Home LiteratureArticle Details
PMID: 16449638 Published · ppublish English Journal Article Research Support, N.I.H., Extramural Research Support, Non-U.S. Gov't

CAND1-mediated substrate adaptor recycling is required for efficient repression of Nrf2 by Keap1.

Molecular and cellular biology ·Vol. 26 ·No. 4 ·2006-02-00 ·Pages 1235-44

Lo SC, Hannink M

Abstract

The bZIP transcription factor Nrf2 controls a genetic program that protects cells from oxidative damage and maintains cellular redox homeostasis. Keap1, a BTB-Kelch protein, is the major upstream regulator of Nrf2. Keap1 functions as a substrate adaptor protein for a Cul3-dependent E3 ubiquitin ligase complex to repress steady-state levels of Nrf2 and Nrf2-dependent transcription. Cullin-dependent ubiquitin ligase complexes have been proposed to undergo dynamic cycles of assembly and disassembly that enable substrate adaptor exchange or recycling. In this report, we have characterized the importance of substrate adaptor recycling for regulation of Keap1-mediated repression of Nrf2. Association of Keap1 with Cul3 was decreased by ectopic expression of CAND1 and was increased by small interfering RNA (siRNA)-mediated knockdown of CAND1. However, both ectopic overexpression and siRNA-mediated knockdown of CAND1 decreased the ability of Keap1 to target Nrf2 for ubiquitin-dependent degradation, resulting in stabilization of Nrf2 and activation of Nrf2-dependent gene expression. Neddylation of Cul3 on Lys 712 is required for Keap1-dependent ubiquitination of Nrf2 in vivo. However, the K712R mutant Cul3 molecule, which is not neddylated, can still assemble with Keap1 into a functional ubiquitin ligase complex in vitro. These results provide support for a model in which substrate adaptor recycling is required for efficient substrate ubiquitination by cullin-dependent E3 ubiquitin ligase complexes.

