The p62/SQSTM1 (sequestosome 1) protein, which acts as a cargo receptor for autophagic degradation of ubiquitinated targets, is up-regulated by various stressors. Induction of the p62 gene by oxidative stress is mediated by NF-E2-related factor 2 (NRF2) and, at the same time, p62 protein contributes to the activation of NRF2, but hitherto the mechanisms involved were not known. Herein, we have mapped an antioxidant response element (ARE) in the p62 promoter that is responsible for its induction by oxidative stress via NRF2. Chromatin immunoprecipitation and gel mobility-shift assays verified that NRF2 binds to this cis-element in vivo and in vitro. Also, p62 docks directly onto the Kelch-repeat domain of Kelch-like ECH-associated protein 1 (KEAP1), via a motif designated the KEAP1 interacting region (KIR), thereby blocking binding between KEAP1 and NRF2 that leads to ubiquitylation and degradation of the transcription factor. The KIR motif in p62 is located immediately C-terminal to the LC3-interacting region (LIR) and resembles the ETGE motif utilized by NRF2 for its interaction with KEAP1. KIR is required for p62 to stabilize NRF2, and inhibition of KEAP1 by p62 occurs from a cytoplasmic location within the cell. The LIR and KIR motifs cannot be engaged simultaneously by LC3 and KEAP1, but because p62 is polymeric the interaction between KEAP1 and p62 leads to accumulation of KEAP1 in p62 bodies, which is followed by autophagic degradation of KEAP1. Our data explain how p62 contributes to activation of NRF2 target genes in response to oxidative stress through creating a positive feedback loop.

Keap1 is a negative regulator of Nrf2, a bZIP transcription factor that mediates adaptation to oxidative stress. Previous studies suggested this negative regulation is a consequence of Keap1 controlling the subcellular distribution of Nrf2. We now report that Keap1 also controls the total cellular level of Nrf2 protein. In the RL34 non-transformed rat liver cell line, Nrf2 was found to accumulate rapidly in response to oxidative stress caused by treatment with sulforaphane, and the accumulation resulted from inhibition of proteasomal-mediated degradation of the bZIP protein. By heterologously expressing in COS1 cells epitope-tagged Nrf2 and an Nrf2 ⌬ETGE mutant lacking the Keap1-binding site, in both the presence and absence of Keap1 we demonstrate that Nrf2 is subject to ubiquitination and proteasomal degradation independently of both Keap1 and the redox environment of the cell. In oxidatively stressed cells, this is the sole mechanism responsible for Nrf2 degradation. However, under homeostatic conditions Nrf2 is subject to a substantially more rapid mode of proteasomal degradation than it is in oxidatively stressed cells, and this rapid turnover of Nrf2 requires it to interact with Keap1. Within Nrf2, the N-terminal Neh2 domain is identified as the redox-sensitive degron. These data suggest that Keap1 negatively regulates Nrf2 by both enhancing its rate of proteasomal degradation and altering its subcellular distribution.

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Regulation of transcription factor Nrf2 (NF-E2-related factor 2) involves redox-sensitive proteasomal degradation via the E3 ubiquitin ligase Keap1/Cul3. However, Nrf2 is controlled by other mechanisms that have not yet been elucidated. We now show that glycogen synthase kinase 3 (GSK-3) phosphorylates a group of Ser residues in the Neh6 domain of mouse Nrf2 that overlap with an SCF/␤-TrCP destruction motif (DSGIS, residues 334 to 338) and promotes its degradation in a Keap1-independent manner. Nrf2 was stabilized by GSK-3 inhibitors in Keap1-null mouse embryo fibroblasts. Similarly, an Nrf2 ⌬ETGE mutant, which cannot be degraded via Keap1, accumulated when GSK-3 activity was blocked. Phosphorylation of a Ser cluster in the Neh6 domain of Nrf2 stimulated its degradation because a mutant Nrf2 ⌬ETGE 6S/6A protein, lacking these Ser residues, exhibited a longer half-life than Nrf2 ⌬ETGE . Moreover, Nrf2 ⌬ETGE 6S/6A was insensitive to ␤-TrCP regulation and exhibited lower levels of ubiquitination than Nrf2⌬ETGE . GSK-3␤ enhanced ubiquitination of Nrf2 ⌬ETGE but not that of Nrf2 ⌬ETGE 6S/6A . The Nrf2 ⌬ETGE protein but not Nrf2 ⌬ETGE 6S/6A coimmunoprecipitated with ␤-TrCP, and this association was enhanced by GSK-3␤. Our results show for the first time that Nrf2 is targeted by GSK-3 for SCF/␤-TrCP-dependent degradation. We propose a "dual degradation" model to describe the regulation of Nrf2 under different pathophysiological conditions.

