Covalent conjugation of proteins by ubiquitin or ubiquitin-like modifiers usually involves a cascade of three enzymatic activities for activating (E1), conjugating (E2), and ligating (E3) ubiquitin or ubiquitin-like modifiers to a substrate. The E3 ubiquitin ligases contain two distinct functions: catalyzing isopeptide bond formation and recruiting the substrate (15
). Two major families of E3 ligases have been described; the HECT domain family that is defined by its homology to E6-associated protein carboxyl terminus (named HECT for homology to E6-associated protein carboxyl terminus) and the RING family that contains either an intrinsic RING finger domain or an associated RING finger protein subunit essential for ubiquitin ligase activity (6
Of several hundred RING finger proteins, ROC1 (named ROC for RING of cullins; also known as Rbx1, Hrt1, and SAG1) is uniquely linked with the ubiquitination of a potentially large number of substrates (8
). Unlike most other RING finger proteins, ROC1 (108 residues) is a small protein with the RING finger taking up 60% of the coding region. Extensive mutational analyses have demonstrated the requirement of the integrity of the RING finger for ubiquitin ligase activity (5
). Purified recombinant ROC1 and ROC2 or their RING finger alone, like that of APC11 (14
), are capable of activating E2-UbcH5 to synthesize polyubiquitin chains in the presence of E1, and removal of N-terminal sequences flanking the RING domain severely reduced ROC1-cullin 1 (CUL1) binding, but not the catalytic function of ROC1 (11
). These results suggest that RING-E2 constitutes the catalytic core of the ubiquitin ligase and that the cullins assemble productive E3 ligases by bringing the RING-E2 catalytic core and substrates together.
The cullins are a family of evolutionarily conserved proteins, which contains three related genes in budding yeast and fission yeast and six related genes in worms, fruit flies, and humans (23
). In addition, three additional proteins, APC2 in all eukaryotes (47
), and PARC and CUL7 in mammals (7
), contain significant sequence homology to cullins over a ~180-amino-acid region involved in binding with ROC1 or APC11. A remarkable aspect of the cullins is that each individual cullin can assemble into multiple distinct E3 ligases by interacting with a protein motif present in multiple proteins.
To recruit specific substrates to CUL1-dependent ligases, the cell utilizes an adaptor protein, SKP1. This adaptor binds simultaneously to an N-terminal domain found in CUL1/Cdc53p, but not other cullins (29
), and to a conserved 40-residue protein motif known as an F-box, which was first recognized in a study of cyclin F and two additional SKP1-binding proteins. F-box proteins contain additional variable protein-protein interaction modules and recruit various substrates, often phosphorylated, to the CUL1-ROC1 catalytic core (2
To recruit specific substrates to CUL2- and possibly CUL5-dependent ligases, a different adaptor is used. A heterodimeric adaptor complex containing elongins B and C binds simultaneously to an analogous N-terminal domain in CUL2 and a conserved 40-residue protein motif, the SOCS box (named SOCS for suppressor of cytokine signaling), which was initially identified in the suppressor of cytokine signaling family of proteins. The SOCS proteins, via their additional protein-protein interaction modules, target various substrates differently to the CUL2- or CUL5-ROC1 catalytic cores (19
). Omitting the adaptor, CUL3 utilizes its N-terminal domain to bind to a conserved 100-residue protein motif known as a BTB domain (named BTB for Drosophila
broad-complex C, Tramtrack, and Bric-a-brac) which was first identified in the Drosophila
broad-complex C (BR-C), Tramtrack (Ttk), and Bric-a-brac (Bab) proteins (53
). BTB proteins, via additional protein-protein interaction domains, then target potentially different substrates to the CUL3-ROC1 catalytic core (10
). The presence of multiple substrate specificity factors—mammals express more than 60 F-box, 40 SOCS, and 200 BTB proteins—suggests that cullins may form by far the largest family of E3 ligases and control the ubiquitination of a potentially large number of substrates. The ability of each cullin-dependent ligase to target the ubiquitination of a large number of substrates may explain various physiological functions linked with individual cullins, such as cell cycle regulation, cell growth control, tumor suppression, and organism development.
In response to oxidative stress and electrophiles, cells express different genes encoding antioxidative and phase II detoxification enzymes. Induction of these genes is regulated largely at the level of transcription, mediated by the antioxidant response element (ARE). The transcriptional activator Nrf2 is a key regulator that interacts with the ARE and is negatively regulated in nonstressed cells by a 75-kDa Kelch-BTB protein, Keap1 (named Keap1 for Kelch-like ECH-associated protein 1) (31
). Targeted deletion of the mouse Keap1
gene resulted in a constitutive accumulation of Nrf2 protein in the nucleus, activation of Nrf2 target genes, and postnatal lethality (in <21 days) that could be rescued by deleting the Nrf2
gene also (43
). These results establish Keap1 as a major regulator of Nrf2 and Nrf2 as a major downstream target of Keap1. The molecular mechanism by which Keap1 negatively regulates Nrf2 function remains incompletely understood. Two different views—sequestering NRF2 in the cytoplasm and promoting NRF2 degradation—are currently being pursued (17
). In this paper, we tested the idea that Keap1 may exert its inhibitory activity toward Nrf2 by targeting it for ubiquitination by the CUL3-ROC1 ligase and subsequent proteasomal degradation.