Dimerization of substrate adaptors can facilitate cullin-mediated ubiquitylation of proteins by a "tethering" mechanism: a two-site interaction model for the Nrf2-Keap1 complex

J Biol Chem. 2006 Aug 25;281(34):24756-68. doi: 10.1074/jbc.M601119200. Epub 2006 Jun 21.

Abstract

The prevalence and mechanistic significance of self-association among substrate adaptors for the Cul-Rbx family of ubiquitin ligases remain unclear. We now report that it is as a homodimer that the substrate adaptor Keap1 interacts with Cul3. The resulting complex facilitates ubiquitylation of the Nrf2 transcription factor but only when this substrate possesses within its Neh2 domain a second cryptic Keap1-binding site, the DLG motif, in addition to its previously described ETGE site. Both motifs recognize overlapping surfaces on Keap1, and the seven lysine residues of Nrf2 that act as ubiquitin acceptors lie between them. Based on these data, we propose a "fixed-ends" model for Nrf2 ubiquitylation in which each binding site becomes tethered to a separate subunit of the Keap1 homodimer. This two-site interaction between Keap1 and Nrf2 constrains the mobility of the target lysine residues in the Neh2 domain, increasing their average concentration in the vicinity of the Rbx-bound ubiquitin-conjugating enzyme, and thus the rate at which the transcription factor is ubiquitylated. We show that self-association is a general feature of Cul3 substrate adaptors and propose that the fixed-ends mechanism is commonly utilized to recruit, orientate, and ubiquitylate substrates upon this family of ubiquitin ligases.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing / chemistry*
  • Adaptor Proteins, Signal Transducing / genetics
  • Adaptor Proteins, Signal Transducing / metabolism
  • Amino Acid Sequence
  • Animals
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cullin Proteins / chemistry
  • Cullin Proteins / genetics
  • Cullin Proteins / metabolism
  • Cytoskeletal Proteins / chemistry*
  • Cytoskeletal Proteins / genetics
  • Cytoskeletal Proteins / metabolism
  • Dimerization
  • Kelch-Like ECH-Associated Protein 1
  • Mice
  • Models, Molecular*
  • Molecular Sequence Data
  • NF-E2-Related Factor 2 / chemistry*
  • NF-E2-Related Factor 2 / genetics
  • NF-E2-Related Factor 2 / metabolism
  • Protein Binding
  • Protein Conformation
  • Protein Structure, Tertiary
  • Sequence Analysis

Substances

  • Adaptor Proteins, Signal Transducing
  • Autophagy-Related Proteins
  • Carrier Proteins
  • Cell Cycle Proteins
  • Cullin Proteins
  • Cytoskeletal Proteins
  • Keap1 protein, mouse
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • UBQLN1 protein, human