CAND1-Mediated Substrate Adaptor Recycling Is Required for Efficient Repression of Nrf2 by Keap1

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.

Activation of Nrf2 by arsenite and monomethylarsonous acid is independent of Keap1-C151: enhanced Keap1–Cul3 interaction

“…Surprisingly, both As(III) and MMA(III) inhibited the Keap1-dependant E3 ubiquitin ligase by augmenting the association of Keap1 with Cul3, which is in sharp contrast with the enhanced dissociation of Keap1 with Cul3 in response to tBHQ and SF. Keap1, functioning as a substrate adaptor for Nrf2, is constantly undergoing assembly/disassembly with the Cul3-Rbx1 core enzyme (Lo and Hannink, 2006). Under the redox balanced conditions, Keap1 brings Nrf2 into the Cul3-Rbx1 E3 ubiquitin ligase complex for ubiquitination and degradation to maintain low constitutive levels of Nrf2 (Cullinan et al, 2004;Kobayashi et al, 2004;Zhang et al, 2004;Furukawa and Xiong, 2005).…”

“…Proper assembly/disassembly of the Keap1-Cul3 E3 complex is important in controlling ubiquitination of Nrf2. Both increased and decreased association of Keap1 with Cul3, as demonstrated by modulating CAND1 levels with CAND1 si-RNA or CAND1 overexpression, impair the ubiquitination of Nrf2, leading to stabilization of Nrf2 (Lo and Hannink, 2006). In addition, Keap1 mutants that have increased association with Cul3 were shown to have a lower ability to target Nrf2 for ubiquitination (Zhang et al, 2004), further demonstrating the importance of having proper affinity of Keap1 for Cul3 to maintain dynamic assembly/ disassembly of the E3 ligase complex.…”

“…Expression plasmids for Keap1-WT, Keap1-C151S, CBD-tagged version of Keap1-WT and Keap1-C151S, HA-Nrf2, HA-Cul3, HA-Cul3-K712R, and Flag-CAND1 have been described (Zhang and Hannink, 2003;Zhang et al, 2004;Lo and Hannink, 2006). The ARE TATA-Inr luciferase reporter plasmid, named mGST-ARE-Luc, was constructed by insertion of a 41 bp ARE sequence from the promoter of the mouse GST-Ya gene into a cloning site of pGL4.22-luc2CP-Puro (Promega).…”

Fungal development and the COP9 signalosome

“…Cand1 (cullin-associated Nedd8-dissociated protein 1) acts as inhibitor of CRLs in vitro, because it stably binds to the deneddylated cullin-RING subcomplex. The protein is necessary to trigger the exchange of F-box proteins of CRLs [26]. This explains what had been termed the CSN paradox: CSN and Cand1 are inhibitors of CRLs in vitro, but are necessary for CRL assembly and disassembly and therefore CRL activity in vivo.…”

Isolation and Characterization ofcul1-7, a Recessive Allele ofCULLIN1That Disrupts SCF Function at the C Terminus of CUL1 inArabidopsis thaliana

“…This cycle of conjugationdeconjugation is, in part, regulated by CAND1 (CullinAssociated-Neddylation-Disassociated 1) Zheng et al 2002a;Oshikawa et al 2003;Goldenberg et al 2004;Min et al 2005;Bornstein et al 2006;Lo and Hannink 2006;Chew and Hagen 2007). Many outstanding questions remain regarding regulation of SCF ligases.…”