MeSH Terms
Adaptor Proteins, Signal Transducing/genetics,metabolism Animals CHO Cells COS Cells Cell Cycle Proteins/genetics,metabolism Cell Line Chlorocebus aethiops Cricetinae Cullin Proteins/genetics,metabolism HeLa Cells Humans NF-E2-Related Factor 2/genetics,metabolism Recombinant Proteins/genetics,metabolism Repressor Proteins/genetics,metabolism Transcription Factors/genetics,metabolism Ubiquitin-Protein Ligases/genetics,metabolism
Chemicals
Adaptor Proteins, Signal Transducing Cell Cycle Proteins Cullin Proteins NF-E2-Related Factor 2 Recombinant Proteins Repressor Proteins Transcription Factors Ubiquitin-Protein Ligases
Authors & Affiliations
2 authors, click to expand affiliations / ORCID
Lo Shih-Ching
Department of Biochemistry, Life Science Center, M121 Medical Sciences Building, University of Missouri-Columbia, Columbia, MO 65212, USA.
Hannink Mark
References (46)
46 references, click to expand
  1. The amyloid precursor protein-binding protein APP-BP1 drives the cell cycle through the S-M checkpoint and causes apoptosis in neurons.
    J Biol Chem. 2000 Mar 24;275(12):8929-35 PMID: 10722740
  2. Function and regulation of cullin-RING ubiquitin ligases.
    Nat Rev Mol Cell Biol. 2005 Jan;6(1):9-20 PMID: 15688063
  3. Oxidative stress, antioxidant defenses, and damage removal, repair, and replacement systems.
    IUBMB Life. 2000 Oct-Nov;50(4-5):279-89 PMID: 11327322
  4. Promotion of NEDD-CUL1 conjugate cleavage by COP9 signalosome.
    Science. 2001 May 18;292(5520):1382-5 PMID: 11337588
  5. NEDD8 recruits E2-ubiquitin to SCF E3 ligase.
    EMBO J. 2001 Aug 1;20(15):4003-12 PMID: 11483504
  6. Direct evidence that sulfhydryl groups of Keap1 are the sensors regulating induction of phase 2 enzymes that protect against carcinogens and oxidants.
    Proc Natl Acad Sci U S A. 2002 Sep 3;99(18):11908-13 PMID: 12193649
  7. Integration and diversity of the regulatory network composed of Maf and CNC families of transcription factors.
    Gene. 2002 Jul 10;294(1-2):1-12 PMID: 12234662
  8. Role of predicted metalloprotease motif of Jab1/Csn5 in cleavage of Nedd8 from Cul1.
    Science. 2002 Oct 18;298(5593):608-11 PMID: 12183637
  9. NEDD8 modification of CUL1 dissociates p120(CAND1), an inhibitor of CUL1-SKP1 binding and SCF ligases.
    Mol Cell. 2002 Dec;10(6):1511-8 PMID: 12504025
  10. CAND1 binds to unneddylated CUL1 and regulates the formation of SCF ubiquitin E3 ligase complex.
    Mol Cell. 2002 Dec;10(6):1519-26 PMID: 12504026
  11. Regulatory mechanisms controlling gene expression mediated by the antioxidant response element.
    Annu Rev Pharmacol Toxicol. 2003;43:233-60 PMID: 12359864
  12. TIP120A associates with unneddylated cullin 1 and regulates its neddylation.
    FEBS Lett. 2003 Apr 24;541(1-3):102-8 PMID: 12706828
  13. TIP120A associates with cullins and modulates ubiquitin ligase activity.
    J Biol Chem. 2003 May 2;278(18):15905-10 PMID: 12609982
  14. Neddylation and deneddylation of CUL-3 is required to target MEI-1/Katanin for degradation at the meiosis-to-mitosis transition in C. elegans.
    Curr Biol. 2003 May 27;13(11):911-21 PMID: 12781129
  15. Proteolytic targeting of transcriptional regulator TIP120B by a HECT domain E3 ligase.
    J Biol Chem. 2003 Jun 27;278(26):23369-75 PMID: 12692129
  16. Oxidative stress and aging: beyond correlation.
    Aging Cell. 2002 Dec;1(2):117-23 PMID: 12882341
  17. Identification and characterization of DEN1, a deneddylase of the ULP family.
    J Biol Chem. 2003 Aug 1;278(31):28892-900 PMID: 12759362
  18. Release of ubiquitin-charged Cdc34-S - Ub from the RING domain is essential for ubiquitination of the SCF(Cdc4)-bound substrate Sic1.
    Cell. 2003 Sep 5;114(5):611-22 PMID: 13678584
  19. COP9 signalosome: a multifunctional regulator of SCF and other cullin-based ubiquitin ligases.
    Cell. 2003 Sep 19;114(6):663-71 PMID: 14505567
  20. Pathways of oxidative damage.
    Annu Rev Microbiol. 2003;57:395-418 PMID: 14527285
  21. Distinct cysteine residues in Keap1 are required for Keap1-dependent ubiquitination of Nrf2 and for stabilization of Nrf2 by chemopreventive agents and oxidative stress.
    Mol Cell Biol. 2003 Nov;23(22):8137-51 PMID: 14585973
  22. Targeting of protein ubiquitination by BTB-Cullin 3-Roc1 ubiquitin ligases.
    Nat Cell Biol. 2003 Nov;5(11):1001-7 PMID: 14528312
  23. Oxidative stress in cardiovascular disease: myth or fact?
    Arch Biochem Biophys. 2003 Dec 15;420(2):217-21 PMID: 14654060
  24. Protection against electrophile and oxidant stress by induction of the phase 2 response: fate of cysteines of the Keap1 sensor modified by inducers.
