Redox-regulated turnover of Nrf2 is determined by at least two separate protein domains, the redox-sensitive Neh2 degron and the redox-insensitive Neh6 degron

J Biol Chem. 2004 Jul 23;279(30):31556-67. doi: 10.1074/jbc.M403061200. Epub 2004 May 13.

Abstract

The Nrf2 transcription factor is more rapidly turned over in cells grown under homeostatic conditions than in those experiencing oxidative stress. The variable turnover of Nrf2 is accomplished through the use of at least two degrons and its redox-sensitive interaction with the Kelch-repeat protein Keap1. In homeostatic COS1 cells, the Neh2 degron confers on Nrf2 a half-life of less than 10 min. Analyses of deletion mutants of a Gal4(HA)mNeh2 fusion protein and full-length mNrf2 indicate that full redox-sensitive Neh2 destabilizing activity depends upon two separate sequences within this N-terminal domain. The DIDLID element (amino acids 17-32) is indispensable for Neh2 activity and appears necessary to recruit a ubiquitin ligase to the fusion protein. A second motif within Neh2, the ETGE tetrapeptide (amino acids 79-82), allows the redox-sensitive recruitment of Nrf2 to Keap1. This interaction, which occurs only in homeostatic cells, enhances the capacity of the Neh2 degron to direct degradation by functioning downstream of ubiquitination mediated by the DIDLID element. By contrast with the situation under homeostatic conditions, the Neh2 degron is neither necessary nor sufficient to account for the characteristic half-life of Nrf2 in oxidatively stressed cells. Instead, the previously uncharacterized, redox-insensitive Neh6 degron (amino acids 329-379) is essential to ensure that the transcription factor is still appropriately turned over in stressed cells, albeit with an increased half-life of 40 min. A model can now be proposed to explain how the turnover of this protein adapts in response to alterations in cellular redox state.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Signal Transducing*
  • Amino Acid Motifs
  • Amino Acid Sequence
  • Animals
  • Base Sequence
  • Biological Transport, Active
  • COS Cells
  • Carrier Proteins / metabolism
  • Cytoskeletal Proteins*
  • DNA-Binding Proteins / chemistry*
  • DNA-Binding Proteins / genetics
  • DNA-Binding Proteins / metabolism*
  • Half-Life
  • Homeostasis
  • In Vitro Techniques
  • Kelch-Like ECH-Associated Protein 1
  • Mice
  • Models, Biological
  • Molecular Sequence Data
  • NF-E2-Related Factor 2
  • Oxidation-Reduction
  • Oxidative Stress
  • Plasmids / genetics
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Deletion
  • Sequence Homology, Amino Acid
  • Trans-Activators / chemistry*
  • Trans-Activators / genetics
  • Trans-Activators / metabolism*

Substances

  • Adaptor Proteins, Signal Transducing
  • Carrier Proteins
  • Cytoskeletal Proteins
  • DNA-Binding Proteins
  • Keap1 protein, mouse
  • Kelch-Like ECH-Associated Protein 1
  • NF-E2-Related Factor 2
  • Nfe2l2 protein, mouse
  • Recombinant Fusion Proteins
  • Trans-Activators