Identification of regulatable mechanisms by which transcription factor NF-E2 p45-related factor 2 (Nrf2) is repressed will allow strategies to be designed that counter drug resistance associated with its up-regulation in tumours that harbour somatic mutations in Kelch-like ECH-associated protein-1 (Keap1), a gene that encodes a joint adaptor and substrate receptor for the Cul3-Rbx1/Roc1 ubiquitin ligase. We now show that mouse Nrf2 contains two binding sites for β-transducin repeat-containing protein (β-TrCP), which acts as a substrate receptor for the Skp1-Cul1-Rbx1/Roc1 ubiquitin ligase complex. Deletion of either binding site in Nrf2 decreased β-TrCP-mediated ubiquitylation of the transcription factor. The ability of one of the two β-TrCP-binding sites to serve as a degron could be both increased and decreased by manipulation of glycogen synthase kinase-3 (GSK-3) activity. Biotinylated-peptide pull-down assays identified DSGIS338 and DSAPGS378 as the two β-TrCP-binding motifs in Nrf2. Significantly, our pull-down assays indicated that β-TrCP binds a phosphorylated version of DSGIS more tightly than its non-phosphorylated counterpart, whereas this was not the case for DSAPGS. These data suggest that DSGIS, but not DSAPGS, contains a functional GSK-3 phosphorylation site. Activation of GSK-3 in Keap1-null mouse embryonic fibroblasts (MEFs), or in human lung A549 cells that contain mutant Keap1, by inhibition of the phosphoinositide 3-kinase (PI3K) – protein kinase B (PKB)/Akt pathway markedly reduced endogenous Nrf2 protein and decreased to 10-50% of normal the levels of mRNA for prototypic Nrf2-regulated enzymes, including the glutamate-cysteine ligase catalytic and modifier subunits, glutathione S-transferases Alpha-1 and Mu-1, heme oxygenase-1 and NAD(P)H:quinone oxidoreductase-1. Pre-treatment of Keap1−/− MEFs or A549 cells with the LY294002 PI3K inhibitor or the MK-2206 PKB/Akt inhibitor increased their sensitivity to acrolein, chlorambucil and cisplatin between 1.9-fold and 3.1-fold, and this was substantially attenuated by simultaneous pre-treatment with the GSK-3 inhibitor CT99021.

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.Ubiquitylation underpins virtually all biological processes as it is the major mechanism regulating the stability of critical effector molecules in eukaryotic cells. It refers to the formation of an isopeptide bond between the C terminus of ubiquitin and the ⑀-NH 2 group of a lysine residue in a target protein. The reaction proceeds in three stages with the final critical step, transfer of activated ubiquitin from an E2 2 ubiquitin-conjugating enzyme to an acceptor lysine, facilitated by E3 ubiquitin ligases (1). Modular complexes based around at least four Cullin-RING box (Cul-Rbx) holoenzymes constitute the largest family of E3 ligases identified to date. These holoenzymes, Cul1-Rbx1, Cul2-Rbx1, Cul3-Rbx1, and Cul5-Rbx2, differ in the nature of the substrate adaptors they bind (2-5). For example, Cul1-Rbx1 recruits protein dimers comprising the S-phase kinase-associated protein 1 (Skp1) bound to the eponymous domain found in over 40 F-box proteins (2). The resulting E3 ligase is termed SCF F-box (Skp1, Cul1, and F-box, with the specific F-box protein identified in supercript). Cul3-Rbx1 recruits Broad complex, Tramtrack, and Bric-a-brac (BTB) proteins to generate a large family of ligases that, by analogy with SCF ligases, are referred to as BC 3 B BTB ubiquitin ligases (6 -9). 3Structural similarities exist among substrate adaptors. For example, Skp1 and BTB proteins all utilize BTB folds to interact with Cul proteins (3). Additionally, the domains utilized to recruit substrates can adopt analogous super-secondary structures; whereas F-box proteins frequently use WD40 domains to recruit substrates and BTB proteins commonly exploit Kelchrepeat domains for this purpose, both domains fold to give sixbladed ␤-propel...

Among pregnant women who have had a cesarean section, major maternal complications are almost twice as likely among those whose deliveries are managed with a trial of labor as among those who undergo an elective second cesarean section.

The cap'n'collar (CNC) bZIP transcription factor Nrf2 controls expression of genes for antioxidant enzymes, metal-binding proteins, drug-metabolising enzymes, drug transporters, and molecular chaperones. Many chemicals that protect against carcinogenesis induce Nrf2-target genes. These compounds are all thiol-reactive and stimulate an adaptive response to redox stress in cells. Such agents induce the expression of genes that posses an antioxidant response element (ARE) in their regulatory regions. Under normal homeostatic conditions, Nrf2 activity is restricted through a Keap1-dependent ubiquitylation by Cul3-Rbx1, which targets the CNC-bZIP transcription factor for proteasomal degradation. However, as the substrate adaptor function of Keap1 is redox-sensitive, Nrf2 protein evades ubiquitylation by Cul3-Rbx1 when cells are treated with chemopreventive agents. As a consequence, Nrf2 accumulates in the nucleus where it heterodimerizes with small Maf proteins and transactivates genes regulated through an ARE. In this review, we describe synthetic compounds and phytochemicals from edible plants that induce Nrf2-target genes. We also discuss evidence for the existence of different classes of ARE (a 16-bp 5'-TMAnnRTGABnnnGCR-3' versus an 11-bp 5'-RTGABnnnGCR-3', with or without the embedded activator protein 1-binding site 5'-TGASTCA-3'), species differences in the ARE-gene battery, and the identity of critical Cys residues in Keap1 required for de-repression of Nrf2 by chemopreventive agents.