive conjugation.

Publication types

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

MeSH terms

  • Anaphase-Promoting Complex-Cyclosome
  • Biocatalysis
  • Cell Line, Tumor
  • Humans
  • I-kappa B Kinase / genetics
  • I-kappa B Kinase / metabolism
  • RNA, Small Interfering / genetics
  • SKP Cullin F-Box Protein Ligases / metabolism*
  • Substrate Specificity
  • Ubiquitin-Conjugating Enzymes / genetics
  • Ubiquitin-Conjugating Enzymes / metabolism*
  • Ubiquitin-Protein Ligase Complexes / genetics
  • Ubiquitin-Protein Ligase Complexes / metabolism*
  • Ubiquitination*

Substances

  • RNA, Small Interfering
  • CDC34 protein, human
  • UBE2D1 protein, human
  • UBE2D2 protein, human
  • UBE2D3 protein, human
  • Ubiquitin-Conjugating Enzymes
  • Ubiquitin-Protein Ligase Complexes
  • Anaphase-Promoting Complex-Cyclosome
  • SKP Cullin F-Box Protein Ligases
  • I-kappa B Kinase