    Proc Natl Acad Sci U S A. 2004 Feb 17;101(7):2040-5 PMID: 14764894
  25. Oxidative stress signalling in Alzheimer's disease.
    Brain Res. 2004 Mar 12;1000(1-2):32-9 PMID: 15053949
  26. Nrf2 signaling in coordinated activation of antioxidant gene expression.
    Free Radic Biol Med. 2004 May 15;36(10):1199-207 PMID: 15110384
  27. Arabidopsis ETA2, an apparent ortholog of the human cullin-interacting protein CAND1, is required for auxin responses mediated by the SCF(TIR1) ubiquitin ligase.
    Plant Cell. 2004 Jul;16(7):1883-97 PMID: 15208392
  28. Oxidative stress sensor Keap1 functions as an adaptor for Cul3-based E3 ligase to regulate proteasomal degradation of Nrf2.
    Mol Cell Biol. 2004 Aug;24(16):7130-9 PMID: 15282312
  29. The Keap1-BTB protein is an adaptor that bridges Nrf2 to a Cul3-based E3 ligase: oxidative stress sensing by a Cul3-Keap1 ligase.
    Mol Cell Biol. 2004 Oct;24(19):8477-86 PMID: 15367669
  30. A new NEDD8-ligating system for cullin-4A.
    Genes Dev. 1998 Aug 1;12(15):2263-8 PMID: 9694792
  31. Keap1 represses nuclear activation of antioxidant responsive elements by Nrf2 through binding to the amino-terminal Neh2 domain.
    Genes Dev. 1999 Jan 1;13(1):76-86 PMID: 9887101
  32. Rbx1, a component of the VHL tumor suppressor complex and SCF ubiquitin ligase.
    Science. 1999 Apr 23;284(5414):657-61 PMID: 10213691
  33. Structure of the Cand1-Cul1-Roc1 complex reveals regulatory mechanisms for the assembly of the multisubunit cullin-dependent ubiquitin ligases.
    Cell. 2004 Nov 12;119(4):517-28 PMID: 15537541
  34. Keap1 is a redox-regulated substrate adaptor protein for a Cul3-dependent ubiquitin ligase complex.
    Mol Cell Biol. 2004 Dec;24(24):10941-53 PMID: 15572695
  35. BTB protein Keap1 targets antioxidant transcription factor Nrf2 for ubiquitination by the Cullin 3-Roc1 ligase.
    Mol Cell Biol. 2005 Jan;25(1):162-71 PMID: 15601839
  36. Crystal structure of the Kelch domain of human Keap1.
    J Biol Chem. 2004 Dec 24;279(52):54750-8 PMID: 15475350
  37. Molecular mechanisms activating the Nrf2-Keap1 pathway of antioxidant gene regulation.
    Antioxid Redox Signal. 2005 Mar-Apr;7(3-4):385-94 PMID: 15706085
  38. Requirement of Cul3 for axonal arborization and dendritic elaboration in Drosophila mushroom body neurons.
    J Neurosci. 2005 Apr 20;25(16):4189-97 PMID: 15843622
  39. Keap1, the sensor for electrophiles and oxidants that regulates the phase 2 response, is a zinc metalloprotein.
    Biochemistry. 2005 May 10;44(18):6889-99 PMID: 15865434
  40. Modifying specific cysteines of the electrophile-sensing human Keap1 protein is insufficient to disrupt binding to the Nrf2 domain Neh2.
    Proc Natl Acad Sci U S A. 2005 Jul 19;102(29):10070-5 PMID: 16006525
  41. CAND1 enhances deneddylation of CUL1 by COP9 signalosome.
    Biochem Biophys Res Commun. 2005 Sep 2;334(3):867-74 PMID: 16036220
  42. Ubiquitination of Keap1, a BTB-Kelch substrate adaptor protein for Cul3, targets Keap1 for degradation by a proteasome-independent pathway.
    J Biol Chem. 2005 Aug 26;280(34):30091-9 PMID: 15983046
  43. Specific patterns of electrophile adduction trigger Keap1 ubiquitination and Nrf2 activation.
    J Biol Chem. 2005 Sep 9;280(36):31768-75 PMID: 15985429
  44. Sequence and structural analysis of BTB domain proteins.
    Genome Biol. 2005;6(10):R82 PMID: 16207353
  45. In vitro cleavage of Nedd8 from cullin 1 by COP9 signalosome and deneddylase 1.
    Methods Enzymol. 2005;398:509-22 PMID: 16275355
  46. Conjugation of Nedd8 to CUL1 enhances the ability of the ROC1-CUL1 complex to promote ubiquitin polymerization.
    J Biol Chem. 2000 Oct 13;275(41):32317-24 PMID: 10921923
Article Info
Journal
Molecular and cellular biology
Abbr.
Mol Cell Biol
ISSN
0270-7306
DOI
10.1128/MCB.26.4.1235-1244.2006
Published
2006-02-00
Pages
1235-44
Language
English
Region
United States
NLM ID
8109087
PMCID
PMC1367193
Subset
IM
Grants
NCI NIH HHS · P50 CA103130 · United States
NCI NIH HHS · CA103130 · United States
NCI NIH HHS · CA106593 · United States
External Links
PubMed source DOI: 10.1128/MCB.26.4.1235-1244.2006 PMC Full Text View journal details
Analysis Services
Analysis Services

Contact

No. 2 Wenbo Road, Zhangqiu District, Jinan, Shandong

Qilu Normal University · Genelibs Bioinformatics Lab

750 Shunhua Rd, Jinan

2F, Bldg F, University Science Park

Tel: 0531-88819269

WeChat Official Account

Follow our WeChat subscription account for real-time updates and the latest in medical and biological research.


Business Email

E-mail: [email protected]

Copyright © 2026.Genelibs Jinan Honghui Biotechnology Co., Ltd. 鲁ICP备20007159号-2